TY - JOUR T1 - Complete genome reconstruction of the global and European regional dispersal history of the lumpy skin disease virus. JF - J Virol Y1 - 2023 A1 - Steven Van Borm A1 - Simon Dellicour A1 - Darren P Martin A1 - Philippe Lemey A1 - Agianniotaki, Eirini I A1 - Chondrokouki, Eleni D A1 - Vidanovic, Dejan A1 - Nikola Vaskovic A1 - Tamaš Petroviċ A1 - Sava Laziċ A1 - Xhelil Koleci A1 - Vodica, Ani A1 - Igor Djadjovski A1 - Kiril Krstevski A1 - Frank Vandenbussche A1 - Andy Haegeman A1 - Kris De Clercq A1 - Elisabeth Mathijs AB -

Lumpy skin disease virus (LSDV) causes a disease of economic importance affecting cattle. Its global epidemiology is complex due to the combination of vector-borne and anthropogenic spread, the circulation of vaccine-like recombinants, and the use of vaccines. The slow molecular evolution of its DNA genome limits the utility of genetic variation for accurate tracing based on evolutionary analyses, but this limitation has not yet been formally assessed. Furthermore, until present, whole genome sequencing in affected areas has remained patchy. This study combines the first fine-grained sampling of LSDV whole genomes from a time-constrained (2015-2017) southeastern European (SEE) LSDV outbreak, which we analyze along with curated public genomes to investigate the global and regional viral dispersal dynamics. First, haplotype networks visualizing the limited genetic variability associated with the SEE LSDV outbreak show intense intermixing between countries. We also assess at which spatial scale a correlation between genetic and geographic distances can be detected for LSDV. On a global scale, we show the importance of accounting for recombination events that can impact phylogenetic and phylogeographic reconstructions. Following the assessment of the temporal signal in the recombination-free alignment, our time-scaled continuous phylogeographic analysis of Kenya-like and recent wild-type viruses confirms the origin and global dissemination history of LSDV. Our analyses highlight the importance of careful selection and application of phylodynamic approaches to DNA viruses, as well as the importance of whole genome sampling in endemic and outbreak areas to improve our understanding of the evolution, epidemiology, and transmission dynamics of DNA viruses. IMPORTANCE Lumpy skin disease virus (LSDV) has a complex epidemiology involving multiple strains, recombination, and vaccination. Its DNA genome provides limited genetic variation to trace outbreaks in space and time. Sequencing of LSDV whole genomes has also been patchy at global and regional scales. Here, we provide the first fine-grained whole genome sequence sampling of a constrained LSDV outbreak (southeastern Europe, 2015-2017), which we analyze along with global publicly available genomes. We formally evaluate the past occurrence of recombination events as well as the temporal signal that is required for calibrating molecular clock models and subsequently conduct a time-calibrated spatially explicit phylogeographic reconstruction. Our study further illustrates the importance of accounting for recombination events before reconstructing global and regional dynamics of DNA viruses. More LSDV whole genomes from endemic areas are needed to obtain a comprehensive understanding of global LSDV dispersal dynamics.

M3 - 10.1128/jvi.01394-23 ER - TY - JOUR T1 - Complete genome reconstruction of the global and European regional dispersal history of the lumpy skin disease virus. JF - J Virol Y1 - 2023 A1 - Steven Van Borm A1 - Simon Dellicour A1 - Darren P Martin A1 - Philippe Lemey A1 - Agianniotaki, Eirini I A1 - Chondrokouki, Eleni D A1 - Vidanovic, Dejan A1 - Nikola Vaskovic A1 - Tamaš Petroviċ A1 - Sava Laziċ A1 - Xhelil Koleci A1 - Vodica, Ani A1 - Igor Djadjovski A1 - Kiril Krstevski A1 - Frank Vandenbussche A1 - Andy Haegeman A1 - Kris De Clercq A1 - Elisabeth Mathijs AB -

Lumpy skin disease virus (LSDV) causes a disease of economic importance affecting cattle. Its global epidemiology is complex due to the combination of vector-borne and anthropogenic spread, the circulation of vaccine-like recombinants, and the use of vaccines. The slow molecular evolution of its DNA genome limits the utility of genetic variation for accurate tracing based on evolutionary analyses, but this limitation has not yet been formally assessed. Furthermore, until present, whole genome sequencing in affected areas has remained patchy. This study combines the first fine-grained sampling of LSDV whole genomes from a time-constrained (2015-2017) southeastern European (SEE) LSDV outbreak, which we analyze along with curated public genomes to investigate the global and regional viral dispersal dynamics. First, haplotype networks visualizing the limited genetic variability associated with the SEE LSDV outbreak show intense intermixing between countries. We also assess at which spatial scale a correlation between genetic and geographic distances can be detected for LSDV. On a global scale, we show the importance of accounting for recombination events that can impact phylogenetic and phylogeographic reconstructions. Following the assessment of the temporal signal in the recombination-free alignment, our time-scaled continuous phylogeographic analysis of Kenya-like and recent wild-type viruses confirms the origin and global dissemination history of LSDV. Our analyses highlight the importance of careful selection and application of phylodynamic approaches to DNA viruses, as well as the importance of whole genome sampling in endemic and outbreak areas to improve our understanding of the evolution, epidemiology, and transmission dynamics of DNA viruses. IMPORTANCE Lumpy skin disease virus (LSDV) has a complex epidemiology involving multiple strains, recombination, and vaccination. Its DNA genome provides limited genetic variation to trace outbreaks in space and time. Sequencing of LSDV whole genomes has also been patchy at global and regional scales. Here, we provide the first fine-grained whole genome sequence sampling of a constrained LSDV outbreak (southeastern Europe, 2015-2017), which we analyze along with global publicly available genomes. We formally evaluate the past occurrence of recombination events as well as the temporal signal that is required for calibrating molecular clock models and subsequently conduct a time-calibrated spatially explicit phylogeographic reconstruction. Our study further illustrates the importance of accounting for recombination events before reconstructing global and regional dynamics of DNA viruses. More LSDV whole genomes from endemic areas are needed to obtain a comprehensive understanding of global LSDV dispersal dynamics.

M3 - 10.1128/jvi.01394-23 ER - TY - JOUR T1 - Duration of Immunity Induced after Vaccination of Cattle with a Live Attenuated or Inactivated Lumpy Skin Disease Virus Vaccine JF - Microorganisms Y1 - 2023 A1 - Andy Haegeman A1 - Ilse De Leeuw A1 - Laurent Mostin A1 - Willem Van Campe A1 - Wannes Philips A1 - Mehdi Elharrak A1 - Nick De Regge A1 - Kris De Clercq KW - duration of immunity KW - Lumpy skin disease KW - vaccines AB -

Vaccines have proven themselves as an efficient way to control and eradicate lumpy skin disease (LSD). In addition to the safety and efficacy aspects, it is important to know the duration for which the vaccines confer protective immunity, as this impacts the design of an efficient control and eradication program. We evaluated the duration of immunity induced by a live attenuated vaccine (LSDV LAV) and an inactivated vaccine (LSDV Inac), both based on LSDV. Cattle were vaccinated and challenged after 6, 12 and 18 months for LSDV LAV or after 6 and 12 months for the LSDV Inac. The LSDV LAV elicited a strong immune response and protection for up to 18 months, as no clinical signs or viremia could be observed after a viral LSDV challenge in any of the vaccinated animals. A good immune response and protection were similarly seen for the LSDV Inac after 6 months. However, two animals developed clinical signs and viremia when challenged after 12 months. In conclusion, our data support the annual booster vaccination when using the live attenuated vaccine, as recommended by the manufacturer, which could potentially even be prolonged. In contrast, a bi-annual vaccination seems necessary when using the inactivated vaccine.

VL - 11 CP - 1 M3 - 10.3390/microorganisms11010210 ER - TY - JOUR T1 - Epidemiological Dynamics of Foot-and-Mouth Disease in the Horn of Africa: The Role of Virus Diversity and Animal Movement. JF - Viruses Y1 - 2023 A1 - Fanos Tadesse Woldemariyam A1 - Kariuki, Christopher Kinyanjui A1 - Joseph Kamau A1 - Annebel De Vleeschauwer A1 - Kris De Clercq A1 - David Lefebvre A1 - Paeshuyse, Jan KW - Africa KW - Animals KW - Animals, Wild KW - Disease Outbreaks KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Livestock KW - Serogroup AB -

The Horn of Africa is a large area of arid and semi-arid land, holding about 10% of the global and 40% of the entire African livestock population. The region's livestock production system is mainly extensive and pastoralist. It faces countless problems, such as a shortage of pastures and watering points, poor access to veterinary services, and multiple endemic diseases like foot-and-mouth disease (FMD). Foot-and-mouth disease is one of the most economically important livestock diseases worldwide and is endemic in most developing countries. Within Africa, five of the seven serotypes of the FMD virus (FMDV) are described, but serotype C is not circulating anymore, a burden unseen anywhere in the world. The enormous genetic diversity of FMDV is favored by an error-prone RNA-dependent RNA polymerase, intra-typic and inter-typic recombination, as well as the quasi-species nature of the virus. This paper describes the epidemiological dynamics of foot-and-mouth disease in the Horn of Africa with regard to the serotypes and topotypes distribution of FMDV, the livestock production systems practiced, animal movement, the role of wildlife, and the epidemiological complexity of FMD. Within this review, outbreak investigation data and serological studies confirm the endemicity of the disease in the Horn of Africa. Multiple topotypes of FMDV are described in the literature as circulating in the region, with further evolution of virus diversity predicted. A large susceptible livestock population and the presence of wild ungulates are described as complicating the epidemiology of the disease. Further, the husbandry practices and legal and illegal trading of livestock and their products, coupled with poor biosecurity practices, are also reported to impact the spread of FMDV within and between countries in the region. The porosity of borders for pastoralist herders fuels the unregulated transboundary livestock trade. There are no systematic control strategies in the region except for sporadic vaccination with locally produced vaccines, while literature indicates that effective control measures should also consider virus diversity, livestock movements/biosecurity, transboundary trade, and the reduction of contact with wild, susceptible ungulates.

VL - 15 CP - 4 M3 - 10.3390/v15040969 ER - TY - JOUR T1 - Evidence of Lumpy Skin Disease Virus Transmission from Subclinically Infected Cattle by Stomoxys calcitrans JF - Viruses Y1 - 2023 A1 - Andy Haegeman A1 - Charlotte Sohier A1 - Laurent Mostin A1 - Ilse De Leeuw A1 - Willem Van Campe A1 - Wannes Philips A1 - Nick De Regge A1 - Kris De Clercq KW - capripox virus; vector transmission; subclinical infection; stable fly M3 - 10.3390/v15061285 ER - TY - JOUR T1 - Evidence of Lumpy Skin Disease Virus Transmission from Subclinically Infected Cattle by . JF - Viruses Y1 - 2023 A1 - Andy Haegeman A1 - Charlotte Sohier A1 - Laurent Mostin A1 - Ilse De Leeuw A1 - Willem Van Campe A1 - Wannes Philips A1 - Nick De Regge A1 - Kris De Clercq KW - Animals KW - capripoxvirus KW - Cattle KW - Cattle Diseases KW - Insect Vectors KW - Lumpy skin disease KW - Lumpy skin disease virus KW - Male KW - Muscidae AB -

Lumpy skin disease virus (LSDV) is a vector-transmitted capripox virus that causes disease in cattle. flies are considered to be important vectors as they are able to transmit viruses from cattle with the typical LSDV skin nodules to naive cattle. No conclusive data are, however, available concerning the role of subclinically or preclinically infected cattle in virus transmission. Therefore, an in vivo transmission study with 13 donors, experimentally inoculated with LSDV, and 13 naïve acceptor bulls was performed whereby flies were fed on either subclinical- or preclinical-infected donor animals. Transmission of LSDV from subclinical donors showing proof of productive virus replication but without formation of skin nodules was demonstrated in two out of five acceptor animals, while no transmission was seen from preclinical donors that developed nodules after flies had fed. Interestingly, one of the acceptor animals which became infected developed a subclinical form of the disease. Our results show that subclinical animals can contribute to virus transmission. Therefore, stamping out only clinically diseased LSDV-infected cattle could be insufficient to completely halt the spread and control of the disease.

VL - 15 CP - 6 M3 - 10.3390/v15061285 ER - TY - JOUR T1 - Evidence of Lumpy Skin Disease Virus Transmission from Subclinically Infected Cattle by Stomoxys calcitrans JF - Viruses Y1 - 2023 A1 - Andy Haegeman A1 - Charlotte Sohier A1 - Laurent Mostin A1 - Ilse De Leeuw A1 - Willem Van Campe A1 - Philips, Wannes A1 - Nick De Regge A1 - Kris De Clercq VL - 15 CP - 6 M3 - 10.3390/v15061285 ER - TY - JOUR T1 - Assessment of the control measures for category A diseases of Animal Health Law: Lumpy Skin Disease JF - EFSA Journal Y1 - 2022 A1 - Søren Saxmose Nielsen A1 - Julio Alvarez A1 - Dominique Joseph Bicout A1 - Paolo Calistri A1 - Elisabetta Canali A1 - Julian Ashley Drewe A1 - Bruno Garin‐Bastuji A1 - José Luis Gonzalez Rojas A1 - Christian Gortázar Schmidt A1 - Mette Herskin A1 - Virginie Michel A1 - Miguel Ángel Miranda Chueca A1 - Barbara Padalino A1 - Paolo Pasquali A1 - Liisa Helena Sihvonen A1 - Hans Spoolder A1 - Ståhl, Karl A1 - Antonio Velarde A1 - Arvo Viltrop A1 - Christoph Winckler A1 - Kris De Clercq A1 - Gubbins, Simon A1 - Klement, Eyal A1 - Jan Arend Stegeman A1 - Sotiria‐Eleni Antoniou A1 - Inma Aznar A1 - Alessandro Broglia A1 - Yves Van der Stede A1 - Gabriele Zancanaro A1 - Helen Clare Roberts KW - disease control measures KW - LSD KW - Lumpy skin disease KW - monitoringperiod KW - protection zone KW - sampling procedures KW - surveillance zone AB -

EFSA received a mandate from the EC to assess the effectiveness of some of the control measuresagainst diseases included in the Category A list according to Regulation (EU) 2016/429 ontransmissible animal diseases (‘Animal Health Law’). This opinion belongs to a series of opinions wherethese control measures are assessed, with this opinion covering the assessment of control measuresfor Lumpy Skin Disease (LSD). In this opinion, EFSA and the AHAW Panel of experts review theeffectiveness of: i) clinical and laboratory sampling procedures, ii) monitoring period and iii) theminimum radius of the protection and surveillance zones, and the minimum length of time thatmeasures should be applied in these zones. The general methodology used for this series of opinionshas been published elsewhere; nonetheless, the transmission kernels used for the assessment of theminimum radius of the protection and surveillance zones are shown. Several scenarios for which thesecontrol measures had to be assessed were designed and agreed prior to the start of the assessment.The monitoring period was assessed as effective, and based on the transmission kernels available, itwas concluded that the protection zone of 20 km radius and the surveillance zone of 50 km radiuswould comprise>99% of the transmission from an affected establishment if transmission occurred.Recommendations provided for each of the assessed scenarios aim to support the EuropeanCommission in the drafting of further pieces of legislation, as well as for plausible ad hoc requests inrelation to LSD.

VL - 20 CP - 1 M3 - 10.2903/j.efsa.2022.7121 ER - TY - JOUR T1 - Identification of diffusion routes of O/EA-3 topotype of foot-and-mouth disease virus in Africa and Western Asia between 1974 and 2019 - a phylogeographic analysis. JF - Transbound Emerg Dis Y1 - 2022 A1 - Laëtitia Canini A1 - Sandra Blaise-Boisseau A1 - Antonello Di Nardo A1 - Shaw, Andrew E A1 - Aurore Romey A1 - Anthony Relmy A1 - Cindy Bernelin-Cottet A1 - Anne-Laure Salomez A1 - Andy Haegeman A1 - Ularamu, Hussaini A1 - Hafsa Madani A1 - Ouoba, Bruno Lalidia A1 - Zerbo, Habibata Lamouni A1 - Mamadou Lamarana Souare A1 - Cyprien Yapi Boke A1 - Ibrahim Eldaghayes A1 - Abdunaser Dayhum A1 - Moina Hasni Ebou A1 - Abouchoaib, Nabil A1 - Soufien Sghaier A1 - David Lefebvre A1 - Kris De Clercq A1 - Valerie Milouet A1 - Brocchi, Emiliana A1 - Giulia Pezzoni A1 - Charles Nfon A1 - Donald King A1 - Durand, Benoit A1 - Nick Knowles A1 - Kassimi, Labib Bakkali- A1 - Souheyla Benfrid KW - Animals KW - Bayes Theorem KW - Cattle KW - Disease Outbreaks KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Nigeria KW - Phylogeny KW - Serogroup AB -

Foot-and-mouth disease (FMD) affects the livestock industry and socioeconomic sustainability of many African countries. The success of FMD control programs in Africa depends largely on understanding the dynamics of FMD virus (FMDV) spread. In light of the recent outbreaks of FMD that affected the North-Western African countries in 2018 and 2019, we investigated the evolutionary phylodynamics of the causative serotype O viral strains all belonging to the East-Africa 3 topotype (O/EA-3). We analyzed a total of 489 sequences encoding the FMDV VP1 genome region generated from samples collected from 25 African and Western Asian countries between 1974 and 2019. Using Bayesian evolutionary models on genomic and epidemiological data, we inferred the routes of introduction and migration of the FMDV O/EA-3 topotype at the inter-regional scale. We inferred a mean substitution rate of 6.64 × 10  nt/site/year and we predicted that the most recent common ancestor for our panel of samples circulated between February 1967 and November 1973 in Yemen, likely reflecting the epidemiological situation in under sampled cattle-exporting East African countries. Our study also reinforces the role previously described of Sudan and South Sudan as a frequent source of FMDVs spread. In particular, we identified two transboundary routes of O/EA-3 diffusion: the first from Sudan to North-East Africa, and from the latter into Israel and Palestine AT; a second from Sudan to Nigeria, Cameroon, and from there to further into West and North-West Africa. This study highlights the necessity to reinforce surveillance at an inter-regional scale in Africa and Western Asia, in particular along the identified migration routes for the implementation of efficient control measures in the fight against FMD.

VL - 69 CP - 5 M3 - 10.1111/tbed.14562 ER - TY - JOUR T1 - Orbivirus Screening from Imported Captive Oryx in the United Arab Emirates Stresses the Importance of Pre-Import and Transit Measures JF - Pathogens Y1 - 2022 A1 - Martinelle, Ludovic A1 - Andy Haegeman A1 - Louis Lignereux A1 - Anne-Lise Chaber A1 - Dal Pozzo, Fabiana A1 - Ilse De Leeuw A1 - Kris De Clercq A1 - Saegerman, Claude KW - arboviruses KW - biosecurity KW - Bluetongue KW - orbivirus KW - oryx KW - vector-borne disease AB -

From 1975 to 2021, the United Arab Emirates (UAE) imported more than 1300 live Arabian oryxes (AOs) and scimitar-horned oryxes (SHOs) for conservation programs. The objective of this study was to estimate the prevalence of orbiviruses Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in AOs and SHOs from captive herds in the UAE. Between October 2014 and April 2015, 16 AOs and 13 SHOs originating from Texas (USA) and 195 out of about 4000 SHOs from two locations in the UAE were blood sampled to be tested by indirect enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcriptase polymerase chain reaction (RT-qPCR) assays. Eight imported AOs (50% CI [24.7–75.4%]) and eight imported SHOs (61.5% CI [31.6–86.1%]) were found BTV seropositive, in contrast with three out of 195 SHOs (1.5% CI [0.3–4.4%]) from the Emirates. BTV-2 genome was detected in 6/16 of the Arabian Oryx, and amongst those, one out of six was seronegative. None of the tested samples was found positive for EHDV. Our results illustrate the wide local variation regarding BTV seroprevalence in domestic and wild ruminants in the Arabian Peninsula. These results stress the need for pre-import risk assessment when considering translocation of wild ruminant species susceptible to orbiviruses not only in the country of destination but also where transit happens

VL - 11 CP - 6 M3 - 10.3390/pathogens11060697 ER - TY - JOUR T1 - Outbreaks of Foot-and-Mouth Disease in Burundi, East Africa, in 2016, Caused by Different Serotypes. JF - Viruses Y1 - 2022 A1 - Andrea Isabel Estevez Garcia A1 - David Lefebvre A1 - Lionel Nyabongo A1 - Andy Haegeman A1 - Canesius Nkundwanayo A1 - Annebel De Vleeschauwer A1 - Désiré Ntakirutimana A1 - Ilse De Leeuw A1 - Deogratias Nsanganiyumwami A1 - Pascal Niyokwizera A1 - Thierry van den Berg A1 - Alfred Niyokwishimira A1 - Kris De Clercq KW - Africa, Eastern KW - Animals KW - Burundi KW - Cattle KW - Cattle Diseases KW - Disease Outbreaks KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Phylogeny KW - Seroepidemiologic Studies KW - Serogroup AB -

Burundi is a small, densely populated country in the African Great Lakes region. In March 2016, several hundreds of cattle were reported with vesicular lesions, suggesting foot-and-mouth disease (FMD). Epithelial samples, saliva, and blood were collected in six of the affected provinces spread over the country. The overall seroprevalence of FMD virus (FMDV) in the affected herds, as determined by antibodies against FMDV non-structural proteins, was estimated at 87%. Antibodies against FMDV serotypes O (52%), A (44%), C (19%), SAT1 (36%), SAT2 (58%), and SAT3 (23%) were detected across the provinces. FMDV genome was detected in samples from five of the six provinces using rRT-PCR. FMDV was isolated from samples from three provinces: in Cibitoke province, serotypes A and SAT2 were isolated, while in Mwaro and Rutana provinces, only serotype SAT2 was isolated. In Bururi and Cankuzo provinces, the serological profile suggested a recent incursion with serotype SAT2, while in Bubanza province, the serological profile suggested past incursions with serotype O and possibly serotype SAT1. The phylogenetic assessments showed the presence of topotypes A/Africa/G-I and SAT2/IV, similarly to previously characterized virus strains from other countries in the region, suggesting a transboundary origin and necessitating a regional approach for vaccination and control of FMD.

VL - 14 CP - 5 M3 - 10.3390/v14051077 ER - TY - JOUR T1 - Recombinant LSDV Strains in Asia: Vaccine Spillover or Natural Emergence? JF - Viruses Y1 - 2022 A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - Wannes Philips A1 - Saduakassova, Meruyert A1 - Ilse De Leeuw A1 - Sultanov, Akhmetzhan A1 - Andy Haegeman A1 - Kris De Clercq VL - 14 CP - 7 M3 - 10.3390/v14071429 ER - TY - JOUR T1 - A robust, cost-effective and widely applicable whole-genome sequencing protocol for capripoxviruses JF - Journal of Virological Methods Y1 - 2022 A1 - Elisabeth Mathijs A1 - Andy Haegeman A1 - Kris De Clercq A1 - Steven Van Borm A1 - Frank Vandenbussche M3 - 10.1016/j.jviromet.2022.114464 ER - TY - JOUR T1 - Assessment of the control measures of the category A diseases of Animal Health Law: sheep and goat pox JF - EFSA Journal Y1 - 2021 A1 - Søren Saxmose Nielsen A1 - Julio Alvarez A1 - Dominique Joseph Bicout A1 - Paolo Calistri A1 - Elisabetta Canali A1 - Julian Ashley Drewe A1 - Bruno Garin‐Bastuji A1 - José Luis Gonzalez Rojas A1 - Christian Gortázar A1 - Mette Herskin A1 - Virginie Michel A1 - Miguel Ángel Miranda Chueca A1 - Barbara Padalino A1 - Paolo Pasquali A1 - Liisa Helena Sihvonen A1 - Hans Spoolder A1 - Ståhl, Karl A1 - Antonio Velarde A1 - Arvo Viltrop A1 - Christoph Winckler A1 - Kris De Clercq A1 - Gubbins, Simon A1 - Inma Aznar A1 - Alessandro Broglia KW - monitoring period KW - protection zone KW - sampling procedures KW - SPP/GTP KW - surveillance zone AB -

EFSA received a mandate from the European Commission to assess the effectiveness of some of thecontrol measures against diseases included in the Category A list according to Regulation (EU) 2016/429on transmissible animal diseases (‘Animal Health Law’). This opinion belongs to a series of opinions wherethese control measures will be assessed, with this opinion covering the assessment of control measuresfor sheep and goat pox. In this opinion, EFSA and the AHAW Panel of experts review the effectiveness of:(i) clinical and laboratory sampling procedures, (ii) monitoring period and (iii) the minimum radii of theprotection and surveillance zones, and the minimum length of time the measures should be applied inthese zones. The general methodology used for this series of opinions has been published elsewhere;nonetheless, the transmission kernels used for the assessment of the minimum radii of the protectionand surveillance zones are shown. Several scenarios for which these control measures had to beassessed were designed and agreed prior to the start of the assessment. Different risk-based samplingprocedures based on clinical visits and laboratory testing are assessed in case of outbreak suspicion,granting animal movements and for repopulation purposes. The monitoring period of 21 days wasassessed as effective. The estimated probability of transmission beyond the protection zone of 3 kmradius from an infectious establishment is 9.6% (95% CI: 3.1–25.8%) and 2.3% (95% CI: 1–5.5%) forthe surveillance zone of 10 km radius. This may be considered sufficient to contain the disease spread(95% probability of containing transmission corresponds to 5.3 Km). To contain 99% of the spread, theradius should be increased to 19.4 km (95% CI: 9.8–26.8). This may increase the number of farms in thesurveillance zone, since the area would increase fourfold.

VL - 19 CP - 12 M3 - 10.2903/j.efsa.2021.6933 ER - TY - JOUR T1 - Bluetongue Virus Infections in Cattle Herds of Manabí Province of Ecuador JF - Pathogens Y1 - 2021 A1 - Euclides De la Torre A1 - Moreira, Nixon A1 - Saegerman, Claude A1 - Kris De Clercq A1 - Maria Salinas A1 - Alex Maldonado A1 - David Jarrín A1 - Maria Sol Vaca A1 - Silvia Pachacama A1 - Jorge Espinoza A1 - Hipatia Delgado A1 - Maritza Barrera KW - Bluetongue KW - BTV KW - Cattle KW - competitive ELISA KW - Ecuador KW - Manabí KW - PCR AB -

Bluetongue (BT) is a viral disease transmitted by Culicoides (Diptera: Ceratopogonidae) to domestic and wild ruminants. Infections in cattle are mainly subclinical, but severe necrotic and hemorrhagic illness and death may occur depending on the strain of the virus and other factors; cattle act as a reservoir for the virus. Although the Ecuadorian coast has climatic conditions that favor the presence of the vector, there are few serologic or virologic BTV studies available. Manabí is a coastal province in which livestock farming is mostly implemented in the northern part. We conducted two studies to assess, for the first time, the presence of active BTV infections in Manabí province. We collected 430 serum samples from 38 randomly selected farms between March and July 2019 to perform BTV competitive ELISA. In addition, six seropositive farms were selected to place eight sentinel BTV-naive calves. All these calves were blood sampled and the presence of BTV RNA and antibodies was tested for by RT-PCR and competitive ELISA, respectively, once a week for 6–8 weeks until seroconversion was evidenced. A high individual seroprevalence (99%) was obtained, and all investigated farms had BTV seropositive animals. All sentinel calves became BTV viremic and seroconverted. The first viremia appeared after 2–5 weeks from arrival at the farm; they seroconverted 1–3 weeks later. We demonstrate for the first time that there is a high level of BTV circulation north of Manabí, with active infections on these farms. Integrated control strategies such as hygienic measures on farms to reduce midge populations would be advisable for the owners as mitigation measures.

VL - 10 CP - 11 M3 - 10.3390/pathogens10111445 ER - TY - JOUR T1 - Coding-Complete Sequences of Recombinant Lumpy Skin Disease Viruses Collected in 2020 from Four Outbreaks in Northern Vietnam. JF - Microbiol Resour Announc Y1 - 2021 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Nguyen, Long A1 - Laetitia Aerts A1 - Nguyen, Tho A1 - Ilse De Leeuw A1 - Quang, Minh A1 - Nguyen, Hoang Dang A1 - Wannes Philips A1 - Dam, Thi Vui A1 - Andy Haegeman A1 - Steven Van Borm A1 - Kris De Clercq AB -

(LSDV) causes a severe, systemic, and economically important disease in cattle. Here, we report coding-complete sequences of recombinant LSDVs from four outbreaks in October and November 2020 in northeastern Vietnam.

VL - 10 CP - 48 M3 - 10.1128/MRA.00897-21 ER - TY - JOUR T1 - Comparative Evaluation of Lumpy Skin Disease Virus-Based Live Attenuated Vaccines. JF - Vaccines (Basel) Y1 - 2021 A1 - Andy Haegeman A1 - Ilse De Leeuw A1 - Laurent Mostin A1 - Willem Van Campe A1 - Laetitia Aerts A1 - Estelle Venter A1 - Tuppurainen, E S M A1 - Saegerman, Claude A1 - Kris De Clercq AB -

Vaccines form the cornerstone of any control, eradication and preventative strategy and this is no different for lumpy skin disease. However, the usefulness of a vaccine is determined by a multiplicity of factors which include stability, efficiency, safety and ease of use, to name a few. Although the vaccination campaign in the Balkans against lumpy skin disease virus (LSDV) was successful and has been implemented with success in the past in other countries, data of vaccine failure have also been reported. It was therefore the purpose of this study to compare five homologous live attenuated LSDV vaccines (LSDV LAV) in a standardized setting. All five LSDV LAVs studied were able to protect against a challenge with virulent LSDV. Aside from small differences in serological responses, important differences were seen in side effects such as a local reaction and a Neethling response upon vaccination between the analyzed vaccines. These observations can have important implications in the applicability in the field for some of these LSDV LAVs.

VL - 9 CP - 5 M3 - 10.3390/vaccines9050473 ER - TY - JOUR T1 - Detection of Clinical and Subclinical Lumpy Skin Disease Using Ear Notch Testing and Skin Biopsies. JF - Microorganisms Y1 - 2021 A1 - Laetitia Aerts A1 - Andy Haegeman A1 - Ilse De Leeuw A1 - Wannes Philips A1 - Willem Van Campe A1 - Isabelle Behaeghel A1 - Laurent Mostin A1 - Kris De Clercq AB -

Lumpy skin disease (LSD) diagnosis is primarily based on clinical surveillance complemented by PCR of lesion crusts or nodule biopsies. Since LSD can be subclinical, the sensitivity of clinical surveillance could be lower than expected. Furthermore, real-time PCR for the detection of LSD viral DNA in blood samples from subclinical animals is only intermittently positive. Therefore, this study aimed to investigate an acceptable, easily applicable and more sensitive testing method for the detection of clinical and subclinical LSD. An animal experiment was conducted to investigate ear notches and biopsies from unaffected skin taken from the neck and dorsal back as alternatives to blood samples. It was concluded that for early LSD confirmation, normal skin biopsies and ear notches are less fit for purpose, as LSDV DNA is only detectable in these samples several days after it is detectable in blood samples. On the other hand, blood samples are less advisable for the detection of subclinical animals, while ear notches and biopsies were positive for LSD viral DNA in all subclinically infected animals by 16 days post infection. In conclusion, ear notches could be used for surveillance to detect subclinical animals after removing the clinical animals from a herd, to regain trade by substantiating the freedom of disease or to support research on LSDV transmission from subclinical animals.

VL - 9 CP - 10 M3 - 10.3390/microorganisms9102171 ER - TY - JOUR T1 - The history of foot-and-mouth disease virus serotype C: the first known extinct serotype?Abstract JF - Virus Evolution Y1 - 2021 A1 - Paton, David J A1 - Antonello Di Nardo A1 - Nick J Knowles A1 - Jemma Wadsworth A1 - Edviges M Pituco A1 - Ottorino Cosivi A1 - Alejandro M Rivera A1 - Kassimi, Labib Bakkali A1 - Brocchi, Emiliana A1 - Kris De Clercq A1 - Consuelo Carrillo A1 - Francois F Maree A1 - Raj K Singh A1 - Wilna Vosloo A1 - Park, Min-Kyung A1 - Keith J Sumption A1 - Anna B Ludi A1 - King, Donald P KW - extinction KW - foot-and-mouth KW - Phylogeny KW - serotype C AB -

Foot-and-mouth disease (FMD) is a highly contagious animal disease caused by an RNA virus subdivided into seven serotypes that are unevenly distributed in Asia, Africa, and South America. Despite the challenges of controlling FMD, since 1996 there have been only two outbreaks attributed to serotype C, in Brazil and in Kenya, in 2004. This article describes the historical distribution and origins of serotype C and its disappearance. The serotype was first described in Europe in the 1920s, where it mainly affected pigs and cattle but as a less common cause of outbreaks than serotypes O and A. No serotype C outbreaks have been reported in Europe since vaccination stopped in 1990. FMD virus is presumed to have been introduced into South America from Europe in the nineteenth century, although whether serotype C evolved there or in Europe is not known. As in Europe, this serotype was less widely distributed and caused fewer outbreaks than serotypes O and A. Since 1994, serotype C had not been reported from South America until four small outbreaks were detected in the Amazon region in 2004. Elsewhere, serotype C was introduced to Asia, in the 1950s to the 1970s, persisting and evolving for several decades in the Indian subcontinent and for eighteen years in the Philippines. Serotype C virus also circulated in East Africa between 1957 and 2004. Many serotype C viruses from European and Kenyan outbreaks were closely related to vaccine strains, including the most recently recovered Kenyan isolate from 2004. International surveillance has not confirmed any serotype C cases, worldwide, for over 15 years, despite more than 2,000 clinical submissions per year to reference laboratories. Serology provides limited evidence for absence of this serotype, as unequivocal interpretation is hampered by incomplete intra-serotype specificity of immunoassays and the continued use of this serotype in vaccines. It is recommended to continue strengthening surveillance in regions of FMD endemicity, to stop vaccination against serotype C and to reduce working with the virus in laboratories, since inadvertent escape of virus during such activities is now the biggest risk for its reappearance in the field.

VL - 7 CP - 1 M3 - 10.1093/ve/veab009 ER - TY - JOUR T1 - The Importance of Quality Control of LSDV Live Attenuated Vaccines for Its Safe Application in the Field. JF - Vaccines (Basel) Y1 - 2021 A1 - Andy Haegeman A1 - Ilse De Leeuw A1 - Saduakassova, Meruyert A1 - Willem Van Campe A1 - Laetitia Aerts A1 - Wannes Philips A1 - Sultanov, Akhmetzhan A1 - Laurent Mostin A1 - Kris De Clercq KW - Lumpy skin disease KW - Quality Control KW - recombinant KW - vaccine AB -

Vaccination is an effective approach to prevent, control and eradicate diseases, including lumpy skin disease (LSD). One of the measures to address farmer hesitation to vaccinate is guaranteeing the quality of vaccine batches. The purpose of this study was to demonstrate the importance of a quality procedure via the evaluation of the LSD vaccine, Lumpivax (Kevevapi). The initial PCR screening revealed the presence of wild type LSD virus (LSDV) and goatpox virus (GTPV), in addition to vaccine LSDV. New phylogenetic PCRs were developed to characterize in detail the genomic content and a vaccination/challenge trial was conducted to evaluate the impact on efficacy and diagnostics. The characterization confirmed the presence of LSDV wild-, vaccine- and GTPV-like sequences in the vaccine vial and also in samples taken from the vaccinated animals. The analysis was also suggestive for the presence of GTPV-LSDV (vaccine/wild) recombinants. In addition, the LSDV status of some of the animal samples was greatly influenced by the differentiating real-PCR used and could result in misinterpretation. Although the vaccine was clinically protective, the viral genomic content of the vaccine (being it multiple Capripox viruses and/or recombinants) and the impact on the diagnostics casts serious doubts of its use in the field.

VL - 9 CP - 9 M3 - 10.3390/vaccines9091019 ER - TY - JOUR T1 - Improving laboratory diagnostic capacities of emerging diseases using knowledge mapping JF - Transboundary and Emerging Diseases Y1 - 2021 A1 - Mickael Cargnel A1 - Juana Bianchini A1 - Sarah Welby A1 - F. Koenen A1 - Yves Van der Stede A1 - Kris De Clercq A1 - Saegerman, Claude VL - 68 CP - 3 M3 - 10.1111/tbed.13768 ER - TY - RPRT T1 - Key considerations for the implementation of high throughput sequencing based metagenomics (mNGS) in diagnostic clinical, food and veterinary labs : a no-nonsense pointer. the Metastava consortium Y1 - 2021 A1 - Steven Van Borm A1 - Frank Vandenbussche A1 - Kris De Clercq A1 - Andy Haegeman A1 - Sigrid C.J. De Keersmaecker A1 - Kevin Vanneste A1 - Michael Peeters A1 - Steven Van Gucht A1 - Yannick Blanchard A1 - Nicole Pavio A1 - Sandra Martin-Latil A1 - Fabrice Touzain A1 - Isabelle Kempf A1 - Liu, Lihong A1 - Mickhayil Hakhverdyan A1 - Mikael Leijon A1 - Alex Bossers A1 - van der Poel, Wim A1 - Marcel Hulst A1 - Bas Oude Munnink A1 - Miranda De Graaf A1 - Sander van Boheemen A1 - Marion Koopmans A1 - Beer, Martin A1 - Anne Pohlmann A1 - Höper, Dirk ER - TY - JOUR T1 - Nearly Complete Genome Sequences of Two Bluetongue Viruses Isolated during the 2020 Outbreak in the Grand Duchy of Luxembourg. JF - Microbiol Resour Announc Y1 - 2021 A1 - Frank Vandenbussche A1 - Manon Bourg A1 - Elisabeth Mathijs A1 - David Lefebvre A1 - Ilse De Leeuw A1 - Andy Haegeman A1 - Laetitia Aerts A1 - Steven Van Borm A1 - Kris De Clercq AB -

Bluetongue is one of the major diseases of ruminants listed by the World Organisation for Animal Health. Bluetongue virus serotype 8 (BTV-8) has been considered enzootic in France since 2018. Here, we report the nearly complete genome sequences of two BTV-8 isolates from the 2020 outbreak in the Grand Duchy of Luxembourg.

VL - 10 CP - 14 M3 - 10.1128/MRA.00210-21 ER - TY - JOUR T1 - Review: Vaccines and Vaccination against Lumpy Skin Disease JF - Vaccines Y1 - 2021 A1 - Eeva Tuppurainen A1 - Klaas Dietze A1 - Janika Wolff A1 - Hannes Bergmann A1 - Daniel Beltran-Alcrudo A1 - Anna Fahrion A1 - Charles Euloge Lamien A1 - Frank Busch A1 - Carola Sauter-Louis A1 - Franz J. Conraths A1 - Kris De Clercq A1 - Hoffmann, Bernd A1 - Sascha Knauf KW - capripoxvirus; KW - Cattle KW - Immunization KW - LSD KW - Lumpy skin disease KW - Vaccination AB -

The geographical distribution of lumpy skin disease (LSD), an economically important cattle disease caused by a capripoxvirus, has reached an unprecedented extent. Vaccination is the only way to prevent the spread of the infection in endemic and newly affected regions. Yet, in the event of an outbreak, selection of the best vaccine is a major challenge for veterinary authorities and farmers. Decision makers need sound scientific information to support their decisions and subsequent actions. The available vaccine products vary in terms of quality, efficacy, safety, side effects, and price. The pros and cons of different types of live attenuated and inactivated vaccines, vaccination strategies, and associated risks are discussed. Seroconversion, which typically follows vaccination, places specific demands on the tools and methods used to evaluate the effectiveness of the LSD vaccination campaigns in the field. We aimed to give a comprehensive update on available vaccines and vaccination against LSD, to better prepare affected and at-risk countries to control LSD and ensure the safe trade of cattle.

VL - 9 CP - 10 M3 - 10.3390/vaccines9101136 ER - TY - JOUR T1 - Scientific Opinion on the assessment of the control measures for category A diseases of Animal Health Law: Foot and Mouth Disease JF - EFSA Journal Y1 - 2021 A1 - Søren Saxmose Nielsen A1 - Julio Alvarez A1 - Dominique Joseph Bicout A1 - Paolo Calistri A1 - Elisabetta Canali A1 - Julian Ashley Drewe A1 - Bruno Garin‐Bastuji A1 - José Luis Gonzalez Rojas A1 - Christian Gortázar Schmidt A1 - Mette Herskin A1 - Virginie Michel A1 - Miguel Ángel Miranda Chueca A1 - Barbara Padalino A1 - Paolo Pasquali A1 - Liisa Helena Sihvonen A1 - Hans Spoolder A1 - Ståhl, Karl A1 - Antonio Velarde A1 - Arvo Viltrop A1 - Christoph Winckler A1 - Kris De Clercq A1 - Gubbins, Simon A1 - Klement, Eyal A1 - Jan Arend Stegeman A1 - Sotiria‐Eleni Antoniou A1 - Inma Aznar A1 - Alessandro Broglia A1 - Alexandra Papanikolaou A1 - Yves Van der Stede A1 - Gabriele Zancanaro A1 - Helen Clare Roberts AB -

EFSA received a mandate from the European Commission to assess the effectiveness of some of thecontrol measures against diseases included in the Category A list according to Regulation (EU) 2016/429 on transmissible animal diseases (‘Animal Health Law’). This opinion belongs to a series ofopinions where these control measures will be assessed, with this opinion covering the assessmentof control measures for foot and mouth disease (FMD). In this opinion, EFSA and the AHAW Panel ofexperts review the effectiveness of: i) clinical and laboratory sampling procedures, ii) monitoring periodand iii) the minimum radius of the protection and surveillance zones, and the minimum length of timethe measures should be applied in these zones. The general methodology used for this series ofopinions has been published elsewhere; nonetheless, the transmission kernels used for the assessmentof the minimum radius of the protection zone of 3 km and of the surveillance zone of 10 km areshown. Several scenarios for which these control measures had to be assessed were designed andagreed prior to the start of the assessment. The monitoring period of 21 days was assessed aseffective, and it was concluded that the protection and the surveillance zones comprise>99% of theinfections from an affected establishment if transmission occurred. Recommendations, provided foreach of the scenarios assessed, aim to support the European Commission in the drafting of furtherpieces of legislation, as well as for plausible ad hoc requests in relation to FMD.

VL - 19 CP - 6 M3 - 10.2903/j.efsa.2021.6632 ER - TY - JOUR T1 - Scientific Opinion on the assessment of the control measures of the category A diseases of Animal Health Law: Highly Pathogenic Avian Influenza JF - EFSA Journal Y1 - 2021 A1 - Søren Saxmose Nielsen A1 - Julio Alvarez A1 - Dominique Joseph Bicout A1 - Paolo Calistri A1 - Klaus Depner A1 - Julian Ashley Drewe A1 - Bruno Garin‐Bastuji A1 - José Luis Gonzalez Rojas A1 - Schmidt, Christian Gortázar A1 - Mette Herskin A1 - Virginie Michel A1 - Miguel Ángel Miranda Chueca A1 - Paolo Pasquali A1 - Helen Clare Roberts A1 - Liisa Helena Sihvonen A1 - Hans Spoolder A1 - Ståhl, Karl A1 - Calvo, Antonio Velarde A1 - Arvo Viltrop A1 - Christoph Winckler A1 - Kris De Clercq A1 - Klement, Eyal A1 - Jan Arend Stegeman A1 - Gubbins, Simon A1 - Sotiria‐Eleni Antoniou A1 - Alessandro Broglia A1 - Yves Van der Stede A1 - Gabriele Zancanaro A1 - Inma Aznar KW - Avian Influenza KW - Control measures KW - HPAI KW - monitoring period KW - protection zone KW - sampling procedures KW - surveillance zone AB -

EFSA received a mandate from the European Commission to assess the effectiveness of some of thecontrol measures against diseases included in the Category A list according to Regulation (EU) 2016/429on transmissible animal diseases (‘Animal Health Law’). This opinion belongs to a series of opinions wherethese control measures will be assessed, with this opinion covering the assessment of control measuresfor Highly Pathogenic Avian Influenza (HPAI). In this opinion, EFSA and the AHAW Panel of expertsreview the effectiveness of: (i) clinical and laboratory sampling procedures, (ii) monitoring period and (iii)the minimum radius of the protection and surveillance zone, and the minimum length of time themeasures should be applied in these zones. The general methodology used for this series of opinions hasbeen published elsewhere; nonetheless, specific details of the model used for the assessment of thelaboratory sampling procedures for HPAI are presented here. Here, also, the transmission kernels usedfor the assessment of the minimum radius of the protection and surveillance zones are shown. Severalscenarios for which these control measures had to be assessed were designed and agreed prior to thestart of the assessment. In summary, sampling procedures as described in the diagnostic manual forHPAI were considered efficient for gallinaceous poultry, whereas additional sampling is advised forAnseriformes. The monitoring period was assessed as effective, and it was demonstrated that thesurveillance zone comprises 95% of the infections from an affected establishment. Recommendationsprovided for each of the scenarios assessed aim to support the European Commission in the drafting offurther pieces of legislation, as well as for plausible ad hoc requests in relation to HPAI.

VL - 19 CP - 1 M3 - 10.2903/j.efsa.2021.6372 ER - TY - RPRT T1 - Scientific Opinion on the assessment of the control measures of the category A diseases of Animal Health Law: African Swine Fever Y1 - 2021 A1 - Søren Saxmose Nielsen A1 - Julio Alvarez A1 - Dominique Joseph Bicout A1 - Paolo Calistri A1 - Klaus Depner A1 - Julian Ashley Drewe A1 - Bruno Garin‐Bastuji A1 - José Luis Gonzalez Rojas A1 - Christian Gortázar Schmidt A1 - Mette Herskin A1 - Virginie Michel A1 - Miguel Ángel Miranda Chueca A1 - Paolo Pasquali A1 - Helen Clare Roberts A1 - Liisa Helena Sihvonen A1 - Hans Spoolder A1 - Ståhl, Karl A1 - Antonio Velarde A1 - Arvo Viltrop A1 - Christoph Winckler A1 - Kris De Clercq A1 - Klement, Eyal A1 - Jan Arend Stegeman A1 - Gubbins, Simon A1 - Sotiria‐Eleni Antoniou A1 - Alessandro Broglia A1 - Yves Van der Stede A1 - Gabriele Zancanaro A1 - Inma Aznar KW - African Swine Fever KW - disease control measures KW - Suids KW - vector borne disease AB -

EFSA received a mandate from the European Commission to assess the effectiveness of some of the control measures against diseases included in the Category A list according to Regulation (EU) 2016/429 on transmissible animal diseases (‘Animal Health Law’). This opinion belongs to a series of opinions where these control measures will be assessed, with this opinion covering the assessment of control measures for African Swine Fever (ASF). In this opinion, EFSA and the AHAW Panel of experts reviewed the effectiveness of: (i) clinical and laboratory sampling procedures, (ii) monitoring period and (iii) the minimum radius of the protection and surveillance zone, and the minimum length of time the measures should be applied in these zones. The general methodology used for this series of opinions has been published elsewhere; nonetheless, specific details of the model used for the assessment of the laboratory sampling procedures for ASF are presented here. Here, also, the transmission kernels used for the assessment of the minimum radius of the protection and surveillance zones are shown. Several scenarios for which these control measures had to be assessed were designed and agreed prior to the start of the assessment. In summary, several sampling procedures as described in the diagnostic manual for ASF were considered ineffective and a suggestion to exclude, or to substitute with more effective procedures was made. The monitoring period was assessed as non-effective for several scenarios and a longer monitoring period was suggested to ensure detection of potentially infected herds. It was demonstrated that the surveillance zone comprises 95% of the infections from an affected establishment, and therefore is considered effective. Recommendations provided for each of the scenarios assessed aim to support the European Commission in the drafting of further pieces of legislation, as well as for plausible ad hoc requests in relation to ASF.

PB - EFSA VL - 19 CP - 1 M3 - 10.2903/j.efsa.2021.6402 ER - TY - JOUR T1 - Scientific Opinion on the assessment of the control measures of the category A diseases of Animal Health Law: African Horse Sickness JF - EFSA Journal Y1 - 2021 A1 - Søren Saxmose Nielsen A1 - Julio Alvarez A1 - Dominique Joseph Bicout A1 - Paolo Calistri A1 - Klaus Depner A1 - Julian Ashley Drewe A1 - Bruno Garin‐Bastuji A1 - José Luis Gonzalez Rojas A1 - Christian Gortázar Schmidt A1 - Mette Herskin A1 - Virginie Michel A1 - Miguel Ángel Miranda Chueca A1 - Paolo Pasquali A1 - Helen Clare Roberts A1 - Liisa Helena Sihvonen A1 - Hans Spoolder A1 - Ståhl, Karl A1 - Antonio Velarde A1 - Arvo Viltrop A1 - Christoph Winckler A1 - Kris De Clercq A1 - Klement, Eyal A1 - Jan Arend Stegeman A1 - Gubbins, Simon A1 - Sotiria‐Eleni Antoniou A1 - Alessandro Broglia A1 - Yves Van der Stede A1 - Gabriele Zancanaro A1 - Inma Aznar KW - African Horse Sickness KW - Equidae KW - Keywords:Disease control measures KW - monitoring period KW - protection and surveillance zones KW - sampling procedures KW - vector borne disease AB -

EFSA received a mandate from the European Commission to assess the effectiveness of some of the control measures against diseases included in the Category A list according to Regulation (EU) 2016/429 on transmissible animal diseases (‘Animal Health Law’). This opinion belongs to a series of opinions where these control measures will be assessed, with this opinion covering the assessment of control measures for African Horse Sickness (AHS). In this opinion, EFSA and the AHAW Panel of experts review the effectiveness of: (i) clinical and laboratory sampling procedures, (ii) monitoring period and (iii) the minimum radius of the protection and surveillance zone, and the minimum duration of measures in these zones. The general methodology used for this series of opinions has been published elsewhere; nonetheless, specific details of the transmission kernels used for the assessment of the minimum radius of the protection and surveillance zones are shown. Several scenarios for which these control measures were assessed were designed and agreed prior to the start of the assessment. In summary, sampling procedures described in the diagnostic manual for AHS were considered efficient for all Equidae considering the high case fatality rate expected. The monitoring period (14 days) was assessed as effective in every scenario, except for those relating to the epidemiological enquiry where the risk manager should consider increasing the monitoring period, based on the awareness of keepers, environmental conditions and the vector abundance in the region. The current protection zone (100 km) comprises more than 95% of the infections from an affected establishment. Both the radius and duration of the zones could be reduced, based on local environmental conditions and the time of year of the first index case. Recommendations provided for each of the scenarios assessed aim to support the European Commission in the drafting of further pieces of legislation relating to AHS.

VL - 19 CP - 2 M3 - 10.2903/j.efsa.2021.6403 ER - TY - JOUR T1 - Selection and use of reference panels: a case study highlighting current gaps in the materials available for foot and mouth disease JF - Revue Scientifique et Technique de l'OIE Y1 - 2021 A1 - A.B. Ludi A1 - V. Mioulet A1 - L.B. Kassimi A1 - David Lefebvre A1 - Kris De Clercq A1 - E. Chitsungo A1 - N. Nwankpa A1 - W. Vosloo A1 - D. J. Paton A1 - D. P. King KW - Assay validation KW - Case study KW - Diagnostic testing KW - Foot and mouth disease KW - Foot and mouth disease vaccine KW - Gaps KW - Inter-laboratory calibration KW - Reagent KW - Reference panel AB -

The World Organisation for Animal Health (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals describes a diverse array of assays that can be used to detect, characterise and monitor the presence of infectious agents of farmed livestock. These methods have been developed in different laboratories, at different times, and often include tests or kits provided by the commercial sector. Reference panels are essential tools that can be used during assay development and in validation exercises to compare the performance of these varied (and sometimes competing) diagnostic technologies. World Organisation for Animal Health Reference Laboratories already provide approved international standard reagents to help calibrate diagnostic tests for a range of diseases, but there remain important gaps in their availability for comparative purposes and the calibration of test results across different laboratories. Using foot and mouth disease (FMD) as an example, this review highlights four specific areas where new reference reagents are required. These are to: reduce bias in estimates of the diagnostic sensitivity and inter-serotypic specificity of tests used to detect diverse strains of FMD virus (FMDV), provide bio-safe positive controls for new point-of-care test formats that can be deployed outside high containment, harmonise FMDV antigens for post-vaccination serology, and address inter-laboratory differences in serological assays used to measure virus-specific FMD antibody responses. Since there are often limited resources to prepare and distribute these materials, sustainable progress in this arena will only be achievable if there is consensus and coordination of these activities among OIE Reference Laboratories.

VL - 40 CP - 1 M3 - 10.20506/rst.40.1.3221 ER - TY - JOUR T1 - Selection and use of reference panels: a case study highlighting current gaps in the materials available for foot and mouth disease. JF - Rev Sci Tech Y1 - 2021 A1 - Ludi, A B A1 - Mioulet, V A1 - L B Kassimi A1 - David Lefebvre A1 - Kris De Clercq A1 - E Chitsungo A1 - N Nwankpa A1 - Vosloo, W A1 - Paton, D J A1 - King, D P KW - Animals KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Livestock KW - Serogroup KW - Vaccination KW - Viral Vaccines AB -

The World Organisation for Animal Health (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals describes a diverse array of assays that can be used to detect, characterise and monitor the presence of infectious agents of farmed livestock. These methods have been developed in different laboratories, at different times, and often include tests or kits provided by the commercial sector. Reference panels are essential tools that can be used during assay development and in validation exercises to compare the performance of these varied (and sometimes competing) diagnostic technologies. World Organisation for Animal Health Reference Laboratories already provide approved international standard reagents to help calibrate diagnostic tests for a range of diseases, but there remain important gaps in their availability for comparative purposes and the calibration of test results across different laboratories. Using foot and mouth disease (FMD) as an example, this review highlights four specific areas where new reference reagents are required. These are to: reduce bias in estimates of the diagnostic sensitivity and inter-serotypic specificity of tests used to detect diverse strains of FMD virus (FMDV), provide bio-safe positive controls for new point-of-care test formats that can be deployed outside high containment, harmonise FMDV antigens for post-vaccination serology, and address inter-laboratory differences in serological assays used to measure virus-specific FMD antibody responses. Since there are often limited resources to prepare and distribute these materials, sustainable progress in this arena will only be achievable if there is consensus and coordination of these activities among OIE Reference Laboratories.

VL - 40 CP - 1 M3 - 10.20506/rst.40.1.3221 ER - TY - JOUR T1 - A TaqMan probe-based multiplex real-time PCR method for the specific detection of wild type lumpy skin disease virus with beta-actin as internal amplification control. JF - Mol Cell Probes Y1 - 2021 A1 - Agianniotaki, Eirini I A1 - Chaintoutis, Serafeim C A1 - Andy Haegeman A1 - Kris De Clercq A1 - Eleni Chondrokouki A1 - Dovas, Chrysostomos I KW - Adverse reactions KW - Internal amplification control KW - Live attenuated vaccine KW - Lumpy skin disease virus KW - Real-time PCR KW - Wild type virus. AB -

Lumpy skin disease (LSD) is a transboundary disease of economic importance affecting cattle and buffaloes. In South-Eastern Europe, immunization of cattle with homologous live attenuated vaccines for LSD control has prevented outbreaks since 2017, but has been associated with adverse reactions resembling disease symptoms. Thus, a diagnostic method suitable for disease surveillance in farms during vaccination campaigns with Neethling (Onderstepoort) and SIS type (Lumpyvax) live attenuated LSDV vaccines in Europe should be able to detect the wild type (WT) LSDV in animals with adverse reactions to the vaccines and samples with potentially high titers of the vaccine LSDV. To this end, a real-time PCR method targeting the EEV gene of LSDV was developed for the specific detection of WT strains, along with the use of beta-actin gene as an internal amplification control (IAC). Amplification efficiency of the WT virus target was 99.0% and 98.6%, in the presence and in the absence of high loads of vaccine LSDV, respectively. In the presence of 10 vaccine LSDV DNA copies, the limit of detection for WT LSDV was 12.6 DNA copies per reaction. The inter-assay CV was 0.04% for WT LSDV and 0.13% for beta-actin. The method can confirm diagnosis in suspect cases irrespective of the presence of the vaccine LSDV DNA by overcoming the masking effect of the WT LSDV. The simultaneous amplification of the beta-actin gene further assures the quality of diagnostic testing. The new method is a surveillance tool, complementing the DIVA real-time PCR during vaccination campaigns and can provide rapid insight on the targeted EEV gene in countries with novel and recombinant LSDV strains.

VL - 60 M3 - 10.1016/j.mcp.2021.101778 ER - TY - JOUR T1 - Transmission of Bluetongue Virus Serotype 8 by Artificial Insemination with Frozen-Thawed Semen from Naturally Infected Bulls. JF - Viruses Y1 - 2021 A1 - Kris De Clercq A1 - Vandaele, Leen A1 - Vanbinst, Tine A1 - Mickaël Riou A1 - Deblauwe, Isra A1 - Wesselingh, Wendy A1 - Anne Pinard A1 - Mieke Van Eetvelde A1 - Olivier Boulesteix A1 - Bart Leemans A1 - Robert Gélineau A1 - Griet Vercauteren A1 - Van der Heyden, Sara A1 - Jean-François Beckers A1 - Saegerman, Claude A1 - Sammin, Donal A1 - de Kruif, Aart A1 - Ilse De Leeuw KW - Abortion, Veterinary KW - Animals KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Female KW - France KW - Insemination, Artificial KW - Male KW - Pregnancy KW - semen KW - Semen Preservation KW - Serogroup AB -

Transmission of bluetongue (BT) virus serotype 8 (BTV-8) via artificial insemination of contaminated frozen semen from naturally infected bulls was investigated in two independent experiments. Healthy, BT negative heifers were hormonally synchronized and artificially inseminated at oestrus. In total, six groups of three heifers received semen from four batches derived from three bulls naturally infected with BTV-8. Each experiment included one control heifer that was not inseminated and that remained BT negative throughout. BTV viraemia and seroconversion were determined in 8 out of 18 inseminated heifers, and BTV was isolated from five of these animals. These eight heifers only displayed mild clinical signs of BT, if any at all, but six of them experienced pregnancy loss between weeks four and eight of gestation, and five of them became BT PCR and antibody positive. The other two infected heifers gave birth at term to two healthy and BT negative calves. The BT viral load varied among the semen batches used and this had a significant impact on the infection rate, the time of onset of viraemia post artificial insemination, and the gestational stage at which pregnancy loss occurred. These results, which confirm unusual features of BTV-8 infection, should not be extrapolated to infection with other BTV strains without thorough evaluation. This study also adds weight to the hypothesis that the re-emergence of BTV-8 in France in 2015 may be attributable to the use of contaminated bovine semen.

VL - 13 CP - 4 M3 - 10.3390/v13040652 ER - TY - JOUR T1 - Complete Coding Sequence of a Lumpy Skin Disease Virus from an Outbreak in Bulgaria in 2016. JF - Microbiol Resour Announc Y1 - 2020 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Emiliya Ivanova A1 - Andy Haegeman A1 - Laetitia Aerts A1 - Ilse De Leeuw A1 - Steven Van Borm A1 - Kris De Clercq AB -

Lumpy skin disease (LSD) is an emerging cattle disease with serious economic consequences. We report the complete coding sequence of LSD virus 210LSD-249/BUL/16, detected in a blood sample from a diseased cow during an outbreak in Bulgaria (Kabile Village, Yambol Region) in June 2016.

VL - 9 CP - 43 M3 - 10.1128/MRA.00977-20 ER - TY - JOUR T1 - Complete Coding Sequence of a Lumpy Skin Disease Virus Strain Isolated during the 2016 Outbreak in Kazakhstan. JF - Microbiol Resour Announc Y1 - 2020 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Meruyert Saduakassova A1 - Tursyn Kabduldanov A1 - Andy Haegeman A1 - Laetitia Aerts A1 - Taskyn Kyzaibayev A1 - Akhmetzhan Sultanov A1 - Steven Van Borm A1 - Kris De Clercq AB -

Lumpy skin disease virus (LSDV) causes an economically important disease in cattle. Here, we report the complete coding sequence of the LSDV isolate Kubash/KAZ/16, detected in a clinical sample from an infected cow from the outbreak reported on 7 July 2016 in Kazakhstan (Atyrau Region).

VL - 9 CP - 4 M3 - 10.1128/MRA.01399-19 ER - TY - JOUR T1 - Complex Circulation of Foot-and-Mouth Disease Virus in Cattle in Nigeria. JF - Front Vet Sci Y1 - 2020 A1 - Hussaini G Ularamu A1 - David Lefebvre A1 - Andy Haegeman A1 - Yiltawe S Wungak A1 - David O Ehizibolo A1 - David D Lazarus A1 - Annebel De Vleeschauwer A1 - Kris De Clercq KW - foot-and-mouth disease (FMD) KW - Foot-and-Mouth Disease Virus KW - Nigeria KW - Phylogeny KW - topotypes. KW - VP1 AB -

Nigeria is a large densely populated country in West Africa. Most of its livestock is raised in a pastoralist production system with typical long distance migration in search of water and feed. As the demand for animal products largely exceeds the domestic production, large numbers of livestock are imported from neighboring countries without sanitary restrictions. In Nigeria, foot-and-mouth disease virus (FMDV) serotypes O, A, and Southern African Territories (SAT)2 are endemic for a long time. Clinical outbreaks of FMD due to serotype SAT1 are described again since 2015, after an absence of more than 30 years. Historically, outbreaks of FMD due to serotypes O, A, SAT1, and SAT2 were each time associated with trade of cattle entering Nigeria from neighboring countries. In the present study, tissue samples from 27 outbreaks of FMD were collected in Nigerian cattle from 2012 until 2017 in six different States and in the Federal Capital Territory. FMDV was isolated and serotyped and further characterized by VP1 sequencing and phylogenetic analysis to gain more knowledge on FMDV circulation in Nigeria. Half of the outbreaks were characterized as FMDV topotype O/EA-3, while outbreaks with other serotypes and topotypes were-in descending order-less prevalent: A/Africa/G-IV, SAT1/X, SAT2/VII, and O/WA. The high dynamics and omnipresence of FMD in Nigeria were illustrated in Plateau State where FMDV serotypes O, SAT1, and SAT2 were isolated during the course of the study, while at some point in the study, outbreaks due to FMDV serotype A were observed in three remote States. The genetic and phylogenetic analysis suggests a mixed origin of FMD outbreaks. Some outbreaks seem to be caused by sustained local transmission of FMDV strains present in Nigeria since a number of years, while other outbreaks seem to be related to recent incursions with new FMDV strains. The role of African buffaloes in the etiology of FMD in Nigeria is unclear, and sampling of wildlife is needed. The results of the present study suggest that systematic sample collection is essential to understand the complex concomitance of FMDV strains in Nigeria and essential to support the implementation of a vaccination-based control plan.

VL - 7 M3 - 10.3389/fvets.2020.00466 ER - TY - JOUR T1 - An Immunoperoxidase Monolayer Assay (IPMA) for the detection of lumpy skin disease antibodies. JF - J Virol Methods Y1 - 2020 A1 - Andy Haegeman A1 - Ilse De Leeuw A1 - Laurent Mostin A1 - Willem Van Campe A1 - Laetitia Aerts A1 - Maria Vastag A1 - Kris De Clercq KW - antibodies KW - IPMA KW - LSDV. AB -

During this study a new Immunoperoxidase Monolayer Assay (IPMA) was developed for the detection of antibodies against lumpy skin disease virus (LSDV) in an easy and low tech setting. Using two dilutions (1:50 and 1:300) in a duplicate format, the test was shown to be highly sensitive, specific and repeatable. In comparison to the VNT and a commercial ELISA, the LSDV-IPMA was able to detect the LSDV antibodies earlier in infected, vaccinated and vaccinated/infected animals. The assay is very flexible as it can be easily adapted for the detection of sheeppox or goatpox antibodies and it can be scaled-up to handle medium size sample sets by preparing the IPMA plates in advance. These plates are safe and can be handled in low biosafety level labs.

VL - 277 M3 - 10.1016/j.jviromet.2019.113800 ER - TY - JOUR T1 - Lumpy Skin Disease Is Characterized by Severe Multifocal Dermatitis With Necrotizing Fibrinoid Vasculitis Following Experimental Infection. JF - Vet Pathol Y1 - 2020 A1 - Beatriz Sanz-Bernardo A1 - Ismar R Haga A1 - Najith Wijesiriwardana A1 - Philippa C Hawes A1 - Jennifer Simpson A1 - Linda R Morrison A1 - Neil MacIntyre A1 - Brocchi, Emiliana A1 - John Atkinson A1 - Andy Haegeman A1 - Kris De Clercq A1 - Darpel, Karin E A1 - Philippa M Beard KW - Animals KW - Asia KW - Cattle KW - Cattle Diseases KW - Communicable Diseases, Emerging KW - Dermatitis KW - Endemic Diseases KW - Europe KW - Lumpy skin disease KW - Lumpy skin disease virus KW - SKIN KW - Vasculitis AB -

Lumpy skin disease is a high-consequence disease in cattle caused by infection with the poxvirus lumpy skin disease virus (LSDV). The virus is endemic in most countries in Africa and an emerging threat to cattle populations in Europe and Asia. As LSDV spreads into new regions, it is important that signs of disease are recognized promptly by animal caregivers. This study describes the gross, microscopic, and ultrastructural changes that occur over time in cattle experimentally challenged with LSDV. Four calves were inoculated with wildtype LSDV and monitored for 19 to 21 days. At 7 days after inoculation, 2 of the 4 cattle developed multifocal cutaneous nodules characteristic of LSD. Some lesions displayed a targetoid appearance. Histologically, intercellular and intracellular edema was present in the epidermis of some nodules. Occasional intracytoplasmic inclusion bodies were identified in keratinocytes. More severe and consistent changes were present in the dermis, with marked histiocytic inflammation and necrotizing fibrinoid vasculitis of dermal vessels, particularly the deep dermal plexus. Chronic lesions consisted of full-thickness necrosis of the dermis and epidermis. Lesions in other body organs were not a major feature of LSD in this study, highlighting the strong cutaneous tropism of this virus. Immunohistochemistry and electron microscopy identified LSDV-infected histiocytes and fibroblasts in the skin nodules of affected cattle. This study highlights the noteworthy lesions of LSDV and how they develop over time.

VL - 57 CP - 3 M3 - 10.1177/0300985820913268 ER - TY - JOUR T1 - Overview of diagnostic tools for Capripox virus infections. JF - Prev Vet Med Y1 - 2020 A1 - Andy Haegeman A1 - Annebel De Vleeschauwer A1 - Ilse De Leeuw A1 - Dejan Vidanović A1 - Milanko Šekler A1 - Tamaš Petrović A1 - Céline Demarez A1 - David Lefebvre A1 - Kris De Clercq KW - Animals KW - capripoxvirus KW - Cattle KW - Goat Diseases KW - Goats KW - Lumpy skin disease KW - Lumpy skin disease virus KW - Poxviridae Infections KW - Sensitivity and Specificity KW - Serologic Tests KW - Sheep KW - Sheep Diseases KW - Sheep, Domestic AB -

Capripox viruses are the causative agents of important animal diseases in cattle (Lumpy Skin Disease), sheep (Sheeppox) and goats (Goatpox) with severe socio-economic impact in case of wide scale outbreaks. Therefore there is a constant need for adequate diagnostic tools. The assays must be fit-for-purpose to identify the virus quickly and correctly and to be useful for surveillance and monitoring at different stages of an epidemic. Different diagnostic performance characteristics are required depending on the situation and the test purpose. The need for high throughput, high specificity/sensitivity and the capability for differentiating field virus strains from vaccine strains drives the development of new and better assays preferably with an advantageous cost-benefit balance. This review aims to look at existing and new virological and serological diagnostic tools used in the control against diseases caused by Capripox viruses.

VL - 181 M3 - 10.1016/j.prevetmed.2019.104704 ER - TY - JOUR T1 - Prioritization of livestock transboundary diseases in Belgium using a multicriteria decision analysis tool based on drivers of emergence JF - Transboundary and Emerging Diseases Y1 - 2020 A1 - Juana Bianchini A1 - Marie‐France Humblet A1 - Mickael Cargnel A1 - Yves Van der Stede A1 - F. Koenen A1 - Kris De Clercq A1 - Saegerman, Claude VL - 67 CP - 1 M3 - 10.1111/tbed.13356 ER - TY - JOUR T1 - Effectiveness and cost‐benefit study to encourage herd owners in a cost sharing vaccination programme against bluetongue serotype‐8 in Belgium JF - Transboundary and Emerging Diseases Y1 - 2019 A1 - Mickael Cargnel A1 - Yves Van der Stede A1 - Andy Haegeman A1 - Ilse De Leeuw A1 - Kris De Clercq A1 - Estelle Méroc A1 - Sarah Welby VL - 66 CP - 1 M3 - 10.1111/tbed.13034 ER - TY - JOUR T1 - Experimental evidence of mechanical lumpy skin disease virus transmission by Stomoxys calcitrans biting flies and Haematopota spp. horseflies. JF - Sci Rep Y1 - 2019 A1 - Charlotte Sohier A1 - Andy Haegeman A1 - Laurent Mostin A1 - Ilse De Leeuw A1 - Willem Van Campe A1 - A De Vleeschauwer A1 - Tuppurainen, E S M A1 - Thierry van den Berg A1 - Nick De Regge A1 - Kris De Clercq AB -

Lumpy skin disease (LSD) is a devastating disease of cattle characterized by fever, nodules on the skin, lymphadenopathy and milk drop. Several haematophagous arthropod species like dipterans and ticks are suspected to play a role in the transmission of LSDV. Few conclusive data are however available on the importance of biting flies and horseflies as potential vectors in LSDV transmission. Therefore an in vivo transmission study was carried out to investigate possible LSDV transmission by Stomoxys calcitrans biting flies and Haematopota spp. horseflies from experimentally infected viraemic donor bulls to acceptor bulls. LSDV transmission by Stomoxys calcitrans was evidenced in 3 independent experiments, LSDV transmission by Haematopota spp. was shown in one experiment. Evidence of LSD was supported by induction of nodules and virus detection in the blood of acceptor animals. Our results are supportive for a mechanical transmission of the virus by these vectors.

VL - 9 CP - 1 M3 - 10.1038/s41598-019-56605-6 ER - TY - JOUR T1 - Failure to Remove Bluetongue Serotype 8 Virus (BTV-8) From Produced and Derived Bovine Embryos and Subsequent Transmission of BTV-8 to Recipient Cows After Embryo Transfer. JF - Front Vet Sci Y1 - 2019 A1 - Andy Haegeman A1 - Vandaele, Leen A1 - Ilse De Leeuw A1 - André P Oliveira A1 - Nauwynck, Hans A1 - Van Soom, Ann A1 - Kris De Clercq KW - Bluetongue virus KW - bovine embryo KW - BTV-8 KW - IETS guidelines KW - Transmission AB -

The behavior of BTV-8 in cattle is different from most other serotypes not only with regards to clinical signs but certainly with respect to virus transmission (transplacental, contact). Therefore, the possibility of virus transmission by means of embryo transfer was examined by exposure of produced and derived bovine blastocysts to BTV-8 followed by different washing protocols, including longer exposure times (up to 120 s) to 0.25% trypsin at room temperature or at 37°C. None of the washing protocols used was successful in removing the viral genome completely from the produced and derived embryos as was demonstrated by real-time PCR. Moreover, BTV-8 virus was transmitted to recipient cows after embryo transfer of derived BTV8-exposed embryos, which had been subjected to routine decontamination as recommended by IETS, consisting of 5 washes in PBS followed by a double treatment of 0.25% trypsin for 45s at 37°C, and an additional 5 washes in PBS with 2% FCS. This study clearly demonstrates the necessity of vigorous application of the directives for screening of potential donors and the collected embryos, especially in regions with BTV-8, to prevent transmission of the disease.

VL - 6 M3 - 10.3389/fvets.2019.00432 ER - TY - JOUR T1 - Complete Genome Sequences of Five Foot-and-Mouth Disease Viruses of Serotype A Isolated from Cattle in Nigeria between 2013 and 2015. JF - Genome Announc Y1 - 2018 A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - Hussaini G Ularamu A1 - David O Ehizibolo A1 - Andy Haegeman A1 - David Lefebvre A1 - Annebel De Vleeschauwer A1 - Steven Van Borm A1 - Kris De Clercq AB -

The complete genome sequences of 5 foot-and-mouth disease viruses of serotype A are reported here. These viruses originate from outbreaks in northern Nigeria in 2013 to 2015 and belong to the A/AFRICA/G-IV lineage.

VL - 6 CP - 7 M3 - 10.1128/genomeA.00039-18 ER - TY - JOUR T1 - Complete Genome Sequence of the Lumpy Skin Disease Virus Isolated from the First Reported Case in Greece in 2015 JF - Genome Announcements Y1 - 2017 A1 - Agianniotaki, Eirini I. A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Tasioudi, Konstantia E. A1 - Andy Haegeman A1 - Iliadou, Peristera A1 - Chaintoutis, Serafeim C. A1 - Dovas, Chrysostomos I. A1 - Steven Van Borm A1 - Chondrokouki, Eleni D. A1 - Kris De Clercq KW - Illumina KW - LSDV KW - Lumpy skin disease KW - NGS KW - WGS VL - 5 CP - 29 M3 - 10.1128/genomeA.00550-17 ER - TY - JOUR T1 - Complete Genome Sequences of Four Foot-and-Mouth Disease Viruses of Serotype South African Territories 1 (SAT 1), Topotype X, Isolated from Cattle in Nigeria in 2015 JF - Genome Announcements Y1 - 2017 A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - Hussaini G. Ularamu A1 - David O. Ehizibolo A1 - Andy Haegeman A1 - David Lefebvre A1 - David D. Lazarus A1 - Yiltawe S. Wungak A1 - Annebel R. De Vleeschauwer A1 - Steven Van Borm A1 - Kris De Clercq VL - 5 CP - 42 M3 - 10.1128/genomeA.01065-17 ER - TY - JOUR T1 - Correction for Mathijs et al., Complete Genome Sequences of the Neethling-Like Lumpy Skin Disease Virus Strains Obtained Directly from Three Commercial Live Attenuated Vaccines JF - Genome Announcements Y1 - 2017 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Andy Haegeman A1 - King, Alasdair A1 - Nthangeni, Bethuel A1 - Potgieter, Christiaan A1 - Maartens, Louis A1 - Steven Van Borm A1 - Kris De Clercq KW - LSDV KW - Lumpy skin disease KW - NGS KW - PacBio KW - WGS VL - 5 CP - 6 M3 - 10.1128/genomeA.01657-16 ER - TY - JOUR T1 - Detection and Molecular Characterization of Foot and Mouth Disease Viruses from Outbreaks in Some States of Northern Nigeria 2013-2015. JF - Transbound Emerg Dis Y1 - 2017 A1 - Ehizibolo, D O A1 - Andy Haegeman A1 - Annebel De Vleeschauwer A1 - Umoh, J U A1 - Kazeem, H M A1 - Okolocha, E C A1 - Steven Van Borm A1 - Kris De Clercq KW - Animals KW - Capsid Proteins KW - Cattle KW - Cattle Diseases KW - Disease Outbreaks KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Nigeria KW - Phylogeny KW - prevalence KW - Sequence Analysis, Protein KW - Seroepidemiologic Studies KW - Serogroup AB -

Control measures for foot and mouth disease (FMD) in Nigeria have not been implemented due to the absence of locally produced vaccines and risk-based analysis resulting from insufficient data on the circulating FMD virus (FMDV) serotypes/strains. In 2013-2015, blood and epithelial samples were collected from reported FMD outbreaks in four states (Kaduna, Kwara, Plateau and Bauchi) in northern Nigeria. FMDV non-structural protein (NSP) seroprevalence for the outbreaks was estimated at 80% (72 of 90) and 70% (131 of 188) post-outbreak. Antibodies against FMDV serotypes O, A, SAT1, SAT2 and SAT3 were detected across the states using solid-phase competitive ELISA. FMDV genome was detected in 99% (73 of 74) of the samples from FMD-affected animals using rRT-PCR, and cytopathic effect was found in cell culture by 59% (44 of 74) of these samples. Three FMDV serotypes O, A and SAT2 were isolated and characterized. The phylogenetic assessments of the virus isolates showed that two topotypes of FMDV serotype O, East Africa-3 (EA-3) and West Africa (WA) topotypes were circulating, as well as FMDV strains belonging to the Africa genotype (G-IV) of serotype A and FMDV SAT2 topotype VII strains. While the serotype O (EA-3) strains from Nigeria were most closely related to a 1999 virus strain from Sudan, the WA strain in Nigeria shares genetic relationship with three 1988 viruses in Niger. The FMDV serotype A strains were closely related to a known virus from Cameroon, and the SAT2 strains were most closely related to virus subtypes in Libya. This study provides evidence of co-occurrence of FMDV serotypes and topotypes in West, Central, East and North Africa, and this has implication for control. The findings help filling the knowledge gap of FMDV dynamics in Nigeria and West Africa subregion to support local and regional development of vaccination-based control plans and international risk assessment.

VL - 64 CP - 6 U1 - https://www.ncbi.nlm.nih.gov/pubmed/28097814?dopt=Abstract M3 - 10.1111/tbed.12602 ER - TY - JOUR T1 - Development and validation of a TaqMan probe-based real-time PCR method for the differentiation of wild type lumpy skin disease virus from vaccine virus strains. JF - J Virol Methods Y1 - 2017 A1 - Agianniotaki, Eirini I A1 - Chaintoutis, Serafeim C A1 - Andy Haegeman A1 - Tasioudi, Konstantia E A1 - Ilse De Leeuw A1 - Katsoulos, Panagiotis-Dimitrios A1 - Sachpatzidis, Achilleas A1 - Kris De Clercq A1 - Alexandropoulos, Thomas A1 - Polizopoulou, Zoe S A1 - Chondrokouki, Eleni D A1 - Dovas, Chrysostomos I AB -

Lumpy skin disease (LSD) is a transboundary viral disease of cattle with severe economic impact. Immunization of cattle with homologous live attenuated vaccines poses a number of diagnostic problems, as it has been associated with adverse reactions resembling disease symptoms. The latter hampers clinical diagnosis and poses challenges in virus identification. To this end, a duplex quantitative real-time PCR method targeting the GPCR gene was developed and validated, for the concurrent detection and differentiation of wild type and vaccine Lumpy skin disease virus (LSDV) strains. The method was evaluated in three laboratories. The evaluation included a panel of 38 poxvirus isolates/strains and the analytical characteristics of the method were determined. Amplification efficiencies were 91.3% and 90.7%, for wild type and vaccine LSDV, respectively; the limit of detection was 8 DNA copies for both targets and the inter-assay CV was 0.30% for wild type and 0.73% for vaccine LSDV. The diagnostic performance was assessed using 163 LSDV-positive samples, including field specimens and samples from experimentally vaccinated/infected animals. The method is able to confirm diagnosis in suspect cases, it differentiates infected from vaccinated animals (DIVA) and can be regarded as an important tool for effective LSD surveillance and eradication during vaccination campaigns.

VL - 249 U1 - http://www.ncbi.nlm.nih.gov/pubmed/28837841?dopt=Abstract M3 - 10.1016/j.jviromet.2017.08.011 ER - TY - JOUR T1 - A dose-escalation study of combretastatin A4-phosphate in healthy dogs. JF - Vet Comp Oncol Y1 - 2017 A1 - Abma, E A1 - Smets, P A1 - Daminet, S A1 - Cornelis, I A1 - Kris De Clercq A1 - Ni, Y A1 - Vlerick, L A1 - de Rooster, H AB -

Combretastatin A4-Phosphate (CA4P) is a vascular disrupting agent revealing promising results in cancer treatments for humans. The aim of this study was to investigate the safety and adverse events of CA4P in healthy dogs as a prerequisite to application of CA4P in dogs with cancer. Ten healthy dogs were included. The effects of escalating doses of CA4P on physical, haematological and biochemical parameters, systolic arterial blood pressure, electrocardiogram, echocardiographic variables and general wellbeing were characterised. Three different doses were tested: 50, 75 and 100 mg m(-2) . At all 3 CA4P doses, nausea, abdominal discomfort as well as diarrhoea were observed for several hours following administration. Likewise, a low-grade neutropenia was observed in all dogs. Doses of 75 and 100 mg m(-2) additionally induced vomiting and elevation of serum cardiac troponine I levels. At 100 mg m(-2) , low-grade hypertension and high-grade neurotoxicity were also observed. In healthy dogs, doses up to 75 mg m(-2) seem to be well tolerated. The severity of the neurotoxicity observed at 100 mg m(-2) , although transient, does not invite to use this dose in canine oncology patients.

U1 - http://www.ncbi.nlm.nih.gov/pubmed/28620942?dopt=Abstract M3 - 10.1111/vco.12327 ER - TY - JOUR T1 - Foot-and-mouth disease virus serotype SAT1 in cattle, Nigeria. JF - Transbound Emerg Dis Y1 - 2017 A1 - Ehizibolo, D O A1 - Andy Haegeman A1 - Annebel De Vleeschauwer A1 - Umoh, J U A1 - Kazeem, H M A1 - Okolocha, E C A1 - Steven Van Borm A1 - Kris De Clercq KW - Animals KW - Cattle KW - Cattle Diseases KW - Disease Outbreaks KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Nigeria KW - Phylogeny KW - Serogroup AB -

The knowledge of foot-and-mouth disease virus (FMDV) dynamics and epidemiology in Nigeria and the West Africa subregion is important to support local and regional control plans and international risk assessment. Foot-and-mouth disease virus serotype South African territories (SAT)1 was isolated, identified and characterized from an FMD outbreak in cattle in Nigeria in 2015, 35 years after the last report of FMDV SAT1 in West Africa. The VP1 coding sequence of the Nigerian 2015 SAT1 isolates diverges from reported SAT1 topotypes resulting in a separate topotype. The reporting of a novel FMDV SAT1 strain in the virus pool 5 (West and Central Africa) highlights the dynamic and complex nature of FMDV in this region of Africa. Sustained surveillance is needed to understand the origin, the extent and distribution of this novel SAT1 topotype in the region as well as to detect and monitor the occurrence of (re-)emerging FMDV strains.

VL - 64 CP - 3 U1 - https://www.ncbi.nlm.nih.gov/pubmed/28224715?dopt=Abstract M3 - 10.1111/tbed.12629 ER - TY - JOUR T1 - Laboratory validation of two real-time RT-PCR methods with 5'-tailed primers for an enhanced detection of foot-and-mouth disease virus. JF - J Virol Methods Y1 - 2017 A1 - Frank Vandenbussche A1 - David Lefebvre A1 - Ilse De Leeuw A1 - Steven Van Borm A1 - Kris De Clercq KW - 5' Untranslated Regions KW - Animals KW - DNA Primers KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Reproducibility of Results KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Sensitivity and Specificity KW - Sheep KW - Swine AB -

The 3D and 5UTR real-time RT-PCR assays (RT-qPCR) from Callahan et al. (2002) and Reid et al. (2002) are commonly used reference methods for the detection of foot-and-mouth disease virus (FMDV). For an optimal detection of FMDV in clinical samples, it is advised to use both assays simultaneously (King et al., 2006). Recently, Vandenbussche et al. (2016) showed that the addition of 5'-tails to the FMDV-specific primers enhances the detection of FMDV in both the 3D and the 5UTR RT-qPCR assay. To validate the 3D and 5UTR RT-qPCR assays with 5'-tailed primers for diagnostic purposes, both assays were run in parallel in a triplex one-step RT-qPCR protocol with beta-actin as an internal control and synthetic RNA as an external control. We obtained low limits of detection and high linearity's, high repeatability and reproducibility, near 100% analytical specificity and >99% diagnostic accuracy for both assays. It was concluded that the 3D and 5UTR RT-qPCR assays with 5'-tailed primers are particularly suited for the detection of FMDV as well as to exclude the presence of FMDV.

VL - 246 U1 - https://www.ncbi.nlm.nih.gov/pubmed/28457784?dopt=Abstract M3 - 10.1016/j.jviromet.2017.04.014 ER - TY - JOUR T1 - Laboratory validation of two real-time RT-PCR methods with 5′-tailed primers for an enhanced detection of foot-and-mouth disease virus JF - Journal of Virological Methods Y1 - 2017 A1 - Frank Vandenbussche A1 - David Lefebvre A1 - Ilse De Leeuw A1 - Steven Van Borm A1 - Kris De Clercq VL - 246 M3 - 10.1016/j.jviromet.2017.04.014 ER - TY - JOUR T1 - Outbreak investigations and molecular characterization of foot-and-mouth disease viruses circulating in south-west Niger. JF - Transbound Emerg Dis Y1 - 2017 A1 - B Souley Kouato A1 - Elliot, F M A1 - King, D P A1 - Hyera, J A1 - Knowles, N J A1 - Ludi, A B A1 - Mioulet, V A1 - Matlho, G A1 - Kris De Clercq A1 - Thys, E A1 - Marichatou, H A1 - Issa, S A1 - Saegerman, C AB -

In Niger, the epidemiological situation regarding foot-and-mouth disease is unclear as many outbreaks are unreported. This study aimed (i) to identify Foot-and-mouth disease virus (FMDV) strains currently circulating in cattle herds, and (ii) to identify risk factors associated with Foot-and-mouth disease (FMD)-seropositive animals in clinical outbreaks. Epithelial tissues (n = 25) and sera (n = 227) were collected from cattle in eight districts of the south-western part of Niger. Testing of clinical material revealed the presence of FMDV serotype O that was characterized within the O/WEST AFRICA topotype. The antigenic relationship between one of the FMDV isolates from Niger (O/NGR/4/2015) and three reference vaccine strains was determined by the two-dimensional virus neutralization test (2dmVNT), revealing a close antigenic match between the field isolate from Niger and three FMDV serotype O vaccine strains. Serological analyses using a non-structural protein (NSP) test provided evidence for previous FMDV infection in 70% (158/227) of the sera tested. Multivariate logistic regression analysis revealed that only the herd composition (presence of both cattle and small ruminants) was significantly associated with FMDV seropositivity as defined by NSP-positive results (p-value = .006). Of these positive sera, subsequent testing by liquid-phase blocking ELISA (LPBE) showed that 86% (136/158) were positive for one (or more) of four FMDV serotypes (A, O, Southern African Territories (SAT) 1 and SAT 2). This study provides epidemiological information about FMD in the south-western part of Niger and highlights the complex transboundary nature of FMD in Africa. These findings may help to develop effective control and preventive strategies for FMD in Niger as well, as other countries in West Africa.

U1 - http://www.ncbi.nlm.nih.gov/pubmed/28345819?dopt=Abstract M3 - 10.1111/tbed.12642 ER - TY - JOUR T1 - Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines. JF - J Virol Y1 - 2017 A1 - Celma, Cristina C A1 - Stewart, Meredith A1 - Wernike, Kerstin A1 - Eschbaumer, Michael A1 - Gonzalez-Molleda, Lorenzo A1 - Bréard, Emmanuel A1 - Schulz, Claudia A1 - Hoffmann, Bernd A1 - Andy Haegeman A1 - Kris De Clercq A1 - Zientara, Stéphan A1 - van Rijn, Piet A A1 - Beer, Martin A1 - Roy, Polly KW - Animals KW - Antibodies, Neutralizing KW - Antibodies, Viral KW - Base Sequence KW - Bluetongue KW - Bluetongue virus KW - Capsid Proteins KW - Cattle KW - Cell Line KW - Drug Stability KW - Drug Storage KW - Female KW - Male KW - Reverse Genetics KW - Serogroup KW - Sheep KW - Vaccination KW - Vaccines, Attenuated KW - Vaccines, Subunit KW - Viral Vaccines KW - Virion AB -

Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV.

IMPORTANCE: Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce highly attenuated, safer vaccines was evident after the development of the BTV reverse-genetics system that allows the introduction of targeted mutations in the virus genome. We targeted an essential gene to develop disabled virus strains as vaccine candidates. The results presented in this report further substantiate our previous evidence and support the suitability of these virus strains as the next-generation BTV vaccines.

VL - 91 CP - 1 U1 - http://www.ncbi.nlm.nih.gov/pubmed/27795442?dopt=Abstract M3 - 10.1128/JVI.01892-16 ER - TY - JOUR T1 - Review: Capripoxvirus Diseases: Current Status and Opportunities for Control. JF - Transbound Emerg Dis Y1 - 2017 A1 - Tuppurainen, E S M A1 - Venter, E H A1 - Shisler, J L A1 - Gari, G A1 - Mekonnen, G A A1 - Juleff, N A1 - Lyons, N A A1 - Kris De Clercq A1 - Upton, C A1 - Bowden, T R A1 - Babiuk, S A1 - Babiuk, L A AB -

Lumpy skin disease, sheeppox and goatpox are high-impact diseases of domestic ruminants with a devastating effect on cattle, sheep and goat farming industries in endemic regions. In this article, we review the current geographical distribution, economic impact of an outbreak, epidemiology, transmission and immunity of capripoxvirus. The special focus of the article is to scrutinize the use of currently available vaccines to investigate the resource needs and challenges that will have to be overcome to improve disease control and eradication, and progress on the development of safer and more effective vaccines. In addition, field evaluation of the efficacy of the vaccines and the genomic database available for poxviruses are discussed.

VL - 64 CP - 3 U1 - http://www.ncbi.nlm.nih.gov/pubmed/26564428?dopt=Abstract M3 - 10.1111/tbed.12444 ER - TY - JOUR T1 - Complete Genome Sequence of Capripoxvirus Strain KSGP 0240 from a Commercial Live Attenuated Vaccine. JF - Genome Announc Y1 - 2016 A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - Andy Haegeman A1 - Al-Majali, Ahmad A1 - Steven Van Borm A1 - Kris De Clercq AB -

Capripoxviruses cause economically important diseases in domestic ruminants in regions endemic for these viruses. We report here the complete genome sequence of the KSGP 0240 vaccine strain from the live attenuated vaccine Kenyavac (JOVAC).

VL - 4 CP - 5 M3 - 10.1128/genomeA.01114-16 ER - TY - JOUR T1 - Complete Genome Sequence of Capripoxvirus Strain KSGP 0240 from a Commercial Live Attenuated Vaccine JF - Genome Announcements Y1 - 2016 A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - Andy Haegeman A1 - Al-Majali, Ahmad A1 - Steven Van Borm A1 - Kris De Clercq KW - capripoxvirus KW - complete genome KW - KSGP 0240 KW - LSDV KW - Lumpy skin disease VL - 4 CP - 5 M3 - 10.1128/genomeA.01114-16 ER - TY - JOUR T1 - Complete Genome Sequence of the Goatpox Virus Strain Gorgan Obtained Directly from a Commercial Live Attenuated Vaccine JF - Genome Announcements Y1 - 2016 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Andy Haegeman A1 - Al-Majali, Ahmad A1 - Kris De Clercq A1 - Steven Van Borm KW - capripoxvirus KW - complete genome KW - goatpox virus KW - gorgan strain KW - GTPV VL - 4 CP - 5 M3 - 10.1128/genomeA.01113-16 ER - TY - JOUR T1 - Complete Genome Sequences of the Neethling-Like Lumpy Skin Disease Virus Strains Obtained Directly from Three Commercial Live Attenuated Vaccines JF - Genome Announcements Y1 - 2016 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Andy Haegeman A1 - King, Alasdair A1 - Nthangeni, Bethuel A1 - Potgieter, Christiaan A1 - Maartens, Louis A1 - Steven Van Borm A1 - Kris De Clercq KW - capripoxviridae KW - long-range PCR KW - Lumpy skin disease KW - NGS KW - pacbio sequencing KW - SMRT KW - WGS VL - 4 CP - 6 M3 - 10.1128/genomeA.01255-16 ER - TY - JOUR T1 - Complete Genome Sequences of Three African Foot-and-Mouth Disease Viruses from Clinical Samples Isolated in 2009 and 2010. JF - Genome Announc Y1 - 2016 A1 - Steven Van Borm A1 - Rosseel, Toon A1 - Andy Haegeman A1 - Fana, Mpolokang Elliot A1 - Latoya Seoke A1 - Hyera, Joseph A1 - Matlho, George A1 - Frank Vandenbussche A1 - Kris De Clercq AB -

The complete genome sequences of three foot-and-mouth disease viruses (one virus of each serotype SAT1, SAT2 and O) were directly sequenced from RNA extracted from clinical bovine samples, demonstrating the feasibility of full-genome sequencing from strong positive samples taken from symptomatic animals.

VL - 4 CP - 3 U1 - https://www.ncbi.nlm.nih.gov/pubmed/27151795?dopt=Abstract M3 - 10.1128/genomeA.00326-16 ER - TY - JOUR T1 - Experimental bluetongue virus superinfection in calves previously immunized with bluetongue virus serotype 8. JF - Vet Res Y1 - 2016 A1 - Martinelle, Ludovic A1 - Dal Pozzo, Fabiana A1 - Sarradin, Pierre A1 - Willem Van Campe A1 - Ilse De Leeuw A1 - Kris De Clercq A1 - Thys, Christine A1 - Thiry, Etienne A1 - Saegerman, Claude KW - Animals KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Female KW - Male KW - Superinfection KW - Viral Vaccines AB -

The effect of a superinfection with bluetongue virus serotype 1 (BTV1) was evaluated on two groups of four calves. One group received a commercial inactivated BTV serotype 8 (BTV8) vaccine. This group and the non-vaccinated group of calves were challenged twice (4 months apart) with the European BTV8 strain isolated during the 2006-2007 epidemics. Calves were then infected with a BTV1 inoculum which was found to be unexpectedly contaminated by BTV serotype 15 (BTV15). BTV1 and BTV15 single infections were performed on two other groups of three BTV naïve calves. A severe clinical picture was obtained after superinfection with BTV1/BTV15 in both vaccinated and non-vaccinated animals and after challenge with BTV8 in non-vaccinated animals. BTV1 and BTV15 single infection caused only very slight clinical signs. After superinfection and at the viraemic peak, there were an average of above 1000 times more BTV15 genomic copies than BTV1 ones. BTV1 RNA could be detected only in the spleen of one calf whereas BTV15 RNA was found in 15 organs of seven different animals. BTV8 immunization whether it was acquired through vaccination and challenges or challenges alone did not change BTV1 or BTV15 RNA detection in superinfected animals. However in these animals a partial cross neutralization between BTV8 and BTV1 might be involved in the lower BTV1 replication versus BTV15. Infection with different serotypes can occur also in the field. Interference between virus strains, genetic reassortment and cross-protection were considered as mechanisms to explain the clinical outcomes and the other virological and immunological findings in the course of BTV1/BTV15 superinfection.

VL - 47 CP - 1 U1 - https://www.ncbi.nlm.nih.gov/pubmed/27465686?dopt=Abstract M3 - 10.1186/s13567-016-0357-6 ER - TY - JOUR T1 - Inter-laboratory evaluation of the performance parameters of a Lateral Flow Test device for the detection of Bluetongue virus-specific antibodies. JF - J Virol Methods Y1 - 2016 A1 - Jean-Baptiste Hanon A1 - Valerie Vandenberge A1 - Deruelle, Matthias A1 - Ilse De Leeuw A1 - Kris De Clercq A1 - Steven Van Borm A1 - F. Koenen A1 - Liu, Lihong A1 - Hoffmann, Bernd A1 - Batten, Carrie Anne A1 - Zientara, Stéphan A1 - Bréard, Emmanuel A1 - Yves Van der Stede KW - Animals KW - Antibodies, Viral KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Chromatography, Affinity KW - Cooperative Behavior KW - Cross Reactions KW - Enzyme-Linked Immunosorbent Assay KW - Hemorrhagic Disease Virus, Epizootic KW - Laboratory Proficiency Testing KW - Reproducibility of Results KW - Ruminants KW - Sensitivity and Specificity KW - Serologic Tests KW - Sheep AB -

Bluetongue (BT) is a viral vector-borne disease affecting domestic and wild ruminants worldwide. In this study, a commercial rapid immuno-chromatographic method or Lateral Flow Test (LFT) device, for the detection of BT virus-specific antibodies in animal serum, was evaluated in an international inter-laboratory proficiency test. The evaluation was done with sera samples of variable background (ruminant species, serotype, field samples, experimental infections, vaccinated animals). The diagnostic sensitivity was 100% (95% C.I. [90.5-100]) and the diagnostic specificity was 95.2% (95% C.I. [76.2-99.9]). The repeatability (accordance) and reproducibility (concordance) were 100% for seropositive samples but were lower for two of the seronegative samples (45% and 89% respectively). The analytical sensitivity, evaluated by testing positive sera at increasing dilutions was better for the BT LFT compared to some commercial ELISAs. Seroconversion of an infected sheep was detected at 4 days post infection. Analytical specificity was impaired by cross-reactions observed with some of the samples seropositive for Epizootic Haemorrhagic Disease Virus (EHDV). The agreement (Cohen's kappa) between the LFT and a commercial BT competitive ELISA was 0.79 (95% CI [0.62-0.95]). Based on these results, it can be concluded that the BT LFT device is a rapid and sensitive first-line serological test that can be used in the field, especially in areas endemic for the disease where there is a lack of diagnostic facilities.

VL - 228 U1 - https://www.ncbi.nlm.nih.gov/pubmed/26687976?dopt=Abstract M3 - 10.1016/j.jviromet.2015.12.001 ER - TY - JOUR T1 - Investigation of a Possible Link Between Vaccination and the 2010 Sheep Pox Epizootic in Morocco. JF - Transbound Emerg Dis Y1 - 2016 A1 - Andy Haegeman A1 - Zro, K A1 - Sammin, D A1 - Frank Vandenbussche A1 - Ennaji, M M A1 - Kris De Clercq KW - Animals KW - capripoxvirus KW - Disease Outbreaks KW - Genotype KW - Morocco KW - Phylogeny KW - polymerase chain reaction KW - Poxviridae Infections KW - Sheep KW - Sheep Diseases KW - Vaccination KW - Vaccines, Attenuated AB -

Sheep pox is endemic in most parts of Northern Africa and has the potential to cause severe economic problems. Live attenuated vaccines are used in Morocco, and in many other countries, to control the disease. Sheep pox virus (SPPV) re-appeared in 2010 causing a nodular clinical form previously not observed in Morocco. The severe clinical signs observed during the course of this outbreak and initial reports citing similarity in nucleotide sequence between the Moroccan vaccine strain and field isolates warranted a more in depth analysis of this epizootic. In this study, sequence analysis showed that isolates obtained from four provinces of eastern Morocco were identical, demonstrating that a single SPPV strain was responsible for the 2010 epizootic. In addition, the genome fragments sequenced and phylogenetic analyses undertaken as part of this study showed significant differences between field isolates and the Moroccan vaccine strain. New PCR methods were developed to differentiate between wild-type isolates and vaccine strains of SPPV. Using these methods, no trace of wild-type SPPV was found in the vaccine and no evidence was found to suggest that the vaccine strain was causing clinical disease.

VL - 63 CP - 6 U1 - https://www.ncbi.nlm.nih.gov/pubmed/25753969?dopt=Abstract M3 - 10.1111/tbed.12342 ER - TY - JOUR T1 - A Refined Guinea Pig Model of Foot-and-Mouth Disease Virus Infection for Assessing the Efficacy of Antiviral Compounds. JF - Transbound Emerg Dis Y1 - 2016 A1 - Annebel De Vleeschauwer A1 - David Lefebvre A1 - Willems, T A1 - Paul, G A1 - Billiet, A A1 - Murao, L E A1 - Neyts, J A1 - Goris, N A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Antiviral Agents KW - Disease Models, Animal KW - Europe KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Guinea Pigs KW - Pyrazines KW - RNA, Viral KW - Vaccination KW - Viral Vaccines AB -

An antiviral containment strategy for foot-and-mouth disease (FMD) outbreaks could support or replace current contingency plans in case of an outbreak in Europe and could spare many healthy animals from being pre-emptively culled. Recently, substantial progress has been made towards the development of small molecule drugs that inhibit FMD virus (FMDV) replication in vitro. For the initial in vivo evaluation of antiviral lead molecules, a refined FMDV-infection model in guinea pigs (GP) is herewith described. This GP model was validated by demonstrating the antiviral effect of T-1105 (an influenza virus inhibitor with reported activity against FMDV). Sixteen animals were orally administered with T-1105 twice daily (400 mg/kg/day) for five consecutive days and inoculated intraplantarly with 100 GPID50 of the GP-adapted FMDV strain O1 Manisa 1 h after the first administration. The efficacy of T-1105 was compared with that of prophylactic vaccination with a highly potent double-oil emulsion-inactivated O1 Manisa vaccine. Ten animals received a single, full (2 ml) cattle vaccine dose and were inoculated 3 weeks later. Fourteen T-1105-treated and all vaccinated GP were completely protected from generalization of vesicular lesions. At 2 dpi, viral RNA was detected in serum of 9/16 T-1105-treated and of 6/10 vaccinated animals. At 4 dpi, viral RNA was detected in serum, organs and oral swabs of half of the T-1105-treated animals and only in the serum of 1/10 of the vaccinated animals. Mean viral RNA levels in serum and organs of T-1105-treated and vaccinated animals were reduced compared to untreated controls (P < 0.01). T-1105 conferred a substantial clinical and virological protection against infection with O1 Manisa, similar to the protection afforded by vaccination. These results validate the suitability of the enhanced GP model for the purpose of initial evaluation of inhibitors of FMDV replication and illustrate the potential of selective inhibitors of viral replication to control FMD outbreaks.

VL - 63 CP - 2 U1 - https://www.ncbi.nlm.nih.gov/pubmed/25164494?dopt=Abstract M3 - 10.1111/tbed.12255 ER - TY - JOUR T1 - A Tale of Tails: Dissecting the Enhancing Effect of Tailed Primers in Real-Time PCR. JF - PLoS One Y1 - 2016 A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - David Lefebvre A1 - Kris De Clercq A1 - Steven Van Borm KW - Animals KW - DNA Primers KW - Foot-and-Mouth Disease Virus KW - Humans KW - Real-Time Polymerase Chain Reaction AB -

Non-specific tail sequences are often added to the 5'-terminus of primers to improve the robustness and overall performance of diagnostic assays. Despite the widespread use of tailed primers, the underlying working mechanism is not well understood. To address this problem, we conducted a detailed in vitro and in silico analysis of the enhancing effect of primer tailing on 2 well-established foot-and-mouth disease virus (FMDV) RT-qPCR assays using an FMDV reference panel. Tailing of the panFMDV-5UTR primers mainly affected the shape of the amplification curves. Modelling of the raw fluorescence data suggested a reduction of the amplification efficiency due to the accumulation of inhibitors. In depth analysis of PCR products indeed revealed the rapid accumulation of forward-primer derived artefacts. More importantly, tailing of the forward primer delayed artefacts formation and concomitantly restored the sigmoidal shape of the amplification curves. Our analysis also showed that primer tailing can alter utilisation patterns of degenerate primers and increase the number of primer variants that are able to participate in the reaction. The impact of tailed primers was less pronounced in the panFMDV-3D assay with only 5 out of 50 isolates showing a clear shift in Cq values. Sequence analysis of the target region of these 5 isolates revealed several mutations in the inter-primer region that extend an existing hairpin structure immediately downstream of the forward primer binding site. Stabilisation of the forward primer with either a tail sequence or cationic spermine units restored the sensitivity of the assay, which suggests that the enhancing effect in the panFMDV-3D assay is due to a more efficient extension of the forward primer. ur results show that primer tailing can alter amplification through various mechanisms that are determined by both the assay and target region. These findings expand our understanding of primer tailing and should enable a more targeted and efficient use of tailed primers.

VL - 11 CP - 10 U1 - https://www.ncbi.nlm.nih.gov/pubmed/27723800?dopt=Abstract M3 - 10.1371/journal.pone.0164463 ER - TY - JOUR T1 - Ulcerative pododermatitis and disseminated erosive lesions associated with cowpox virus infection in a domestic cat JF - Veterinary Record Case Reports Y1 - 2016 A1 - Louisa Ludwig A1 - Jéromine Bohn A1 - Isabelle Remy A1 - Andy Haegeman A1 - Marianne Heimann A1 - Mauroy, Axel A1 - Kris De Clercq A1 - Thiry, Etienne AB -

The authors report on a case of feline cowpox virus infection associated with severe ulcerative dermatitis of a paw and disseminated erosive lesions. While the anamnesis of the cat being a known rodent-hunter, a typical seasonality of infection and the progression of clinical signs from a primary anterior lesion (forelimb) indicated a possible cowpox virus infection, the differential diagnosis was complicated by the resemblance of clinical signs to those induced by feline herpesvirus-dermatitis or feline calicivirus infection. These differential diagnoses were excluded by means of immunostaining and PCR, respectively. Detection of eosinophilic intracytoplasmic inclusion bodies in cells from biopsy material and positive PCR and sequencing results confirmed the diagnosis of cowpox virus infection. Genetic characterisation of the isolate, based on the highly diverse haemagglutinin gene, showed that the strain (Liege 2015; GenBank accession number: KU726584) clustered with other European isolates, mostly from exotic zoo animals

VL - 4 CP - 1 M3 - 10.1136/vetreccr-2016-000331 ER - TY - JOUR T1 - Bluetongue virus RNA detection by real-time rt-PCR in post-vaccination samples from cattle. JF - Transbound Emerg Dis Y1 - 2015 A1 - Ilse De Leeuw A1 - Garigliany, M A1 - Bertels, G A1 - Willems, T A1 - Desmecht, D A1 - Kris De Clercq KW - Animals KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Europe KW - Real-Time Polymerase Chain Reaction KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Vaccination KW - Vaccines, Inactivated KW - Viral Vaccines AB -

Bluetongue virus serotype 8 (BTV-8) was responsible for a large outbreak among European ruminant populations in 2006-2009. In spring 2008, a massive vaccination campaign was undertaken, leading to the progressive disappearance of the virus. During surveillance programmes in Western Europe in 2010-2011, a low but significant number of animals were found weakly positive using BTV-specific real-time RT-PCR, raising questions about a possible low level of virus circulation. An interference of the BTV-8 inactivated vaccine on the result of the real-time RT-PCR was also hypothesized. Several studies specifically addressed the potential association between a recent vaccination and BTV-8 RNA detection in the blood of sheep. Results were contradictory and cattles were not investigated. To enlighten this point, a large study was performed to determine the risks of detection of bluetongue vaccine-associated RNA in the blood and spleen of cattle using real-time RT-PCR. Overall, the results presented clearly demonstrate that vaccine viral RNA can reach the blood circulation in sufficient amounts to be detected by real-time RT-PCR in cattle. This BTV-8 vaccine RNA carriage appears as short lasting.

VL - 62 CP - 2 U1 - https://www.ncbi.nlm.nih.gov/pubmed/23611408?dopt=Abstract M3 - 10.1111/tbed.12100 ER - TY - JOUR T1 - Evaluation of adaptive immune responses and heterologous protection induced by inactivated bluetongue virus vaccines. JF - Vaccine Y1 - 2015 A1 - Bréard, Emmanuel A1 - Belbis, Guillaume A1 - Viarouge, Cyril A1 - Nomikou, Kyriaki A1 - Andy Haegeman A1 - Kris De Clercq A1 - Hudelet, Pascal A1 - Hamers, Claude A1 - Moreau, Francis A1 - Lilin, Thomas A1 - Durand, Benoit A1 - Mertens, Peter A1 - Vitour, Damien A1 - Sailleau, Corinne A1 - Zientara, Stéphan KW - Animals KW - Antibodies, Viral KW - Bluetongue KW - Bluetongue virus KW - Cross Protection KW - Immunity, Heterologous KW - Random Allocation KW - Severity of Illness Index KW - Sheep KW - Vaccines, Inactivated KW - Viral Load KW - Viral Vaccines KW - Viremia AB -

Eradication of bluetongue virus is possible, as has been shown in several European countries. New serotypes have emerged, however, for which there are no specific commercial vaccines. This study addressed whether heterologous vaccines would help protect against 2 serotypes. Thirty-seven sheep were randomly allocated to 7 groups of 5 or 6 animals. Four groups were vaccinated with commercial vaccines against BTV strains 2, 4, and 9. A fifth positive control group was given a vaccine against BTV-8. The other 2 groups were unvaccinated controls. Sheep were then challenged by subcutaneous injection of either BTV-16 (2 groups) or BTV-8 (5 groups). Taken together, 24/25 sheep from the 4 experimental groups developed detectable antibodies against the vaccinated viruses. Furthermore, sheep that received heterologous vaccines showed significantly reduced viraemia and clinical scores for BTV-16 when compared to unvaccinated controls. Reductions in clinical signs and viraemia among heterologously vaccinated sheep were not as common after challenge with BTV-8. This study shows that heterologous protection can occur, but that it is difficult to predict if partial or complete protection will be achieved following inactivated-BTV vaccination.

VL - 33 CP - 4 U1 - https://www.ncbi.nlm.nih.gov/pubmed/25500308?dopt=Abstract M3 - 10.1016/j.vaccine.2014.11.053 ER - TY - JOUR T1 - Full-Genome Sequencing of Four Bluetongue Virus Serotype 11 Viruses. JF - Transbound Emerg Dis Y1 - 2015 A1 - Frank Vandenbussche A1 - Sailleau, C A1 - Rosseel, T A1 - Desprat, A A1 - Viarouge, C A1 - Richardson, J A1 - Eschbaumer, M A1 - Hoffmann, B A1 - Kris De Clercq A1 - Bréard, E A1 - Zientara, S KW - Animals KW - Base Sequence KW - Bluetongue KW - Bluetongue virus KW - Europe KW - Genome, Viral KW - Molecular Sequence Data KW - Phylogeny KW - Serogroup KW - Sheep KW - Vaccination KW - Viral Vaccines AB -

Recently, a contamination incident was described in which the challenge inoculum used in a bluetongue virus serotype 8 (BTV-8) vaccination trial was contaminated with a BTV-11 virus that was closely related to the Belgian BTV-11 virus from 2008. This study reports the first complete genome sequences of four BTV-11 viruses: the BTV-11 contaminant, BTV-11 reference strain, BTV-11 vaccine strain and a recently isolated BTV-11 field strain from Martinique. Full-genome analysis showed that these viruses belong to serotype 11/nucleotype A and cluster together with other western topotype bluetongue viruses. Detailed comparisons of the genomes further indicated that the contaminant was derived from the BTV-11 reference strain, as they were distinguished by a single synonymous nucleotide substitution. The previously reported partial sequence of genome segment 2 of the Belgian BTV-11 was found to be identical to that of the BTV-11 vaccine strain, indicating that it most likely was the BTV-11 vaccine strain. These findings also suggest that the BTV-11 contaminant and the Belgian BTV-11 are not the same viruses. Finally, comparison of the reference and vaccine strain did not allow determining the amino acid substitutions that contribute to the attenuated phenotype.

VL - 62 CP - 5 U1 - https://www.ncbi.nlm.nih.gov/pubmed/24750582?dopt=Abstract M3 - 10.1111/tbed.12178 ER - TY - JOUR T1 - The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family. JF - PLoS Pathog Y1 - 2015 A1 - van der Linden, Lonneke A1 - Vives-Adrián, Laia A1 - Selisko, Barbara A1 - Ferrer-Orta, Cristina A1 - Liu, Xinran A1 - Lanke, Kjerstin A1 - Ulferts, Rachel A1 - De Palma, Armando M A1 - Tanchis, Federica A1 - Goris, Nesya A1 - David Lefebvre A1 - Kris De Clercq A1 - Leyssen, Pieter A1 - Lacroix, Céline A1 - Pürstinger, Gerhard A1 - Coutard, Bruno A1 - Canard, Bruno A1 - Boehr, David D A1 - Arnold, Jamie J A1 - Cameron, Craig E A1 - Verdaguer, Nuria A1 - Neyts, Johan A1 - van Kuppeveld, Frank J M KW - Animals KW - Antiviral Agents KW - Binding Sites KW - Cardiovirus KW - Chlorocebus aethiops KW - Enterovirus B, Human KW - HeLa Cells KW - Humans KW - Poliovirus KW - RNA-Dependent RNA Polymerase KW - Viral Proteins AB -

The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.

VL - 11 CP - 3 U1 - https://www.ncbi.nlm.nih.gov/pubmed/25799064?dopt=Abstract M3 - 10.1371/journal.ppat.1004733 ER - TY - JOUR T1 - Serologic screening for 13 infectious agents in roe deer (Capreolus capreolus) in Flanders. JF - Infect Ecol Epidemiol Y1 - 2015 A1 - Tavernier, Paul A1 - Sys, Stanislas U A1 - Kris De Clercq A1 - Ilse De Leeuw A1 - Ann Brigitte Cay A1 - Miet De Baere A1 - Nick De Regge A1 - David Fretin A1 - Virginie Roupie A1 - Govaerts, Marc A1 - Heyman, Paul A1 - Vanrompay, Daisy A1 - Yin, Lizi A1 - Kalmar, Isabelle A1 - Vanessa Suin A1 - Bernard Brochier A1 - Alexandre Dobly A1 - Stéphane De Craeye A1 - Sophie Roelandt A1 - Goossens, Els A1 - S. Roels AB -

INTRODUCTION: In order to investigate the role of roe deer in the maintenance and transmission of infectious animal and human diseases in Flanders, we conducted a serologic screening in 12 hunting areas.

MATERIALS AND METHODS: Roe deer sera collected between 2008 and 2013 (n=190) were examined for antibodies against 13 infectious agents, using indirect enzyme-linked immunosorbent assay, virus neutralisation, immunofluorescence, or microagglutination test, depending on the agent.

RESULTS AND DISCUSSION: High numbers of seropositives were found for Anaplasma phagocytophilum (45.8%), Toxoplasma gondii (43.2%) and Schmallenberg virus (27.9%), the latter with a distinct temporal distribution pattern following the outbreak in domestic ruminants. Lower antibody prevalence was found for Chlamydia abortus (6.7%), tick-borne encephalitis virus (5.1%), Neospora caninum (4.8%), and Mycobacterium avium subsp paratuberculosis (4.1%). The lowest prevalences were found for Leptospira (1.7%), bovine viral diarrhoea virus 1 (1.3%), and Coxiella burnetii (1.2%). No antibodies were found against Brucella sp., bovine herpesvirus 1, and bluetongue virus. A significant difference in seroprevalence between ages (higher in adults >1 year) was found for N. caninum. Four doubtful reacting sera accounted for a significant difference in seroprevalence between sexes for C. abortus (higher in females).

CONCLUSIONS: Despite the more intensive landscape use in Flanders, the results are consistent with other European studies. Apart from maintaining C. abortus and MAP, roe deer do not seem to play an important role in the epidemiology of the examined zoonotic and domestic animal pathogens. Nevertheless, their meaning as sentinels should not be neglected in the absence of other wild cervid species.

VL - 5 U1 - http://www.ncbi.nlm.nih.gov/pubmed/26609692?dopt=Abstract M3 - 10.3402/iee.v5.29862 ER - TY - JOUR T1 - Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases. JF - Antiviral Res Y1 - 2014 A1 - van der Linden, Lonneke A1 - Ulferts, Rachel A1 - Nabuurs, Sander B A1 - Kusov, Yuri A1 - Liu, Hong A1 - George, Shyla A1 - Lacroix, Céline A1 - Goris, Nesya A1 - David Lefebvre A1 - Lanke, Kjerstin H W A1 - Kris De Clercq A1 - Hilgenfeld, Rolf A1 - Neyts, Johan A1 - van Kuppeveld, Frank J M KW - 3C Viral Proteases KW - Animals KW - Antiviral Agents KW - Cell Line KW - Cysteine Endopeptidases KW - Drug Evaluation, Preclinical KW - Genes, Reporter KW - Humans KW - Luciferases, Firefly KW - Picornaviridae KW - Protease Inhibitors KW - Viral Proteins AB -

Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases and peptide substrates. To deal with the disadvantages associated with in vitro assays, we modified a cell-based protease assay for picornavirus proteases. The assay is based on the induction of expression of a firefly luciferase reporter by a chimeric transcription factor in which the viral protease and cleavage sites are inserted between the GAL4 binding domain and the VP16 activation domain. Firefly luciferase expression is dependent on cleavage of the transcription factor by the viral protease. This biosafe assay enables testing the effect of compounds on protease activity in cells while circumventing the need for infection. We designed the assay for 3C proteases (3C(pro)) of various enteroviruses as well as of viruses of several other picornavirus genera, and show that the assay is amenable for use in a high-throughput setting. Furthermore, we show that the spectrum of activity of 3C(pro) inhibitor AG7088 (rupintrivir) not only encompasses enterovirus 3C(pro) but also 3C(pro) of foot-and-mouth disease virus (FMDV), an aphthovirus. In contrary, AG7404 (compound 1), an analogue of AG7088, had no effect on FMDV 3C(pro) activity, for which we provide a structural explanation.

VL - 103 U1 - https://www.ncbi.nlm.nih.gov/pubmed/24393668?dopt=Abstract M3 - 10.1016/j.antiviral.2013.12.012 ER - TY - JOUR T1 - False-positive results in metagenomic virus discovery: a strong case for follow-up diagnosis. JF - Transbound Emerg Dis Y1 - 2014 A1 - Rosseel, T A1 - Pardon, B A1 - Kris De Clercq A1 - Orkun Ozhelvaci A1 - Steven Van Borm KW - Animals KW - Cattle KW - Cattle Diseases KW - DNA, Viral KW - False Positive Reactions KW - Female KW - Metagenomics KW - Parvoviridae Infections KW - Real-Time Polymerase Chain Reaction KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - specimen handling KW - Viruses AB -

A viral metagenomic approach using virion enrichment, random amplification and next-generation sequencing was used to investigate an undiagnosed cluster of dairy cattle presenting with high persistent fever, unresponsive to anti-microbial and anti-inflammatory treatment, diarrhoea and redness of nose and teat. Serum and whole blood samples were taken in the predicted hyperviraemic state of an animal that a few days later presented with these clinical signs. Bioinformatics analysis of the resulting data from the DNA virus identification workflow (a total of 32 757 sequences with average read length 335 bases) initially demonstrated the presence of parvovirus-like sequences in the tested blood sample. Thorough follow-up using specific real-time RT-PCR assays targeting the detected sequence fragments confirmed the presence of these sequences in the original sample as well as in a sample of an additional animal, but a contamination with an identical genetic signature in negative extraction controls was demonstrated. Further investigation using an alternative extraction method identified a contamination of the originally used Qiagen extraction columns with parvovirus-like nucleic acids or virus particles. Although we did not find any relevant virus that could be associated with the disease, these observations clearly illustrate the importance of using a proper control strategy and follow-up diagnostic tests in any viral metagenomic study.

VL - 61 CP - 4 U1 - https://www.ncbi.nlm.nih.gov/pubmed/24912559?dopt=Abstract M3 - 10.1111/tbed.12251 ER - TY - JOUR T1 - In vitro surrogate models to aid in the development of antivirals for the containment of foot-and-mouth disease outbreaks. JF - Antiviral Res Y1 - 2014 A1 - Osiceanu, Ana-Maria A1 - Murao, Lyre Espada A1 - Kollanur, Denny A1 - Swinnen, Jan A1 - Annebel De Vleeschauwer A1 - David Lefebvre A1 - Kris De Clercq A1 - Neyts, Johan A1 - Goris, Nesya KW - Animals KW - Antiviral Agents KW - Aphthovirus KW - Benzimidazoles KW - Cattle KW - Cell Line KW - Chlorocebus aethiops KW - Cytidine KW - Cytopathogenic Effect, Viral KW - Drug Discovery KW - Foot-and-Mouth Disease KW - Models, Theoretical KW - Oximes KW - Ribavirin KW - RNA, Viral KW - Sulfonamides KW - Virus Cultivation KW - Virus Replication AB -

Foot-and-mouth disease virus (FMDV) is a highly pathogenic member of the genus Aphthovirus (family Picornaviridae) that is only to be manipulated in high-containment facilities, thus complicating research on and discovery of antiviral strategies against the virus. Bovine rhinitis B virus (BRBV) and equine rhinitis A virus (ERAV), phylogenetically most closely related to FMDV, were explored as surrogates for FMDV in antiviral studies. Although no efficient cell culture system has been reported so far for BRBV, we demonstrate that infection of primary bovine kidney cells resulted in an extensive but rather poorly-reproducible induction of cytopathic effect (CPE). Madin-Darby bovine kidney cells on the other hand supported viral replication in the absence of CPE. Antiviral tests were developed for ERAV in Vero A cells employing a viral RNA-reduction assay and CPE-reduction assay; the latter having a Z' factor of 0.83±0.07. The BRBV and ERAV models were next used to assess the anti-aphthovirus activity of two broad-spectrum antiviral agents 2'-C-methylcytidine (2CMC) and ribavirin, as well as of the enterovirus-specific inhibitor enviroxime. The effects of the three compounds in the CPE-reduction (ERAV) and viral RNA-reduction assays (BRBV and ERAV) were comparable. Akin to 2CMC, compound A, a recently-discovered non-nucleoside pan-serotype FMDV inhibitor, also inhibited the replication of both BRBV and ERAV, whereas enviroxime was devoid of activity. The BRBV and ERAV surrogate models reported here can be manipulated in BSL-2 laboratories and may facilitate studies to unravel the mechanism of action of novel FMDV inhibitors.

VL - 105 U1 - https://www.ncbi.nlm.nih.gov/pubmed/24583031?dopt=Abstract M3 - 10.1016/j.antiviral.2014.02.009 ER - TY - JOUR T1 - Proof of concept for the inhibition of foot-and-mouth disease virus replication by the anti-viral drug 2'-C-methylcytidine in severe combined immunodeficient mice. JF - Transbound Emerg Dis Y1 - 2014 A1 - David Lefebvre A1 - Annebel De Vleeschauwer A1 - Goris, N A1 - Kollanur, D A1 - Billiet, A A1 - Murao, L A1 - Neyts, J A1 - Kris De Clercq KW - Animals KW - Antiviral Agents KW - Cytidine KW - Disease Models, Animal KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - mice KW - Mice, SCID KW - RNA, Viral AB -

Recent European contingency plans envisage emergency vaccination as an animal-friendly control strategy for foot-and-mouth disease (FMD). Anti-viral drugs may be used as an alternative or complementary measure. We here demonstrate that the nucleoside analogue 2'-C-methylcytidine (2'CMC) protects severe combined immunodeficient (SCID) mice against lethal FMD virus infection. In brief, SCID mice were inoculated with serotype A FMD virus and treated for five consecutive days with 2'CMC. All 15 treated mice remained healthy until the end of the study at 14 days post-infection (dpi). At that time, viral RNA was no longer detected in 13 of 15 treated mice. All eight untreated mice suffered from an acute generalized disease and were euthanized for ethical reasons on average at 4 dpi. These results illustrate the potential of small molecules to control FMD.

VL - 61 CP - 6 U1 - https://www.ncbi.nlm.nih.gov/pubmed/23480064?dopt=Abstract M3 - 10.1111/tbed.12069 ER - TY - JOUR T1 - A thiazepino[4,5-a]benzimidazole derivative hampers the RNA replication of Eurasian serotypes of foot-and-mouth disease virus. JF - Biochem Biophys Res Commun Y1 - 2014 A1 - David Lefebvre A1 - Annebel De Vleeschauwer A1 - Goris, Nesya A1 - Steven Van Borm A1 - Chimirri, Alba A1 - Monforte, Anna Maria A1 - Valdazo-Gonzalez, Begona A1 - King, Donald P A1 - Neyts, Johan A1 - Kris De Clercq KW - Animals KW - Antiviral Agents KW - Benzimidazoles KW - Cell Line KW - Cell Survival KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Mutation KW - Sequence Analysis, DNA KW - Serogroup KW - Swine KW - Thiazepines KW - Virus Replication AB -

The stamping-out policy for the control of foot-and-mouth disease virus (FMDV) in countries that are free from FMD without vaccination has a dramatic socio-economic impact, huge animal welfare issues and may result in the loss of farm animal genetic resources. As an alternative to pre-emptive culling or emergency vaccination we further explore the possibility to use antiviral drugs in the event of an FMD outbreak. In the present study, we tested the in vitro cytotoxicity and anti-FMDV activity of 1,2,4,5-tetrahydro-[1,4]thiazepino[4,5-a]benzimidazole. The molecule was shown to inhibit the replication of reference strains of the Eurasian FMDV serotypes O, A, C and Asia but not the FMDV serotypes from the South African Territories (SAT) neither a related picornavirus, i.e. swine vesicular disease virus. The molecule can be added until 2h post inoculation in a 'single replication cycle experiment' without losing its antiviral activity. The genetic characterization of progressively selected resistant FMD viruses shows that the molecule presumably interacts with the non-structural 2C protein of FMDV. Further studies are required on the use of this molecule in vivo.

VL - 455 CP - 3-4 U1 - https://www.ncbi.nlm.nih.gov/pubmed/25446115?dopt=Abstract M3 - 10.1016/j.bbrc.2014.11.023 ER - TY - JOUR T1 - The use of serosurveys following emergency vaccination, to recover the status of "foot-and-mouth disease free where vaccination is not practised". JF - Vaccine Y1 - 2014 A1 - Paton, D J A1 - Füssel, A-E A1 - Vosloo, W A1 - Dekker, A A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Cattle KW - Cattle Diseases KW - Epidemiologic Methods KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Serologic Tests KW - Vaccination KW - Viral Vaccines AB -

To eliminate incursions of foot-and-mouth disease (FMD) quickly, a combination of measures, including emergency vaccination, can help block the spread of infection. For the earliest recovery of the FMD-free status for trade, without the slaughter of uninfected vaccinated animals, a serosurvey for antibodies to FMD virus non-structural proteins (NSP) must be used to substantiate absence of occult virus infections. Areas of doubt over requirements for post-vaccination serosurveillance and its feasibility include the required and achievable confidence, the amount of sampling necessary, and the appropriate responses to and consequences of different seropositive findings. This derives largely from uncertainty over the extent of localised pockets of virus infection that may remain within vaccinated populations and the circumstances that permit this. The question therefore remains whether tests are sufficiently sensitive and specific to detect and eliminate infected animals, without excessive culling of uninfected animals, before vaccinated animals mix with non-vaccinated livestock when movement restrictions are lifted. It is recommended to change the rationale for serosurveillance after emergency vaccination. Only when emergency vaccination is used in limited outbreaks is it possible to test and cull comprehensively, an approach compatible with a three-month minimum period to recover the FMD-free status. In other situations, where emergency vaccination is used, such as dealing with large outbreaks in animal-dense regions and where the onset of vaccination has been delayed, post-vaccination serosurveys should be targeted and focus on providing an assurance to detect higher levels of infection, in case of inadequate control measures. As this provides less assurance of absence of infection, the approach would be compatible with a six-month waiting period for free-status recovery and should be complemented by other methods to provide evidence that vaccination and control measures have been effectively implemented, as these are the best guarantee against continuing virus transmission.

VL - 32 CP - 52 U1 - https://www.ncbi.nlm.nih.gov/pubmed/25444827?dopt=Abstract M3 - 10.1016/j.vaccine.2014.10.064 ER - TY - JOUR T1 - Bluetongue surveillance system in Belgium: a stochastic evaluation of its risk-based approach effectiveness. JF - Prev Vet Med Y1 - 2013 A1 - Sarah Welby A1 - Estelle Méroc A1 - Faes, Christel A1 - Kris De Clercq A1 - Hooyberghs, Jozef A1 - Mintiens, Koen A1 - Yves Van der Stede KW - Animals KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Disease Outbreaks KW - Enzyme-Linked Immunosorbent Assay KW - Models, Theoretical KW - polymerase chain reaction KW - Population Surveillance KW - Risk Assessment KW - Seasons KW - Sensitivity and Specificity KW - Sentinel Surveillance KW - Sheep KW - Stochastic Processes AB -

BACKGROUND: The aim of this study was to assess the sensitivity of the four major bluetongue surveillance components implemented in Belgium in 2007 for farmed animals and prescribed by the European Union regulation; winter serological screening, sentinel system, passive clinical surveillance, export testing. Scenario tree methodology was used to evaluate the relative sensitivity of detection and targeted approach of each component in terms of early detection and freedom of infection substantiation. Field data collected from the previous year's outbreaks in Belgium were used to determine the risk groups to be considered.

RESULTS: The best sensitivities at herd level, taking into account the diagnostic test sensitivity, design prevalence and the number of animals tested within a herd were obtained with the winter screening and sentinel component. The sensitivities at risk group level, taking into account the obtained herd sensitivity, effective probabilities of infection and number of herds tested were high in all components, except for the export component. Component sensitivities ranged between 0.77 and 1 for all components except for the export component with a mean value of 0.22 (0.17-0.26). In terms of early detection, the probability of detection was best using the passive clinical component or the sentinel component. Sensitivity analysis showed that the passive clinical component sensitivity was mostly affected by the diagnostic process and the number of herds sampled. The sentinel and export components sensitivity were mainly affected by the relative risk estimates whereas the winter screening component was mainly affected by the assumptions about the design prevalence.

CONCLUSIONS: This study revealed interesting features regarding the sensitivity of detection and early detection of infection in the different surveillance components and their risk based approach as requested by the international standards.

VL - 112 CP - 1-2 U1 - https://www.ncbi.nlm.nih.gov/pubmed/23938251?dopt=Abstract M3 - 10.1016/j.prevetmed.2013.07.005 ER - TY - JOUR T1 - Clinical pattern characterization of cattle naturally infected by BTV-8. JF - Transbound Emerg Dis Y1 - 2013 A1 - Zanella, G A1 - Martinelle, L A1 - Guyot, H A1 - Mauroy, A A1 - Kris De Clercq A1 - Saegerman, C KW - Animals KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Conjunctivitis, Viral KW - Enzyme-Linked Immunosorbent Assay KW - France KW - Lethargy KW - Nasal Mucosa KW - Real-Time Polymerase Chain Reaction KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Messenger AB -

Forty-one cattle from seven Belgian farms and two French farms confirmed as infected with bluetongue virus serotype 8 (BTV-8) were monitored from the onset of clinical signs to describe the disease pattern and estimate the duration of blood RT-qPCR and competitiveELISA positivity under field conditions. On each visit, blood samples were taken, and a standardized clinical form was filled in for each animal. A clinical score was calculated for every week until the end of clinical signs. A classification and regression tree (CART) analysis was conducted to determine the most important clinical signs every week for the first 7 weeks. The highest scores were recorded within 2 weeks of clinical onset. The first recorded clinical signs were quite obviously visible (lethargy, conjunctivitis, lesions of nasal mucosa, nasal discharge). Skin lesions, a drop in milk production and weight loss appeared later in the course of the disease. A biphasic pattern regarding nasal lesions was noticed: the first peak concerned mainly congestive and ulcerative lesions, whereas the second peak mainly concerned crusty lesions. The median time estimated by survival analysis to obtain negative RT-qPCR results from the onset of clinical signs was 195 days (range 166-213 days) in the 23 cattle included in the analysis. Serological results remained strongly positive until the end of the study. These results should ensure more accurate detection of an emerging infectious disease and are of prime importance in improving the modelling of BTV-8 persistence in Europe.

VL - 60 CP - 3 U1 - https://www.ncbi.nlm.nih.gov/pubmed/22571462?dopt=Abstract M3 - 10.1111/j.1865-1682.2012.01334.x ER - TY - JOUR T1 - Development and validation of three Capripoxvirus real-time PCRs for parallel testing. JF - J Virol Methods Y1 - 2013 A1 - Andy Haegeman A1 - Zro, K A1 - Frank Vandenbussche A1 - Demeestere, L A1 - Willem Van Campe A1 - Ennaji, M M A1 - Kris De Clercq KW - Animals KW - capripoxvirus KW - Poxviridae Infections KW - Real-Time Polymerase Chain Reaction KW - Sensitivity and Specificity KW - Veterinary Medicine AB -

Capripoxviruses have the potential to cause outbreaks with a severe socio-economic impact. The latter, combined with an altered virus dissemination pattern, warrants its status as an important emerging disease. Disease control or eradication programmes can only be applied successfully if the necessary diagnostic tools are available allowing clear and unequivocal identification of the pathogen. Real-time PCR combines high sensitivity/specificity with a reduced analysis time and is thus a proven useful tool for identification of many pathogens, including Capripoxviruses. In order for a real-time PCR to be used in a diagnostic capacity, the different analytical and diagnostic parameters need to be evaluated to assure data quality. The implementation of parallel testing using multiple real-time PCRs with similar characteristics can improve further Capripoxvirus diagnosis. It was therefore the purpose of this study to develop a triplet real-time PCR panel with similar high sensitivity/specificity and provide sufficient validation data regarding the performance characteristics that the panel can be used in parallel, depending on the purpose and local situation.

VL - 193 CP - 2 U1 - https://www.ncbi.nlm.nih.gov/pubmed/23850698?dopt=Abstract M3 - 10.1016/j.jviromet.2013.07.010 ER - TY - JOUR T1 - Experimental co-infections of calves with bluetongue virus serotypes 1 and 8. JF - Vet Microbiol Y1 - 2013 A1 - Dal Pozzo, Fabiana A1 - Martinelle, Ludovic A1 - Thys, Christine A1 - Sarradin, Pierre A1 - Ilse De Leeuw A1 - Willem Van Campe A1 - Kris De Clercq A1 - Thiry, Etienne A1 - Saegerman, Claude KW - Animals KW - Antibodies, Neutralizing KW - Antibodies, Viral KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Coinfection KW - Viremia AB -

The contemporary circulation of multiple bluetongue virus (BTV) serotypes or strains within the same territory can imply the co-infection of the ruminant and/or the vector populations. As a consequence, the clinical and pathological outcomes of co-infections as well as the biological properties of the viral progeny could be influenced and exhibit relevant variation. In this study, two independent co-infection experiments were carried out in calves using European strains of BTV serotypes 1 and 8 (BTV-1 and BTV-8, respectively), with the objective of studying the clinical and virological outcomes in comparison with BTV-1 and BTV-8 single infections. Synchronous co-infections using the same titre for the two viral strains were performed and the clinical signs were quantified using a standardized clinical form. Serotype-specific real-time RT-PCRs and viral isolation were used to monitor the course of viraemia. Neutralizing antibody titres were measured during the experiments, and necropsy with viral detection in the affected organs was performed. In the co-infected calves, a high BTV-8 viraemia was detected, while BTV-1 viraemia was irregular and sporadic. During BTV-1 single infection the development of viraemia and high titres of anti-BTV-1 neutralizing antibodies proved that the inoculum was infectious and the detection protocols were efficient. Several hypotheses could explain the predominant detection of BTV-8 in the co-infected calves, such as the occurrence of a privileged BTV-8 segment 2 reassortment, as recently described during in vitro BTV-1/BTV-8 co-infections; interference between the two viral strains; or a higher BTV-8 tropism for the bovine species.

VL - 165 CP - 1-2 U1 - https://www.ncbi.nlm.nih.gov/pubmed/23415033?dopt=Abstract M3 - 10.1016/j.vetmic.2013.01.016 ER - TY - JOUR T1 - Pulmonary artery haemorrhage in newborn calves following bluetongue virus serotype 8 experimental infections of pregnant heifers. JF - Vet Microbiol Y1 - 2013 A1 - Martinelle, Ludovic A1 - Dal Pozzo, Fabiana A1 - Sarradin, Pierre A1 - Ilse De Leeuw A1 - Kris De Clercq A1 - Thys, Christine A1 - Thiry, Etienne A1 - Saegerman, Claude KW - Animals KW - Antibodies, Neutralizing KW - Antibodies, Viral KW - Bluetongue KW - Bluetongue virus KW - brain KW - Cattle KW - Cattle Diseases KW - Europe KW - Female KW - milk KW - Placenta KW - Pregnancy KW - Pregnancy Complications, Infectious KW - Pulmonary Artery KW - RNA, Viral KW - Sheep KW - Vaccination AB -

The emergence of bluetongue disease (BT) among livestock in Europe in 2006 raised many questions including the occurrence and epidemiological significance of foetal infections in cattle. To clarify these aspects, vaccinated and unvaccinated pregnant heifers were sequentially infected twice in an isolation facility (biosafety level 3) with a northern European outbreak strain of Bluetongue virus serotype 8 (BTV-8). The study was terminated 2 months after calving with necropsy of the dams and their offspring. The cattle were monitored throughout the study by clinical scoring and for the presence of circulating neutralising antibodies, and after calving for the presence of infectious virus and viral RNA in blood and milk. Four calves, one born from a vaccinated dam and three from non-vaccinated ones, that were infected at 120 days of gestation had obvious haemorrhage of the pulmonary artery at necropsy. Although haemorrhage of the pulmonary artery is highly characteristic of BT, viral RNA was not detected in any of these calves. Furthermore, although none of the calves born from heifers infected prior to mid-gestation had teratogenic BTV typical brain lesions, some had lesions at birth suggestive of in utero BTV infection. Despite the lack of viral RNA detection, the presence of haemorrhage of the pulmonary artery deserves to be reported as a new observation in the context of the multiple investigations having as main subject the BTV placental crossing in cattle.

VL - 167 CP - 3-4 U1 - https://www.ncbi.nlm.nih.gov/pubmed/24035481?dopt=Abstract M3 - 10.1016/j.vetmic.2013.08.016 ER - TY - JOUR T1 - Rapid generation of replication-deficient monovalent and multivalent vaccines for bluetongue virus: protection against virulent virus challenge in cattle and sheep. JF - J Virol Y1 - 2013 A1 - Celma, Cristina C P A1 - Boyce, Mark A1 - van Rijn, Piet A A1 - Eschbaumer, Michael A1 - Wernike, Kerstin A1 - Hoffmann, Bernd A1 - Beer, Martin A1 - Andy Haegeman A1 - Kris De Clercq A1 - Roy, Polly KW - Animals KW - Antibodies, Neutralizing KW - Antibodies, Viral KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Disease Outbreaks KW - Europe KW - Female KW - Reassortant Viruses KW - RNA, Viral KW - Serotyping KW - Sheep, Domestic KW - Vaccines, Subunit KW - Viral Vaccines KW - Virus Replication AB -

Since 1998, 9 of the 26 serotypes of bluetongue virus (BTV) have spread throughout Europe, and serotype 8 has suddenly emerged in northern Europe, causing considerable economic losses, direct (mortality and morbidity) but also indirect, due to restriction in animal movements. Therefore, many new types of vaccines, particularly subunit vaccines, with improved safety and efficacy for a broad range of BTV serotypes are currently being developed by different laboratories. Here we exploited a reverse genetics-based replication-deficient BTV serotype 1 (BTV-1) (disabled infectious single cycle [DISC]) strain to generate a series of DISC vaccine strains. Cattle and sheep were vaccinated with these viruses either singly or in cocktail form as a multivalent vaccine candidate. All vaccinated animals were seroconverted and developed neutralizing antibody responses to their respective serotypes. After challenge with the virulent strains at 21 days postvaccination, vaccinated animals showed neither any clinical reaction nor viremia. Further, there was no interference with protection with a multivalent preparation of six distinct DISC viruses. These data indicate that a very-rapid-response vaccine could be developed based on which serotypes are circulating in the population at the time of an outbreak.

VL - 87 CP - 17 U1 - https://www.ncbi.nlm.nih.gov/pubmed/23824810?dopt=Abstract M3 - 10.1128/JVI.01514-13 ER - TY - JOUR T1 - Bluetongue sentinel surveillance program and cross-sectional serological survey in cattle in Belgium in 2010-2011. JF - Prev Vet Med Y1 - 2012 A1 - Vangeel, I A1 - Ilse De Leeuw A1 - Estelle Méroc A1 - Frank Vandenbussche A1 - Riocreux, F A1 - Hooyberghs, J A1 - Raemaekers, M A1 - Houdart, P A1 - Y Van der Stede A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - cross-sectional studies KW - Enzyme-Linked Immunosorbent Assay KW - Female KW - Population Surveillance KW - prevalence KW - Reverse Transcriptase Polymerase Chain Reaction KW - Seasons KW - Seroepidemiologic Studies AB -

Bluetongue virus serotype 8 (BTV-8) emerged in Central Western Europe in 2006 causing a large scale epidemic in 2007 that involved several European Union (EU) countries including Belgium. As in several other EU member states, vaccination against BTV-8 with inactivated vaccines was initiated in Belgium in spring 2008 and appeared to be successful. Since 2009, no clinical cases of Bluetongue (BT) have been reported in Belgium and BTV-8 circulation seemed to have completely disappeared by spring 2010. Therefore, a series of repeated cross-sectional surveys, the BT sentinel surveillance program, based on virus detection in blood samples by means of real-time RT-PCR (RT-qPCR) were carried out in dairy cattle from the end of 2010 onwards with the aim to demonstrate the absence of BTV circulation in Belgium. This paper describes the results of the first two sampling rounds of this BT sentinel surveillance program carried out in October-November 2010 and January-February 2011. In addition, the level of BTV-specific maternal antibodies in young non-vaccinated animals was monitored and the level of herd immunity against BTV-8 after 3 consecutive years of compulsory BTV-8 vaccination was measured by ELISA. During the 1st sampling round of the BT sentinel surveillance program, 15 animals tested positive and 2 animals tested doubtful for BTV RNA by RT-qPCR. During the 2nd round, 17 animals tested positive and 5 animals tested doubtful. The positive/doubtful animals in both rounds were re-sampled 2-4 weeks after the original sampling and then all tested negative by RT-qPCR. These results demonstrate the absence of BTV circulation in Belgium in 2010 at a minimum expected prevalence of 2% and 95% confidence level. The study of the maternal antibodies in non-vaccinated animals showed that by the age of 7 months maternal antibodies against BTV had disappeared in most animals. The BTV seroprevalence at herd level after 3 years of compulsory BTV-8 vaccination was very high (97.4% [95% CI: 96.2-98.2]). The overall true within-herd BTV seroprevalence in 6-24 month old Belgian cattle in early 2011 was estimated at 73.4% (95% CI: 71.3-75.4).

VL - 106 CP - 3-4 U1 - https://www.ncbi.nlm.nih.gov/pubmed/22483650?dopt=Abstract M3 - 10.1016/j.prevetmed.2012.03.011 ER - TY - JOUR T1 - Characteristics of serology-based vaccine potency models for foot-and-mouth disease virus. JF - Vaccine Y1 - 2012 A1 - Willems, Tom A1 - David Lefebvre A1 - Goris, Nesya A1 - Diev, Vyacheslav I A1 - Kremenchugskaya, Svetlana R A1 - Paul, Guntram A1 - Haas, Bernd A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Cattle KW - Enzyme-Linked Immunosorbent Assay KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Logistic Models KW - Male KW - Neutralization Tests KW - Predictive Value of Tests KW - Reproducibility of Results KW - Roc Curve KW - Statistics as Topic KW - Viral Vaccines AB -

BACKGROUND: Foot-and-mouth disease (FMD) vaccine potency testing involves hundreds of animals each year. Despite considerable efforts during the past decades, a challenge-free alternative vaccine potency test to replace the European protective dose 50% test (PD(50)) has not been implemented yet. The aim of the present study was to further characterize the properties of serological vaccine potency models.

METHODS: Logistic regression models were built for 5 serological assays from 3 different laboratories. The serum samples originated from 5 repeated PD(50) vaccine potency trials with a highly potent A/IRN/11/96 vaccine. Receiver Operating Characteristic analysis was used to determine a serological pass mark for predicting in vivo protected animals. Subsequently, an estimated PD(50) was calculated and the serotype dependency of the logistic models was investigated.

RESULTS: Although differences were observed between the laboratories and the serological assays used, the logistic models accurately predicted the in vivo protection status of the animals in 74-93% of the cases and the antibody pass levels corresponded to 84-97% of protection, depending on the serological assay used. For logistic models that combine different serotypes, the model fit can be increased by inclusion of a serotype factor in the logistic regression function.

CONCLUSIONS: The in vitro estimated PD(50) method may be at least as precise as the in vivo PD(50) test and may accurately predict the PD(50) content of a vaccine. However, the laboratory-effect and the serotype-dependency should be further investigated.

VL - 30 CP - 40 U1 - https://www.ncbi.nlm.nih.gov/pubmed/22824343?dopt=Abstract M3 - 10.1016/j.vaccine.2012.07.015 ER - TY - JOUR T1 - Viral RNA load in semen from bluetongue serotype 8-infected rams: relationship with sperm quality. JF - Vet J Y1 - 2012 A1 - Leemans, Jérôme A1 - Raes, Marianne A1 - Vanbinst, Tine A1 - Kris De Clercq A1 - Saegerman, Claude A1 - Kirschvink, Nathalie KW - Animals KW - Belgium KW - Biomarkers KW - Bluetongue KW - Bluetongue virus KW - Disease Outbreaks KW - Male KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - semen KW - Semen Analysis KW - Sheep KW - Spermatozoa AB -

This study investigated if viral RNA was detectable in the semen of rams clinically infected with bluetongue virus serotype 8 (BTV-8) by RT-qPCR, and to what extent the amount detected may be predictive of sperm quality. Semen samples were collected on six occasions from 93 BTV-8 infected rams involved in two longitudinal (n=12 and 27, respectively) and one cross-sectional (n=54) field study. Semen quality was assessed in terms of mass motility, concentration of spermatozoa, percentage of living and dead spermatozoa as well as cytological features. An overall semen quality score (SQS) was established. Depending upon the studied population, BTV RNA was detected in 75-100% of semen samples at initial testing 25-57 days post-observation (DPO) of clinical signs, and was detectable up to 116 DPO in a proportion of rams undergoing repeated sampling. Semen quality variables were significantly altered following natural BTV-8 infection and correlated with the amount of BTV RNA present. The SQS did not return to normal when virus was no longer detectable, suggesting that clearance of BTV precedes full recovery of sperm quality. In conclusion, viral RNA may be transiently recovered from the semen of BTV-8 affected rams and may serve as an indicator in predicting ram breeding potential following natural infection.

VL - 192 CP - 3 U1 - https://www.ncbi.nlm.nih.gov/pubmed/21802323?dopt=Abstract M3 - 10.1016/j.tvjl.2011.06.028 ER - TY - JOUR T1 - Cattle, sheep and pigs vaccinated against foot and mouth disease: does trade in these animals and their products present a risk of transmitting the disease? JF - Rev Sci Tech Y1 - 2011 A1 - Garland, A J M A1 - Kris De Clercq KW - Animals KW - Carrier State KW - Cattle KW - Cattle Diseases KW - Commerce KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Goat Diseases KW - Goats KW - Risk Assessment KW - Risk Factors KW - Sheep KW - Sheep Diseases KW - Swine KW - Swine Diseases KW - Vaccination KW - Viral Vaccines AB -

The foot and mouth disease (FMD) status of a country or region has a profound bearing on access to export markets for live animals and animal products. In countries without FMD-free status, and in accordance with the international standards of the World Organisation for Animal Health (OIE), restrictions may be applied to trade in both vaccinated and unvaccinated animals and their products. Available information suggests that, provided there is compliance with essential criteria concerning vaccines, vaccination and other zoosanitary measures (especially quarantine and ante- and post-mortem inspection), the risk of spreading FMD through the importation of vaccinated cattle, sheep and pigs is extremely small. The risk from products derived from vaccinated animals is even smaller, provided that appropriate risk mitigation measures are applied. Knowledge of the zoosanitary status of the exporting country is critical for risk assessment, but can be difficult to verify. Although empirical evidence and practical experience strongly indicate low risk, it is not possible to assert that the risk is zero for vaccinated animals or their products. In the absence of key factual data, risk analysis is only practicable on a qualitative or semi-quantitative basis. However, a very low level of risk is both unavoidable and acceptable if such trade is to be conducted.

VL - 30 CP - 1 U1 - https://www.ncbi.nlm.nih.gov/pubmed/21809764?dopt=Abstract M3 - 10.20506/rst.30.1.2023 ER - TY - JOUR T1 - Diagnostic performance and application of two commercial cell viability assays in foot-and-mouth disease research. JF - J Virol Methods Y1 - 2011 A1 - Willems, Tom A1 - David Lefebvre A1 - Neyts, Johan A1 - Kris De Clercq KW - Animals KW - Antibodies, Neutralizing KW - Antibodies, Viral KW - Automation KW - Cattle KW - Cattle Diseases KW - Cell Survival KW - Colorimetry KW - Fluorometry KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Sensitivity and Specificity KW - virology AB -

Cell-based assays are still used widely in foot-and-mouth disease (FMD) research, despite the existence of a wide variety of molecular techniques. The aim of this study was to validate an automated, quantitative spectrometric reading to replace the time-consuming and subjective microscopic (MIC) evaluation of the FMD virus-induced cytopathic effect (CPE). Therefore, the diagnostic performance of two commercial cell viability assays (CellTiter 96(®) AQueous One Solution Cell Proliferation Assay (MTS) and CellTiter-Blue(®) Cell Viability Assay (CTB), both from Promega, Leiden, The Netherlands) was evaluated. Following optimization of the assay protocols and using the MIC results as a reference standard, the absorbance-read MTS assay, the fluorescence-read CTB assay and the absorbance-read CTB (CTB(abs)) assay demonstrated similar high sensitivities (97%, 99% and 98%, respectively), specificities (100%, 98% and 99%, respectively), accuracy measures (0.99, 0.98 and 0.98, respectively), precision measures (1.00, 0.98 and 0.99, respectively) and Cohen kappa agreement indices (0.97, 0.97 and 0.96, respectively) for detecting CPE in cell cultures. Due to its performance, cost effectiveness and ease of use, the CTB(abs) assay was selected for further evaluation of its ability to detect virus neutralization and to screen antiviral compounds. The CTB(abs) assay had 99% sensitivity and 100% specificity for the detection of neutralizing antibodies in sera from cattle infected with FMDV and in sera from unvaccinated, uninfected cattle and resulted in a mean Z'-factor of 0.85 for antiviral compound test plates. The CTB(abs) assay is now used routinely in the Belgian FMD reference laboratory for serological testing and high-throughput antiviral compound screening.

VL - 173 CP - 1 U1 - https://www.ncbi.nlm.nih.gov/pubmed/21295609?dopt=Abstract M3 - 10.1016/j.jviromet.2011.01.015 ER - TY - JOUR T1 - The impact of naturally-occurring, trans-placental bluetongue virus serotype-8 infection on reproductive performance in sheep. JF - Vet J Y1 - 2011 A1 - Saegerman, Claude A1 - Bolkaerts, Benoît A1 - Baricalla, Christine A1 - Raes, Marianne A1 - Wiggers, Laetitia A1 - Ilse De Leeuw A1 - Frank Vandenbussche A1 - Zimmer, Jean-Yves A1 - Haubruge, Eric A1 - Cassart, Dominique A1 - Kris De Clercq A1 - Kirschvink, Nathalie KW - Abortion, Veterinary KW - Animals KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Disease Outbreaks KW - Female KW - Infectious Disease Transmission, Vertical KW - Placenta KW - Pregnancy KW - Pregnancy Complications, Infectious KW - Serotyping KW - Sheep AB -

Infection with bluetongue virus serotype (BTV)-8 occurred in ruminants in 2006 in Central-Western Europe. The trans-placental passage of this virus has been demonstrated in naturally- and experimentally-infected cattle and in experimentally-infected sheep. Trans-placental transmission is potentially important in the 'over-wintering' of this virus and its subsequent impact on reproductive performance. This epidemiological study was carried out on a sheep flock in Belgium that had experienced a severe outbreak of BTV-8 infection, and where the seroprevalence had increased from 1.3% to 88% between January and November 2007. In total, 476 lambs and 26 aborted fetuses from 300 ewes, lambing at four distinct time periods, were investigated between November 2007 and May 2008. The following evidence suggested that BTV-8 infection occurred in utero: (1) positive PCR results from splenic tissue from aborted fetuses (n=4); (2) fetal malformations suggestive of BTV infection (n=10); (3) positive PCR results from red blood cells in-lambs (n=7), and (4) the presence of antibody at birth in viable lambs prior to the intake of colostrum (n=9). The evidence provided by this investigation strongly suggests that trans-placental BTV-8 infection occurs in naturally-infected sheep and the impact of infection on the reproductive performance of such a naïve flock was considerable, with up to 25% of ewes aborting and with flock fertility reduced by 50%. The contribution of in utero-infected lambs to the over-wintering of BTV appears limited.

VL - 187 CP - 1 U1 - https://www.ncbi.nlm.nih.gov/pubmed/20061168?dopt=Abstract M3 - 10.1016/j.tvjl.2009.11.012 ER - TY - JOUR T1 - The presence of bluetongue virus serotype 8 RNA in Belgian cattle since 2008. JF - Transbound Emerg Dis Y1 - 2011 A1 - Garigliany, M A1 - Ilse De Leeuw A1 - Kleijnen, D A1 - Frank Vandenbussche A1 - Callens, J A1 - Van Loo, H A1 - Lebrun, M A1 - Saulmont, M A1 - Desmecht, D A1 - Kris De Clercq KW - Animals KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Carrier State KW - Cattle KW - Cattle Diseases KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Spleen KW - Time Factors KW - Viral Vaccines AB -

After a short winter break, bluetongue virus serotype 8 was responsible in 2007 for a large-scale epidemic among ruminant populations in Western Europe. Little is known about the mechanisms allowing the virus to survive winter conditions. A yearly mass vaccination of cattle and sheep started in spring 2008, which was recognized as successful in terms of clinical protection, but occult circulation of the bluetongue virus has not been adequately addressed. We studied the carriage of bluetongue RNA in the spleen of cattle in the vector-free period and the circulation of bluetongue virus in cattle populations in Belgium since the introduction of vaccination programmes. Overall, the results presented here show evidence for the long-term carriage of bluetongue virus RNA in the spleen of cattle and demonstrated a low but significant circulation and transplacental transmission of bluetongue virus in Belgian cattle in 2009, with apparent disappearance in 2010.

VL - 58 CP - 6 U1 - https://www.ncbi.nlm.nih.gov/pubmed/21605347?dopt=Abstract M3 - 10.1111/j.1865-1682.2011.01230.x ER - TY - JOUR T1 - Susceptibility of in vitro produced hatched bovine blastocysts to infection with bluetongue virus serotype 8. JF - Vet Res Y1 - 2011 A1 - Vandaele, Leen A1 - Wesselingh, Wendy A1 - Kris De Clercq A1 - Ilse De Leeuw A1 - Favoreel, Herman A1 - Van Soom, Ann A1 - Nauwynck, Hans KW - Animals KW - Antigens, Viral KW - Apoptosis KW - Blastocyst KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Fluorescent Antibody Technique, Indirect AB -

Bluetongue virus serotype 8 (BTV-8), which caused an epidemic in ruminants in central Western Europe in 2006 and 2007, seems to differ from other bluetongue serotypes in that it can spread transplacentally and has been associated with an increased incidence of abortion and other reproductive problems. For these reasons, and also because BTV-8 is threatening to spread to other parts of the world, there is a need for more information on the consequences of infection during pregnancy. The aim of the present study was to investigate whether hatched (i.e. zona pellucida-free) in vitro produced bovine blastocysts at 8-9 days post insemination are susceptible to BTV-8 and whether such infection induces cell death as indicated by apoptosis. Exposure of hatched in vitro produced bovine blastocysts for 1 h to a medium containing 10(3.8) or 10(4.9) TCID50 of the virus resulted in active viral replication in between 25 and 100% of the cells at 72 h post exposure. The infected blastocysts also showed growth arrest as evidenced by lower total cell numbers and a significant level of cellular apoptosis. We conclude from this in vitro study that some of the reproductive problems that are reported when cattle herds are infected with BTV-8 may be attributed to direct infection of blastocysts and other early-stage embryos in utero.

VL - 42 CP - 1 U1 - https://www.ncbi.nlm.nih.gov/pubmed/21314973?dopt=Abstract M3 - 10.1186/1297-9716-42-14 ER - TY - JOUR T1 - Two alternative inocula to reproduce bluetongue virus serotype 8 disease in calves. JF - Vaccine Y1 - 2011 A1 - Martinelle, Ludovic A1 - Dal Pozzo, Fabiana A1 - Sarradin, Pierre A1 - Ilse De Leeuw A1 - Kris De Clercq A1 - Thys, Christine A1 - Ziant, Dominique A1 - Thiry, Etienne A1 - Saegerman, Claude KW - Animals KW - Antibody Formation KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Female KW - Immunity, Humoral KW - Interferon-gamma KW - Interleukin-4 KW - RNA, Viral KW - Viremia AB -

The aim of this study was to investigate the consequences in calves of two forms of inocula alternative to the use of wild type infectious blood. Two groups of five calves were infected with low cell-passaged virus and infectious blood issued from one animal passage of the same strain. A longitudinal study was implemented and characterised by clinical standardised observations, haematology, BTV RNA detection and viral isolation from blood, detection of serogroup and neutralising antibodies, cytokine expression and post-mortem examination 46 days post-infection (PI). Both tested inocula were able to reproduce clinical expression of the disease, in the bloodstream viral genome was detected until the end of the experiment while virus isolation was possible between days 7 and 31 PI. Humoral immune response developed earlier in calves infected with low cell-passaged virus, while in both groups a massive antibody production was confirmed by the immune balance between IL-4 and IFN-γ expression. Both tested inocula are presented as valid alternative to the use of wild type infectious blood in the study of the pathogenesis of BTV-8 or the efficacy of current and future vaccines.

VL - 29 CP - 19 U1 - https://www.ncbi.nlm.nih.gov/pubmed/21376798?dopt=Abstract M3 - 10.1016/j.vaccine.2011.02.055 ER - TY - JOUR T1 - Bluetongue virus in wild deer, Belgium, 2005-2008. JF - Emerg Infect Dis Y1 - 2010 A1 - Linden, Annick A1 - Gregoire, Fabien A1 - Nahayo, Adrien A1 - Hanrez, David A1 - Mousset, Benedicte A1 - Massart, Audrey Laurent A1 - Ilse De Leeuw A1 - Vandemeulebroucke, Elise A1 - Frank Vandenbussche A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Deer KW - Disease Reservoirs KW - Geography KW - RNA, Viral KW - Seroepidemiologic Studies KW - Serotyping KW - Sheep KW - Spleen AB -

To investigate bluetongue virus serotype 8 infection in Belgium, we conducted a virologic and serologic survey on 2,416 free-ranging cervids during 2005-2008. Infection emerged in 2006 and spread over the study area in red deer, but not in roe deer.

VL - 16 CP - 5 U1 - https://www.ncbi.nlm.nih.gov/pubmed/20409376?dopt=Abstract M3 - 10.3201/eid1605.091217 ER - TY - JOUR T1 - Confidence in indirect assessment of foot-and-mouth disease vaccine potency and vaccine matching carried out by liquid phase ELISA and virus neutralization tests. JF - Vaccine Y1 - 2010 A1 - Robiolo, Blanca A1 - La Torre, José A1 - Maradei, Eduardo A1 - Beascoechea, Claudia Perez A1 - Perez, Alejandro A1 - Seki, Cristina A1 - Smitsaart, Eliana A1 - Fondevila, Norberto A1 - Palma, Eduardo A1 - Goris, Nesya A1 - Kris De Clercq A1 - Mattion, Nora KW - Animals KW - Cattle KW - Cattle Diseases KW - Cell Line KW - Cricetinae KW - Cross Protection KW - Enzyme-Linked Immunosorbent Assay KW - Foot-and-Mouth Disease KW - Neutralization Tests KW - Reproducibility of Results KW - Viral Vaccines AB -

The necessity of avoiding the use of animals in vaccine potency testing has been widely recognized. The repeatability and reproducibility of the Expected Percentage of Protection (EPP) as a serological potency surrogate for A24 Cruzeiro foot-and-mouth disease virus (FMDV) strain was assessed, and compared with the results obtained with challenge in the Protection against Podal Generalization (PPG) test. To determine the EPPs, the serum titers obtained by liquid phase blocking competitive ELISA (lpELISA) and virus neutralization (VNT) in 10 potency trials using the same A24 Cruzeiro vaccine, were interpolated into previously validated logit transformation curves that correlate PPG with serology. Indirect serological assessment of vaccine matching between the serotype A FMDV strains A24 Cruzeiro and A/Argentina/01 was also carried out by lpELISA and VNT. The results obtained in this study strongly support the replacement of challenge tests for vaccine potency by indirect serological assays, at least for A24 Cruzeiro FMDV strain. While determination of EPPs by lpELISA titers showed an excellent repeatability, reproducibility and concordance with PPG for vaccine potency, assessments of cross-protection by VNT titers were more consistent with the PPG outcome.

VL - 28 CP - 38 U1 - https://www.ncbi.nlm.nih.gov/pubmed/20643090?dopt=Abstract M3 - 10.1016/j.vaccine.2010.07.012 ER - TY - JOUR T1 - Development of a foot-and-mouth disease infection model in severe combined immunodeficient mice for the preliminary evaluation of antiviral drugs. JF - Transbound Emerg Dis Y1 - 2010 A1 - David Lefebvre A1 - Neyts, J A1 - Kris De Clercq KW - Animals KW - Antiviral Agents KW - Disease Models, Animal KW - Female KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Immunocompromised Host KW - Male KW - mice KW - Mice, Inbred BALB C AB -

Recent European guidelines facilitate the use of emergency vaccines during outbreaks of foot-and-mouth disease. Antiviral drugs could be used as a complementary measure. This study aimed at developing a small animal model to assess the in vivo activity of early antiviral lead molecules with anti-foot-and-mouth disease virus (FMDV) activity in vitro. In a first attempt, several FMDV strains were titrated in Balb/c mice. Inoculations with O₁ Manisa or C₁ Noville did not induce clinical disease, whereas Asia1 Shamir induced death too rapidly [i.e. within 4 days post-inoculation (dpi)]. Therefore, we switched to severe combined immunodeficient mice which are frequently used as a model for viral infections and experimental therapeutics. Strain O₁ Manisa did not induce clinical disease, but titrations with A₂₂ Iraq, C₁ Noville or Asia1 Shamir resulted in virus-induced morbidity (including respiratory problems and weight loss) with subsequent mortality. Inoculations with strain A₂₂ Iraq resulted in a reproducible mean time of death of 6 dpi (this was shorter for the other strains). In this newly developed rodent model, strain A₂₂ Iraq seems the most suited to assess the in vivo anti-FMDV activity of selective inhibitors of FMDV.

VL - 57 CP - 6 U1 - https://www.ncbi.nlm.nih.gov/pubmed/21029400?dopt=Abstract M3 - 10.1111/j.1865-1682.2010.01169.x ER - TY - JOUR T1 - A duplex real-time RT-PCR for the detection of bluetongue virus in bovine semen. JF - J Virol Methods Y1 - 2010 A1 - Vanbinst, Tine A1 - Frank Vandenbussche A1 - Dernelle, Eric A1 - Kris De Clercq KW - Animals KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Reproducibility of Results KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - semen KW - Sensitivity and Specificity KW - virology AB -

The control measures prescribed by the World Organization for Animal Health (OIE) for international trade in extended semen implicate repeated free testing of the donor's blood for bluetongue virus (BTV). The aim of this study was to validate a real-time RT-PCR for the direct testing of semen for artificial insemination (AI). The amplification of the BTV target was combined with an internal control target in duplex format. Optimal RNA recovery and efficient removal of PCR inhibitors was established using Trizol-based RNA extraction. The total assay was highly repeatable, the preliminary analysis of the specificity was 100% (95% CI: 92-100%) and the limit of detection was -0.36 log(10)TCID(50) ml(-1) (95% CI: -0.53 to -0.18 log(10)TCID(50) ml(-1)) in BTV-8 spiked extended semen. The protocol was evaluated using 89 extended semen samples from 19 bulls showing typical clinical signs of a natural BTV-8 infection. Forty-eight samples were positive, 30 were doubtful and 11 were negative. Infectious BTV-8 was isolated. Based on varying real-time RT-PCR results of additional straws from cut-off samples it is highly recommended to analyse at least five straws per semen batch before declaring semen free of BTV. In conclusion, the partially validated assay presented has the potential to be used for the control of semen for international trade through direct testing of the semen.

VL - 169 CP - 1 U1 - https://www.ncbi.nlm.nih.gov/pubmed/20674609?dopt=Abstract M3 - 10.1016/j.jviromet.2010.07.019 ER - TY - JOUR T1 - Haemorrhagic diathesis in neonatal calves: an emerging syndrome in Europe. JF - Transbound Emerg Dis Y1 - 2010 A1 - Pardon, B A1 - Steukers, L A1 - Dierick, J A1 - Ducatelle, R A1 - Saey, V A1 - Maes, S A1 - Vercauteren, G A1 - Kris De Clercq A1 - Callens, J A1 - De Bleecker, K A1 - Deprez, P KW - Animals KW - Animals, Newborn KW - Autopsy KW - Bone Marrow KW - Cattle KW - Cattle Diseases KW - Europe KW - Hemorrhage KW - Hemorrhagic Disorders KW - Kidney KW - Melena KW - Pancytopenia KW - polymerase chain reaction KW - Purpura KW - Syndrome AB -

In 2008 and 2009 a large number of cases of haemorrhagic diathesis (HD) in neonatal calves were reported in different European countries. In Flanders, 84 cases of neonatal HD in 30 herds were reported in this period. The disease typically affects calves younger than 1 month old from different breed and gender. Prominent clinical signs are cutaneous bleeding, petechiae on all mucosae, melena and often high fever. Early in the disease, the mental state of the animals is uncompromised. The typical haematological finding is pancytopenia, with severe to complete thrombocytopenia being the cause of the increased susceptibility to bleeding. In seven of the affected herds blood samples of calves of the same age group as the clinical case were collected and on six of those farms at least one subclinical case could be identified. Necropsy findings were generalized petechiae, ecchymoses or haemorrhages and variable lymphadenopathy. Histopathology of haemorrhagic lesions revealed multifocal extravasation of red blood cells (haemorrhage) with preservation of tissue architecture and absence of other abnormalities. Total bone marrow aplasia and depletion of all lymphoid tissue was the most prominent finding on histology. Activated macrophages and haemophagocytosis were seen on bone marrow cytology from two live calves. Polymerase chain reaction for bovine viral diarrhoea virus, bluetongue and epizootic haemorrhagic disease virus was negative. Several attempts to isolate a viral agent were unsuccessful.

VL - 57 CP - 3 U1 - https://www.ncbi.nlm.nih.gov/pubmed/20202175?dopt=Abstract M3 - 10.1111/j.1865-1682.2010.01098.x ER - TY - JOUR T1 - The most likely time and place of introduction of BTV8 into Belgian ruminants. JF - PLoS One Y1 - 2010 A1 - Saegerman, Claude A1 - Mellor, Philip A1 - Uyttenhoef, Aude A1 - Jean-Baptiste Hanon A1 - Kirschvink, Nathalie A1 - Haubruge, Eric A1 - Delcroix, Pierre A1 - Houtain, Jean-Yves A1 - Pourquier, Philippe A1 - Frank Vandenbussche A1 - Verheyden, Bart A1 - Kris De Clercq A1 - Czaplicki, Guy KW - Animals KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Ceratopogonidae KW - Geography KW - Insect Vectors KW - Population Density KW - Retrospective Studies KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Ruminants KW - Serotyping KW - Sheep KW - Sheep Diseases KW - Time Factors AB -

BACKGROUND: In northern Europe, bluetongue (BT) caused by the BT virus (BTV), serotype 8, was first notified in August 2006 and numerous ruminant herds were affected in 2007 and 2008. However, the origin and the time and place of the original introduction have not yet been determined.

METHODS AND PRINCIPAL FINDINGS: Four retrospective epidemiological surveys have been performed to enable determination of the initial spatiotemporal occurrence of this emerging disease in southern Belgium: investigations of the first recorded outbreaks near to the disease epicenter; a large anonymous, random postal survey of cattle herds and sheep flocks; a random historical milk tank survey of samples tested with an indirect ELISA and a follow-up survey of non-specific health indicators. The original introduction of BTV into the region probably occurred during spring 2006 near to the National Park of Hautes Fagnes and Eifel when Culicoides become active.

CONCLUSIONS/SIGNIFICANCE: The determination of the most likely time and place of introduction of BTV8 into a country is of paramount importance to enhance awareness and understanding and, to improve modeling of vector-borne emerging infectious diseases.

VL - 5 CP - 2 U1 - https://www.ncbi.nlm.nih.gov/pubmed/20195379?dopt=Abstract M3 - 10.1371/journal.pone.0009405 ER - TY - JOUR T1 - Oesophageal paresis associated with bluetongue virus serotype 8 in cattle. JF - Vet Rec Y1 - 2010 A1 - Pardon, B A1 - Valerie Vandenberge A1 - Maes, S A1 - Kris De Clercq A1 - Ducatelle, R A1 - Deprez, P KW - Animals KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Disease Outbreaks KW - Esophageal Diseases KW - Female KW - Male KW - Serotyping VL - 167 CP - 15 U1 - https://www.ncbi.nlm.nih.gov/pubmed/21257422?dopt=Abstract M3 - 10.1136/vr.c4979 ER - TY - JOUR T1 - A proposed validation method for automated nucleic acid extraction and RT-qPCR analysis: an example using Bluetongue virus. JF - J Virol Methods Y1 - 2010 A1 - Vandemeulebroucke, Elise A1 - Kris De Clercq A1 - Yves Van der Stede A1 - Frank Vandenbussche KW - Animals KW - Automation KW - Bluetongue virus KW - Cell Line KW - Chick Embryo KW - Clinical Laboratory Techniques KW - Cricetinae KW - Diagnostic Errors KW - nucleic acids KW - Reference Standards KW - Reproducibility of Results KW - Reverse Transcriptase Polymerase Chain Reaction KW - Validation Studies as Topic AB -

This study proposes a validation strategy for an automated extraction procedure, followed by RT-qPCR analysis. To avoid false-negative results, a triplex RT-qPCR was used which detects the target viral RNA, an internal and an external control. The methods to determine the validation parameters such as linearity, efficiency, analytical sensitivity, analytical specificity and intra- and interrun variability are described in detail. Special attention is given to the analytical sensitivity, which is determined by probit analysis. The limit of detection was set at the input concentration resulting in a positive result in 95% of the repeats. The intra- and interrun variability was analysed profoundly by testing samples covering a broad range of viral loads, from strong positive to weak positive. To increase the diagnostic capacity, the extraction protocol was automated with a JANUS Automated Workstation (PerkinElmer, Waltham, MA), which can extract 186 samples in 2h and 30 min. The automation of the extraction protocol implied some additional validation parameters to be determined such as position-effect, absence of cross-contamination and comparison with the manual protocol. These parameters give essential information about the performance of the robot and are of great importance when the automated assay is used in an accreditation system.

VL - 165 CP - 1 U1 - https://www.ncbi.nlm.nih.gov/pubmed/20116401?dopt=Abstract M3 - 10.1016/j.jviromet.2010.01.007 ER - TY - JOUR T1 - Simultaneous detection of bluetongue virus RNA, internal control GAPDH mRNA, and external control synthetic RNA by multiplex real-time PCR. JF - Methods Mol Biol Y1 - 2010 A1 - Frank Vandenbussche A1 - Vandemeulebroucke, Elise A1 - Kris De Clercq KW - Animals KW - Bluetongue virus KW - Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) KW - Reference Standards KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Sensitivity and Specificity AB -

Bluetongue is an insect-borne disease of domestic and wild ruminants that requires strict monitoring by sensitive, reproducible and robust methods. Real-time reverse transcription polymerase chain reaction (RT-qPCR) analysis has become the method of choice for routine viral diagnosis. As false-negative test results can have serious implications; an internal/external control system should be incorporated in each analysis to detect RT-qPCR failure due to poor sample quality, improper nucleic acid extraction and/or PCR inhibition. To increase the diagnostic capacity and reduce costs, it is recommended to use a multiplex strategy which enables the amplification of multiple targets in a single reaction. This chapter describes the application of a triplex RT-qPCR for the simultaneous detection of bluetongue viral RNA, an internal control and an external control. The primer and probe sequences of the BTV RT-qPCR were taken from Toussaint et al. (J Virol Methods 140:115-123, 2007), whereas the internal and external RT-qPCRs were specifically designed to detect endogenous glyceraldehyde-3-phosphate dehydrogenase mRNA and a synthetic RNA, respectively. To maximize the sensitivity of the assay, the primer concentrations of the internal/external control reactions were limited and the amount of Taq DNA polymerase was increased. A comparison of the singleplex versus triplex RT-qPCR indicated that the triplex RT-qPCR exhibits a higher analytical sensitivity. Due to the incorporation of the internal/external control system, the triplex RT-qPCR allows an even more reliable and rapid diagnosis of bluetongue than the previously described singleplex RT-qPCR (J Virol Methods 140:115-123, 2007).

VL - 630 U1 - https://www.ncbi.nlm.nih.gov/pubmed/20300993?dopt=Abstract M3 - 10.1007/978-1-60761-629-0_7 ER - TY - JOUR T1 - Bluetongue control in Europe--new challenges and achievements. JF - Berl Munch Tierarztl Wochenschr Y1 - 2009 A1 - Makoschey, B A1 - Maclachlan, J A1 - v Wuijckhuise, L A1 - Kirschvink, N A1 - dal Pozzo, F A1 - Petit, H A1 - Kaandorp-Huber, C A1 - P van Rijn A1 - Sellal, E A1 - Oura, C A1 - Boinas, F A1 - Cavirani, S A1 - Kris De Clercq A1 - Lucientes, J A1 - Meijjes, C Posthumus A1 - Zientara, S A1 - Meyer, G A1 - Thiry, E KW - Animals KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Costs and Cost Analysis KW - Europe KW - Female KW - Male KW - reproduction KW - Sheep KW - Vaccination KW - Viral Vaccines VL - 122 CP - 7-8 U1 - http://www.ncbi.nlm.nih.gov/pubmed/19681404?dopt=Abstract ER - TY - JOUR T1 - Bluetongue in Belgium: episode II. JF - Transbound Emerg Dis Y1 - 2009 A1 - Estelle Méroc A1 - Herr, C A1 - Verheyden, B A1 - Hooyberghs, J A1 - Houdart, P A1 - Raemaekers, M A1 - Frank Vandenbussche A1 - Kris De Clercq A1 - Mintiens, K KW - Abortion, Veterinary KW - Animals KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Disease Outbreaks KW - Female KW - Goat Diseases KW - Goats KW - Male KW - Pregnancy KW - Serotyping KW - Sheep AB -

Bluetongue (BT) is an arthropod-borne viral disease of ruminants. In August 2006, domestic ruminant populations in Northern Europe became infected with BT virus serotype 8 (BTV-8). The first BTV-8-case of the year 2007 in Belgium was notified in July. This case was the starting point of a second wave of BT outbreaks. The main objective of this study was to describe the evolution and the clinical impact of the second episode of BT in Belgium. In addition, the main differences with the previous episode (August-December 2006) are reported. Both outbreak and rendering plant data were analysed. Overall cumulative incidence at herd level was estimated at 11.5 (11.2-11.8) and 7.5 (7.3-7.8) per cent in cattle and sheep populations respectively. The findings went in favour of a negative association between within-herd prevalence in 2006 and the risk of showing clinical signs of BT in 2007 (via protective immunity). A high level of correlation was demonstrated between BT incidence and small ruminant mortality data when shifting the latter of 1-week backwards. This result supports the hypothesis that the high increase in small ruminant mortality observed in 2007 was the consequence of the presence of BT. For cattle, the correlation was not as high. An increase in cattle foetal mortality was also observed during the year 2007 and a fair correlation was found between BT incidence and foetal mortality.

VL - 56 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/19200297?dopt=Abstract M3 - 10.1111/j.1865-1682.2008.01063.x ER - TY - JOUR T1 - Bluetongue virus detection by real-time RT-PCR in Culicoides captured during the 2006 epizootic in Belgium and development of an internal control. JF - Transbound Emerg Dis Y1 - 2009 A1 - Vanbinst, T A1 - Frank Vandenbussche A1 - Vandemeulebroucke, E A1 - Ilse De Leeuw A1 - Deblauwe, I A1 - De Deken, G A1 - Madder, M A1 - Haubruge, E A1 - Losson, B A1 - Kris De Clercq KW - Animals KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Ceratopogonidae KW - Disease Outbreaks KW - Female KW - Insect Vectors KW - Male KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Sheep AB -

After the emergence of bluetongue (BT) in Belgium in 2006, two types of entomological surveys were initiated, the one to identify the local vector species, and the other to study their population dynamics. In the vector study, Culicoides were captured near farms with recently infected cattle or sheep; in the population study Culicoides were captured in two meadows situated in the BT-affected region. A total of 130 pools of parous, non-blood engorged female midges (with a mean of 7.5 midges per pool) were analysed with real-time reverse transcription PCR (RT-qPCR) targeting bluetongue virus (BTV) segment 5. To ensure the RNA integrity of the samples, all pools were also tested in a second RT-qPCR targeting Culicoides 18S rRNA, which served as an internal control. Seventeen pools with negative results for both 18S and BTV were excluded, most of which originated from the population survey. In the vector survey near outbreak sites, female midges of the obsoletus complex, including C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, dominated the black-light trap collections with 19 of 89 pools being BTV-positive. Moreover, all the collections from the vector survey included at least one positive pool of the obsoletus complex compared with only 20% collections (C. obsoletus/C. scoticus) in the population survey. The current study also revealed the presence of BTV RNA in one of five pools of C. pulicaris females captured near recent BT outbreaks, suggesting that this species might have played a role in transmission. Finally, the use of RT-qPCR for the recognition of new potential BTV vector species and the impact of an appropriate monitoring method and internal control are discussed.

VL - 56 CP - 5 U1 - http://www.ncbi.nlm.nih.gov/pubmed/19432638?dopt=Abstract M3 - 10.1111/j.1865-1682.2009.01077.x ER - TY - JOUR T1 - Emergence of bluetongue serotypes in Europe, part 1: description and validation of four real-time RT-PCR assays for the serotyping of bluetongue viruses BTV-1, BTV-6, BTV-8 and BTV-11. JF - Transbound Emerg Dis Y1 - 2009 A1 - Frank Vandenbussche A1 - Ilse De Leeuw A1 - Vandemeulebroucke, E A1 - Kris De Clercq KW - Animals KW - Bluetongue virus KW - Cell Line KW - Cricetinae KW - Europe KW - Reproducibility of Results KW - Reverse Transcriptase Polymerase Chain Reaction KW - Sensitivity and Specificity KW - Serotyping AB -

The control of bluetongue virus (BTV) in Central-Western Europe is greatly complicated by the coexistence of several BTV serotypes. Rapid, sensitive and specific assays are therefore needed to correctly identify the currently circulating BTV serotypes in field samples. In the present study, four serotype-specific real-time RT-PCR assays (RT-qPCR) are described for the detection of the BTV-1, BTV-6, BTV-8 and BTV-11 serotypes. The analytical sensitivity of the BTV-1/S2, BTV-6/S2, BTV-8/S2 and BTV-11/S2 serotype-specific RT-qPCR assays is comparable to the earlier described serogroup-specific pan-BTV/S5 RT-qPCR assay. In silico and in vitro analyses indicated that none of the assays cross-react with viruses which are symptomatically or genetically related to BTV and only detect the intended BTV serotypes. All assays exhibited a linear range of at least 0.05-3.80 log(10) TCID(50) ml(-1) and a PCR-efficiency approaching the ideal amplification factor of two per PCR cycle. Both intra- and inter-run variations were found to be low with a total coefficient of variation of 1-2% for clear positive samples and <10% for very weak positive samples. Finally, the performance of the described assays was compared with commercially available kits for the detection of BTV-1, BTV-6 and BTV-8. Three in-house assays gave exactly the same diagnostic result (positive/negative) as the commercial assays and can thus be used interchangeably. Together with the earlier described serogroup-specific pan-BTV/S5, the serotype-specific RT-qPCR assays form a flexible and properly validated set of tools to detect and differentiate the BTV serotypes currently circulating in Central-Western Europe.

VL - 56 CP - 9-10 U1 - https://www.ncbi.nlm.nih.gov/pubmed/19824952?dopt=Abstract M3 - 10.1111/j.1865-1682.2009.01093.x ER - TY - JOUR T1 - Emergence of bluetongue serotypes in Europe, part 2: the occurrence of a BTV-11 strain in Belgium. JF - Transbound Emerg Dis Y1 - 2009 A1 - Kris De Clercq A1 - Mertens, P A1 - Ilse De Leeuw A1 - Oura, C A1 - Houdart, P A1 - Potgieter, A C A1 - Maan, S A1 - Hooyberghs, J A1 - Batten, C A1 - Vandemeulebroucke, E A1 - Wright, I M A1 - Maan, N A1 - Riocreux, F A1 - Sanders, A A1 - Yves Van der Stede A1 - Nomikou, K A1 - Raemaekers, M A1 - Bin-Tarif, A A1 - Shaw, A A1 - Henstock, M A1 - Bréard, E A1 - E. Dubois A1 - Gastaldi-Thiéry, C A1 - Zientara, S A1 - Verheyden, B A1 - Frank Vandenbussche KW - Animals KW - Antibodies, Viral KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - cross-sectional studies KW - Dairying KW - Female KW - Population Surveillance KW - Pregnancy KW - Pregnancy Complications KW - RNA, Viral KW - Seasons KW - Sheep AB -

An EDTA-blood sample from a cow without clinical signs, which gave early birth to a newborn calf that died soon after delivery, was shown to be positive for bluetongue virus (BTV)-RNA using a group-specific real-time RT-PCR (RT-qPCR). In-house serotype-specific RT-qPCR assays for bluetongue virus serotype 1 (BTV-1), -6 and -8 all gave negative results. Subsequent assays were carried out using conventional (gel-based) RT-PCR primers for all 25 BTV serotypes and only two primer sets, both specific for BTV-11, gave bands of the expected size. The cDNAs generated were sequenced and comparisons of the genome segment 2 sequence with that of the modified 'live' vaccine strain of BTV-11 from South Africa showed 100% identity. A survey of all ruminants in a 1-km area around the first positive farm using a BTV-11 serotype-specific RT-qPCR revealed five other holdings with in total nine BTV-11 positive animals. A cross-sectional monitoring of dairy cattle in Belgium showed an overall prevalence of 3.8% on herd level and 0.2% on animal level. A BTV-11 has been introduced into the Belgian cattle herd during the 2008 vector season. The source of the infection and the way by which the virus was introduced are unknown.

VL - 56 CP - 9-10 U1 - http://www.ncbi.nlm.nih.gov/pubmed/19909474?dopt=Abstract M3 - 10.1111/j.1865-1682.2009.01092.x ER - TY - JOUR T1 - Experimental reproduction of bluetongue virus serotype 8 clinical disease in calves. JF - Vet Microbiol Y1 - 2009 A1 - dal Pozzo, F A1 - Kris De Clercq A1 - Guyot, H A1 - Vandemeulebroucke, E A1 - Sarradin, P A1 - Frank Vandenbussche A1 - Thiry, E A1 - Saegerman, C KW - Animals KW - Antibodies, Viral KW - Bluetongue KW - Bluetongue virus KW - Body Temperature KW - Cattle KW - Cattle Diseases KW - Enzyme-Linked Immunosorbent Assay KW - Female KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Viremia AB -

Cattle are commonly subclinically infected following natural or experimental infection with bluetongue virus (BTV). The introduction of BTV serotype 8 (BTV-8) in Europe has been characterized by the manifestation of clinical signs in infected cattle. In order to study the pathogenesis of BTV-8 in this host, an animal model able to reproduce the clinical manifestations of the disease is required. In this work, two calves were subcutaneously and intravenously injected with a low passage cell-adapted strain of BTV-8. Both calves showed typical bluetongue clinical signs, including pyrexia, ocular discharge, conjunctivitis, oral mucosal congestion, development of ulcers and necrotic lesions on the lips and tongue, submandibular oedema, coronitis and oedema of the coronet and pastern region. A score was assigned depending on the severity of the lesions and a total clinical score was calculated for each animal daily and at the end of the experiment. Both calves became viraemic 24h post-infection and seroconversion occurred between 7 and 11 days P.I. In this study we present the development of a protocol of infection in calves able to reproduce the severity of the lesions observed with BTV-8 in field conditions.

VL - 136 CP - 3-4 U1 - http://www.ncbi.nlm.nih.gov/pubmed/19128895?dopt=Abstract M3 - 10.1016/j.vetmic.2008.11.012 ER - TY - JOUR T1 - Modulating mouse innate immunity to RNA viruses by expressing the Bos taurus Mx system. JF - Transgenic Res Y1 - 2009 A1 - Garigliany, M-M A1 - Cloquette, K A1 - Mathias Leroy A1 - Decreux, A A1 - Goris, N A1 - Kris De Clercq A1 - Desmecht, D KW - Animals KW - Cattle KW - Cells, Cultured KW - GTP-Binding Proteins KW - Immunity, Innate KW - mice KW - Mice, Transgenic KW - Myxovirus Resistance Proteins KW - RNA Viruses AB -

Mx proteins are interferon-induced members of the dynamin superfamily of large guanosine triphosphatases. These proteins have attracted much attention because some display antiviral activity against pathogenic RNA viruses, such as members of the orthomyxoviridae, bunyaviridae, and rhabdoviridae families. Among the diverse mammalian Mx proteins examined so far, we have recently demonstrated in vitro that the Bos taurus isoform 1 (boMx1) is endowed with exceptional anti-rabies-virus activity. This finding has prompted us to seek an appropriate in vivo model for confirming and evaluating gene therapy strategies. Using a BAC transgene, we have generated transgenic mouse lines expressing the antiviral boMx1 protein and boMx2 proteins under the control of their natural promoter and short- and long-range regulatory elements. Expressed boMx1 and boMx2 are correctly assembled, as deduced from mRNA sequencing and western blotting. Poly-I/C-subordinated expression of boMx1 was detected in various organs by immunohistochemistry, and transgenic lines were readily classified as high- or low-expression lines on the basis of tissue boMx1 concentrations measured by ELISA. Poly-I/C-induced Madin-Darby bovine kidney cells, bovine turbinate cells, and cultured cells from high-expression line of transgenic mice were found to contain about the same concentration of boMx1, suggesting that this protein is produced at near-physiological levels. Furthermore, insertion of the bovine Mx system rendered transgenic mice resistant to vesicular-stomatitis-virus-associated morbidity and mortality, and embryonic fibroblasts derived from high-expression transgenic mice were far less permissive to the virus. These results demonstrate that the Bos taurus Mx system is a powerful anti-VSV agent in vivo and suggest that the transgenic mouse lines generated here constitute a good model for studying in vivo the various antiviral functions-known and yet to be discovered-exerted by bovine Mx proteins, with priority emphasis on the antirabic function of boMx1.

VL - 18 CP - 5 U1 - http://www.ncbi.nlm.nih.gov/pubmed/19387858?dopt=Abstract M3 - 10.1007/s11248-009-9268-x ER - TY - JOUR T1 - Quantification of foot-and-mouth disease virus transmission rates using published data. JF - ALTEX Y1 - 2009 A1 - Goris, Nesya E A1 - Eblé, Phaedra L A1 - de Jong, Mart C M A1 - Kris De Clercq KW - Animal Testing Alternatives KW - Animals KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Sheep KW - Sheep Diseases KW - Swine KW - Swine Diseases AB -

Foot-and-mouth disease is an extremely infectious and devastating disease affecting all species of cloven-hoofed animals. To understand the epidemiology of the causative virus and predict viral transmission dynamics, quantified transmission parameters are essential to decision makers and modellers alike. However, such quantified parameters are scarcely available, and recently a series of animal experiments was set up to obtain such data experimentally. In this communication, however, we report on the use of data from an animal experiment conducted 10 years ago to quantify transmission of foot-and-mouth disease virus between non-vaccinated sheep and from sub-clinically infected sheep to in-contact pigs. This new analysis utilises a state-of-the-art Generalised Linear Model to estimate the transmission rate. From the obtained results it is concluded that meta-analysis of "old" experiments using newly developed techniques can provide useful data to replace, reduce and refine future foot-and-mouth disease transmission experiments, thereby minimising animal suffering for research purposes.

VL - 26 CP - 1 U1 - http://www.ncbi.nlm.nih.gov/pubmed/19326033?dopt=Abstract ER - TY - JOUR T1 - Some guidelines for determining foot-and-mouth disease vaccine strain matching by serology. JF - Vaccine Y1 - 2009 A1 - Mattion, Nora A1 - Goris, Nesya A1 - Willems, Tom A1 - Robiolo, Blanca A1 - Maradei, Eduardo A1 - Beascoechea, Claudia Perez A1 - Perez, Alejandro A1 - Smitsaart, Eliana A1 - Fondevila, Norberto A1 - Palma, Eduardo A1 - Kris De Clercq A1 - La Torre, José KW - Animals KW - Antibodies, Viral KW - Argentina KW - Cattle KW - Cross Reactions KW - Disease Outbreaks KW - Enzyme-Linked Immunosorbent Assay KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Immunoassay KW - Neutralization Tests KW - Viral Vaccines AB -

The selection of matching strains for use in outbreaks of foot-and-mouth disease (FMD) virus can be assessed in vivo or by serological r-value determination. Sera from animals involved in vaccine potency and cross-protection trials performed using the "Protection against Podal Generalization" (PPG) test for two serotype A strains were collected and analyzed by the virus neutralization test (VNT) and liquid-phase ELISA (lpELISA) in three laboratories. The average VNT r-values for medium and high serum titer classes from the A(24) Cruzeiro vaccinated animals were in line with the A/Arg/01 heterologous PPG outcome for all testing laboratories, suggesting that the vaccine strain A(24) Cruzeiro is unlikely to protect against the field isolate A/Arg/01. The corresponding lpELISA r-values were slightly higher and indicate a closer relationship between both strains. Pooling of serum samples significantly reduced the inter-animal and inter-trial variation. The results suggest that a suitable reference serum for vaccine matching r-value experiments might be a pool or a medium to high VNT or lpELISA titer serum. Furthermore, the VNT seems to produce the most reproducible inter-laboratory results. More work is, however, needed in order to substantiate these claims.

VL - 27 CP - 5 U1 - http://www.ncbi.nlm.nih.gov/pubmed/19041355?dopt=Abstract M3 - 10.1016/j.vaccine.2008.11.026 ER - TY - JOUR T1 - Validation of two real-time RT-PCR methods for foot-and-mouth disease diagnosis: RNA-extraction, matrix effect, uncertainty of measurement and precision. JF - J Virol Methods Y1 - 2009 A1 - Goris, Nesya A1 - Frank Vandenbussche A1 - Herr, Cécile A1 - Villers, Jérôme A1 - Yves Van der Stede A1 - Kris De Clercq KW - Animals KW - blood KW - Diagnostic Errors KW - Foot KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Sensitivity and Specificity AB -

Real-time reverse transcription polymerase chain reaction (rRT-PCR) assays are being used routinely for diagnosing foot-and-mouth disease virus (FMDV). Although most laboratories determine analytical and diagnostic sensitivity and specificity, a thorough validation in terms of establishing optimal RNA-extraction conditions, matrix effect, uncertainty of measurement and precision is not performed or reported generally. In this study, different RNA-extraction procedures were compared for two FMDV rRT-PCRs. The NucleoSpin columns available commercially combined high extraction efficiency with ease-of-automation. Furthermore, six different FMDV-negative matrices were spiked with a dilution series of FMDV SAT1 ZIM 25/89. Compared to cell-culture-spiked viral control samples, no matrix effect on the analytical sensitivity was found for blood or foot epithelium. Approximately 1log(10) reduction in detection limit was noted for faecal and tongue epithelium samples, whereas a 3log(10) decrease was observed for spleen samples. By testing the same dilution series in duplicate on 10 different occasions, an estimation of uncertainty of measurement and precision was obtained using blood as matrix. Both rRT-PCRs produced highly precise results emphasising their potential to replace conventional virological methods. The uncertainty measurement, as described in this study, proved to be a useful tool to evaluate the probability of making a wrong decision.

VL - 160 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/19447138?dopt=Abstract M3 - 10.1016/j.jviromet.2009.05.005 ER - TY - JOUR T1 - Assessment of the diagnostic potential of immuno-RCA in 96-well ELISA plates for foot-and-mouth disease virus. JF - J Virol Methods Y1 - 2008 A1 - Wesley Van Dessel A1 - Frank Vandenbussche A1 - Staes, Mik A1 - Goris, Nesya A1 - Kris De Clercq KW - Animals KW - Cattle KW - Cattle Diseases KW - Enzyme-Linked Immunosorbent Assay KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus AB -

The need for fast and very early detection of foot-and-mouth disease virus (FMDV) infection has yielded different types of diagnostic tools over the past decades: whereas very sensitive techniques such as virus isolation (VI) and more recently also real-time RT-PCR can provide evidence for the presence of low virus quantities, VI requires additional confirmation of the nature of the virus strain and both techniques (currently) lack the ability for direct serotyping. The latter usually depends on ELISA, which is a far less sensitive method and may require virus culturing. This paper elaborates on experimental efforts towards the development of an 'immuno-rolling circle amplification (RCA)' assay in 96-well plates, the aim being to increase the sensitivity of immunological FMDV detection and serotyping by means of RCA. The study attempts to explain the encountered hurdles and the complexity of the different setups tested. Conclusively, immuno-RCA in 96-well plates as a reliable diagnostic assay for FMDV seems very difficult to achieve.

VL - 147 CP - 1 U1 - https://www.ncbi.nlm.nih.gov/pubmed/17913251?dopt=Abstract M3 - 10.1016/j.jviromet.2007.08.020 ER - TY - JOUR T1 - A Bayesian evaluation of six diagnostic tests for foot-and-mouth disease for vaccinated and non-vaccinated cattle. JF - Prev Vet Med Y1 - 2008 A1 - Engel, Bas A1 - Buist, Willem A1 - Orsel, Karin A1 - Dekker, Aldo A1 - Kris De Clercq A1 - Grazioli, Santina A1 - van Roermund, Herman KW - Animals KW - Antibodies, Viral KW - Antigens, Viral KW - Bayes Theorem KW - Cattle KW - Cattle Diseases KW - Enzyme-Linked Immunosorbent Assay KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Reference Values KW - Reproducibility of Results KW - Sensitivity and Specificity KW - Vaccination KW - Viral Nonstructural Proteins AB -

The sensitivity and specificity of six ELISA tests for foot-and-mouth disease (FMD) to discriminate between sero-converted (for non-structural FMD virus proteins) and non-sero-converted cattle were evaluated for vaccinated and unvaccinated cattle. Since none of the tests could be considered as a proper reference test and for about half of the tested sera the true status (sero-converted or not for non-structural proteins, i.e. presence of antibodies) of the animals was unknown, a Bayesian analysis employing a latent class model was used that did not rely on the use of a reference test or gold standard. Prior information about prevalence for subsets of the data and specificity of the tests was incorporated into the analysis. The specificity of the six tests for vaccinated and non-vaccinated cattle ranged from 96 to 99%. For vaccinated cattle, one test stood out with an estimated sensitivity of 94% (95% CI from 89.8 to 98.1%). Second best for vaccinated cattle were two tests with estimated sensitivities of 85% (95% CI from 78.9 to 89.7%) and 92% (95% CI from 86.2 to 95.6%). For non-vaccinated cattle, the sensitivities of these three tests were around 97%. The remaining three tests showed lower estimated sensitivity for vaccinated cattle, ranging from 57 to 79%.

VL - 86 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18455817?dopt=Abstract M3 - 10.1016/j.prevetmed.2008.03.009 ER - TY - JOUR T1 - Bluetongue control--a new challenge for Europe. JF - Berl Munch Tierarztl Wochenschr Y1 - 2008 A1 - Makoschey, Birgit A1 - Beer, M A1 - Zientara, S A1 - Haubruge, E A1 - Rinaldi, L A1 - Dercksen, D A1 - Millemann, Y A1 - von Rijn, P A1 - Kris De Clercq A1 - Oura, C A1 - Saegerman, C A1 - Domingo, M A1 - Sanchez-Vizcaino, J M A1 - Mehlhorn, H A1 - Tamba, M A1 - Thiry, E KW - Animals KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Ceratopogonidae KW - Europe KW - Insect Vectors KW - Sheep KW - Viral Vaccines VL - 121 CP - 7-8 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18712267?dopt=Abstract ER - TY - JOUR T1 - Bluetongue in captive yaks. JF - Emerg Infect Dis Y1 - 2008 A1 - Mauroy, Axel A1 - Guyot, Hugues A1 - Kris De Clercq A1 - Cassart, Dominique A1 - Thiry, Etienne A1 - Saegerman, Claude KW - Animals KW - Bluetongue KW - Cattle KW - Cattle Diseases KW - Female VL - 14 CP - 4 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18394296?dopt=Abstract M3 - 10.3201/eid1404.071416 ER - TY - JOUR T1 - Bluetongue in Eurasian lynx. JF - Emerg Infect Dis Y1 - 2008 A1 - Jauniaux, Thierry P A1 - Kris De Clercq A1 - Cassart, Dominique E A1 - Kennedy, Seamus A1 - Frank Vandenbussche A1 - Vandemeulebroucke, Elise L A1 - Vanbinst, Tine M A1 - Verheyden, Bart I A1 - Goris, Nesya E A1 - Coignoul, Freddy L KW - Animals KW - Animals, Zoo KW - Bluetongue KW - Lung KW - Lynx VL - 14 CP - 9 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18760034?dopt=Abstract M3 - 10.3201/eid1409.080434 ER - TY - JOUR T1 - Bluetongue virus serotype 8-associated congenital hydranencephaly in calves. JF - Transbound Emerg Dis Y1 - 2008 A1 - Vercauteren, G A1 - Miry, C A1 - Frank Vandenbussche A1 - Ducatelle, R A1 - Van der Heyden, S A1 - Vandemeulebroucke, E A1 - Ilse De Leeuw A1 - Deprez, P A1 - Chiers, K A1 - Kris De Clercq KW - Animals KW - Animals, Newborn KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Female KW - Hydranencephaly KW - Infectious Disease Transmission, Vertical KW - Male KW - Pregnancy KW - Pregnancy Complications, Infectious KW - RNA, Viral AB -

Hydranencephaly, the almost complete absence of the cerebral parenchyma, induced by infection with modified live bluetongue virus (BTV) crossing the placenta has previously been reported in sheep and rarely in cattle in the USA and in South Africa. The current study describes 29 cases of hydranencephaly in bovine foetuses and 'dummy' calves up to 3 months of age in Belgium associated with natural BTV serotype 8 infection very early in gestation. Histological examination of the remaining cerebral parenchyma showed moderate to severe atrophy of the neural tissue. The lesions observed support the hypothesis of BTV-induced destruction of precursor cells. However, in several calves a slight infiltration of the walls of venules and arterioles with T lymphocytes (vasculitis) was observed as well, which seems to be responsible for at least some of the lesions. Bluetongue viral RNA was detected in 15 animals using a BTV-specific real-time RT-PCR with a much higher success rate in brain tissues compared with blood and spleen samples. Virus isolation in embryonated eggs was unsuccessful. In conclusion, hydranencephaly in calves can be associated with natural wild-type BTV-8 intra-uterine infection.

VL - 55 CP - 7 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18503510?dopt=Abstract M3 - 10.1111/j.1865-1682.2008.01034.x ER - TY - JOUR T1 - Effect of pooling and multiplexing on the detection of bluetongue virus RNA by real-time RT-PCR. JF - J Virol Methods Y1 - 2008 A1 - Frank Vandenbussche A1 - Vanbinst, T A1 - Vandemeulebroucke, E A1 - Goris, N A1 - Sailleau, C A1 - Zientara, S A1 - Kris De Clercq KW - Actins KW - Animals KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Sheep AB -

Real-time RT-PCR (RT-qPCR) was used routinely for laboratory diagnosis during the 2006/2007 bluetongue virus (BTV) serotype 8 epidemic. In the present study the impact of pooling and multiplexing strategies on RT-qPCR are assessed. To avoid any bias in the pooling experiments, 121 BTV-8 positive blood samples with a low to high viral load were selected and pooled individually with nine negative blood samples. Analyses of the individually and pooled samples indicated an overall mean difference of 4.32 Ct-values. The most pronounced differences were observed in samples with the lowest viral load of which 70% could no longer be detected after pooling. The pooling strategy is therefore not suitable for BTV detection at the individual level since animals infected recently may be missed. An alternative approach to reduce costs and workload is to apply a multiplexing strategy in which the viral RNA and internal beta-actin control RNA are detected in a single reaction. Parallel analysis (singleplex versus multiplex) of a 10-fold dilution series and 546 field samples proved that the sensitivity of the BTV RT-qPCR was not affected whereas the beta-actin reaction was reduced only slightly. Without the use of an internal control, 0.6% of 1985 field samples is at risk of being diagnosed incorrectly as negative.

VL - 152 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18590769?dopt=Abstract M3 - 10.1016/j.jviromet.2008.06.005 ER - TY - JOUR T1 - Establishing the spread of bluetongue virus at the end of the 2006 epidemic in Belgium. JF - Vet Microbiol Y1 - 2008 A1 - Estelle Méroc A1 - Faes, C A1 - Herr, C A1 - Staubach, C A1 - Verheyden, B A1 - Vanbinst, T A1 - Frank Vandenbussche A1 - Hooyberghs, J A1 - Aerts, M A1 - Kris De Clercq A1 - Mintiens, K KW - Animals KW - Bayes Theorem KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - cross-sectional studies KW - Diagnosis, Differential KW - Disease Outbreaks KW - Linear Models KW - Logistic Models KW - Seasons KW - Sensitivity and Specificity KW - Seroepidemiologic Studies KW - Sheep KW - Sheep Diseases AB -

Bluetongue (BT) was notified for the first time in several Northern European countries in August 2006. The first reported outbreaks of BT were confirmed in herds located near the place where Belgium, The Netherlands and Germany share borders. The disease was rapidly and widely disseminated throughout Belgium in both sheep and cattle herds. During the epidemic, case reporting by the Veterinary Authorities relied almost exclusively on the identification of herds with confirmed clinical infected ruminants. A cross-sectional serological survey targeting all Belgian ruminants was then undertaken during the vector-free season. The first objective of this study was to provide unbiased estimates of BT-seroprevalence for different regions of Belgium. Since under-reporting was suspected during the epidemic, a second goal was to compare the final dispersion of the virus based on the seroprevalence estimates to the dispersion of the confirmed clinical cases which were notified in Belgium, in order to estimate the accuracy of the case detection based on clinical suspicion. True within-herd seroprevalence was estimated based on a logistic-normal regression model with prior specification on the diagnostic test's sensitivity and specificity. The model was fitted in a Bayesian framework. Herd seroprevalence was estimated using a logistic regression model. To study the linear correlation between the BT winter screening data and the case-herds data, the linear predicted values for the herd prevalence were compared and the Pearson correlation coefficient was estimated. The overall herd and true within-herd seroprevalences were estimated at 83.3 (79.2-87.0) and 23.8 (20.1-28.1)%, respectively. BT seropositivity was shown to be widely but unevenly distributed throughout Belgium, with a gradient decreasing towards the south and the west of the country. The analysis has shown there was a strong correlation between the outbreak data and the data from the survey (r=0.73, p<0.0001). The case detection system based on clinical suspicion underestimated the real impact of the epidemic, but indicated an accurate spatial distribution of the virus at the end of the epidemic.

VL - 131 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18479845?dopt=Abstract M3 - 10.1016/j.vetmic.2008.03.012 ER - TY - JOUR T1 - Evaluation of antibody-ELISA and real-time RT-PCR for the diagnosis and profiling of bluetongue virus serotype 8 during the epidemic in Belgium in 2006. JF - Vet Microbiol Y1 - 2008 A1 - Frank Vandenbussche A1 - Vanbinst, Tine A1 - Verheyden, Bart A1 - Wesley Van Dessel A1 - Demeestere, Lien A1 - Houdart, Philippe A1 - Bertels, Guido A1 - Praet, Nicolas A1 - Berkvens, Dirk A1 - Mintiens, Koen A1 - Goris, Nesya A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Disease Outbreaks KW - Enzyme-Linked Immunosorbent Assay KW - Reverse Transcriptase Polymerase Chain Reaction KW - Sheep AB -

In 2006 bluetongue (BT) emerged for the first time in North-Western Europe. Reliable diagnostic tools are essential in controlling BT but data on the diagnostic sensitivity (Se) and specificity (Sp) are often missing. This paper aims to describe and analyse the results obtained with the diagnostics used in Belgium during the 2006 BT crisis. The diagnosis was based on a combination of antibody detection (competitive ELISA, cELISA) and viral RNA detection by real-time RT-PCR (RT-qPCR). The performance of the cELISA as a diagnostic tool was assessed on field results obtained during the epidemic and previous surveillance campaigns. As the infectious status of the animals is unknown during an epidemic, a Bayesian analysis was performed. Both assays were found to be equally specific (RT-qPCR: 98.5%; cELISA: 98.2%) while the diagnostic sensitivity of the RT-qPCR (99.5%) was superior to that of the cELISA (87.8%). The assumption of RT-qPCR as standard of comparison during the bluetongue virus (BTV) epidemic proved valid based on the results of the Bayesian analysis. A ROC analysis of the cELISA, using RT-qPCR as standard of comparison, showed that the cut-off point with the highest accuracy occurred at a percentage negativity of 66, which is markedly higher than the cut-off proposed by the manufacturer. The analysis of the results was further extended to serological and molecular profiling and the possible use of profiling as a rapid epidemiological marker of the BTV in-field situation was assessed. A comparison of the serological profiles obtained before, during and at the end of the Belgian epidemic clearly showed the existence of an intermediate zone which appears soon after BTV (re)enters the population. The appearance or disappearance of this intermediate zone is correlated with virus circulation and provides valuable information, which would be entirely overlooked if only positive and negative results were considered.

VL - 129 CP - 1-2 U1 - https://www.ncbi.nlm.nih.gov/pubmed/18093753?dopt=Abstract M3 - 10.1016/j.vetmic.2007.10.029 ER - TY - JOUR T1 - FMD vaccines: reflections on quality aspects for applicability in European disease control policy. JF - Transbound Emerg Dis Y1 - 2008 A1 - Kris De Clercq A1 - Goris, N A1 - Barnett, P V A1 - MacKay, D K KW - Animals KW - Europe KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - HEALTH POLICY KW - Quality Control KW - Viral Vaccines AB -

Most foot-and-mouth disease (FMD) vaccines used around the world are inactivated vaccines for prophylactic or emergency use, generally manufactured by the same basic methodology outlined in the OIE Manual and, for Europe, in the European Pharmacopoeia, and for the EU Member States in compliance with Directive 2001/82/EC of the European Parliament and of the Council of 6 November 2001 on the Community code relating to veterinary medicinal products as amended by Directive 2004/28/EC. Most of the requirements that apply to all immunological veterinary medicinal products apply equally to FMD vaccines. There are, however, some unique features of the disease and vaccines used against it that require a different approach to fulfil the requirements of the relevant legislation, if a vaccinate-to-live policy will be applied with 'authorized' vaccines. Several aspects of vaccine efficacy and safety are elaborated with emphasis on quality assurance/quality control (QA/QC). The purity of the vaccine in respect of the presence of non-structural protein antibodies could be checked indirectly by serology after vaccination. The viability of a vaccine bank approach was greatly aided by the principle of storing inactivated concentrated FMD viral antigen (Ag) over liquid nitrogen for subsequent formulation into vaccine. A worldwide Ag bank network might be an option for the far future and a solution to the problem of covering many different FMDV serotypes and strains. The producers should respect the strict FMD biosecurity rules worked out by the FAO EUFMD and described in Council Directive 2003/85/EC. Making the experience related to vaccine QA/QC available to all countries will reduce the risk of an FMD outbreak within these countries and consequently will reduce the FMD risk around the world.

VL - 55 CP - 1 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18397508?dopt=Abstract M3 - 10.1111/j.1865-1682.2007.01012.x ER - TY - JOUR T1 - Foot-and-mouth disease vaccine potency testing in cattle using homologous and heterologous challenge strains: precision of the "Protection against Podal Generalisation" test. JF - Vaccine Y1 - 2008 A1 - Goris, N A1 - Maradei, E A1 - D'Aloia, R A1 - Fondevila, N A1 - Mattion, N A1 - Perez, A A1 - Smitsaart, E A1 - Nauwynck, H J A1 - La Torre, J A1 - Palma, E A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Cattle KW - Cattle Diseases KW - Cross Reactions KW - Enzyme-Linked Immunosorbent Assay KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Viral Vaccines AB -

The level of protection conferred by foot-and-mouth disease (FMD) vaccines in primovaccinated animals primarily depends on the potency of the vaccine and the relatedness of the vaccine strain and circulating field isolate. The "Gold Standard" FMD vaccine potency test is the in vivo test performed in the target species. The objective of the study was to determine the precision of the in vivo "Protection against Podal Generalisation" (PPG) FMD vaccine potency test in cattle using homologous (vaccine quality control) and heterologous (vaccine matching) viral challenge. The overall level of protection induced by the A(24) Cruzeiro/Brazil/55 vaccine used in six homologous PPG tests was 88.5%. Vaccine accordance (VACC) and vaccine concordance (VCON) were estimated to be 75.9% and 73.7%, respectively. In four heterologous challenge PPG tests, the overall level of cross-protection induced by the A(24) Cruzeiro/Brazil/55 vaccine against A Argentina/2001 challenge was 26.6%, with VACC and VCON values of 65.7% and 59.2%, respectively. Results indicate that the homologous PPG test is more reliable than the European Pharmacopoeia potency test, but that a larger number of animals should be used in order to increase the test's statistical power. In this regard, indirect alternative tests for vaccine potency and vaccine matching merit consideration.

VL - 26 CP - 27-28 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18499310?dopt=Abstract M3 - 10.1016/j.vaccine.2008.04.034 ER - TY - JOUR T1 - Impact of human interventions on the spread of bluetongue virus serotype 8 during the 2006 epidemic in north-western Europe. JF - Prev Vet Med Y1 - 2008 A1 - Mintiens, K A1 - Estelle Méroc A1 - Faes, C A1 - Abrahantes, J Cortiñas A1 - Hendrickx, G A1 - Staubach, C A1 - Gerbier, G A1 - Elbers, A R W A1 - Aerts, M A1 - Kris De Clercq KW - Animals KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Disease Outbreaks KW - Europe KW - Humans KW - Models, Biological KW - Sheep AB -

Bluetongue virus (BTV) can be spread by movement or migration of infected ruminants. Infected midges (Culicoides sp.) can be dispersed with livestock or on the wind. Transmissions of infection from host to host by semen and trans-placental infection of the embryo from the dam have been found. As for any infectious animal disease, the spread of BTV can be heavily influenced by human interventions preventing or facilitating the transmission pathways. This paper describes the results of investigations that were conducted on the potential role of the above-mentioned human interventions on the spread of BTV-8 during the 2006 epidemic in north-western Europe. Data on surveillance and control measures implemented in the affected European Union (EU) Member States (MS) were extracted from the legislation and procedures adopted by the national authorities in Belgium, France, Germany, and The Netherlands. The impact of the control measures on the BTV-incidence in time and space was explored. Data on ruminant transports leaving the area of first infection (AFI) to other areas within and beyond the affected MS were obtained from the national identification and registration systems of the three initially affected MS (Belgium, Germany, The Netherlands) and from the Trade Control and Expert System (TRACES) of the European Commission. The association between the cumulative number of cases that occurred in a municipality outside the AFI and the number of movements or the number of animals moved from the AFI to that municipality was assessed using a linear negative binomial regression model. The results of this study indicated that the control measures which were implemented in the affected MS (in accordance with EU directives) were not able to fully stop further spread of BTV and to control the epidemic. This finding is not surprising because BT is a vector-borne disease and it is difficult to limit vector movements. We could not assess the consequences of not taking control measures at all but it is possible, if not most likely, that this would have resulted in even wider spread. The study also showed an indication of the possible involvement of animal movements in the spread of BTV during the epidemic. Therefore, the prevention of animal movements remains an important tool to control BTV outbreaks. The extension of the epidemic to the east cannot be explained by the movement of animals, which mainly occurred in a north-western direction. This indicates that it is important to consider other influential factors such as dispersal of infected vectors depending on wind direction, or local spread.

VL - 87 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18649960?dopt=Abstract M3 - 10.1016/j.prevetmed.2008.06.010 ER - TY - JOUR T1 - The importance of quality assurance/quality control of diagnostics to increase the confidence in global foot-and-mouth disease control. JF - Transbound Emerg Dis Y1 - 2008 A1 - Kris De Clercq A1 - Goris, N A1 - Barnett, P V A1 - MacKay, D K KW - Animals KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Global Health KW - International Cooperation KW - Laboratories KW - Quality Control AB -

The last decade international trade in animals and animal products was liberated and confidence in this global trade can increase only if appropriate control measures are applied. As foot-and-mouth disease (FMD) diagnostics will play an essential role in this respect, the Food and Agriculture Organization European Commission for the Control of Foot-and-Mouth Disease (EUFMD) co-ordinates, in collaboration with the European Commission, several programmes to increase the quality of FMD diagnostics. A quality assurance (QA) system is deemed essential for laboratories involved in certifying absence of FMDV or antibodies against the virus. Therefore, laboratories are encouraged to validate their diagnostic tests fully and to install a continuous quality control (QC) monitoring system. Knowledge of performance characteristics of diagnostics is essential to interpret results correctly and to calculate sample rates in regional surveillance campaigns. Different aspects of QA/QC of classical and new FMD virological and serological diagnostics are discussed in respect to the EU FMD directive (2003/85/EC). We recommended accepting trade certificates only from laboratories participating in international proficiency testing on a regular basis.

VL - 55 CP - 1 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18397507?dopt=Abstract M3 - 10.1111/j.1865-1682.2007.01011.x ER - TY - JOUR T1 - Indirect foot-and-mouth disease vaccine potency testing based on a serological alternative. JF - Vaccine Y1 - 2008 A1 - Goris, Nesya A1 - Willems, Tom A1 - Diev, Vyacheslav I A1 - Merkelbach-Peters, Petra A1 - Vanbinst, Tine A1 - Yves Van der Stede A1 - Kraft, Horst-Peter A1 - Zakharov, Valery M A1 - Borisov, Vladimir V A1 - Nauwynck, Hans J A1 - Haas, Bernd A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Cattle KW - Enzyme-Linked Immunosorbent Assay KW - Foot-and-Mouth Disease KW - Logistic Models KW - Neutralization Tests KW - Predictive Value of Tests KW - Reproducibility of Results KW - Roc Curve KW - Statistics as Topic KW - Viral Vaccines AB -

Foot-and-mouth disease (FMD) vaccine potency testing has historically been performed by experimentally infecting vaccinated cattle. A few alternative approaches to the in vivo challenge test based on the correlation between serum titres of primo-vaccinated cattle and protection against infection have been proposed, but none have been accepted by the European Pharmacopoeia (Ph.Eur.) due to the lack of statistical power and the pooling of data over time. The present study addresses these issues and presents data of 150 cattle vaccinated according to Ph.Eur. standards. Four laboratories took part in the serological testing and different serological assays were used, including virus neutralisation assays and ELISA formats. Models correlating specific anti-FMD virus antibody titres to protection were built using logistic regression followed by Receiver Operating Characteristic (ROC) analysis. The best models accurately predicted the in vivo protection status in 80.0% of the cases. Although differences were observed between laboratories and assays used, the majority of antibody pass-levels, determined using ROC analysis, corresponded to at least 75.0% probability of protection. The indirect potency assessment procedure proposed is at least as precise (repeatability=65.8%, reproducibility=60.7%) as the in vivo test, can be standardised and results in a quantitative PD50 value. The validity of the procedure was also demonstrated.

VL - 26 CP - 31 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18555565?dopt=Abstract M3 - 10.1016/j.vaccine.2008.05.013 ER - TY - JOUR T1 - Possible routes of introduction of bluetongue virus serotype 8 into the epicentre of the 2006 epidemic in north-western Europe. JF - Prev Vet Med Y1 - 2008 A1 - Mintiens, K A1 - Estelle Méroc A1 - Mellor, P S A1 - Staubach, C A1 - Gerbier, G A1 - Elbers, A R W A1 - Hendrickx, G A1 - Kris De Clercq KW - Animals KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Disease Outbreaks KW - Europe KW - Sheep AB -

In August 2006, bluetongue (BT) was notified in The Netherlands on several animal holdings. This was the onset of a rapidly spreading BT-epidemic in north-western Europe (latitude >51 degrees N) that affected cattle and sheep holdings in The Netherlands, Belgium, Germany, France and Luxembourg. The outbreaks were caused by bluetongue virus (BTV) serotype 8, which had not been identified in the European Union before. Bluetongue virus can be introduced into a free area by movement of infected ruminants, infected midges or by infected semen and embryos. In this study, information on animal movements or transfer of ruminant germ plasms (semen and embryos) into the Area of First Infection (AFI), which occurred before and during the onset of the epidemic, were investigated in order to establish the conditions for the introduction of this virus. All inbound transfers of domestic or wild ruminants, non-susceptible mammal species and ruminant germ plasms into the AFI during the high-risk period (HRP), registered by the Trade Control and Expert System (TRACES) of the EC, were obtained. Imports originating from countries with a known or suspected history of BTV-incidence of any serotype were identified. The list of countries with a reported history of BTV incidence was obtained from the OIE Handistatus II for the period from 1996 until 2004. No ruminants were imported from a Member State (MS) with a known history of BTV-8 or from any other country with a known or suspected history of BTV incidence of any serotype. Of all non-susceptible mammal species only 233 horses were transported directly into the AFI during the HRP. No importations of semen or embryos into the AFI were registered in TRACES during the period of interest. An obvious source for the introduction of BTV-8, such as import of infected ruminants, could not be identified and the exact origin and route of the introduction of BTV-8 thus far remains unknown. However, the absence of legal import of ruminants from outside the EU into the AFI and the absence of BTV-8 in southern Europe suggest that, the introduction of the BTV-8 infection into the north-western part of Europe took place via another route. Specifically, in relation to this, the potential for Culicoides to be imported along with or independently of the import of animals, plants or other 'materials', and the effectiveness of measures to reduce such a possibility, merit further study.

VL - 87 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18667252?dopt=Abstract M3 - 10.1016/j.prevetmed.2008.06.011 ER - TY - JOUR T1 - Potential of antiviral therapy and prophylaxis for controlling RNA viral infections of livestock. JF - Antiviral Res Y1 - 2008 A1 - Goris, Nesya A1 - Frank Vandenbussche A1 - Kris De Clercq KW - Animals KW - Animals, Domestic KW - Antiviral Agents KW - Cattle KW - Chemoprevention KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - RNA Virus Infections KW - RNA Viruses KW - Viral Vaccines AB -

With intensification of trade, livestock are increasingly exposed to severe animal diseases caused by a range of RNA viruses. Recent prime examples include outbreaks of foot-and-mouth disease (FMD), peste des petits ruminants, Rift Valley fever and bluetongue. To minimise their impact, controlling the spread of virus is of utmost importance. Good quality, reliable vaccines exist for some, although not all, of these diseases, but suffer from a set of drawbacks, not the least of which being the time needed to trigger the immune response (i.e. "immunity-gap"). Effective, rapid control tools are, therefore, urgently needed and antiviral compounds could serve this purpose. Although limited in vitro and in vivo research has been performed, encouraging results for FMD suggest that livestock could be protected against infection within 24h following antiviral treatment and up to 12h post-infection. Such prophylactic/therapeutic antiviral drugs could complement emergency vaccination in a previously disease-free setting or be applied to treat valuable zoological collections and breeding stocks in endemic and previously disease-free regions alike. This paper will primarily focus on the effects of FMD on livestock and other sectors, and on appropriate control tools. The outlined principles can be extrapolated to other RNA viral diseases.

VL - 78 CP - 1 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18035428?dopt=Abstract M3 - 10.1016/j.antiviral.2007.10.003 ER - TY - JOUR T1 - Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparison to other European strains. JF - Virology Y1 - 2008 A1 - Maan, Sushila A1 - Maan, Narender S A1 - Ross-smith, Natalie A1 - Batten, Carrie A A1 - Shaw, Andrew E A1 - Anthony J Simon A1 - Samuel, Alan R A1 - Darpel, Karin E A1 - Veronesi, Eva A1 - Oura, Chris A L A1 - Singh, Karam P A1 - Nomikou, Kyriaki A1 - Potgieter, Abraham C A1 - Attoui, Houssam A1 - van Rooij, Eugene A1 - van Rijn, Piet A1 - Kris De Clercq A1 - Frank Vandenbussche A1 - Zientara, Stéphan A1 - Bréard, Emmanuel A1 - Sailleau, Corinne A1 - Beer, Martin A1 - Hoffman, Bernd A1 - Mellor, Philip S A1 - Mertens, Peter P C KW - Animals KW - Base Sequence KW - Bluetongue KW - Bluetongue virus KW - Capsid Proteins KW - Europe KW - Genome, Viral KW - Molecular Sequence Data KW - Netherlands KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Double-Stranded KW - RNA, Viral KW - Sequence Analysis KW - Serotyping AB -

During 2006 the first outbreak of bluetongue ever recorded in northern Europe started in Belgium and the Netherlands, spreading to Luxemburg, Germany and north-east France. The virus overwintered (2006-2007) reappearing during May-June 2007 with greatly increased severity in affected areas, spreading further into Germany and France, reaching Denmark, Switzerland, the Czech Republic and the UK. Infected animals were also imported into Poland, Italy, Spain and the UK. An initial isolate from the Netherlands (NET2006/04) was identified as BTV-8 by RT-PCR assays targeting genome segment 2. The full genome of NET2006/04 was sequenced and compared to selected European isolates, South African vaccine strains and other BTV-8 strains, indicating that it originated in sub-Saharan Africa. Although NET2006/04 showed high levels of nucleotide identity with other 'western' BTV strains, it represents a new introduction and was not derived from the BTV-8 vaccine, although its route of entry into Europe has not been established.

VL - 377 CP - 2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18570969?dopt=Abstract M3 - 10.1016/j.virol.2008.04.028 ER - TY - JOUR T1 - Transplacental bluetongue infection in cattle. JF - Vet Rec Y1 - 2008 A1 - Kris De Clercq A1 - Frank Vandenbussche A1 - Vandemeulebroucke, E A1 - Vanbinst, T A1 - Ilse De Leeuw A1 - Verheyden, B A1 - Goris, N A1 - Mintiens, K A1 - Estelle Méroc A1 - Herr, C A1 - Hooybergs, J A1 - Houdart, P A1 - Sustronck, B A1 - De Deken, R A1 - Maquet, G A1 - Bughin, J A1 - Saulmont, M A1 - Lebrun, M A1 - Bertels, G A1 - Miry, C KW - Animals KW - Animals, Newborn KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Female KW - Infectious Disease Transmission, Vertical KW - Pregnancy KW - Pregnancy Complications, Infectious KW - RNA, Viral VL - 162 CP - 17 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18441360?dopt=Abstract ER - TY - JOUR T1 - Transplacental infection and apparently immunotolerance induced by a wild-type bluetongue virus serotype 8 natural infection. JF - Transbound Emerg Dis Y1 - 2008 A1 - Kris De Clercq A1 - Ilse De Leeuw A1 - Verheyden, B A1 - Vandemeulebroucke, E A1 - Vanbinst, T A1 - Herr, C A1 - Estelle Méroc A1 - Bertels, G A1 - Steurbaut, N A1 - Miry, C A1 - De Bleecker, K A1 - Maquet, G A1 - Bughin, J A1 - Saulmont, M A1 - Lebrun, M A1 - Sustronck, B A1 - De Deken, R A1 - Hooyberghs, J A1 - Houdart, P A1 - Raemaekers, M A1 - Mintiens, K A1 - Pierre Kerkhofs A1 - Goris, N A1 - Frank Vandenbussche KW - Abortion, Veterinary KW - Animals KW - Animals, Newborn KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Female KW - Infectious Disease Transmission, Vertical KW - Pregnancy KW - Pregnancy Complications, Infectious KW - RNA, Viral KW - Serotyping AB -

Until recently, bluetongue (BT) virus (BTV) serotypes reportedly causing transplacental infections were all ascribed to the use of modified live virus strains. During the 2007 BT epidemic in Belgium, a significant increase in the incidence of abortions was reported. A study including 1348 foetuses, newborns and young animals with or without suspicion of BTV infection, was conducted to investigate the occurrence of natural transplacental infection caused by wild-type BTV-8 and to check the immunocompetence of newborns. BTV RNA was present in 41% and 18.5% of aborted foetuses from dams with or without suspected BTV involvement during pregnancy, respectively. The results of dam/calf pairs sampled before colostrum uptake provide evidence of almost 10% transplacental BTV infection in newborns. Apparently immunotolerant calves were found at a level of 2.4%. The current study concludes that the combined serological and real-time PCR (RT-qPCR) result of pregnant dams gives no indication of the infection status of the offspring except in the case of a double negative result. In a group of 109 calves with clinical suspicion of BT, born during the vector-free period, 11% were found to be RT-qPCR positive. The true prevalence was estimated to be 2.3%, indicating the extent of transplacental infection in a group of 733 calves of one to 4 months of age without BT suspicion. Moreover, virus isolation was successful for two newborn calves, emphasizing the need for restricting trade to BT-free regions of pregnant dams possibly infected during gestation, even if they are BTV RT-qPCR negative.

VL - 55 CP - 8 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18673339?dopt=Abstract M3 - 10.1111/j.1865-1682.2008.01044.x ER - TY - JOUR T1 - Use of continuous results to compare ELISAs for the detection of antibodies to non-structural proteins of foot-and-mouth disease virus. JF - Vaccine Y1 - 2008 A1 - Dekker, Aldo A1 - Sammin, Donal A1 - Greiner, Matthias A1 - Bergmann, Ingrid A1 - Paton, David A1 - Grazioli, Santina A1 - Kris De Clercq A1 - Brocchi, Emiliana KW - Animals KW - Antibodies, Viral KW - Cattle KW - Cattle Diseases KW - Enzyme-Linked Immunosorbent Assay KW - Foot-and-Mouth Disease Virus KW - ITALY KW - Models, Statistical KW - Sensitivity and Specificity KW - Viral Nonstructural Proteins AB -

Six tests for detection of antibodies against the non-structural proteins of foot-and-mouth disease virus (FMDV) were compared at an international workshop in Brescia, Italy in 2004 on the basis of dichotomous test results. However, as results from all of these assays were also available on a continuous scale, validation was extended by calculating and subsequently analysing the receiver-operator characteristic (ROC) curves and likelihood ratios (LR) for each test method. For the purposes of these analyses, test results for a total of 1337 sera were selected from the Brescia workshop dataset, 237 sera that had been obtained from cattle exposed to FMDV and 1100 sera obtained from cattle that were not exposed to the virus; sera from "exposed" cattle were considered to be "true positives" and sera from "non-exposed" cattle were considered to be "true negatives". Analysis of ROC curves showed that at specificities of both 99 and 99.5%, the IZS-Brescia and the Ceditest ELISA had significantly better detection rates in exposed cattle than the other ELISAs. The ROC analysis confirms the previous finding that the IZS-Brescia and the Ceditest ELISAs have both better detection rates in exposed cattle combined with a high specificity. The analysis of likelihood ratios provides information that may be very useful in the interpretation of test results, and a working example is presented to show how these likelihood ratios might be used in an objective approach to deciding the true infection status of surveyed populations.

VL - 26 CP - 22 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18450336?dopt=Abstract M3 - 10.1016/j.vaccine.2008.03.052 ER - TY - JOUR T1 - Vector monitoring at Belgian outbreak sites during the bluetongue epidemic of 2006. JF - Prev Vet Med Y1 - 2008 A1 - De Deken, G A1 - Madder, M A1 - Deblauwe, I A1 - Kris De Clercq A1 - Fassotte, C A1 - Losson, B A1 - Haubruge, E A1 - De Deken, R KW - Animals KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Ceratopogonidae KW - cross-sectional studies KW - Disease Outbreaks KW - Insect Vectors KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Sheep AB -

In response to the first bluetongue outbreak in Belgium a monitoring programme was started at the end of August 2006 to identify possible vectors transmitting the disease. Black light traps were deployed at 36 outbreak sites and captured 1959 Culicoides specimens belonging to 16 different species. Eighty four percent of the biting midges captured belonged to the C. obsoletus complex, among them C. obsoletus s.s., C. dewulfi and C. scoticus, three suspected bluetongue vectors. The Veterinary and Agrochemical Research Centre detected viral RNA in pools of individuals belonging to this complex. Culicoides pulicaris, a potential bluetongue vector in Italy, should yet not be excluded as a possible vector in Belgium as this species was frequently found around outbreak sites, notwithstanding this species is not easily captured with the trapping techniques used during this survey.

VL - 87 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18640732?dopt=Abstract M3 - 10.1016/j.prevetmed.2008.06.006 ER - TY - JOUR T1 - 2'-C-methylcytidine as a potent and selective inhibitor of the replication of foot-and-mouth disease virus. JF - Antiviral Res Y1 - 2007 A1 - Goris, Nesya A1 - De Palma, Armando A1 - Toussaint, Jean-François A1 - Musch, Ina A1 - Neyts, Johan A1 - Kris De Clercq KW - Animals KW - Antiviral Agents KW - Cricetinae KW - Cytidine KW - Enterovirus B, Human KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Swine KW - Swine Vesicular Disease KW - Virus Replication AB -

We report on the potent and selective in vitro antiviral activity of 2'-C-methylcytidine (2'-C-MetCyt) against foot-and-mouth disease virus (FMDV). FMDV belongs to the Picornaviridae and has the potential to cause devastating epidemics in livestock. The 50% and 90% effective concentrations (EC50 and EC90) for inhibition of the FMDV-induced cytopathic effect (CPE) formation were 6.4+/-3.8 and 10.8+/-5.4 microM. Comparable EC50 values for inhibition of viral RNA synthesis were observed. Treatment of FMDV-infected BHK-21 cells with 77 microM 2'-C-MetCyt resulted in a (1.6-3.2)x10(3)-fold reduction of infectious virus yield. Time-of-drug addition experiments suggest that 2'-C-MetCyt interacts with viral replication at a time point that coincides with the onset of intracellular viral RNA synthesis. In contrast to emergency vaccination, a potent and selective antiviral agent may provide almost immediate (prophylactic/therapeutic) protection against infection and thus constitute an important alternative/supplementary option to contain outbreaks such as those caused by FMDV.

VL - 73 CP - 3 U1 - http://www.ncbi.nlm.nih.gov/pubmed/17055073?dopt=Abstract M3 - 10.1016/j.antiviral.2006.09.007 ER - TY - JOUR T1 - Bluetongue in Belgium, 2006. JF - Emerg Infect Dis Y1 - 2007 A1 - Toussaint, Jean-François A1 - Sailleau, Corinne A1 - Mast, Jan A1 - Houdart, Philippe A1 - Czaplicki, Guy A1 - Demeestere, Lien A1 - VandenBussche, Frank A1 - van Dessel, Wesley A1 - Goris, Nesya A1 - Bréard, Emmanuel A1 - Bounaadja, Lotfi A1 - Etienne, Thiry A1 - Zientara, Stéphan A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Cell Line KW - Cricetinae KW - Enzyme-Linked Immunosorbent Assay KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Sheep KW - Viral Load AB -

Bluetongue has emerged recently in Belgium. A bluetongue virus strain was isolated and characterized as serotype 8. Two new real-time reverse transcription-quantitative PCRs (RT-qPCRs) that amplified 2 different segments of bluetongue virus detected this exotic strain. These 2 RT-qPCRs detected infection earlier than a competitive ELISA for antibody detection.

VL - 13 CP - 4 U1 - https://www.ncbi.nlm.nih.gov/pubmed/17553280?dopt=Abstract M3 - 10.3201/eid1304.061136 ER - TY - JOUR T1 - Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments. JF - J Virol Methods Y1 - 2007 A1 - Toussaint, J F A1 - Sailleau, C A1 - Bréard, E A1 - Zientara, S A1 - Kris De Clercq KW - Animals KW - Bluetongue KW - Bluetongue virus KW - Cell Line KW - Cricetinae KW - DNA Primers KW - DNA Probes KW - Genome, Viral KW - Nucleic Acid Amplification Techniques KW - Reproducibility of Results KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Messenger KW - RNA, Viral KW - Serotyping KW - Sheep Diseases KW - Sheep, Domestic KW - Time Factors AB -

The detection of the bluetongue virus (BTV) by conventional methods is especially difficult and labour-intensive. Molecular diagnosis is also complex because of the high genetic diversity between and within the 24 serotypes of BTV. In the present study, two laboratories joined forces to develop and validate two new RT-qPCRs detecting and amplifying BTV segments 1 and 5. The 2 assays detect strains from all 24 serotypes. They both have a detection limit of 0.01 ECE50 and all 114 samples from BTV-free goats, sheep and cattle were negative. The two assays resulted in similar C(t) values when testing biological samples collected in sheep infected experimentally with a field strain of BTV from the Mediterranean basin. On average, the C(t) values obtained with the 2 methods applied to the 24 serotypes were not significantly different from each other, but some moderate to high differences were seen with a few strains. Therefore these two methods are complementary and could be used in parallel to confirm the diagnosis of a possible new introduction of BTV. An RT-qPCR amplifying a fragment of the beta-actin mRNA was also developed and validated as internal control for the bluetongue specific assays. The three assays described allow a reliable and rapid detection of BTV.

VL - 140 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/17196266?dopt=Abstract M3 - 10.1016/j.jviromet.2006.11.007 ER - TY - JOUR T1 - European Pharmacopoeia foot-and-mouth disease vaccine potency testing in cattle: between test variability and its consequences. JF - Vaccine Y1 - 2007 A1 - Goris, N A1 - Merkelbach-Peters, P A1 - Diev, V I A1 - Verloo, D A1 - Zakharov, V M A1 - Kraft, H-P A1 - Kris De Clercq KW - Animals KW - Cattle KW - Dose-Response Relationship, Immunologic KW - Europe KW - Foot-and-Mouth Disease Virus KW - Male KW - Pharmacopoeias as Topic KW - Reproducibility of Results KW - Viral Vaccines AB -

In the event of a foot-and-mouth (FMD) outbreak in a densely populated livestock area within the European Community, emergency vaccination will most likely be employed. The objective of the present study was to support the European FMD control policy by evaluating the between test variability of the European accepted method for assessing the potency, a major determinant in vaccine choice, of an FMD vaccine batch. The test system suffers from low in vivo repeatability and reproducibility (67.6 and 58.8%, respectively). Consequently, the results of 10 identical, individual vaccine potency tests using an FMD virus O1 Manisa vaccine batch indicate that the obtained potency of a vaccine with an overall 50% protective dose (PD(50)) value of 9.99 may vary from 4.59 to 24.25 PD(50).

VL - 25 CP - 17 U1 - http://www.ncbi.nlm.nih.gov/pubmed/17227691?dopt=Abstract M3 - 10.1016/j.vaccine.2006.12.049 ER - TY - JOUR T1 - Foot-and-mouth disease non-structural protein serology in cattle: use of a Bayesian framework to estimate diagnostic sensitivity and specificity of six ELISA tests and true prevalence in the field. JF - Vaccine Y1 - 2007 A1 - Goris, N A1 - Praet, N A1 - Sammin, D A1 - Yadin, H A1 - Paton, D A1 - Brocchi, E A1 - Berkvens, D A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Antigens, Viral KW - Bayes Theorem KW - Cattle KW - Enzyme-Linked Immunosorbent Assay KW - Foot-and-Mouth Disease KW - Israel KW - prevalence KW - Sensitivity and Specificity KW - Viral Nonstructural Proteins KW - Zimbabwe AB -

The diagnostic performance of six foot-and-mouth disease (FMD) assays for detection of antibodies to the non-structural proteins (NSP) of the FMD virus (FMDV) was estimated using a Bayesian analysis on field sera from cattle of unknown infection status originating from post-FMDV outbreak situations in Israel and Zimbabwe. Estimations of the disease prevalence in both populations were also obtained. The diagnostic sensitivity estimates did not differ between both field studies, although overall Bayesian estimates were markedly higher than those previously reported based on sera from comparable experimentally infected (vaccinated) cattle populations. All NSP-based assays demonstrated a lower diagnostic specificity when applied to the Zimbabwean sera compared to both published specificities and similar Bayesian specificity estimates derived for the Israeli dataset. In Israel, the disease prevalence was estimated at 23.9% (95% credibility interval: 19.5-28.8%), whereas 65.4% (59.0-72.5%) was found in Zimbabwe. The need for reliable diagnostic test performance estimates and the benefits of Bayesian analysis in obtaining them are also addressed.

VL - 25 CP - 41 U1 - http://www.ncbi.nlm.nih.gov/pubmed/17709157?dopt=Abstract M3 - 10.1016/j.vaccine.2007.07.023 ER - TY - JOUR T1 - Uncertainty of measurement for competitive and indirect ELISAs. JF - Rev Sci Tech Y1 - 2007 A1 - Toussaint, J F A1 - Assam, P A1 - Ann Brigitte Cay A1 - Dekeyser, F A1 - K Knapen A1 - Hein Imberechts A1 - Goris, N A1 - Molenberghs, G A1 - Mintiens, K A1 - Kris De Clercq KW - Analysis of Variance KW - Animals KW - Data Interpretation, Statistical KW - Diagnosis, Differential KW - Enzyme-Linked Immunosorbent Assay KW - Evaluation Studies as Topic KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Logistic Models KW - Predictive Value of Tests KW - Quality Control KW - Reference Values KW - Reproducibility of Results KW - Sensitivity and Specificity KW - uncertainty AB -

A method for the estimation of the uncertainty of measurements for Gaussian outcomes of enzyme-linked immunosorbent assay (ELISA) is described using competitive and indirect foot and mouth disease (FMD) ELISAs. Assay repeatability was determined by random effects analysis of variance, and the normality of the residuals was checked. The standard errors of the individual predicted values were transformed into confidence intervals around the corresponding observed values and further transformed into probabilities of being above/below a cut-off. Logistic regression models were subsequently used to interpolate probability values for the whole range of possible assay values. The uncertainty of measurement of a test result was finally defined as the probability of not observing the same qualitative test result when retesting the same sample. For the competitive ELISA any sample with a percent inhibition 4% above the cut-off value had an uncertainty level (probability of a negative result in the case of retest) below 5%. In the indirect ELISA with a cut-off OD of 0.1, the uncertainty was below 5% for any sample with a normalised OD value above 0.22.

VL - 26 CP - 3 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18293613?dopt=Abstract ER - TY - JOUR T1 - Application of non-structural protein antibody tests in substantiating freedom from foot-and-mouth disease virus infection after emergency vaccination of cattle. JF - Vaccine Y1 - 2006 A1 - Paton, David J A1 - Kris De Clercq A1 - Greiner, Matthias A1 - Dekker, Aldo A1 - Brocchi, Emiliana A1 - Bergmann, Ingrid A1 - Sammin, Donal J A1 - Gubbins, Simon A1 - Parida, Satya KW - Animals KW - Antibodies, Viral KW - Cattle KW - Cattle Diseases KW - Emergency Medical Services KW - False Positive Reactions KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Population Surveillance KW - Vaccination KW - Viral Vaccines AB -

There has been much debate about the use of the so-called "vaccinate-to-live" policy for the control of foot-and-mouth disease (FMD) in Europe, according to which, spread of the FMD virus (FMDV) from future outbreaks could be controlled by a short period of "emergency" vaccination of surrounding herds, reducing the need for large-scale preemptive culling of at-risk animals. Since vaccinated animals may become subclinically infected with FMDV following challenge exposure, it is necessary to either remove all vaccinates (vaccinate-to-kill) or to detect and remove vaccinates in which virus is circulating or has established persistent infections (vaccinate-to-live), in order to rapidly regain the most favoured trading status of FMD-free without vaccination. The latter approach can be supported by testing vaccinated animals for the presence of antibodies to certain non-structural proteins (NSP) of FMDV, which are induced by infection with the virus, but not by vaccination with purified FMD vaccines. Using test sensitivity and specificity data established at a recent workshop on NSP assays [Brocchi E, Bergmann I, Dekker A, Paton DJ, Sammin DJ, Greiner M, et al. Comparative performance of six ELISAs for antibodies to the non-structural proteins of foot-and-mouth disease. Vaccine, in press], this paper examines the ways in which serological testing with NSP ELISAs can be used and interpreted and the effect that this will have on the confidence with which freedom from infection can be demonstrated within guidelines specified by the World Animal Health Organisation and the European Commission.

VL - 24 CP - 42-43 U1 - http://www.ncbi.nlm.nih.gov/pubmed/16872727?dopt=Abstract M3 - 10.1016/j.vaccine.2006.06.032 ER - TY - JOUR T1 - Bluetongue in northern Europe. JF - Vet Rec Y1 - 2006 A1 - Thiry, Etienne A1 - Saegerman, Claude A1 - Guyot, Hugues A1 - Kirten, Philippe A1 - Losson, Bertrand A1 - Rollin, Frédéric A1 - Bodmer, Michèle A1 - Czaplicki, Guy A1 - Toussaint, Jean-François A1 - Kris De Clercq A1 - Dochy, Jean-Marie A1 - Dufey, Jacques A1 - Gilleman, Jean-Luc A1 - Messeman, Karl KW - Animals KW - Belgium KW - Bluetongue KW - Cattle KW - Cattle Diseases KW - Ceratopogonidae KW - Disease Outbreaks KW - Europe KW - Insect Vectors KW - Sheep AB -

NA

VL - 159 CP - 10 U1 - http://www.ncbi.nlm.nih.gov/pubmed/16950894?dopt=Abstract M3 - 10.1136/vr.159.10.327 ER - TY - JOUR T1 - Comparative evaluation of six ELISAs for the detection of antibodies to the non-structural proteins of foot-and-mouth disease virus. JF - Vaccine Y1 - 2006 A1 - Brocchi, E A1 - Bergmann, I E A1 - Dekker, A A1 - Paton, D J A1 - Sammin, D J A1 - Greiner, M A1 - Grazioli, S A1 - De Simone, F A1 - Yadin, H A1 - Haas, B A1 - Bulut, N A1 - Malirat, V A1 - Neitzert, E A1 - Goris, N A1 - Parida, S A1 - Sørensen, K A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Antibody Specificity KW - Carrier State KW - Cattle KW - Databases, Factual KW - Enzyme-Linked Immunosorbent Assay KW - Foot-and-Mouth Disease Virus KW - Reproducibility of Results KW - Sheep KW - Swine KW - Vaccination KW - Viral Proteins AB -

To validate the use of serology in substantiating freedom from infection after foot-and-mouth disease (FMD) outbreaks have been controlled by measures that include vaccination, 3551 sera were tested with six assays that detect antibodies to the non-structural proteins of FMD virus. The sera came from naïve, vaccinated, infected and vaccinated-and-infected animals; two-thirds from cattle, the remainder from sheep and pigs. The assays were covariant for sensitivity, but not necessarily for specificity. A commercial kit from Cedi-diagnostics and an in-house assay from IZS-Brescia were comparable to the NCPanaftosa-screening index method described in the Diagnostic Manual of the World Animal Health Organisation. Using these three tests the specificity and sensitivity for the detection of carriers in vaccinated cattle approaches or exceeds 99% and 90%, respectively.

VL - 24 CP - 47-48 U1 - http://www.ncbi.nlm.nih.gov/pubmed/16753241?dopt=Abstract M3 - 10.1016/j.vaccine.2006.04.050 ER - TY - JOUR T1 - Foot-and-mouth disease virus: a first inter-laboratory comparison trial to evaluate virus isolation and RT-PCR detection methods. JF - Vet Microbiol Y1 - 2006 A1 - Ferris, N P A1 - King, D P A1 - Reid, S M A1 - Hutchings, G H A1 - Shaw, A E A1 - Paton, D J A1 - Goris, N A1 - Haas, B A1 - Hoffmann, B A1 - Brocchi, E A1 - Bugnetti, M A1 - Dekker, A A1 - Kris De Clercq KW - Animals KW - Cells, Cultured KW - Clinical Laboratory Techniques KW - Enzyme-Linked Immunosorbent Assay KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Reproducibility of Results KW - Reverse Transcriptase Polymerase Chain Reaction KW - Sensitivity and Specificity KW - Swine KW - Time Factors KW - Virus Cultivation AB -

Five European reference laboratories participated in an exercise to evaluate the sensitivity and specificity of their routinely employed RT-PCR tests and cell cultures for the detection and isolation of foot-and-mouth disease (FMD) virus. Five identical sets of 20 coded samples were prepared from 10 vesicular epithelia, which were derived from submissions from suspect cases of FMD or swine vesicular disease (SVD). Sixteen samples were derived from six FMD virus positive epithelia representing four different serotypes (two each of types O and A and one each of types Asia 1 and SAT 2), two from samples which had been found to be negative by antigen ELISA and virus isolation (VI) in cell culture and two from SVD virus positive epithelia. Some of the FMD virus positive samples were prepared from 10-fold serial dilutions of three of the initial suspensions. Each laboratory tested the samples by one or more of its available RT-PCR procedures and inoculated cell cultures that it routinely uses for FMD diagnosis in attempts to isolate virus, the specificity of which was confirmed by antigen ELISA. The best of the RT-PCR assays used in each laboratory gave comparable results while the sensitivity of cell cultures was variable from high in one laboratory, moderate in two and low in two others. This prototype panel of samples would appear suitable for external quality assurance of these tests but would benefit from the inclusion of more negative samples and an extension in the serial dilution range of one or more of the FMD positive sample titration series.

VL - 117 CP - 2-4 U1 - http://www.ncbi.nlm.nih.gov/pubmed/16846700?dopt=Abstract M3 - 10.1016/j.vetmic.2006.06.001 ER - TY - JOUR T1 - Quality assurance/quality control of foot and mouth disease solid phase competition enzyme-linked immunosorbent assay--Part II. Quality control: comparison of two charting methods to monitor assay performance. JF - Rev Sci Tech Y1 - 2005 A1 - Goris, N A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Cattle KW - Clinical Laboratory Techniques KW - Commerce KW - Enzyme-Linked Immunosorbent Assay KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Guidelines as Topic KW - International Cooperation KW - Quality Control KW - Reference Standards KW - Reproducibility of Results KW - Sensitivity and Specificity KW - Vaccines, Marker KW - Viral Vaccines AB -

Diagnostic laboratories are increasingly required to meet stringent quality standards, and validated assays are needed to achieve formal accreditation. Validation of test methods is often considered to be finalised when the assay parameters and characteristics have been established. However, like any process, diagnostic assays are subject to random variation resulting in shifts in the mean test values. Continuous monitoring of assays using control charts will alert the interpreter of changes in performance. For this purpose, several charting methods have been developed and implemented. This paper compares the Shewhart and the exponentially weighted moving average (EWMA) control charts with respect to the day to day monitoring of internal quality control samples for the foot and mouth disease solid phase competition enzyme-linked immunosorbent assay. Both chart types are equally sensitive to shifts, but the EWMA method seems to provide the best balance between false rejection and false acceptance.

VL - 24 CP - 3 U1 - http://www.ncbi.nlm.nih.gov/pubmed/16642771?dopt=Abstract ER - TY - JOUR T1 - Quality assurance/quality control of foot and mouth disease solid phase competition enzyme-linked immunosorbent assay--Part I. Quality assurance: development of secondary and working standards. JF - Rev Sci Tech Y1 - 2005 A1 - Goris, N A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Cattle KW - Commerce KW - Enzyme-Linked Immunosorbent Assay KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Quality Control KW - Reference Standards KW - Vaccines, Marker KW - Viral Vaccines AB -

International movement in animals and animal products has urged organisations like the World Organisation for Animal Health (OIE) to draw up guidelines to regulate and facilitate trade between Member Countries. However, as the global market continues to grow, further standardisation and harmonisation of antibody detection assays for infectious diseases are needed, especially regarding the development and use of reference materials. For OIE notifiable diseases for which primary or international reference standards are available or under development, National or Regional Reference Laboratories are encouraged to establish their own secondary and/or working standards. This paper describes the development of standards for the foot and mouth disease (FMD) solid phase competition enzyme-linked immunosorbent assay using positive serum obtained from calves vaccinated against the FMD virus. The procedure outlined in this manuscript can easily be extrapolated to similar serological assays and should lead to further international harmonisation of assays and test results.

VL - 24 CP - 3 U1 - http://www.ncbi.nlm.nih.gov/pubmed/16642770?dopt=Abstract ER - TY - JOUR T1 - Conditional expression of type I interferon-induced bovine Mx1 GTPase in a stable transgenic vero cell line interferes with replication of vesicular stomatitis virus. JF - J Interferon Cytokine Res Y1 - 2004 A1 - Baise, Etienne A1 - Pire, Grégory A1 - Leroy, Michaël A1 - Gérardin, Joël A1 - Goris, Nesya A1 - Kris De Clercq A1 - Pierre Kerkhofs A1 - Desmecht, Daniel KW - Animals KW - Cattle KW - Cercopithecus aethiops KW - Clone Cells KW - DNA, Complementary KW - Gene Expression KW - Gene Expression Regulation KW - GTP-Binding Proteins KW - Humans KW - Interferon Type I KW - Myxovirus Resistance Proteins KW - Transgenes KW - Vero Cells KW - Vesicular stomatitis Indiana virus KW - Vesiculovirus KW - Virus Replication AB -

In some vertebrate species, type I interferon(IFN)-induced Mx gene expression has been shown to confer resistance to some single-stranded RNA (ssRNA) viruses in vitro. Because the bovine species is subject to an exceptionally wide array of infections caused by such viruses, it is anticipated that an antiviral allele should have been retained by evolution at the bovine Mx locus. The identification of such allele may help in evaluating the real significance of the Mx genotype for disease resistance in vivo, in deciphering host-virus molecular interactions involved, or in improving innate disease resistance of livestock through marker-assisted selection. We validated a double transgenic Vero cell clone in which the bovine Mx1 reference allele is placed under control of the human cytomegalovirus (CMV) enhancer-promoter sequence containing elements from the bacterial tetracycline resistance operon to regulate transcription. In the selected clone, transgene repression was very tight, and derepression by doxycycline led to homogeneous 48-h duration expression of physiologic levels of bovine Mx1. Expression of the transgene caused a dramatic decrease in cytopathic efficiency and a 500-5000-fold yield reduction of the Indiana and New Jersey serotypes of vesicular stomatitis virus (VSV). To our knowledge, the transgenic clone developed here is the first ever reported that allows conditional expression of an Mx protein, thus providing a valuable tool for studying functions of Mx proteins in general and that of bovine Mx1 in particular. This latter may henceforward be included in the group of Mx proteins with authenticated anti-VSV activity, which offers new research avenues into the field of host-virus interactions.

VL - 24 CP - 9 U1 - http://www.ncbi.nlm.nih.gov/pubmed/15450127?dopt=Abstract M3 - 10.1089/jir.2004.24.513 ER - TY - JOUR T1 - Extending the foot-and-mouth disease module to the control of other diseases. JF - Dev Biol (Basel) Y1 - 2004 A1 - Kris De Clercq A1 - Goris, N KW - Animals KW - Australia KW - Communicable Disease Control KW - Disease Outbreaks KW - Drug Storage KW - Emergency Treatment KW - Euthanasia, Animal KW - Foot-and-Mouth Disease KW - International Cooperation KW - Viral Vaccines AB -

During the recent devastating epidemics of foot-and-mouth disease (FMD), bluetongue (BT), the highly pathogenic avian influenza (HPAI) and New Castle disease, more than 115 million animals were culled. The mass slaughter of animals raised serious ethical questions. These epidemics showed that the use of emergency vaccination is an essential element in disease control. During the last decade the FMD antigen banks have proved to be effective and this module should be extended. An international vaccine stock should be considered for classical swine fever and HPAI. Agreements with vaccine producers should be made easily available, with instant access to a vaccine reserve for rinderpest, peste des petits ruminants, BT, African horse sickness and Rift valley fever. These vaccines should meet international standards and should allow distinction between vaccinated and infected animals. Information should be gathered proactively on the use of vaccines for lumpy skin disease, sheep and goat pox and contagious bovine pleuropneumonia.

VL - 119 U1 - http://www.ncbi.nlm.nih.gov/pubmed/15742644?dopt=Abstract ER - TY - JOUR T1 - Diagnosis of foot-and-mouth disease by RT-PCR: use of phylogenetic data to evaluate primers for the typing of viral RNA in clinical samples. JF - Arch Virol Y1 - 2001 A1 - Reid, S M A1 - Ferris, N P A1 - Hutchings, G H A1 - Kris De Clercq A1 - Newman, B J A1 - Knowle, N J A1 - Samuel, A R KW - Animals KW - DNA Primers KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Phylogeny KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Sensitivity and Specificity KW - Sequence Analysis, DNA KW - Serotyping AB -

The results of type-specific RT-PCR diagnostic assays on foot-and-mouth disease (FMD) viruses in clinical samples were mapped onto serotype-specific dendrograms representing the degree of nucleotide sequence variation between the FMD virus isolates. This novel approach assisted the selection of suitable PCR primer sets for the diagnosis of FMD virus isolates belonging to different topotypes within each serotype. These interpretations were qualified by using a universal (FMD virus group) specific primer to confirm that FMD virus RNA had been extracted from the samples under investigation. The analyses showed that the design of primer sets for the detection of FMD virus serotypes O, A, Asia 1, SAT 1 and SAT 3 were generally satisfactory, as most virus isolates within the major virus sub-groupings were successfully detected. However, the FMD virus serotype C and SAT 2 specific primers were less efficient as certain virus sub-groups were not detected. This identified the need for additional or alternative primers to improve RT-PCR procedures for more comprehensive detection of divergent virus strains within these serotypes. There were some examples where not all virus isolates from the same outbreak reacted with particular type-specific primers which suggested that either further minor refinements may be necessary in the primer design or that there were shortcomings in the RT-PCR methodology.

VL - 146 CP - 12 U1 - http://www.ncbi.nlm.nih.gov/pubmed/11811689?dopt=Abstract ER - TY - JOUR T1 - Diagnosis of foot-and-mouth disease by RT-PCR: evaluation of primers for serotypic characterisation of viral RNA in clinical samples. JF - J Virol Methods Y1 - 1999 A1 - Reid, S M A1 - Hutchings, G H A1 - Ferris, N P A1 - Kris De Clercq KW - Animals KW - Aphthovirus KW - DNA Primers KW - Enzyme-Linked Immunosorbent Assay KW - Evaluation Studies as Topic KW - Foot-and-Mouth Disease KW - Genome, Viral KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Sensitivity and Specificity KW - Serotyping KW - virology AB -

Multiple primers designed from the 1D and 2AB regions of the foot-and-mouth disease (FMD) viral genome were evaluated extensively for the detection of all seven serotypes of the virus by reverse transcription polymerase chain reaction (RT-PCR) at the OIE/FAO World Reference Laboratory for FMD (WRL), Pirbright. The primers had been characterised previously elsewhere on a relatively small number of cell culture grown isolates and epithelial suspensions and had been shown to identify and differentiate all seven serotypes of FMD virus. The extended study evaluated several RT-PCR protocols on epithelial suspensions and supernatant fluids, resulting from their passage in cell culture, derived from clinical samples of diverse molecular characteristics. Each of the serotype-specific primers in selected RT-PCR protocols demonstrated suitable specificity and detected cell culture passaged isolates with some success but were not adequate for the serotyping of suspensions prepared from clinical samples of epithelium. The results showed that the primers can be used in RT-PCR procedures in conjunction with the routine detection methods of virus isolation and ELISA for the diagnosis and serotyping of FMD virus.

VL - 83 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/10598089?dopt=Abstract ER - TY - JOUR T1 - Highly sensitive detection of swine vesicular disease virus based on a single tube RT-PCR system and DIG-ELISA detection. JF - J Virol Methods Y1 - 1999 A1 - Callens, M A1 - Kris De Clercq KW - Animals KW - Cells, Cultured KW - Digoxigenin KW - DNA Probes KW - Enterovirus KW - Enzyme-Linked Immunosorbent Assay KW - Epithelial Cells KW - Feces KW - Reverse Transcriptase Polymerase Chain Reaction KW - Sensitivity and Specificity KW - Swine KW - Swine Vesicular Disease KW - Virus Cultivation AB -

A highly sensitive detection of swine vesicular disease virus (SVDV) based on a single tube RT-PCR system and digoxigenin (DIG)-PCR-ELISA detection was developed. Using a one tube RT-PCR system, optimisation of the PCR conditions and optimisation of the microwell hybridisation and colourimetric detection of the amplicons resulted in a method that could detect viral RNA in infected tissue culture fluid with a titre as low as 0.1 TCID50/100 microl. The same sensitivity was obtained with SVDV-spiked faeces, if the samples were pre-treated with 1,1,2-trichlorotrifluoroethane/chloroform and subsequently concentrated using an ultrafiltration system and RNA extracted with the Purescript kit. The specificity of the test was validated on 27 SVDV strains belonging to four different groups. No cross-reactivity with genetically and symptomatically related viruses was detected using RNA of foot-and-mouth disease virus (FMDV), porcine enterovirus (PEV), vesicular stomatitis virus (VSV), Coxsackie B5 virus (CV-B5) and encephalomyocarditis virus (EMCV). The test was validated successfully on clinical samples, being slightly more sensitive and much faster than virus isolation on cell cultures. Moreover the possibility of automating the procedure will allow the processing of large numbers of clinical samples.

VL - 77 CP - 1 U1 - http://www.ncbi.nlm.nih.gov/pubmed/10029329?dopt=Abstract ER - TY - JOUR T1 - Detection of foot-and-mouth disease by reverse transcription polymerase chain reaction and virus isolation in contact sheep without clinical signs of foot-and-mouth disease. JF - Vet Q Y1 - 1998 A1 - Callens, M A1 - Kris De Clercq A1 - Gruia, M A1 - Danes, M AB -

Summary Two non-vaccinated sheep were experimentally, infected with FMDV and one day later 4 other sheep were brought in contact. Although the contact sheep showed no clinical signs, serology indicated that all sheep became infected. Various secretion samples, taken over a period of at least one month, and various tissue samples were examined for the presence of FMDV by RT-PCR and by virus isolation. FMDV was most often found in saliva (mouth swabs), followed by nasal secretion and sera. Faecal material, wool and milk were less suitable. The period of detection with the highest frequency of positive isolations was between 2 to 4 days pi for the infected sheep and between 5 to 10 days pc for the contact animals. It was established that in subclinically infected sheep, with a very low amount of virus present, FMD viral RNA could be detected by a sensitive RT-PCR-ELISA although virus isolation and standard RT-PCR remained negative. Moreover there was some evidence of active spreading of FMDV from the contact sheep to two sentinel pigs. This indicates that serologically positive contact sheep without clinical signs may be considered as a danger for the transmission of FMDV.

VL - 20 CP - sup2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/22077296?dopt=Abstract M3 - 10.1080/01652176.1998.9694964 ER - TY - JOUR T1 - Implementation of quality assurance in national foot and mouth disease laboratories, based on the guidelines of the Office International des Epizooties. JF - Rev Sci Tech Y1 - 1998 A1 - Kris De Clercq KW - Accreditation KW - Animals KW - Foot-and-Mouth Disease KW - International Cooperation KW - Laboratories KW - Quality Control KW - Veterinary Medicine AB -

The final act of the Uruguay Round of the General Agreement on Tariffs and Trade, 1994, contains the Agreement on the Application of Sanitary and Phytosanitary Measures, which aims to reduce the negative effects of health barriers on international trade to a minimum. Therefore, the Office International des Epizooties (OIE) Regional Commission for Europe proposed that an accreditation system based on the EN 45000 standard should be applied to achieve international recognition of certificates and testing laboratories. To this end, the OIE Standards Commission published a series of guidelines for the evaluation of laboratory quality and proficiency testing. In 1995, the Food and Agriculture Organization European Commission for the Control of Foot and Mouth Disease recommended that the OIE proposal should be applied in all foot and mouth disease (FMD) diagnostic laboratories in Europe. This review summarises the EN 45001 standard and the OIE guidelines, and proposes that these guidelines should be taken as a basis for the implementation of a quality assurance programme in FMD laboratories.

VL - 17 CP - 3 U1 - http://www.ncbi.nlm.nih.gov/pubmed/9850551?dopt=Abstract ER - TY - JOUR T1 - Reduction of singleton reactors against swine vesicular disease virus by a combination of virus neutralisation test, monoclonal antibody-based competitive ELISA and isotype specific ELISA. JF - J Virol Methods Y1 - 1998 A1 - Kris De Clercq KW - Animals KW - Antibodies, Monoclonal KW - Antibodies, Viral KW - Enteroviruses, Porcine KW - Enzyme-Linked Immunosorbent Assay KW - False Positive Reactions KW - Immunoglobulin Isotypes KW - Neutralization Tests KW - Sensitivity and Specificity KW - Swine KW - Swine Vesicular Disease AB -

Pigs which are serologically positive for swine vesicular disease virus (SVDV) but which show no clinical signs and for which there is neither a relevant history of the disease on the holding nor contact with a known outbreak are considered as singleton reactors. False positive serological results for an epizootic disease, like SVD, in a non-vaccinated population or in imported animals are of great concern to international trade. For the virus neutralisation test, the gold standard for SVD, singleton reactors are found at a level of 1-3/1000. Singleton reactors also occur when other serological testing methods are used. The number of animals finally considered as singleton reactors can be reduced considerably by performing three different serological tests (virus neutralisation test, monoclonal antibody-based competitive ELISA and isotype specific ELISA) on the same serum. A serological profile of the animal can be derived by analysing the results in greater detail. This procedure can reduce considerably the number of pig holdings on which a prohibition of movement and trade needs to be imposed without requiring analysis of supplementary samples.

VL - 70 CP - 1 U1 - http://www.ncbi.nlm.nih.gov/pubmed/9506808?dopt=Abstract ER - TY - JOUR T1 - Differentiation of the seven serotypes of foot-and-mouth disease virus by reverse transcriptase polymerase chain reaction. JF - J Virol Methods Y1 - 1997 A1 - Callens, M A1 - Kris De Clercq KW - Animals KW - Aphthovirus KW - Capsid KW - Capsid Proteins KW - Cross Reactions KW - DNA Primers KW - Moloney murine leukemia virus KW - polymerase chain reaction KW - RNA, Viral KW - RNA-Directed DNA Polymerase KW - Serotyping KW - Species Specificity AB -

A strategy for reverse transcriptase polymerase chain reaction (RT-PCR) using multiple primers was developed to detect and to differentiate the seven serotypes of foot-and-mouth disease virus (FMDV) simultaneously, quickly and accurately. The development of the test was carried out on virus isolates grown in tissue culture prior to cDNA synthesis and PCR using various sets of primers. Primers P33 and P32 were used for the consensus PCR to detect FMDV regardless of the serotype. Positive cDNA was assayed in two multi-primer PCR mixes containing type-specific primers capable of distinguishing between the seven serotypes. The serotype-specific primers were selected to correspond to regions of the genome coding for parts of the VP1 polypeptide that is responsible for the antigenic diversity of the virus group. Multi-primer mix P33-P(A-C-O-ASIA 1) gave products of 732, 596, 402, 292 bp for the A,C,O and ASIA 1 serotypes, respectively, and no target products for South African Territories serotypes (SAT 1, SAT 2 and SAT 3). The multi-primer mix P33-P(A-C-O-ASIA 1) was also capable of detecting a mixture of two different serotypes. Multi-primer mix P1-P(SAT 1-3-2) gave products of 246, 201 and 75 bp for the SAT 1, SAT 3 and SAT 2 serotypes, respectively, and no specific products for serotypes A, C, O and ASIA 1. This is the first PCR assay to be described that differentiates between the SAT serotypes of FMDV. The method has been applied to 25 cell-culture-derived isolates of the SAT serotypes of FMDV and the results were totally compatible with the standard techniques for FMDV detection and serotyping.

VL - 67 CP - 1 U1 - http://www.ncbi.nlm.nih.gov/pubmed/9274816?dopt=Abstract ER - TY - JOUR T1 - Rapid & sensitive polymerase chain reaction based detection & typing of foot-and-mouth disease virus in clinical samples and cell culture isolates, combined with a simultaneous differentiation with other genomically and/or symptomatically related viruses JF - Arch Virol Y1 - 1996 A1 - Vangrysperre, W A1 - Kris De Clercq KW - Animals KW - Aphthovirus KW - Base Sequence KW - Cattle KW - Cell Line KW - Cells, Cultured KW - Cercopithecus aethiops KW - Cricetinae KW - DNA Primers KW - Foot-and-Mouth Disease KW - Genome, Viral KW - Kidney KW - Male KW - Molecular Sequence Data KW - polymerase chain reaction KW - RNA-Directed DNA Polymerase KW - Sensitivity and Specificity KW - Sequence Homology, Nucleic Acid KW - Serotyping KW - Sheep KW - Testis KW - Vero Cells AB -

Reverse transcription followed by the polymerase chain reaction method (PCR) allowed the detection of foot-and-mouth disease virus (FMDV), regardless of the serotype. A primer set corresponding to highly conserved regions of the 2B sequence was selected. By combining in a single reaction tube specific primer pairs for FMDV, swine vesicular disease virus, (SVDV), encephalomyocarditis virus (EMCV) and bovine viral diarrhea virus (BVDV), all four viruses could be identified and differentiated in one amplification reaction, based on the different lengths of the respective amplified segments. In a similar way, FMDV types O, A, C, SAT 2 and Asia 1 could be identified and differentiated, using primers selected from the ID (VP1) genome region. All results were confirmed by direct sequencing of the PCR product. The very fast RNA extraction, reverse transcription and PCR permitted us to read the agarose gels within three hours after samples (cell culture isolates as well as clinical material) arrived, which is of great importance in case of an FMDV suspicion. Furthermore, a very high sensitivity was achieved (less than one PFU). Therefore, our powerful detection assay by means of PCR for FMDV as well as for SVDV, EMCV and BVDV, has advantages compared to the presently used procedures.

VL - 141 CP - 2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/8634024?dopt=Abstract M3 - NA ER - TY - JOUR T1 - Reproductive failure in sows following experimental infection with a Belgian EMCV isolate. JF - Vet Microbiol Y1 - 1994 A1 - F. Koenen A1 - Kris De Clercq A1 - Lefebvre, J A1 - Strobbe, R KW - Animals KW - Antibodies, Viral KW - Belgium KW - Cardiovirus Infections KW - Encephalomyocarditis virus KW - Female KW - Fetal Death KW - Heart KW - Pregnancy KW - Pregnancy Complications, Infectious KW - Spleen KW - Swine KW - Swine Diseases AB -

In this study, a transplacental infection with fetal death was demonstrated following inoculation of pregnant sows with a Belgian encephalomyocarditis virus (EMCV) isolate. Eight multiparus sows were inoculated between 60 and 92 days of gestation with this EMCV-isolate to investigate its ability to cause reproductive failure in sows. Virus persistence and antibody titre in their offspring were also studied. Only the two sows inoculated at 60 days of gestation showed premature farrowing, but all sows seroconverted to EMCV. Virus was recovered from the offspring of all sows at the time of farrowing, but not from every piglet born. One month after birth EMCV could be isolated from all the piglets examined. These results can help in a better understanding of the spread of the disease in piggeries.

VL - 39 CP - 1-2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/8203116?dopt=Abstract ER - TY - JOUR T1 - Serological response to a booster foot-and-mouth disease vaccination with strains different from the primary vaccine. JF - Vet Res Commun Y1 - 1989 A1 - Kris De Clercq A1 - Strobbe, R A1 - Vanopdenbosch, E A1 - Debecq, J A1 - Theys, H KW - Animals KW - Antibodies, Viral KW - Aphthovirus KW - Cattle KW - Immunization, Secondary KW - Neutralization Tests KW - Viral Vaccines AB -

Young calves were vaccinated with belgian foot-and-mouth disease (FMD) vaccine and revaccinated with either the same vaccine or with a foreign FMD vaccine. There was a significant serological response to the primary vaccine strains after the first vaccination which was greater following revaccination. At one and two months after revaccination there was no significant difference between the responses to revaccination with vaccine identical to the primary vaccine or with the foreign FMD vaccine. It was concluded that revaccination of young calves is effective even with an FMD vaccine different from the primary vaccine.

VL - 13 CP - 3 U1 - http://www.ncbi.nlm.nih.gov/pubmed/2551092?dopt=Abstract ER - TY - JOUR T1 - The serological response to foot-and-mouth disease vaccination is not affected by simultaneous infectious bovine rhinotracheitis/adenovirus/parainfluenza 3 vaccination. JF - Arch Exp Veterinarmed Y1 - 1989 A1 - Kris De Clercq A1 - Strobbe, R A1 - van Opdenbosch, E A1 - Wellemans, G A1 - Theys, H A1 - Debecq, J KW - Adenoviridae KW - Animals KW - Antibodies, Viral KW - Aphthovirus KW - Cattle KW - Herpesvirus 1, Bovine KW - Parainfluenza Virus 3, Human KW - Viral Vaccines AB -

Young calves were simultaneously vaccinated by subcutaneous route against foot-and-mouth disease (FMD) and against infectious bovine rhinotracheitis/adenovirus/parainfluenza-3 (IBR/Adeno/PI-3) by intranasal route. The serological response against the 3 FMD virus types of the FMD vaccine was clearly positive. There was no significant difference between results of simultaneous FMD and IBR/Adeno/PI-3 vaccination and FMD vaccination only.

VL - 43 CP - 3 U1 - http://www.ncbi.nlm.nih.gov/pubmed/2549906?dopt=Abstract ER - TY - JOUR T1 - Simultaneous vaccination of piglets against foot-and-mouth disease and classical swine fever. JF - Vet Microbiol Y1 - 1989 A1 - Kris De Clercq A1 - F. Koenen A1 - Strobbe, R A1 - Debecq, J KW - Animals KW - Antibodies, Viral KW - Antigens, Viral KW - Classical Swine Fever KW - Dose-Response Relationship, Immunologic KW - Fluorescent Antibody Technique KW - Foot-and-Mouth Disease KW - Swine KW - Vaccines, Attenuated KW - Vaccines, Inactivated KW - Viral Vaccines AB -

Six-week-old piglets, born of unvaccinated sows, were vaccinated against foot-and-mouth disease (FMD) with a trivalent, inactivated vaccine containing an adjuvant or vaccinated against classical swine fever (CSF) with a live attenuated vaccine or against both diseases simultaneously at two different sites. The antibody response to the FMD vaccine was not significantly influenced by the simultaneous vaccination against CSF. FMD vaccine administered simultaneously with the CSF vaccine produced a significantly higher antibody response to CSF than occurred with CSF vaccination only.

VL - 20 CP - 3 U1 - http://www.ncbi.nlm.nih.gov/pubmed/2672549?dopt=Abstract ER - TY - JOUR T1 - Estimation of maternally acquired immunity in suckling mice to evaluate foot-and-mouth disease vaccine. JF - Arch Exp Veterinarmed Y1 - 1988 A1 - Strobbe, R A1 - Kris De Clercq A1 - Debecq, J KW - Animals KW - Aphthovirus KW - Cattle KW - Cattle Diseases KW - Female KW - Foot-and-Mouth Disease KW - Immunity, Maternally-Acquired KW - mice KW - Pregnancy KW - Vaccination KW - Viral Vaccines VL - 42 CP - 3 U1 - http://www.ncbi.nlm.nih.gov/pubmed/2845880?dopt=Abstract ER - TY - JOUR T1 - Experimental transmission of enzootic bovine leukosis to cattle, sheep and goats: infectious doses of blood and incubation period of the disease. JF - Leuk Res Y1 - 1987 A1 - Mammerickx, M A1 - Portetelle, D A1 - Kris De Clercq A1 - Burny, A KW - Animals KW - Antibodies, Viral KW - Cattle KW - Goats KW - Leukemia Virus, Bovine KW - Leukemia, Experimental KW - Leukocytosis KW - Retroviridae KW - Sheep AB -

A group of 49 BLV-free recipient animals (24 cattle, 15 sheep and 10 goats) were inoculated intradermally with serial dilutions of blood collected on two BLV-positive donor cows. One donor had a high lymphocytosis and high antibody titers to gpBLV antigens; these two parameters were low for the second donor. The number of lymphocytes which induced BLV infection in recipient animals varied widely with the donor. The high infectivity of a donor seemed to be correlated with high lymphocytosis and high antibody titers to gpBLV antigens. Identification and removal of infectious animals would reduce or stop the spread of the infection in a herd, and could be used in the strategy to eradicate the disease. A given inoculum can be infectious in sheep and, at the same time, harmless in cattle. The incubation periods, apparently shorter in sheep, were generally in the range from 2 to 5 weeks for the three species, and exceptionally above.

VL - 11 CP - 4 U1 - http://www.ncbi.nlm.nih.gov/pubmed/3031388?dopt=Abstract ER -