%0 Journal Article %J Microorganisms %D 2023 %T Belgian Anopheles plumbeus Mosquitoes Are Competent for Japanese Encephalitis Virus and Readily Feed on Pigs, Suggesting a High Vectorial Capacity %A Claudia Van den Eynde %A Charlotte Sohier %A Severine Matthijs %A Nick De Regge %K Anopheles plumbeus; field-collected mosquitoes; vector competence; vectorial capacity; Japanese encephalitis virus %B Microorganisms %V 11 %8 Jan-06-2023 %G eng %N 6 %R 10.3390/microorganisms11061386 %0 Journal Article %J Microorganisms %D 2023 %T Belgian Mosquitoes Are Competent for Japanese Encephalitis Virus and Readily Feed on Pigs, Suggesting a High Vectorial Capacity. %A Claudia Van den Eynde %A Charlotte Sohier %A Severine Matthijs %A Nick De Regge %X

, a day-active mosquito known to feed aggressively on humans, was reported as a nuisance species near an abandoned pigsty in Belgium. Since Japanese encephalitis virus (JEV) is an emerging zoonotic flavivirus which uses pigs as amplification hosts, we investigated (1) whether would feed on pigs and (2) its vector competence for JEV, to investigate whether this species could be a potential vector. Three- to seven-day-old F0-generation adult mosquitoes, emerged from field-collected larvae, were fed on a JEV genotype 3 Nakayama strain spiked blood meal. Blood-fed mosquitoes were subsequently incubated for 14 days at two temperature conditions: a constant 25 °C and a 25/15 °C day/night temperature gradient. Our results show that is a competent vector for JEV at the 25 °C condition and this with an infection rate of 34.1%, a dissemination rate of 67.7% and a transmission rate of 14.3%. The vector competence showed to be influenced by temperature, with a significantly lower dissemination rate (16.7%) and no transmission when implementing the temperature gradient. Moreover, we demonstrated that readily feeds on pigs when the opportunity occurs. Therefore, our results suggest that Belgian mosquitoes may play an important role in the transmission of JEV upon an introduction into our region if temperatures increase with climate change.

%B Microorganisms %V 11 %8 2023 May 25 %G eng %N 6 %R 10.3390/microorganisms11061386 %0 Journal Article %J Viruses %D 2023 %T Comparison of Serological Methods for Tick-Borne Encephalitis Virus-Specific Antibody Detection in Wild Boar and Sheep: Impact of the Screening Approach on the Estimated Seroprevalence. %A Gabrielle Trozzi %A Nadjah Radia Adjadj %A Vervaeke, Muriel %A Severine Matthijs %A Charlotte Sohier %A Nick De Regge %K Animals %K antibodies %K Encephalitis Viruses, Tick-Borne %K Seroepidemiologic Studies %K Sheep %K Sus scrofa %K Swine %X

Tick-borne encephalitis virus (TBEV) is a flavivirus transmitted by ticks. Serological screenings in animals are performed to estimate the prevalence and distribution of TBEV. Most screenings consist of a primary screening by ELISA, followed by confirmation of positive samples by plaque reduction neutralization tests (PRNTs). In this study, 406 wild boar sera were tested with 2 regularly used commercial ELISAs for flavivirus screening in animals (Immunozym FSME (TBEV) IgG All Species (Progen) and ID Screen West Nile Competition (Innovative Diagnostics)) and PRNTs for TBEV and USUTU virus. The results showed that the Immunozym and IDScreen ELISAs had low relative sensitivities of 23% and 20%, respectively, compared to the PRNT results. The relative specificities were 88% and 84% due to cross reactions with USUTU virus-specific antibodies. The minimal TBEV prevalence in our sample set was 8.6% when determined by PRNT. When the screening approach of ELISA testing followed by PRNT confirmation was applied, a TBEV seroprevalence of only 2.0% and 1.7% was found. The suboptimal performance of the ELISAs was confirmed by testing sera collected from experimentally TBEV-infected sheep. While the PRNT detected TBEV specific antibodies in 94% of samples collected between 7 and 18 days post-infection, the ELISAs classified only 50% and 31% of the samples as positive. Both routinely used ELISAs for TBEV antibody screening in animal sera were shown to have a low sensitivity, potentially leading to an underestimation of the true prevalence, and furthermore cross-react with other flavivirus antibodies.

%B Viruses %V 15 %8 2023 Feb 06 %G eng %N 2 %R 10.3390/v15020459 %0 Journal Article %J Viruses %D 2023 %T Comparison of Serological Methods for Tick-Borne Encephalitis Virus-Specific Antibody Detection in Wild Boar and Sheep: Impact of the Screening Approach on the Estimated Seroprevalence %A Gabrielle Trozzi %A Nadjah Radia Adjadj %A Vervaeke, Muriel %A Severine Matthijs %A Charlotte Sohier %A Nick De Regge %B Viruses %V 15 %8 Jan-02-2023 %G eng %N 2 %R 10.3390/v15020459 %0 Journal Article %J Viruses %D 2023 %T Comparison of Serological Methods for Tick-Borne Encephalitis Virus-Specific Antibody Detection in Wild Boar and Sheep: Impact of the Screening Approach on the Estimated Seroprevalence %A Gabrielle Trozzi %A Nadjah Radia Adjadj %A Vervaeke, Muriel %A Severine Matthijs %A Charlotte Sohier %A Nick De Regge %K tick-borne encephalitis virus; diagnostic; ELISA; seroprevalence %B Viruses %G eng %R 10.3390/v15020459 %0 Journal Article %J Viruses %D 2023 %T Development and Validation of a New DIVA Real-Time PCR Allowing to Differentiate Wild-Type Lumpy Skin Disease Virus Strains, Including the Asian Recombinant Strains, from Neethling-Based Vaccine Strains. %A Andy Haegeman %A Ilse De Leeuw %A Wannes Philips %A Nick De Regge %K Animals %K capripoxvirus %K Cattle %K Lumpy skin disease %K Lumpy skin disease virus %K Real-Time Polymerase Chain Reaction %K Vaccines, Attenuated %K Viral Vaccines %X

The current epidemic in Asia, driven by LSDV recombinants, poses difficulties to existing DIVA PCR tests, as these do not differentiate between homologous vaccine strains and the recombinant strains. We, therefore, developed and validated a new duplex real-time PCR capable of differentiating Neethling-based vaccine strains from classical and recombinant wild-type strains that are currently circulating in Asia. The DIVA potential of this new assay, seen in the in silico evaluation, was confirmed on samples from LSDV infected and vaccinated animals and on isolates of LSDV recombinants (n = 12), vaccine (n = 5), and classic wild-type strains (n = 6). No cross-reactivity or a-specificity with other capripox viruses was observed under field conditions in non-capripox viral stocks and negative animals. The high analytical sensitivity is translated into a high diagnostic specificity as more than 70 samples were all correctly detected with Ct values very similar to those of a published first-line pan capripox real-time PCR. Finally, the low inter- and intra-run variability observed shows that the new DIVA PCR is very robust which facilitates its implementation in the lab. All validation parameters that are mentioned above indicate the potential of the newly developed test as a promising diagnostic tool which could help to control the current LSDV epidemic in Asia.

%B Viruses %V 15 %8 2023 Mar 28 %G eng %N 4 %R 10.3390/v15040870 %0 Journal Article %J Microorganisms %D 2023 %T Duration of Immunity Induced after Vaccination of Cattle with a Live Attenuated or Inactivated Lumpy Skin Disease Virus Vaccine %A Andy Haegeman %A Ilse De Leeuw %A Laurent Mostin %A Willem Van Campe %A Wannes Philips %A Mehdi Elharrak %A Nick De Regge %A Kris De Clercq %K duration of immunity %K Lumpy skin disease %K vaccines %X

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.

%B Microorganisms %V 11 %8 Jan-01-2023 %G eng %N 1 %R 10.3390/microorganisms11010210 %0 Journal Article %J Viruses %D 2023 %T Evidence of Lumpy Skin Disease Virus Transmission from Subclinically Infected Cattle by Stomoxys calcitrans %A Andy Haegeman %A Charlotte Sohier %A Laurent Mostin %A Ilse De Leeuw %A Willem Van Campe %A Philips, Wannes %A Nick De Regge %A Kris De Clercq %B Viruses %V 15 %8 Jan-06-2023 %G eng %N 6 %R 10.3390/v15061285 %0 Journal Article %J Viruses %D 2023 %T Evidence of Lumpy Skin Disease Virus Transmission from Subclinically Infected Cattle by . %A Andy Haegeman %A Charlotte Sohier %A Laurent Mostin %A Ilse De Leeuw %A Willem Van Campe %A Wannes Philips %A Nick De Regge %A Kris De Clercq %K Animals %K capripoxvirus %K Cattle %K Cattle Diseases %K Insect Vectors %K Lumpy skin disease %K Lumpy skin disease virus %K Male %K Muscidae %X

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.

%B Viruses %V 15 %8 2023 May 30 %G eng %N 6 %R 10.3390/v15061285 %0 Journal Article %J Viruses %D 2023 %T Evidence of Lumpy Skin Disease Virus Transmission from Subclinically Infected Cattle by Stomoxys calcitrans %A Andy Haegeman %A Charlotte Sohier %A Laurent Mostin %A Ilse De Leeuw %A Willem Van Campe %A Wannes Philips %A Nick De Regge %A Kris De Clercq %K capripox virus; vector transmission; subclinical infection; stable fly %B Viruses %G eng %R 10.3390/v15061285 %0 Journal Article %J Viruses %D 2023 %T First Expert Elicitation of Knowledge on Possible Drivers of Observed Increasing Human Cases of Tick-Borne Encephalitis in Europe %A Saegerman, Claude %A Marie-France Humblet %A Marc Leandri %A Gaëlle Gonzalez %A Heyman, Paul %A Sprong, Hein %A Monique L’Hostis %A Sara Moutailler %A Sarah I. Bonnet %A Nadia Haddad %A Nathalie Boulanger %A Stephen L. Leib %A Thierry Hoch %A Thiry, Etienne %A Laure Bournez %A Jana Kerlik %A Aurélie Velay %A Jore, Solveig %A Elsa Jourdain %A Emmanuelle Gilot-Fromont %A Katharina Brugger %A Julia Geller %A Marie Studahl %A Nataša Knap %A Tatjana Avšič-Županc %A Daniel Růžek %A Tizza P. Zomer %A René Bødker %A Thomas F.H. Berger %A Sandra Martin-Latil %A Nick De Regge %A Alice Raffetin %A Sandrine A. Lacour %A Matthias Klein %A Tinne Lernout %A Elsa Quillery %A Hubálek, Zdeněk %A Francisco Ruiz-Fons %A Agustín Estrada-Peña %A Philippe Fravalo %A Pauline Kooh %A Florence Etore %A Céline M. Gossner %A Bethan Purse %B Viruses %V 15 %8 Jan-03-2023 %G eng %N 3 %R 10.3390/v15030791 %0 Journal Article %J Viruses %D 2023 %T Lumpy Skin Disease Virus Genome Sequence Analysis: Putative Spatio-Temporal Epidemiology, Single Gene versus Whole Genome Phylogeny and Genomic Evolution. %A Floris C Breman %A Andy Haegeman %A Nina Krešić %A Philips, Wannes %A Nick De Regge %K Animals %K Cattle %K Disease Outbreaks %K Evolution, Molecular %K Genomics %K Humans %K Lumpy skin disease %K Lumpy skin disease virus %K Phylogeny %X

is a poxvirus from the genus that mainly affects bovines and it causes severe economic losses to livestock holders. The is currently dispersing in Asia, but little is known about detailed phylogenetic relations between the strains and genome evolution. We reconstructed a whole-genome-sequence (WGS)-based phylogeny and compared it with single-gene-based phylogenies. To study population and spatiotemporal patterns in greater detail, we reconstructed networks. We determined that there are strains from multiple clades within the previously defined cluster 1.2 that correspond with recorded outbreaks across Eurasia and South Asia (Indian subcontinent), while strains from cluster 2.5 spread in Southeast Asia. We concluded that using only a single gene (cheap, fast and easy to routinely use) for sequencing lacks phylogenetic and spatiotemporal resolution and we recommend to create at least one WGS whenever possible. We also found that there are three gene regions, highly variable, across the genome of LSDV. These gene regions are located in the 5' and 3' flanking regions of the LSDV genome and they encode genes that are involved in immune evasion strategies of the virus. These may provide a starting point to further investigate the evolution of the virus.

%B Viruses %V 15 %8 2023 Jun 28 %G eng %N 7 %R 10.3390/v15071471 %0 Journal Article %J Viruses %D 2023 %T Relevant Day/Night Temperatures Simulating Belgian Summer Conditions Reduce Japanese Encephalitis Virus Dissemination and Transmission in Belgian Field-Collected Culex pipiens Mosquitoes %A Claudia Van den Eynde %A Charlotte Sohier %A Severine Matthijs %A Nick De Regge %K Japanese encephalitis virus; vector competence; field-caught mosquitoes; Culex pipiens %B Viruses %V 15 %8 Jan-03-2023 %G eng %N 3 %R 10.3390/v15030764 %0 Journal Article %J Virologie (Montrouge) %D 2022 %T [Foot and mouth disease virus: transmission, pathogenesis, diagnosis and surveillance]. %A Morgan Sarry %A Aurore Romey %A David Lefebvre %A Souheyla Benfrid %A Barbara Dufour %A Durand, Benoit %A Gina Zanella %A Nick De Regge %A Zientara, Stéphan %A Labib Bakkali Kassimi %A Sandra Blaise-Boisseau %K Animals %K Artiodactyla %K Europe %K Foot-and-Mouth Disease %K Foot-and-Mouth Disease Virus %K Serogroup %X

Foot-and-mouth disease (FMD) is one of the most contagious viral animal diseases. It is an old disease which still poses a permanent threat of re-emergence for free zones. Foot-and-Mouth Disease Virus (FMDV), a Picornavirus belonging to genus Aphthovirus affects domestic and wild artiodactyls. FMD has a considerable socio-economic impact on agricultural production and trade in endemic regions, but also when incursions occur into FMD free areas, as in Europe in 2001. FMDV is historically one of the most studied viruses. Due to its high genetic and antigenic variability, the absence of cross-immunity between its seven serotypes, its ability to survive in the environment, its high contagiousness, its wide range of hosts and its particular biology, FMDV remains of major interest in animal health and the subject of many research projects. This review presents different aspects of FMDV infection, ranging from basic biology to diagnosis, surveillance and control.

%B Virologie (Montrouge) %V 26 %8 2022 Sep 01 %G eng %N 5 %R 10.1684/vir.2022.0972 %0 Journal Article %J Pathogens %D 2022 %T Japanese Encephalitis Virus Interaction with Mosquitoes: A Review of Vector Competence, Vector Capacity and Mosquito Immunity %A Claudia Van den Eynde %A Charlotte Sohier %A Severine Matthijs %A Nick De Regge %K Japanese encephalitis virus; vector competence; vector capacity; vector immunity; virus–vector interactions; arboviruses %B Pathogens %8 3 March 2022 %G eng %R 10.3390/pathogens11030317 %0 Journal Article %J Pathogens %D 2022 %T Japanese Encephalitis Virus Interaction with Mosquitoes: A Review of Vector Competence, Vector Capacity and Mosquito Immunity %A Claudia Van den Eynde %E Charlotte Sohier %E Severine Matthijs %E Nick De Regge %B Pathogens %V 11 %8 Jan-03-2022 %G eng %N 3 %R 10.3390/pathogens11030317 %0 Journal Article %J Viruses %D 2022 %T Tick-Borne Encephalitis Virus Prevalence in Sheep, Wild Boar and Ticks in Belgium. %A Nadjah Radia Adjadj %A Vervaeke, Muriel %A Charlotte Sohier %A Mickael Cargnel %A Nick De Regge %K Animals %K Antibodies, Viral %K Belgium %K Encephalitis Viruses, Tick-Borne %K Encephalitis, Tick-Borne %K prevalence %K Seroepidemiologic Studies %K Sheep %K Sus scrofa %K Swine %K Ticks %X

Tick-borne encephalitis virus (TBEV) is the most important tick-borne zoonotic virus in Europe. In Belgium, antibodies to TBEV have already been detected in wildlife and domestic animals, but up-to-date prevalence data for TBEV are lacking, and no studies have assessed its seroprevalence in sheep. Serum samples of 480 sheep from all over Belgium and 831 wild boar hunted in Flanders (northern Belgium) were therefore screened for TBEV antibodies by ELISA and plaque reduction neutralization test (PRNT), respectively. The specificity of positive samples was assessed by PRNTs for TBEV and the Louping Ill, West Nile, and Usutu viruses. TBEV seroprevalence was 0.42% (2/480, CI 95%: 0.11-1.51) in sheep and 9.27% (77/831, CI 95%: 7.48-11.43) in wild boar. TBEV seroprevalence in wild boar from the province of Flemish Brabant was significantly higher (22.38%, 15/67) compared to Limburg (7.74%, 34/439) and Antwerp (8.61%, 28/325). Oud-Heverlee was the hunting area harboring the highest TBEV seroprevalence (33.33%, 11/33). In an attempt to obtain a Belgian TBEV isolate, 1983 ticks collected in areas showing the highest TBEV seroprevalence in wild boars were tested by real-time qPCR. No TBEV-RNA-positive tick was detected. The results of this study suggest an increase in TBEV prevalence over the last decade and highlight the need for One-Health surveillance in Belgium.

%B Viruses %V 14 %8 2022 Oct 26 %G eng %N 11 %R 10.3390/v14112362 %0 Journal Article %J Transboundary and Emerging Diseases %D 2021 %T Risk assessment of SARS‐CoV‐2 infection in free‐ranging wild animals in Belgium %A Myriam Logeot %A Mauroy, Axel %A Thiry, Etienne %A Nick De Regge %A Vervaeke, Muriel %A Olivier Beck %A Valérie De Waele %A Thierry van den Berg %B Transboundary and Emerging Diseases %8 Feb-05-2023 %G eng %0 Journal Article %J Transboundary and Emerging Diseases %D 2021 %T Semi‐quantitative risk assessment by expert elicitation of potential introduction routes of African swine fever from wild reservoir to domestic pig industry and subsequent spread during the Belgian outbreak (2018–2019) %A Mauroy, Axel %A Depoorter, Pieter %A Saegerman, Claude %A Ann Brigitte Cay %A Nick De Regge %A Maria‐Eleni Filippitzi %A Claude Fischer %A Martine Laitat %A Maes, Dominiek %A Kevin Morelle %A Nauwynck, Hans %A Xavier Simons %A Thierry van den Berg %A Van Huffel, Xavier %A Thiry, Etienne %A Dewulf, Jeroen %B Transboundary and Emerging Diseases %V 68 %8 Jan-09-2021 %G eng %N 5 %R 10.1111/tbed.14067 %0 Journal Article %J Int J Mol Sci %D 2021 %T Species-Specific Humoral Immune Responses in Sheep and Goats upon Small Ruminant Lentivirus Infections Inversely Correlate with Protection against Virus Replication and Pathological Lesions. %A Rodolphe Michiels %A S. Roels %A Nick Vereecke %A Elisabeth Mathijs %A Laurent Mostin %A Nick De Regge %K Animals %K Antibodies, Viral %K Arthritis-Encephalitis Virus, Caprine %K Female %K Genotype %K Goat Diseases %K Goats %K Immunity, Humoral %K Lung %K Mammary Glands, Animal %K Pneumonia, Progressive Interstitial, of Sheep %K Sheep %K Species Specificity %K Viral Load %K Virus Replication %K Visna-maedi virus %X

Maedi-Visna-like genotype A strains and Caprine arthritis encephaltis-like genotype B strains are small ruminant lentiviruses (SRLV) which, for incompletely understood reasons, appear to be more virulent in sheep and goats, respectively. A 9-month in vivo infection experiment using Belgian genotype A and B SRLV strains showed that almost all homologous (genotype A in sheep; genotype B in goats) and heterologous (genotype A in goats; genotype B in sheep) intratracheal inoculations resulted in productive infection. No differences in viremia and time to seroconversion were observed between homologous and heterologous infections. Higher viral loads and more severe lesions in the mammary gland and lung were however detected at 9 months post homologous compared to heterologous infection which coincided with strongly increased IFN-γ mRNA expression levels upon homologous infection. Pepscan analysis revealed a strong antibody response against immune-dominant regions of the capsid and surface proteins upon homologous infection, which was absent after heterologous infection. These results inversely correlated with protection against virus replication in target organs and observed histopathological lesions, and thus require an in-depth evaluation of a potential role of antibody dependent enhancement in SRLV infection. Finally, no horizontal intra- and cross-species SRLV transmission to contact animals was detected.

%B Int J Mol Sci %V 22 %8 2021 Sep 11 %G eng %N 18 %R 10.3390/ijms22189824 %0 Journal Article %J Parasites & Vectors %D 2021 %T Temperature and food sources influence subadult development and blood-feeding response of Culicoides obsoletus (sensu lato) under laboratory conditions %A Claudia Van den Eynde %A Charlotte Sohier %E Severine Matthijs %E Nick De Regge %B Parasites & Vectors %V 14 %8 Jan-12-2021 %G eng %N 1 %R 10.1186/s13071-021-04781-8 %0 Journal Article %J J Neuroinflammation %D 2020 %T Efficient control of Japanese encephalitis virus in the central nervous system of infected pigs occurs in the absence of a pronounced inflammatory immune response. %A Valerie Redant %A Favoreel, Herman W %A Kai Dallmeier %A Willem Van Campe %A Nick De Regge %X

BACKGROUND: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. JEV infection of mice and humans can lead to an uncontrolled inflammatory response in the central nervous system (CNS), resulting in a detrimental outcome. Pigs act as important amplification and reservoir hosts, and JEV infection of pigs is mostly subclinical. Information on virus spread in the CNS and immune responses controlling JEV infection in the CNS of pigs, however remains scarce.

METHODS: Nine-week-old pigs were inoculated intranasal or intradermal with a relevant dose of 10 TCID of JEV genotype 3 Nakayama strain. Clinical signs were assessed daily, and viral spread was followed by RT-qPCR. mRNA expression profiles were determined to study immune responses in the CNS.

RESULTS: Besides a delay of 2 days to reach the peak viremia upon intranasal compared to intradermal inoculation, the overall virus spread via both inoculation routes was highly similar. JEV appearance in lymphoid and visceral organs was in line with a blood-borne JEV dissemination. JEV showed a particular tropism to the CNS but without the induction of neurological signs. JEV entry in the CNS probably occurred via different hematogenous and neuronal pathways, but replication in the brain was mostly efficiently suppressed and associated with a type I IFN-independent activation of OAS1 expression. In the olfactory bulb and thalamus, where JEV replication was not completely controlled by this mechanism, a short but strong induction of chemokine gene expression was detected. An increased IFNy expression was simultaneously observed, probably originating from infiltrating T cells, correlating with a fast suppression of JEV replication. The chemokine response was however not associated with the induction of a strong inflammatory response, nor was an induction of the NLRP3 inflammasome observed.

CONCLUSIONS: These findings indicate that an adequate antiviral response and an attenuated inflammatory response contribute to a favorable outcome of JEV infection in pigs and help to explain the limited neurological disease compared to other hosts. We show that the NLRP3 inflammasome, a key mediator of neurologic disease in mice, is not upregulated in pigs, further supporting its important role in JEV infections.

%B J Neuroinflammation %V 17 %8 2020 Oct 23 %G eng %N 1 %R 10.1186/s12974-020-01974-3 %0 Journal Article %J Transbound Emerg Dis %D 2020 %T An expert opinion assessment of blood-feeding arthropods based on their capacity to transmit African swine fever virus in Metropolitan France. %A Saegerman, Claude %A Sarah Bonnet %A Emilie Bouhsira %A Nick De Regge %A Johanna Fite %A Florence Etoré %A Mutien-Marie Garigliany %A Ferran Jori %A Laetitia Lempereur %A Le Potier, Marie-Frédérique %A Elsa Quillery %A Timothée Vergne %A Laurence Vial %X

To deal with the limited literature data on the vectorial capacity of blood-feeding arthropods (BFAs) and their role in the transmission of African swine fever virus (ASFV) in Metropolitan France, a dedicated working group of the French Agency for Food, Environmental and Occupational Health & Safety performed an expert knowledge elicitation. In total, 15 different BFAs were selected as potential vectors by the ad hoc working group involved. Ten criteria were considered to define the vectorial capacity: vectorial competence, current abundance, expected temporal abundance, spatial distribution, longevity, biting rate, active dispersal capacity, trophic preferences for Suidae, probability of contact with domestic pigs and probability of contact with wild boar. Fourteen experts participated to the elicitation. For each BFA, experts proposed a score (between 0 and 3) for each of the above criteria with an index of uncertainty (between 1 and 4). Overall, all experts gave a weight for all criteria (by distributing 100 marbles). A global weighted sum of score per BFA was calculated permitting to rank the different BFAs in decreasing order. Finally, a regression tree analysis was used to group those BFAs with comparable likelihood to play a role in ASF transmission. Out of the ten considered criteria, the experts indicated vectorial competence, abundance and biting rate as the most important criteria. In the context of Metropolitan France, the stable fly (Stomoxys calcitrans) was ranked as the most probable BFA to be a vector of ASFV, followed by lice (Haematopinus suis), mosquitoes (Aedes, Culex and Anopheles), Culicoides and Tabanidea. Since scientific knowledge on their vectorial competence for ASF is scarce and associated uncertainty on expert elicitation moderate to high, more studies are however requested to investigate the potential vector role of these BFAs could have in ASFV spread, starting with Stomoxys calcitrans.

%B Transbound Emerg Dis %8 2020 Aug 04 %G eng %R 10.1111/tbed.13769 %0 Journal Article %J Transbound Emerg Dis %D 2020 %T Mechanical transmission of African swine fever virus by Stomoxys calcitrans: Insights from a mechanistic model. %A Timothée Vergne %A Mathieu Andraud %A Sarah Bonnet %A Nick De Regge %A Marc Desquesnes %A Johanna Fite %A Florence Etoré %A Mutien-Marie Garigliany %A Ferran Jori %A Laetitia Lempereur %A Le Potier, Marie-Frédérique %A Elsa Quillery %A Saegerman, Claude %A Laurence Vial %A Emilie Bouhsira %X

African swine fever (ASF) represents a global threat with huge economic consequences for the swine industry. Even though direct contact is likely to be the main transmission route from infected to susceptible hosts, recent epidemiological investigations have raised questions regarding the role of haematophagous arthropods, in particular the stable fly (Stomoxys calcitrans). In this study, we developed a mechanistic vector-borne transmission model for ASF virus (ASFV) within an outdoor domestic pig farm in order to assess the relative contribution of stable flies to the spread of the virus. The model was fitted to the ecology of the vector, its blood-feeding behaviour and pig-to-pig transmission dynamic. Model outputs suggested that in a context of low abundance (<5 flies per pig), stable flies would play a minor role in the spread of ASFV, as they are expected to be responsible for around 10% of transmission events. However, with abundances of 20 and 50 stable flies per pig, the vector-borne transmission would likely be responsible for almost 30% and 50% of transmission events, respectively. In these situations, time to reach a pig mortality of 10% would be reduced by around 26% and 40%, respectively. The sensitivity analysis emphasized that the expected relative contribution of stable flies was strongly dependent on the volume of blood they regurgitated and the infectious dose for pigs. This study identified crucial knowledge gaps that need to be filled in order to assess more precisely the potential contribution of stable flies to the spread of ASFV, including a quantitative description of the populations of haematophagous arthropods that could be found in pig farms, a better understanding of blood-feeding behaviours of stable flies and the quantification of the probability that stable flies partially fed with infectious blood transmit the virus to a susceptible pig during a subsequent blood-feeding attempt.

%B Transbound Emerg Dis %8 2020 Sep 10 %G eng %R 10.1111/tbed.13824 %0 Journal Article %J Viruses %D 2020 %T Nationwide Screening for Bee Viruses and Parasites in Belgian Honey Bees. %A Severine Matthijs %A Valérie De Waele %A Valerie Vandenberge %A Bénédicte Verhoeven %A Jacqueline Evers %A Marleen Brunain %A Saegerman, Claude %A Paul J J De Winter %A S. Roels %A De Graaf, Dirk C %A Nick De Regge %X

The health of honey bees is threatened by multiple factors, including viruses and parasites. We screened 557 honey bee () colonies from 155 beekeepers distributed all over Belgium to determine the prevalence of seven widespread viruses and two parasites ( sp. and sp.). Deformed wing virus B (DWV-B), black queen cell virus (BQCV), and sacbrood virus (SBV) were highly prevalent and detected by real-time RT-PCR in more than 95% of the colonies. Acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV) and deformed wing virus A (DWV-A) were prevalent to a lower extent (between 18 and 29%). Most viruses were only present at low or moderate viral loads. Nevertheless, about 50% of the colonies harbored at least one virus at high viral load (>10 genome copies/bee). mites and sp. were found in 81.5% and 59.7% of the honey bee colonies, respectively, and all were identified as by real time PCR. Interestingly, we found a significant correlation between the number of mites and DWV-B viral load. To determine the combined effect of these and other factors on honey bee health in Belgium, a follow up of colonies over multiple years is necessary.

%B Viruses %V 12 %8 2020 08 14 %G eng %N 8 %R 10.3390/v12080890 %0 Journal Article %J Pathogens %D 2020 %T Phylogenetic Analysis of Belgian Small Ruminant Lentiviruses Supports Cross Species Virus Transmission and Identifies New Subtype B5 Strains. %A Michiels, Rodolphe %A Nadjah Radia Adjadj %A Nick De Regge %X

Small ruminant lentiviruses (SRLV) are a group of highly divergent viruses responsible for global and fatal infections in sheep and goats. Since the current phylogenetic classification of these viruses was proposed in 2004, it nowadays consists out of 5 genotypes and 28 subtypes. In support of our national SRLV control program, we performed the genetic characterization of SRLV strains circulating in the Belgian sheep and goat population. Fourteen sheep and 9 goat strains were sequenced in the and regions using the method described by Shah. Most SRLV strains from sheep and goats belonged to prototype A1 and B1 subtypes, respectively. We, however, also found indications for cross-species transmission of SRLV strains between sheep and goats and vice versa, and identified a new subtype designated as B5. An in-depth analysis of the current SRLV phylogeny revealed that many subtypes have been defined over the years based on limited sequence information. To keep phylogeny as a useful tool, we advocate to apply more rigorous sequencing standards to ensure the correct classification of current and new emerging strains. The genetic characterization of Belgian SRLV strains will help in the development of appropriate diagnostic tools to assist the national control program.

%B Pathogens %V 9 %8 2020 Mar 03 %G eng %N 3 %R 10.3390/pathogens9030183 %0 Journal Article %J Viruses %D 2020 %T Putative Role of Arthropod Vectors in African Swine Fever Virus Transmission in Relation to Their Bio-Ecological Properties. %A Sarah I Bonnet %A Emilie Bouhsira %A Nick De Regge %A Johanna Fite %A Florence Etoré %A Mutien-Marie Garigliany %A Ferran Jori %A Laetitia Lempereur %A Le Potier, Marie-Frédérique %A Elsa Quillery %A Saegerman, Claude %A Timothée Vergne %A Laurence Vial %X

African swine fever (ASF) is one of the most important diseases in Suidae due to its significant health and socioeconomic consequences and represents a major threat to the European pig industry, especially in the absence of any available treatment or vaccine. In fact, with its high mortality rate and the subsequent trade restrictions imposed on affected countries, ASF can dramatically disrupt the pig industry in afflicted countries. In September 2018, ASF was unexpectedly identified in wild boars from southern Belgium in the province of Luxembourg, not far from the Franco-Belgian border. The French authorities rapidly commissioned an expert opinion on the risk of ASF introduction and dissemination into metropolitan France. In Europe, the main transmission routes of the virus comprise direct contact between infected and susceptible animals and indirect transmission through contaminated material or feed. However, the seasonality of the disease in some pig farms in Baltic countries, including outbreaks in farms with high biosecurity levels, have led to questions on the possible involvement of arthropods in the transmission of the virus. This review explores the current body of knowledge on the most common arthropod families present in metropolitan France. We examine their potential role in spreading ASF-by active biological or mechanical transmission or by passive transport or ingestion-in relation to their bio-ecological properties. It also highlights the existence of significant gaps in our knowledge on vector ecology in domestic and wild boar environments and in vector competence for ASFV transmission. Filling these gaps is essential to further understanding ASF transmission in order to thus implement appropriate management measures.

%B Viruses %V 12 %8 2020 07 20 %G eng %N 7 %R 10.3390/v12070778 %0 Journal Article %J Sci Rep %D 2019 %T Experimental evidence of mechanical lumpy skin disease virus transmission by Stomoxys calcitrans biting flies and Haematopota spp. horseflies. %A Charlotte Sohier %A Andy Haegeman %A Laurent Mostin %A Ilse De Leeuw %A Willem Van Campe %A A De Vleeschauwer %A Tuppurainen, E S M %A Thierry van den Berg %A Nick De Regge %A Kris De Clercq %X

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.

%B Sci Rep %V 9 %8 2019 Dec 27 %G eng %N 1 %R 10.1038/s41598-019-56605-6 %0 Journal Article %J Viruses %D 2019 %T (Non-)Sense of Milk Testing in Small Ruminant Lentivirus Control Programs in Goats. Comparative Analysis of Antibody Detection and Molecular Diagnosis in Blood and Milk. %A Nadjah Radia Adjadj %A Vicca, Jo %A Michiels, Rodolphe %A Nick De Regge %K Animals %K Antibodies, Viral %K Antibody Specificity %K Enzyme-Linked Immunosorbent Assay %K Goat Diseases %K Goats %K Lentivirus %K Lentivirus Infections %K milk %K Real-Time Polymerase Chain Reaction %K Sensitivity and Specificity %K Serologic Tests %X

Small ruminant lentivirus (SRLV) control programs are mainly based on diagnostic tests performed on blood samples collected from sheep and goats. Since blood sampling is costly and stressful for the animals, we evaluated whether milk could be used as an inexpensive and easily collectable matrix for SRLV detection. We therefore compared SRLV detection via two commercial enzyme-linked immunosorbent assays (ELISAs) and quantitative polymerase chain reaction (qPCR) in blood and corresponding milk samples from 321 goats originating from eight different SRLV-infected farms in Flanders (Belgium). The IDscreen ELISA had a better relative sensitivity (97% vs 93%) and specificity (100% and 97%) than the Elitest ELISA for SRLV-specific antibody detection in milk compared to serum. The higher sensitivity correlates with a 10-fold higher analytical sensitivity of the IDscreen test. In contrast to the overall good ELISA results, qPCR on milk cell pellets lacked sensitivity (81%) and specificity (88%), compared to molecular detection in blood leucocyte pellets. Our results show that serology is more suitable than qPCR for SRLV diagnosis, and that milk may represent an interesting matrix for a preliminary evaluation of a herd's infection status. Serum remains however the sample of choice for control programs where it is important to identify positive animals with the highest sensitivity.

%B Viruses %V 12 %8 2019 12 18 %G eng %N 1 %R 10.3390/v12010003 %0 Journal Article %J Parasites & Vectors %D 2019 %T Prevalence of pathogens in ticks collected from humans through citizen science in Belgium %A Tinne Lernout %A Nick De Regge %A Katrien Tersago %A Manoj Fonville %A Vanessa Suin %A Sprong, Hein %B Parasites & Vectors %V 12 %8 Jan-12-2019 %G eng %N 1 %R 10.1186/s13071-019-3806-z %0 Journal Article %J Viruses %D 2018 %T Comparative Analysis of Different Serological and Molecular Tests for the Detection of Small Ruminant Lentiviruses (SRLVs) in Belgian Sheep and Goats. %A Michiels, Rodolphe %A Van Mael, Eva %A Quinet, Christian %A Nadjah Radia Adjadj %A Ann Brigitte Cay %A Nick De Regge %X

Countries rely on good diagnostic tests and appropriate testing schemes to fight against economically important small ruminant lentivirus (SRLV) infections. We undertook an extensive comparative analysis of seven commercially available serological tests and one in-house real-time PCR (qPCR) detecting genotype A and B strains using a large panel of representative Belgian field samples and samples from experimentally infected sheep and goats. ELISAs generally performed well and detected seroconversion within three weeks post experimental infection. Two enzyme-linked immunosorbent assays (ELISAs) (Elitest and IDscreen kits) showed the highest sensitivities (>96%) and specificities (>95%) in both species, and their combined use allowed to correctly identify the infection status of all animals. Individual agar gel immunodiffusion (AGIDs) kits lacked sensitivity, but interestingly, the combined use of both kits had a sensitivity and specificity of 100%. qPCRs detected SRLV infection before seroconversion at two weeks post infection and showed a specificity of 100%. Sensitivity however remained suboptimal at 85%. These results allow to propose a faster and cheaper diagnostic testing strategy for Belgium by combining a first ELISA screening, followed by confirmation of positive samples in AGID and/or a second ELISA. Since genotypes A and B strains are predominant in many countries, these results are interesting for other countries implementing SRLV control programs.

%B Viruses %V 10 %8 2018 12 08 %G eng %N 12 %R 10.3390/v10120696 %0 Journal Article %J Parasit Vectors %D 2018 %T Longitudinal monitoring of Culicoides in Belgium between 2007 and 2011: local variation in population dynamics parameters warrant cautious use of monitoring data. %A Charlotte Sohier %A Deblauwe, Isra %A De Deken, Reginald %A Madder, Maxime %A Fassotte, Christiane %A Losson, Bertrand %A Nick De Regge %K Animals %K Belgium %K Bluetongue %K Bluetongue virus %K Bunyaviridae Infections %K Ceratopogonidae %K Epidemiological Monitoring %K Female %K Insect Vectors %K Livestock %K Longitudinal Studies %K Male %K Orthobunyavirus %K Population Dynamics %K Risk Assessment %K Seasons %K Species Specificity %X

BACKGROUND: Several European countries suffered important economic losses during the past decade due to the emergence of bluetongue and Schmallenberg viruses. Both are viruses of veterinary importance and are spread by Culicoides spp. This triggered many European countries to start Culicoides population monitoring. Recently a one year monitoring study at 16 sites in Belgium revealed that important variation existed in Culicoides abundance and species diversity between collection sites. In order to analyze whether this variation is consistent over years, a detailed analysis of monitoring data collected at seven locations in Belgium between 2007 and 2011 was performed in this study. At all locations, biting midges were collected with OVI black light traps set-up in close proximity to livestock.

RESULTS: In total, 42 different Culicoides species were morphologically identified. Species of the subgenus Avaritia represented 83% of all collected midges. Nevertheless, important differences in species composition were found between sites. Furthermore, statistical differences between sites were found for the total and maximum annual abundance, showing that a consistent higher or lower number of Culicoides could be collected depending on the selected collection site. Yearly, up to 16 and 30-fold differences in total and maximum annual abundances between sites, respectively, were found. Also the month in which most Culicoides were collected varied greatly between years, both at local (from May to October) and country level [May (2008), June (2010), July (2009), August (2011), October (2007)]. Finally, the average vector-free period over all sites and years was 173 days and could roughly be defined between November and the end of April. Interestingly, important yearly variations of up to two months in the duration of the vector-free period were found between the studied collection sites. In contrast to the abundance parameters, no specific sites could however be identified where monitoring consistently showed shorter or longer vector-free periods.

CONCLUSIONS: In conclusion, our results show that the selection of collection sites for Culicoides monitoring, even in a small country such as Belgium, strongly influences abundance parameters and that yearly variation in seasonality occurs. This emphasizes that care should be taken when using such parameters in risk assessments for transmission of Culicoides-borne diseases and that more clear and strict guidelines for Culicoides monitoring should be considered when monitoring data are used for legislative purposes.

%B Parasit Vectors %V 11 %8 2018 Sep 17 %G eng %N 1 %R https://doi.org/10.1186/s13071-018-3082-3 %0 Journal Article %J Front Immunol %D 2018 %T Porcine NK Cells Stimulate Proliferation of Pseudorabies Virus-Experienced CD8 and CD4CD8 T Cells. %A Steffi De Pelsmaeker %A Devriendt, Bert %A Nick De Regge %A Favoreel, Herman W %X

Natural killer (NK) cells belong to the innate immune system and play a central role in the defense against viral infections and cancer development, but also contribute to shaping adaptive immune responses. NK cells are particularly important in the first line defense against herpesviruses, including alphaherpesviruses. In addition to their ability to kill target cells and produce interferon-γ, porcine and human NK cell subsets have been reported to display features associated with professional antigen presenting cells (APC), although it is currently unclear whether NK cells may internalize debris of virus-infected cells and whether this APC-like activity of NK cells may stimulate proliferation of antiviral T cells. Here, using the porcine alphaherpesvirus pseudorabies virus (PRV), we show that vaccination of pigs with a live attenuated PRV vaccine strain triggers expression of MHC class II on porcine NK cells, that porcine NK cells can internalize debris from PRV-infected target cells, and that NK cells can stimulate proliferation of CD8 and CD4CD8 PRV-experienced T cells. These results highlight the potential of targeting these NK cell features in future vaccination strategies.

%B Front Immunol %V 9 %8 2018 %G eng %R 10.3389/fimmu.2018.03188 %0 Journal Article %J Prev Vet Med %D 2018 %T Seroprevalence and risk factors related to small ruminant lentivirus infections in Belgian sheep and goats. %A Michiels, Rodolphe %A Van Mael, Eva %A Quinet, Christian %A Sarah Welby %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Arthritis-Encephalitis Virus, Caprine %K Belgium %K Goat Diseases %K Goats %K Lentivirus Infections %K prevalence %K Risk Factors %K Seroepidemiologic Studies %K Sheep %K Sheep Diseases %K Visna-maedi virus %X

Maedi-Visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are two prototype members of the group of small ruminant lentiviruses (SRLVs). Both result in progressive and persistent infections of sheep and goats that impact animal health and cause economic losses. In Belgium, the sheep and goat sector is small and consists mostly of hobbyist farmers keeping few animals. A voluntary control program however exists, but less than 2% of the farmers participate to the program. The current lack of SRLV seroprevalence data and knowledge on risk factors related to SRLV seropositivity in this hobbyist sector makes it difficult to evaluate the risk of SRLV transmission from non-certified to SRLV free certified farms. We performed a nationwide SRLV seroprevalence study based on a stratified sampling proportional to the number of sheep and goat holders per province. Randomly selected sheep and goat owners were invited to participate and subject to a short questionnaire to collect information about flock size, animal health condition, age, flock constitution and housing conditions. Samples were collected from maximum 7 animals per farm and tested in a commercial ELISA. In total, we received samples from 87 sheep and 76 goat farms. Sheep flocks showed an overall seroprevalence of 9% (CI : 5-15) and a between-herd seroprevalence of 17% (CI :11-27). Seroprevalence at animal level in goat flocks was 6% (CI : 3-12) and the between-herd seroprevalence was 13% (CI : 7-23). Multiple sheep and goat breeds were found SRLV seropositive. Answers provided during the questionnaire confirmed the mostly hobbyist nature of the sector and showed that more than 65% of sheep and goat farmers had never heard of the disease. The only risk factor found to be related to SRLV seroprevalence was flock size. Herds of more than 10 goats had significantly higher chance to harbor seropositive animals (OR: 4.36; CI: 1.07; 17.73). In conclusion, it was shown that participants to the SRLV free certification program are at risk for reintroduction of the disease in their herds since SRLVs are present on about 15%-20% of non-certified farms. Except from flock size, no clear risk factors were found that are helpfull to identify flocks at risk. Greater effort should be made to inform sheep and goat farmers about the existence and consequences of this disease in order to promote the voluntary control program and further reduce the disease prevalence.

%B Prev Vet Med %V 151 %8 2018 Mar 01 %G eng %R 10.1016/j.prevetmed.2017.12.014 %0 Journal Article %J J Virol %D 2017 %T Age-Dependent Differences in Pseudorabies Virus Neuropathogenesis and Associated Cytokine Expression. %A Verpoest, Sara %A Ann Brigitte Cay %A Favoreel, Herman %A Nick De Regge %K Age factors %K Animals %K Brain Stem %K Cytokines %K DNA, Viral %K Female %K Gene Expression %K Herpesvirus 1, Suid %K Olfactory Bulb %K Pseudorabies %K Swine %K Swine Diseases %K Trigeminal Ganglion %K virulence %X

The severity of clinical symptoms induced by pseudorabies virus (PRV) infection of its natural host is inversely related to the age of the pig. During this study, 2- and 15-week-old pigs were inoculated with PRV strain NIA3. This resulted in important clinical disease, although the associated morbidity and mortality were lower in older pigs. Quantitative PCR analysis of viral DNA in different organs confirmed the general knowledge on PRV pathogenesis. Several new findings and potential explanations for the observed age-dependent differences in virulence, however, were determined from the study of viral and cytokine mRNA expression at important sites of neuropathogenesis. First, only limited viral and cytokine mRNA expression was detected in the nasal mucosa, suggesting that other sites may serve as the primary replication site. Second, PRV reached the trigeminal ganglion (TG) and brain stem rapidly upon infection but, compared to 2-week-old pigs, viral replication was less pronounced in 15-week-old pigs, and the decrease in viral mRNA expression was not preceded by or associated with an increased cytokine expression. Third, extensive viral replication associated with a robust expression of cytokine mRNA was detected in the olfactory bulbs of pigs from both age categories and correlated with the observed neurological disease. Our results suggest that age-dependent differences in PRV-induced clinical signs are probably due to enhanced viral replication and associated immunopathology in immature TG and the central nervous system neurons of 2-week-old pigs and that neurological disease is related with extensive viral replication and an associated immune response in the olfactory bulb.

IMPORTANCE: It is well known that alphaherpesvirus infections of humans and animals result in more severe clinical disease in newborns than in older individuals and that this is probably related to differences in neuropathogenesis. The underlying mechanisms, however, remain unclear. Pseudorabies virus infection of its natural host, the pig, provides a suitable infection model to study this more profoundly. We show here that the severe neurological disease observed in 2-week-old pigs does not appear to be related to a hampered innate immune response but is more likely to reflect the immature development state of the trigeminal ganglia (TG) and central nervous system (CNS) neurons, resulting in an inefficient suppression of viral replication. In 15-week-old pigs, viral replication was efficiently suppressed in the TG and CNS without induction of an extensive immune response. Furthermore, our results provide evidence that neurological disease could, at least in part, be related to viral replication and associated immunopathology in the olfactory bulb.

%B J Virol %V 91 %8 2017 Jan 15 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/27852848?dopt=Abstract %R 10.1128/JVI.02058-16 %0 Journal Article %J Curr Opin Virol %D 2017 %T Akabane, Aino and Schmallenberg virus-where do we stand and what do we know about the role of domestic ruminant hosts and Culicoides vectors in virus transmission and overwintering? %A Nick De Regge %K Aino virus %K Akabane virus %K Culicoides %K overwintering %K Schmallenberg virus %X

Akabane, Aino and Schmallenberg virus belong to the Simbu serogroup of Orthobunyaviruses and depend on Culicoides vectors for their spread between ruminant hosts. Infections of adults are mostly asymptomatic or associated with only mild symptoms, while transplacental crossing of these viruses to the developing fetus can have important teratogenic effects. Research mainly focused on congenital malformations has established a correlation between the developmental stage at which a fetus is infected and the outcome of an Akabane virus infection. Available data suggest that a similar correlation also applies to Schmallenberg virus infections but is not yet entirely conclusive. Experimental and field data furthermore suggest that Akabane virus is more efficient in inducing congenital malformations than Aino and Schmallenberg virus, certainly in cattle. The mechanism by which these Simbu viruses cross-pass yearly periods of very low vector abundance in temperate climate zones remains undefined. Yearly wind-borne reintroductions of infected midges from tropical endemic regions with year-round vector activity have been proposed, just as overwintering in long-lived adult midges. Experimental and field data however indicate that a role of vertical virus transmission in the ruminant host currently cannot be excluded as an overwintering mechanism. More studies on Culicoides biology and specific groups of transplacentally infected newborn ruminants without gross malformations are needed to shed light on this matter.

%B Curr Opin Virol %V 27 %8 2017 Oct 30 %G eng %R 10.1016/j.coviro.2017.10.004 %0 Report %D 2017 %T European OneHealth/EcoHealth workshop report - Brussels, 6-7 October 2016. %A Keune, Hans %A Flandroy, Lucette %A Thys, Séverine %A Nick De Regge %A Marcella Mori %A Thierry van den Berg %A Antoine-Moussiaux, Nicolas %A Vanhove, Maarten P M %A Javiera Rebolledo %A Steven Van Gucht %A Deblauwe, Isra %A Biot, Pierre %A Hiemstra, Wim %A Häsler, Barbara %A Binot, Aurélie %K 2016 %K European One Health / Eco Health Workshop %I Biodiversity & Health Community of Practice and the Belgian Biodiversity Platform. %C Brussels, Belgium %P 48 %8 201 %G eng %M NA %0 Journal Article %J Transboundary and Emerging Diseases %D 2017 %T Evidence of extensive renewed Schmallenberg virus circulation in Belgium during summer of 2016 - increase in arthrogryposis-hydranencephaly cases expected %A Charlotte Sohier %A I. Deblauwe %A T. Van Loo %A Jean-Baptiste Hanon %A Ann Brigitte Cay %A Nick De Regge %X

A seroprevalence study carried out between June and September 2016 in the Belgian sheep population showed a significant increase in overall (from 25% to 62%) and between-herd (from 60% to 96%) seroprevalence against Schmallenberg virus (SBV) during this period, indicating the most extensive recirculation of SBV since its original emergence in 2011. SBV recirculation was confirmed by the detection of SBV RNA-positive Culicoides obsoletus complex midges collected in the region of Antwerp in August 2016, reaching a minimum infection rate of 3%. The recirculation of SBV in the largely unprotected ruminant population during summer 2016 will likely cause an increase in the number of arthrogryposis-hydranencephaly cases in newborn ruminants during the coming months.

%B Transboundary and Emerging Diseases %V 64 %8 Jan-08-2017 %G eng %N 4 %R 10.1111/tbed.12655 %0 Journal Article %J J Gen Virol %D 2017 %T Genetically stable infectious Schmallenberg virus persists in foetal envelopes of pregnant ewes. %A Poskin, Antoine %A Martinelle, Ludovic %A Yves Van der Stede %A Saegerman, Claude %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Antibodies, Viral %K Base Sequence %K Bunyaviridae Infections %K Chorion %K Female %K Orthobunyavirus %K Placenta %K Pregnancy %K RNA, Viral %K Sequence Analysis, RNA %K Sheep %K Sheep Diseases %X

Schmallenberg virus (SBV) is a recently emerged vector-borne virus, inducing congenital defects in bovines, ovines and caprines. Here we have shown that infectious SBV is capable of persisting until the moment of birth in the foetal envelopes of ewes infected with SBV-infectious serum at day 45 (1/5 positive) and 60 (4/6 positive) of gestation. This persistence of at least 100 days is a new aspect of the SBV pathogenesis that could help to explain how SBV overwinters the cold season in temperate climate zones. Furthermore, sequencing of the M segment shows that the persisting virus in the foetal envelopes is genetically stable since only a few mutations compared to the inoculum were found. This supports the hypothesis that persisting virus could start the infection of new hosts. Finally, neutralization tests showed that infectious SBV present in the foetal envelopes at birth can be neutralized by the humoral immunity present in the infected ewes.

%B J Gen Virol %V 98 %P 1630-1635 %8 2017 Jul %G eng %N 7 %1 https://www.ncbi.nlm.nih.gov/pubmed/28699878?dopt=Abstract %R 10.1099/jgv.0.000841 %0 Journal Article %J Arch Public Health %D 2017 %T The need for European OneHealth/EcoHealth networks. %A Keune, Hans %A Flandroy, Lucette %A Thys, Séverine %A Nick De Regge %A Marcella Mori %A Antoine-Moussiaux, Nicolas %A Vanhove, Maarten P M %A Javiera Rebolledo %A Steven Van Gucht %A Deblauwe, Isra %A Hiemstra, Wim %A Häsler, Barbara %A Binot, Aurélie %A Savic, Sara %A Ruegg, Simon R %A De Vries, Sjerp %A Garnier, Julie %A Thierry van den Berg %K Community of Practice %K Crosssectorial %K EcoHealth %K Europe %K Interdisciplinarity %K One Health %K Transdisciplinarity %X

Elaborating from the European One Health/Ecohealth (OH/EH) workshop that took place in fall 2016 and aimed to bring together different communities and explore collaborative potential, the creation of European networks focusing on the development of important OH/EH perspectives was a direct output from discussions at the end of some sessions, in particular: - A network on transdisciplinary One Health education. - A network integrating inputs from social sciences in One Health/EcoHealth actions and networks. - A network aiming at translating research findings on the Environment-Microbiome-Health axis into policy making, with a view to make healthy ecosystems a cost-effective disease prevention healthcare strategy. It was also suggested that a European Community of Practice could be initiated in order to support these several concrete networking initiatives, and to help to promote the building of other emerging initiatives.

%B Arch Public Health %V 75 %8 2017 %G eng %& 64 %R 10.1186/s13690-017-0232-6 %0 Journal Article %J Virulence %D 2017 %T Reduced virulence of a pseudorabies virus isolate from wild boar origin in domestic pigs correlates with hampered visceral spread and age-dependent reduced neuroinvasive capacity. %A Verpoest, Sara %A Valerie Redant %A Ann Brigitte Cay %A Favoreel, Herman %A Nick De Regge %X

Morbidity and mortality associated with pseudorabies virus (PRV) infection are dependent on the age of the pig and the virulence of the strain. PRV strains circulating in wild boar are considered to be low virulent, but no mechanistic explanation for their reduced virulence is available. Here infection of 2- and 15-week-old domestic pigs with the PRV wild boar strain BEL24043 did not induce clinical symptoms in 15-week-old pigs, but resulted in important neurological and respiratory disease in 2-week-old piglets. A detailed study of the (neuro) pathogenesis and associated cytokine mRNA expression showed that the reduced virulence of the wild boar strain, compared to what was previously reported for the virulent domestic NIA3 strain, is due to a severely hampered spread to visceral organs in pigs of both age categories and to an efficient suppression of viral replication at primary replication sites of 15-week-old pigs and to a lesser extent in those of 2-week-old piglets. The age-dependent difference in induced symptoms seems to be due to an immature development state of the immune and/or nervous system in 2-week-old pigs. An extensive viral replication associated with a robust expression of cytokine-related mRNA was found in the olfactory bulb of 2-week-old piglets, correlating with observed neurological disease. Neuroinvasion also occurred via the trigeminal route in 2-week-old pigs, but viral replication was efficiently suppressed in the trigeminal ganglion in the presence of a moderate induction of cytokine-related mRNA. Viral replication in the peripheral and central nervous system of 15-week-old pigs was limited and efficiently suppressed.

%B Virulence %P 1-14 %8 2017 Sep 05 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28873002?dopt=Abstract %R 10.1080/21505594.2017.1368941 %0 Journal Article %J Transbound Emerg Dis %D 2017 %T Resurgence of Schmallenberg Virus in Belgium after 3 Years of Epidemiological Silence. %A Delooz, L %A Saegerman, C %A Quinet, C %A Petitjean, T %A Nick De Regge %A Ann Brigitte Cay %X

In spring 2016, three years after the last reported outbreak of Schmallenberg virus (SBV) in Belgium, an abortion was notified in a two year old Holstein heifer that previously had not been vaccinated against SBV. The autopsy of the eight-month-old malformed foetus revealed hydrocephalus, torticollis and arthrogryposis. Foetal brain tissue and blood were found to be SBV-positive by RT-PCR and ELISA tests, respectively. Evidencing the circulation of SBV in Belgium in the autumn 2015 is important to anticipate future outbreaks and advise veterinarians about the risks associated with calving, as more bovine foetuses might have been infected.

%B Transbound Emerg Dis %V 64 %P 1641-1642 %8 2017 Oct %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/27485019?dopt=Abstract %R 10.1111/tbed.12552 %0 Journal Article %J Transbound Emerg Dis %D 2017 %T Three Different Routes of Inoculation for Experimental Infection with Schmallenberg Virus in Sheep. %A Martinelle, L %A Poskin, A %A dal Pozzo, F %A Laurent Mostin %A Willem Van Campe %A Ann Brigitte Cay %A Nick De Regge %A Saegerman, C %K Administration, Intranasal %K Animals %K Bunyaviridae Infections %K Female %K Injections, Intradermal %K Injections, Subcutaneous %K Lymph Nodes %K Orthobunyavirus %K Sheep %K Sheep Diseases %K Spleen %K Vaccination %X

Schmallenberg virus (SBV) is an emerging Orthobunyavirus affecting European domestic ruminants. In this study, three groups of ewes (n = 3) were inoculated with 1 ml of an SBV infectious serum, via the subcutaneous (SC), intradermal (ID) or intranasal (IN) route. The ewes were monitored for 10 days and no clinical signs were reported. IN inoculation failed to generate any detectable RNAemia. SC and ID inoculation induced typical SBV RNAemia and seroconversion upon day 6 post-inoculation in 3/3 and 2/3 sheep, respectively. In all the animals that showed RNAemia, the viral genome could be detected in spleen and mesenteric lymph nodes. Both the SC and ID routes seem suitable to properly reproduce field conditions, as comparable observations were reported regarding RNAemia, seroconversion and viral genome detection in organs.

%B Transbound Emerg Dis %V 64 %P 305-308 %8 2017 Feb %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/25891033?dopt=Abstract %R 10.1111/tbed.12356 %0 Journal Article %J Research in Veterinary Science %D 2017 %T Unchanged Schmallenberg virus seroprevalence in the Belgian sheep population after the vector season of 2014 and 2015 despite evidence of virus circulation %A Charlotte Sohier %A Michiels, Rodolphe %A Kapps, Elena %A Van Mael, Eva %A Quinet, Christian %A Ann Brigitte Cay %A Nick De Regge %B Research in Veterinary Science %V 114 %8 Jan-10-2017 %G eng %R 10.1016/j.rvsc.2017.04.011 %0 Journal Article %J J Gen Virol %D 2016 %T Age- and strain-dependent differences in the outcome of experimental infections of domestic pigs with wild boar pseudorabies virus isolates. %A Verpoest, Sara %A Ann Brigitte Cay %A Willem Van Campe %A Laurent Mostin %A Sarah Welby %A Favoreel, Herman %A Nick De Regge %K Animals %K Disease Models, Animal %K Disease Transmission, Infectious %K Europe %K Herpesvirus 1, Suid %K Pseudorabies %K Sus scrofa %K Swine %K Swine Diseases %K Treatment Outcome %X

Although pseudorabies virus (PRV) has been eradicated in domestic swine in many countries, its presence in wild boars remains a threat for a reintroduction into the currently unprotected swine population. To assess the possible impact of such a reintroduction in a naive herd, an in vivo infection study using two genetically characterized wild boar PRV isolates (BEL24043 and BEL20075) representative for wild boar strains circulating in south-western and central Europe and the virulent NIA3 reference strain was performed in 2- and 15-week-old domestic pigs. Our study revealed an attenuated nature of both wild boar strains in 15-week-old pigs. In contrast, it showed the capacity of strain BEL24043 to induce severe clinical symptoms and mortality in young piglets, thereby confirming that the known age dependency of disease outcome after PRV infection also holds for wild boar isolates. Despite the absence of clinical disease in 15-week-old sows, both wild boar PRV strains were able to induce seroconversion, but to a different extent. Importantly, differences in infection and transmission capacity of both strains were observed in 15-week-old sows. Strain BEL24043 induced a more prolonged and disseminated infection than strain BEL20075 and was able to spread efficiently to contact animals, indicative of its capacity to induce a sustained infection. In conclusion, it was shown that a reintroduction of a wild boar isolate into the domestic swine population could have serious economic consequences due to the induction of clinical symptoms in piglets and by jeopardizing the PRV-negative status.

%B J Gen Virol %V 97 %P 487-495 %8 2016 Feb %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/26589961?dopt=Abstract %R 10.1099/jgv.0.000347 %0 Journal Article %J PLoS One %D 2016 %T Comparison of PRRSV Nucleic Acid and Antibody Detection in Pen-Based Oral Fluid and Individual Serum Samples in Three Different Age Categories of Post-Weaning Pigs from Endemically Infected Farms. %A Nick De Regge %A Ann Brigitte Cay %K Animals %K Antibodies, Viral %K Belgium %K farms %K nucleic acids %K Porcine Reproductive and Respiratory Syndrome %K Porcine respiratory and reproductive syndrome virus %K Saliva %K Sensitivity and Specificity %K Swine %K Weaning %X

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of an economically important disease in swine. Since it has been shown that PRRSV and PRRSV specific antibodies can be detected in oral fluid, many different aspects have been studied to show that oral fluid could be a worthy alternative diagnostic sample to serum for monitoring and surveillance of this disease. Thorough field evaluations are however missing to convincingly show its usefulness under representative field conditions.

METHODOLOGY: Pen-based oral fluid samples and serum samples from all individual pigs in the corresponding pens were collected from post-weaning pigs of three different age categories in eight endemically PRRSV infected farms and one PRRSV free farm in Belgium. All samples were tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and ELISA to detect PRRSV RNA and PRRSV specific antibodies, respectively.

RESULTS: While the relative specificity of PRRSV detection by qRT-PCR in pen-based oral fluid compared to serum collected from individual pigs was high in all age categories (>90%), the relative sensitivity decreased with the age of the pigs (89, 93 and 10% in 8-12w, 16-20w and 24-28w old pigs, respectively). The latter correlated with a lower percentage of PRRSV positive pigs in serum/pen in the different age categories (55, 29 and 6%, respectively). Irrespective of the age category, pen-based oral fluid samples were always found PCR positive when at least 30% of the individual pigs were positive in serum. PRRSV specific antibody detection in oral fluid by ELISA showed a 100% relative sensitivity to detection in serum since oral fluid samples were always positive as soon as one pig in the pen was positive in serum. On the other hand, two false positive oral fluid samples in 11 pens without serum positive pigs were found, resulting in a relative specificity of 82%. Indications are however present that the oral fluid result indicated the correct infection status but the absence of a golden standard test makes it difficult to define definitive test characteristics.

CONCLUSIONS: Overall it can be concluded that oral fluid seems to be a useful matrix for diagnosis of PRRSV under field conditions and that differences in kinetics of PRRSV and PRRSV specific antibody detection in oral fluid and serum of individual pigs can also be reflected in pen-based oral fluid results.

%B PLoS One %V 11 %P e0166300 %8 2016 %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/27820859?dopt=Abstract %R 10.1371/journal.pone.0166300 %0 Journal Article %J Genome Announc %D 2016 %T Complete Genome Sequence of Pseudorabies Virus Reference Strain NIA3 Using Single-Molecule Real-Time Sequencing. %A Elisabeth Mathijs %A Frank Vandenbussche %A Verpoest, Sara %A Nick De Regge %A Steven Van Borm %X

Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease in pigs. PRV strains are also used as model organisms for the study of alphaherpesvirus biology or for neuronal pathway studies. We present here the complete genome of the virulent wild-type PRV reference strain NIA3, determined by single-molecule real-time sequencing.

%B Genome Announc %V 4 %8 2016 May 26 %G eng %N 3 %1 https://www.ncbi.nlm.nih.gov/pubmed/27231370?dopt=Abstract %R 10.1128/genomeA.00440-16 %0 Journal Article %J J Gen Virol %D 2016 %T Pseudorabies virus isolates from domestic pigs and wild boars show no apparent in vitro differences in replication kinetics and sensitivity to interferon-induced antiviral status. %A Verpoest, Sara %A Ann Brigitte Cay %A Favoreel, Herman %A Nick De Regge %K Animals %K Antiviral Agents %K Cells, Cultured %K Herpesvirus 1, Suid %K Interferons %K Sus scrofa %K Viral Plaque Assay %K Virus Replication %X

Pseudorabies virus is the causative agent of Aujeszky's disease. Domestic pigs and wild boars are its natural hosts, and strains circulating within both populations differ in their capacity to induce clinical disease. Cell biological and molecular explanations for the observed differences in virulence are, however, lacking. Different virulence determinants that can be assessed in vitro were determined for five domestic swine strains, four wild boar strains and the NIA3 reference strain. Replication kinetics and plaque formation capacity in continuous swine testicular cells and different primary porcine cell lines were highly similar for isolates from both populations. Treatment of these cell lines with IFNα, IFNγ or a combination of both provoked similar plaque-reducing effects for all strains. In conclusion, our results indicate that isolates from domestic swine and wild boar differ neither in intrinsic replication and dissemination capacity nor in sensitivity to antiviral effects of IFNs.

%B J Gen Virol %V 97 %P 473-9 %8 2016 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/26590089?dopt=Abstract %R 10.1099/jgv.0.000348 %0 Journal Article %J Vet Microbiol %D 2016 %T Reconstruction of the Schmallenberg virus epidemic in Belgium: Complementary use of disease surveillance approaches. %A Poskin, Antoine %A Théron, Léonard %A Jean-Baptiste Hanon %A Saegerman, Claude %A Vervaeke, Muriel %A Yves Van der Stede %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Animals, Wild %K Belgium %K Bunyaviridae Infections %K Cattle %K Cattle Diseases %K Ceratopogonidae %K Computer Simulation %K Orthobunyavirus %K Population Surveillance %K Reverse Transcriptase Polymerase Chain Reaction %K Seroepidemiologic Studies %X

Schmallenberg virus (SBV) emerged across Europe in 2011 and Belgium was among the first countries affected. In this study, published findings are combined with new data from veterinary surveillance networks and the Belgian reference laboratory for SBV at the Veterinary and Agrochemical Research centre (CODA-CERVA) to reconstruct the epidemic in Belgium. First retrospective cases of SBV were reported by veterinarians that observed decreased milk yield and fever in dairy cattle in May 2011. The number of SBV suspicions subsequently increased in adult cattle in August 2011. That month, first SBV positive pools of Culicoides were detected and extensive virus circulation occurred in Belgium during late summer and autumn 2011. As a consequence, most pregnant ruminants were infected and their fetuses exposed to the virus. This resulted in an outbreak of abortions, still-births and malformed new-borns observed between January and April 2012. The number of cases drastically diminished in 2012-2013, although multiple lines of evidence obtained from cross-sectional serological surveys, analyses on aborted foetuses, sentinel herd surveillance and surveillance of SBV in vectors prove that SBV was still circulating in Belgium at that time. Virus circulation was then probably strongly reduced in 2013-2014, while increasing evidence indicates its recirculation in 2014-2015 in Belgium. Based on the experience gathered with the closely related Akabane virus, recurrent outbreaks of congenital events can be expected for a long period. Vaccination of seronegative animals before the first mating could be used to prevent the deleterious effects of SBV. During this epidemic, different surveillance approaches including syndromic surveillance, sentinel herd surveillance, cross-sectional seroprevalence studies and pathogen surveillance in vectors have proven their utility and should be considered to continue in the future.

%B Vet Microbiol %V 183 %P 50-61 %8 2016 Feb 01 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26790935?dopt=Abstract %R 10.1016/j.vetmic.2015.11.036 %0 Journal Article %J Vet Rec %D 2016 %T Schmallenberg virus: on its way out or due for a comeback? %A Nick De Regge %K Animals %K Bunyaviridae Infections %K Ceratopogonidae %K Insect Vectors %K Orthobunyavirus %K United Kingdom %B Vet Rec %V 179 %8 2016 Oct 29 %G eng %N 17 %R 10.1136/vr.i5548 %0 Journal Article %J Med Vet Entomol %D 2015 %T Culicoides monitoring in Belgium in 2011: analysis of spatiotemporal abundance, species diversity and Schmallenberg virus detection. %A Nick De Regge %A De Deken, R %A Fassotte, C %A Losson, B %A Deblauwe, I %A Madder, M %A Vantieghem, P %A Tomme, M %A Smeets, F %A Ann Brigitte Cay %K Animals %K Belgium %K Bunyaviridae Infections %K Ceratopogonidae %K Insect Vectors %K Orthobunyavirus %K polymerase chain reaction %K Population Density %K Species Specificity %X

In 2011, Culicoides (Diptera: Ceratopogonidae) were collected at 16 locations covering four regions of Belgium with Onderstepoort Veterinary Institute (OVI) traps and at two locations with Rothamsted suction traps (RSTs). Quantification of the collections and morphological identification showed important variations in abundance and species diversity between individual collection sites, even for sites located in the same region. However, consistently higher numbers of Culicoides midges were collected at some sites compared with others. When species abundance and diversity were analysed at regional level, between-site variation disappeared. Overall, species belonging to the subgenus Avaritia together with Culicoides pulicaris (subgenus Culicoides) were the most abundant, accounting for 80% and 96% of all midges collected with RSTs and OVI traps, respectively. Culicoides were present during most of the year, with Culicoides obsoletus complex midges found from 9 February until 27 December. Real-time reverse-transcription polymerase chain reaction screening for Schmallenberg virus in the heads of collected midges resulted in the first detection of the virus in August 2011 and identified C. obsoletus complex, Culicoides chiopterus and Culicoides dewulfi midges as putative vector species. At Libramont in the south of Belgium, no positive pools were identified.

%B Med Vet Entomol %V 29 %P 263-75 %8 2015 Sep %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/25761054?dopt=Abstract %R 10.1111/mve.12109 %0 Journal Article %J PLoS One %D 2015 %T Detection and Isolation of Swine Influenza A Virus in Spiked Oral Fluid and Samples from Individually Housed, Experimentally Infected Pigs: Potential Role of Porcine Oral Fluid in Active Influenza A Virus Surveillance in Swine. %A Decorte, Inge %A Mieke Steensels %A Bénédicte Lambrecht %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Chick Embryo %K Influenza A Virus, H1N1 Subtype %K Influenza A Virus, H3N2 Subtype %K Real-Time Polymerase Chain Reaction %K Reverse Transcriptase Polymerase Chain Reaction %K Saliva %K Swine %X

BACKGROUND: The lack of seasonality of swine influenza A virus (swIAV) in combination with the capacity of swine to harbor a large number of co-circulating IAV lineages, resulting in the risk for the emergence of influenza viruses with pandemic potential, stress the importance of swIAV surveillance. To date, active surveillance of swIAV worldwide is barely done because of the short detection period in nasal swab samples. Therefore, more sensitive diagnostic methods to monitor circulating virus strains are requisite.

METHODS: qRT-PCR and virus isolations were performed on oral fluid and nasal swabs collected from individually housed pigs that were infected sequentially with H1N1 and H3N2 swIAV strains. The same methods were also applied to oral fluid samples spiked with H1N1 to study the influence of conservation time and temperature on swIAV infectivity and detectability in porcine oral fluid.

RESULTS: All swIAV infected animals were found qRT-PCR positive in both nasal swabs and oral fluid. However, swIAV could be detected for a longer period in oral fluid than in nasal swabs. Despite the high detectability of swIAV in oral fluid, virus isolation from oral fluid collected from infected pigs was rare. These results are supported by laboratory studies showing that the PCR detectability of swIAV remains unaltered during a 24 h incubation period in oral fluid, while swIAV infectivity drops dramatically immediately upon contact with oral fluid (3 log titer reduction) and gets lost after 24 h conservation in oral fluid at ambient temperature.

CONCLUSIONS: Our data indicate that porcine oral fluid has the potential to replace nasal swabs for molecular diagnostic purposes. The difficulty to isolate swIAV from oral fluid could pose a drawback for its use in active surveillance programs.

%B PLoS One %V 10 %P e0139586 %8 2015 %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/26431039?dopt=Abstract %R 10.1371/journal.pone.0139586 %0 Journal Article %J J Vet Diagn Invest %D 2015 %T Diagnosis of the Lelystad strain of Porcine reproductive and respiratory syndrome virus infection in individually housed pigs: comparison between serum and oral fluid samples for viral nucleic acid and antibody detection. %A Decorte, Inge %A Willem Van Campe %A Laurent Mostin %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Antibodies, Viral %K Enzyme-Linked Immunosorbent Assay %K Housing, Animal %K Mouth %K Porcine Reproductive and Respiratory Syndrome %K Porcine respiratory and reproductive syndrome virus %K Reverse Transcriptase Polymerase Chain Reaction %K Swine %X

There has been a developing interest in the use of oral fluid for the diagnosis of different pathogens such as Porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV and PRRSV-specific antibodies have been shown to be present in oral fluid samples, but the correlation between diagnostic results in oral fluid and serum samples has been insufficiently addressed. Studies investigating this correlation focused on boars older than 6 months and type 2 strains, but it is known that the outcome of a PRRSV infection is age and strain dependent. To address this gap, the current study reports on the detection of PRRSV and PRRSV-specific antibodies in serum and oral fluid samples collected over a 6-week period after an experimental infection of 8-week-old individually housed pigs with Lelystad virus, the type 1 prototype strain. Quantitative reverse transcription polymerase chain reaction analysis showed that significantly more serum samples were PRRSV RNA-positive than oral fluid until 5 days postinfection (dpi). Between 7 and 21 dpi, PRRSV RNA detection was similar in both samples but higher detection rates in oral fluid were found from 28 dpi. Compared with existing literature, this highlights that detection rates at particular time points postinfection might vary in function of strain virulence and animal age and provides useful information for the interpretation of pen-based oral fluid results. An excellent agreement between the oral fluid and serum enzyme-linked immunosorbent assay results was observed at every time point, further supporting the usefulness of oral fluid as a diagnostic sample for antibody detection.

%B J Vet Diagn Invest %V 27 %P 47-54 %8 2015 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/25525137?dopt=Abstract %R 10.1177/1040638714561252 %0 Journal Article %J PLoS One %D 2015 %T Experimental Infection of Sheep at 45 and 60 Days of Gestation with Schmallenberg Virus Readily Led to Placental Colonization without Causing Congenital Malformations. %A Martinelle, Ludovic %A Poskin, Antoine %A Dal Pozzo, Fabiana %A Nick De Regge %A Ann Brigitte Cay %A Saegerman, Claude %K Animals %K Animals, Newborn %K Antibodies, Viral %K Bunyaviridae Infections %K Cercopithecus aethiops %K Congenital Abnormalities %K Female %K Gestational Age %K Infectious Disease Transmission, Vertical %K Male %K Orthobunyavirus %K Placenta %K Pregnancy %K Reverse Transcriptase Polymerase Chain Reaction %K RNA, Viral %K Sheep %K Sheep Diseases %K Time Factors %K Vero Cells %X

BACKGROUND: Main impact of Schmallenberg virus (SBV) on livestock consists in reproductive disorders, with teratogenic effects, abortions and stillbirths. SBV pathogenesis and viral placental crossing remain currently poorly understood. Therefore, we implemented an experimental infection of ewes, inoculated with SBV at 45 or 60 days of gestation (dg).

METHODOLOGY: "Mourerous" breed ewes were randomly separated in three groups: eight and nine ewes were subcutaneously inoculated with 1 ml of SBV infectious serum at 45 and 60 dg, respectively (G45 and G60). Six other ewes were inoculated subcutaneously with sterile phosphate buffer saline as control group. All SBV inoculated ewes showed RNAemia consistent with previously published studies, they seroconverted and no clinical sign was reported. Lambs were born at term via caesarian-section, and right after birth they were blood sampled and clinically examined. Then both lambs and ewes were euthanatized and necropsied.

PRINCIPAL FINDINGS/SIGNIFICANCE: No lambs showed any malformation suggestive of SBV infection and none of them had RNAemia or anti-SBV antibodies prior to colostrum uptake. Positive SBV RNA detection in organs was rare in both G45 and G60 lambs (2/11 and 1/10, respectively). Nevertheless most of the lambs in G45 (9/11) and G60 (9/10) had at least one extraembryonic structure SBV positive by RTqPCR. The number of positive extraembryonic structures was significantly higher in G60 lambs. Time of inoculation (45 or 60 dg) had no impact on the placental colonization success rate but affected the frequency of detecting the virus in the offspring extraembryonic structures by the time of lambing. SBV readily colonized the placenta when ewes were infected at 45 or 60 dg but infection of the fetuses was limited and did not lead to congenital malformations.

%B PLoS One %V 10 %P e0139375 %8 2015 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/26418420?dopt=Abstract %R 10.1371/journal.pone.0139375 %0 Journal Article %J Transbound Emerg Dis %D 2015 %T Follow-up of the Schmallenberg Virus Seroprevalence in Belgian Cattle. %A Estelle Méroc %A Poskin, A %A Van Loo, H %A Van Driessche, E %A Czaplicki, G %A Quinet, C %A Riocreux, F %A Nick De Regge %A Ann Brigitte Cay %A Thierry van den Berg %A Hooyberghs, J %A Yves Van der Stede %K Animals %K Belgium %K Bunyaviridae Infections %K Cattle %K Cattle Diseases %K cross-sectional studies %K Follow-Up Studies %K Orthobunyavirus %K Risk Factors %K Seasons %K Seroepidemiologic Studies %X

Schmallenberg virus (SBV), which emerged in Northwestern Europe in 2011, is an arthropod-borne virus affecting primarily ruminants. Based on the results of two cross-sectional studies conducted in the Belgian ruminant population during winter 2011-2012, we concluded that at the end of 2011, almost the whole population had already been infected by SBV. A second cross-sectional serological study was conducted in the Belgian cattle population during winter 2012-2013 to examine the situation after the 2012 transmission period and to analyse the change in immunity after 1 year. A total of 7130 blood samples collected between 1st January and 28 February 2013 in 188 herds were tested for the presence of SBV-specific antibodies. All sampled herds tested positive and within-herd seroprevalence was estimated at 65.66% (95% CI: 62.28-69.04). A statistically significant decrease was observed between the beginning and the end of 2012. On the other hand, age-cohort-specific seroprevalence stayed stable from 1 year to the other. During winter 2012-2013, calves between 6 and 12 months had a seroprevalence of 20.59% (95% CI: 15.34-25.83), which seems to be an indication that SBV was still circulating at least in some parts of Belgium during summer-early autumn 2012. Results showed that the level of immunity against SBV of the animals infected has not decreased and remained high after 1 year and that the spread of the virus has slowed down considerably during 2012. This study also indicated that in the coming years, there are likely to be age cohorts of unprotected animals.

%B Transbound Emerg Dis %V 62 %P e80-4 %8 2015 Oct %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/24330658?dopt=Abstract %R 10.1111/tbed.12202 %0 Journal Article %J Vet Res %D 2015 %T Persistence of the protective immunity and kinetics of the isotype specific antibody response against the viral nucleocapsid protein after experimental Schmallenberg virus infection of sheep. %A Poskin, Antoine %A Verite, Stephanie %A Comtet, Loïc %A Yves Van der Stede %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Antibodies, Neutralizing %K Antibodies, Viral %K Bunyaviridae Infections %K Enzyme-Linked Immunosorbent Assay %K Female %K Immunity, Innate %K Neutralization Tests %K Nucleocapsid Proteins %K Orthobunyavirus %K Real-Time Polymerase Chain Reaction %K RNA, Viral %K Sheep %K Sheep Diseases %X

Schmallenberg virus (SBV) is an Orthobunyavirus that induces abortion, stillbirths and congenital malformations in ruminants. SBV infection induces a long lasting seroconversion under natural conditions. The persistence of the protective immunity and the isotype specific antibody response upon SBV infection of sheep has however not been studied in detail. Five sheep were kept in BSL3 facilities for more than 16 months and subjected to repeated SBV infections. Blood was regularly sampled and organs were collected at euthanasia. The presence of SBV RNA in serum and organs was measured with quantitative real-time PCR. The appearance and persistence of neutralizing and SBV nucleoprotein (N) isotype specific antibodies was determined with virus neutralization tests (VNT) and ELISAs. The primo SBV infection protected ewes against clinical signs, viraemia and virus replication in organs upon challenge infections more than 15 months later. Production of neutralizing SBV specific antibodies was first detected around 6 days post primo-inoculation with VNT and correlated with the appearance of SBV-N specific IgM antibodies. These IgM antibodies remained present for 2 weeks. SBV-N specific IgG antibodies were first detected between 10 and 21 dpi and reached a plateau at 28 dpi. This plateau remained consistently high and no significant decrease in titre was found over a period of more than 1 year. Similar results were found for the neutralising antibody response. In conclusion, the SBV specific IgM response probably eliminates SBV from the blood and the protective immunity induced by SBV infection protects sheep against reinfection for at least 16 months.

%B Vet Res %V 46 %P 119 %8 2015 Oct 15 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26472116?dopt=Abstract %R 10.1186/s13567-015-0260-6 %0 Journal Article %J Infect Ecol Epidemiol %D 2015 %T Serologic screening for 13 infectious agents in roe deer (Capreolus capreolus) in Flanders. %A Tavernier, Paul %A Sys, Stanislas U %A Kris De Clercq %A Ilse De Leeuw %A Ann Brigitte Cay %A Miet De Baere %A Nick De Regge %A David Fretin %A Virginie Roupie %A Govaerts, Marc %A Heyman, Paul %A Vanrompay, Daisy %A Yin, Lizi %A Kalmar, Isabelle %A Vanessa Suin %A Bernard Brochier %A Alexandre Dobly %A Stéphane De Craeye %A Sophie Roelandt %A Goossens, Els %A S. Roels %X

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.

%B Infect Ecol Epidemiol %V 5 %P 29862 %8 2015 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26609692?dopt=Abstract %R 10.3402/iee.v5.29862 %0 Journal Article %J BMC Vet Res %D 2014 %T Bluetongue, Schmallenberg - what is next? Culicoides-borne viral diseases in the 21st Century. %A Koenraadt, Constantianus Jm %A Balenghien, Thomas %A Carpenter, Simon %A Ducheyne, Els %A Elbers, Armin Rw %A Fife, Mark %A Garros, Claire %A Ibáñez-Justicia, Adolfo %A Kampen, Helge %A Kormelink, Richard Jm %A Losson, Bertrand %A van der Poel, Wim Hm %A Nick De Regge %A van Rijn, Piet A %A Sanders, Christopher %A Schaffner, Francis %A Sloet van Oldruitenborgh-Oosterbaan, Marianne M %A Takken, Willem %A Werner, Doreen %A Seelig, Frederik %K Animals %K Bluetongue %K Bluetongue virus %K Bunyaviridae Infections %K Cattle %K Cattle Diseases %K Ceratopogonidae %K Communicable Diseases, Emerging %K education %K Europe %K Orthobunyavirus %K Sheep %X

In the past decade, two pathogens transmitted by Culicoides biting midges (Diptera: Ceratopogonidae), bluetongue virus and Schmallenberg virus, have caused serious economic losses to the European livestock industry, most notably affecting sheep and cattle. These outbreaks of arboviral disease have highlighted large knowledge gaps on the biology and ecology of indigenous Culicoides species. With these research gaps in mind, and as a means of assessing what potential disease outbreaks to expect in the future, an international workshop was held in May 2013 at Wageningen University, The Netherlands. It brought together research groups from Belgium, France, Germany, Spain, Switzerland, United Kingdom and The Netherlands, with diverse backgrounds in vector ecology, epidemiology, entomology, virology, animal health, modelling, and genetics. Here, we report on the key findings of this workshop.

%B BMC Vet Res %V 10 %8 2014 Mar 31 %G eng %R 10.1186/1746-6148-10-77 %0 Journal Article %J BMC Vet Res %D 2014 %T Detection of total and PRRSV-specific antibodies in oral fluids collected with different rope types from PRRSV-vaccinated and experimentally infected pigs. %A Decorte, Inge %A Van Breedam, Wander %A Yves Van der Stede %A Nauwynck, Hans J %A Nick De Regge %A Ann Brigitte Cay %K Animals %K Antibodies, Viral %K cannabis %K Cotton Fiber %K Female %K Immunoglobulin Isotypes %K Nylons %K Polyesters %K Porcine Reproductive and Respiratory Syndrome %K Porcine respiratory and reproductive syndrome virus %K Saliva %K specimen handling %K Swine %K Viral Vaccines %X

BACKGROUND: Oral fluid collected by means of ropes has the potential to replace serum for monitoring and surveillance of important swine pathogens. Until now, the most commonly used method to collect oral fluid is by hanging a cotton rope in a pen. However, concerns about the influence of rope material on subsequent immunological assays have been raised. In this study, we evaluated six different rope materials for the collection of oral fluid and the subsequent detection of total and PRRSV-specific antibodies of different isotypes in oral fluid collected from PRRSV-vaccinated and infected pigs.

RESULTS: An initial experiment showed that IgA is the predominant antibody isotype in porcine saliva. Moreover, it was found that synthetic ropes may yield higher amounts of IgA, whereas all rope types seemed to be equally suitable for IgG collection. Although IgA is the predominant antibody isotype in porcine oral fluid, the PRRSV-specific IgA-based IPMA and ELISA tests were clearly not ideal for sensitive detection of PRRSV-specific IgA antibodies. In contrast, PRRSV-specific IgG in oral fluids was readily detected in PRRSV-specific IgG-based IPMA and ELISA tests, indicating that IgG is a more reliable isotype for monitoring PRRSV-specific antibody immunity in vaccinated/infected animals via oral fluids with the currently available tests.

CONCLUSIONS: Since PRRSV-specific IgG detection seems more reliable than PRRSV-specific IgA detection for monitoring PRRSV-specific antibody immunity via oral fluids, and since all rope types yield equal amounts of IgG, it seems that the currently used cotton ropes are an appropriate choice for sample collection in PRRSV monitoring.

%B BMC Vet Res %V 10 %P 134 %8 2014 Jun 17 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/24938323?dopt=Abstract %R 10.1186/1746-6148-10-134 %0 Journal Article %J Transbound Emerg Dis %D 2014 %T Distribution of Schmallenberg virus and seroprevalence in Belgian sheep and goats. %A Estelle Méroc %A Nick De Regge %A Riocreux, F %A Ann Brigitte Cay %A Thierry van den Berg %A Yves Van der Stede %K Animals %K Belgium %K Bunyaviridae Infections %K Epidemics %K Goat Diseases %K Goats %K Orthobunyavirus %K Seroepidemiologic Studies %K Sheep %K Sheep Diseases %X

A serological survey to detect Schmallenberg virus (SBV)-specific antibodies by ELISA was organized in the Belgian sheep population to study the seroprevalence at the end of the epidemic. One thousand eighty-two sheep samples which were collected from 83 herds all over Belgium between November 2011 and April 2012 were tested. The overall within-herd seroprevalence and the intraclass correlation coefficient were estimated at 84.31% (95% CI: 84.19-84.43) and 0.34, respectively. The overall between-herd seroprevalence was 98.03% (95% CI: 97.86-98.18). A spatial cluster analysis identified a cluster of six farms with significantly lower within-herd seroprevalence in the south of Belgium compared with the rest of the population (P = 0.04). It was shown that seroprevalence was associated to flock density and that the latter explained the presence of the spatial cluster. Additionally, 142 goat samples from eight different herds were tested for SBV-specific antibodies. The within-herd seroprevalence in goats was estimated at 40.68% (95% CI: 23.57-60.4%). The results of the current study provided evidence that almost every Belgian sheep herd has been in contact with SBV during 2011 and should be taken into consideration as part of comprehensive SBV surveillance and control strategies.

%B Transbound Emerg Dis %V 61 %P 425-31 %8 2014 Oct %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/23305427?dopt=Abstract %R 10.1111/tbed.12050 %0 Journal Article %J Vet J %D 2014 %T Dose-dependent effect of experimental Schmallenberg virus infection in sheep. %A Poskin, A %A Martinelle, L %A Laurent Mostin %A Willem Van Campe %A dal Pozzo, F %A Saegerman, C %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Bunyaviridae Infections %K Cattle %K Enzyme-Linked Immunosorbent Assay %K Female %K Orthobunyavirus %K polymerase chain reaction %K Sheep %K Sheep Diseases %K Viremia %X

Schmallenberg virus (SBV) is an orthobunyavirus affecting European domestic ruminants. In this study, the dose-dependent effect of experimental infection of sheep with SBV was evaluated. Four groups of three ewes were each inoculated subcutaneously with 1 mL of successive 10-fold dilutions of an SBV infectious serum. The ewes were monitored for 10 days, but no clinical signs were observed. The number of productively infected animals within each group, as evidenced by viraemia, seroconversion and viral RNA in the organs, depended on the inoculated dose, indicating that a critical dose has to be administered to obtain a homogeneous response in infected animals under experimental conditions. In the productively infected animals, no statistical differences between the different inoculation doses were found in the duration or quantity of viral RNA circulating in blood, nor in the amount of viral RNA present in virus positive lymphoid organs.

%B Vet J %V 201 %P 419-22 %8 2014 Sep %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/24954869?dopt=Abstract %R 10.1016/j.tvjl.2014.05.031 %0 Journal Article %J Vet Microbiol %D 2014 %T Experimental Schmallenberg virus infection of pigs. %A Poskin, Antoine %A Willem Van Campe %A Laurent Mostin %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Antibodies, Neutralizing %K Antibodies, Viral %K Bunyaviridae Infections %K Feces %K Orthobunyavirus %K Swine %X

Schmallenberg virus (SBV) is a newly emerged virus responsible for an acute non-specific syndrome in adult cattle including high fever, decrease in milk production and severe diarrhea. It also causes reproductive problems in cattle, sheep and goat including abortions, stillbirths and malformations. The role of pigs in the epidemiology of SBV has not yet been evaluated while this could be interesting seen their suggested role in the epidemiology of the closely related Akabane virus. To address this issue, four 12 week old seronegative piglets were subcutaneously infected with 1 ml of SBV infectious serum (FLI) and kept into contact with four non-infected piglets to examine direct virus transmission. Throughout the experiment blood, swabs and feces samples were collected and upon euthanasia at 28 dpi different organs (cerebrum, cerebellum, brain stem, lung, liver, iliac lymph nodes, kidney and spleen) were sampled. No clinical impact was observed and all collected samples tested negative for SBV in rRT-PCR. Despite the absence of viremia and virus transmission, low and short lasting amounts of neutralizing antibodies were found in 2 out of 4 infected piglets. The limited impact of SBV infection in pigs was further supported by the absence of neutralizing anti-SBV antibodies in field collected sera from indoor housed domestic pigs (n=106). In conclusion, SBV infection of pigs can induce seroconversion but is ineffective in terms of virus replication and transmission indicating that pigs have no obvious role in the SBV epidemiology.

%B Vet Microbiol %V 170 %P 398-402 %8 2014 Jun 04 %G eng %N 3-4 %1 http://www.ncbi.nlm.nih.gov/pubmed/24679959?dopt=Abstract %R 10.1016/j.vetmic.2014.02.026 %0 Journal Article %J European Journal of Wildlife Research %D 2014 %T Isolation and characterization of pseudorabies virus from a wolf (Canis lupus) from Belgium %A Verpoest, Sara %A Ann Brigitte Cay %A Bertrand, Olivier %A Saulmont, Marc %A Nick De Regge %K Belgium %K pseudorabies virus %K wolf %B European Journal of Wildlife Research %V 6010574930126969143277316113815614693318912315733 %8 Jan-02-2014 %G eng %N 1 %R 10.1007/s10344-013-0774-z %0 Journal Article %J Vet Microbiol %D 2014 %T Molecular characterization of Belgian pseudorabies virus isolates from domestic swine and wild boar. %A Verpoest, Sara %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Belgium %K Disease Reservoirs %K Genotype %K Herpesvirus 1, Suid %K Phylogeny %K Pseudorabies %K Sus scrofa %K Swine %K Swine Diseases %X

Aujeszky's disease is an economically important disease in domestic swine caused by suid herpesvirus 1, also called pseudorabies virus (PRV). In several European countries, including Belgium, the virus has successfully been eradicated from the domestic swine population. The presence of PRV in the wild boar population however poses a risk for possible reintroduction of the virus into the domestic pig population. It is therefore important to assess the genetic relatedness between circulating strains and possible epidemiological links. In this study, nine historical Belgian domestic swine isolates that circulated before 1990 and five recent wild boar isolates obtained since 2006 from Belgium and the Grand Duchy of Luxembourg were genetically characterized by restriction fragment length polymorphism (RFLP) analysis and phylogenetic analysis. While all wild boar isolates were characterized as type I RFLP genotypes, the RFLP patterns of the domestic swine isolates suggest that a shift from genotype I to genotype II might have occurred in the 1980s in the domestic population. By phylogenetic analysis, Belgian wild boar isolates belonging to both clade A and B were observed, while all domestic swine isolates clustered within clade A. The joint phylogenetic analysis of both wild boar and domestic swine strains showed that some isolates with identical sequences were present within both populations, raising the question whether these strains represent an increased risk for reintroduction of the virus into the domestic population.

%B Vet Microbiol %V 172 %P 72-7 %8 2014 Aug 06 %G eng %N 1-2 %1 http://www.ncbi.nlm.nih.gov/pubmed/24908275?dopt=Abstract %R 10.1016/j.vetmic.2014.05.001 %0 Journal Article %J PLoS One %D 2014 %T Schmallenberg virus circulation in culicoides in Belgium in 2012: field validation of a real time RT-PCR approach to assess virus replication and dissemination in midges. %A Nick De Regge %A Madder, Maxime %A Deblauwe, Isra %A Losson, Bertrand %A Fassotte, Christiane %A Demeulemeester, Julie %A Smeets, François %A Tomme, Marie %A Ann Brigitte Cay %K Animals %K Belgium %K Bunyaviridae Infections %K Ceratopogonidae %K Insect Vectors %K Orthobunyavirus %K Real-Time Polymerase Chain Reaction %K Reverse Transcriptase Polymerase Chain Reaction %K RNA, Viral %K Virus Replication %X

Indigenous Culicoides biting midges are suggested to be putative vectors for the recently emerged Schmallenberg virus (SBV) based on SBV RNA detection in field-caught midges. Furthermore, SBV replication and dissemination has been evidenced in C. sonorensis under laboratory conditions. After SBV had been detected in Culicoides biting midges from Belgium in August 2011, it spread all over the country by the end of 2011, as evidenced by very high between-herd seroprevalence rates in sheep and cattle. This study investigated if a renewed SBV circulation in midges occurred in 2012 in the context of high seroprevalence in the animal host population and evaluated if a recently proposed realtime RT-PCR approach that is meant to allow assessing the vector competence of Culicoides for SBV and bluetongue virus under laboratory conditions was applicable to field-caught midges. Therefore midges caught with 12 OVI traps in four different regions in Belgium between May and November 2012, were morphologically identified, age graded, pooled and tested for the presence of SBV RNA by realtime RT-PCR. The results demonstrate that although no SBV could be detected in nulliparous midges caught in May 2012, a renewed but short lived circulation of SBV in parous midges belonging to the subgenus Avaritia occured in August 2012 at all four regions. The infection prevalence reached up to 2.86% in the south of Belgium, the region where a lower seroprevalence was found at the end of 2011 than in the rest of the country. Furthermore, a frequency analysis of the Ct values obtained for 31 SBV-S segment positive pools of Avaritia midges showed a clear bimodal distribution with peaks of Ct values between 21-24 and 33-36. This closely resembles the laboratory results obtained for SBV infection of C. sonorensis and implicates indigenous midges belonging to the subgenus Avaritia as competent vectors for SBV.

%B PLoS One %V 9 %P e87005 %8 2014 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/24466312?dopt=Abstract %R 10.1371/journal.pone.0087005 %0 Journal Article %J J Virol Methods %D 2013 %T Development, validation and evaluation of added diagnostic value of a q(RT)-PCR for the detection of genotype A strains of small ruminant lentiviruses. %A Nick De Regge %A Ann Brigitte Cay %K Animals %K Belgium %K Goat Diseases %K Goats %K Lentivirus %K Lentivirus Infections %K Molecular Diagnostic Techniques %K Molecular Sequence Data %K Real-Time Polymerase Chain Reaction %K RNA, Viral %K Sensitivity and Specificity %K Sequence Analysis, DNA %K Sheep %K Sheep Diseases %K Veterinary Medicine %X

Small ruminant lentiviruses (SRLV) infect sheep and goats. Diagnosis of SRLV infection mostly relies on serological testing but more recently, also PCR is regarded as a useful complementary tool in SRLV diagnosis. The goal of this study was to develop and validate a quantitative PCR capable to detect a broad range of SRLV strains from genotype A, including strains circulating in Belgium. The developed q(RT)-PCR targets a region of the gag gene and showed to be highly sensitive and specific with a limit of detection of 6 DNA and 40 RNA copies/reaction respectively. SRLV sequences could be detected in lung samples and leukocytes pellets. The q(RT)-PCR identified SRLV positive animals in Belgian sheep flocks, but also SRLV isolates and samples from Scotland, The Netherlands, Spain, Portugal, UK, Iceland, Finland and USA were found positive. Samples known to contain 'CAEV like' SRLV from France and Spain were not identified as positive. Combined serological and PCR analysis of a limited number (n=35) of Belgian sheep underlined the usefulness of the described PCR as a complementary diagnostic tool since 3 seronegative animals were found positive by the PCR. In conclusion, the validated q(RT)-PCR shows excellent analytical characteristics and is capable to detect SRLV strains belonging to genotype A from various countries.

%B J Virol Methods %V 194 %P 250-7 %8 2013 Dec %G eng %N 1-2 %1 http://www.ncbi.nlm.nih.gov/pubmed/24045043?dopt=Abstract %R 10.1016/j.jviromet.2013.09.001 %0 Journal Article %J Vet Microbiol %D 2013 %T Diagnosis of Schmallenberg virus infection in malformed lambs and calves and first indications for virus clearance in the fetus. %A Nick De Regge %A Thierry van den Berg %A Georges, Laura %A Ann Brigitte Cay %K Abortion, Veterinary %K Animals %K Antibodies, Neutralizing %K Antibodies, Viral %K Brain Stem %K Bunyaviridae Infections %K Cattle %K Cattle Diseases %K Congenital Abnormalities %K Female %K Neutralization Tests %K Orthobunyavirus %K Pregnancy %K Pregnancy Complications, Infectious %K Real-Time Polymerase Chain Reaction %K Sheep %K Sheep Diseases %K Sheep, Domestic %K Stillbirth %X

Since mid-December 2011, samples from malformed lambs and calves are sent to CODA-CERVA in Belgium for diagnosis of Schmallenberg virus (SBV), a novel Orthobunyavirus that was first detected by researchers of the Friedrich-Loeffler-Institut (FLI, Germany) in German cattle in autumn 2011 and was later shown to be involved in congenital malformations in lambs, goat kids and calves. Surprisingly, by making use of real time RT-PCR (rRT-PCR) assays developed by the FLI, presence of SBV RNA could only be confirmed in part of the SBV suspected newborns examined. To investigate possible causes for non-confirmation by rRT-PCR, a comparative analysis between different organs and tissues (cerebrum, cerebellum, brain stem, spinal cord, thymus, spleen, lymph nodes, meconium) originating from respectively 90 and 81 malformed lambs and calves was undertaken. Furthermore, thoracic fluids of respectively 55 malformed lambs and calves were examined by a virus neutralization test (VNT) to evaluate the presence of neutralizing anti-SBV antibodies in these animals. Our results show that among the different organs tested by rRT-PCR, brain stem material is the most appropriate tissue for SBV detection while it could also be detected in all other tissues but to a more variable degree. The VNT test showed that 95% of the malformed lambs were positive for anti-SBV neutralizing antibodies while this was only the case for 44% of malformed calves. These immunological data suggest that a humoral immune response could assist in the clearance of SBV from the fetus during gestation and that SBV specific antibody testing should be considered together with rRT-PCR analysis for confirmation of SBV infection.

%B Vet Microbiol %V 162 %P 595-600 %8 2013 Mar 23 %G eng %N 2-4 %1 http://www.ncbi.nlm.nih.gov/pubmed/23265245?dopt=Abstract %R 10.1016/j.vetmic.2012.11.029 %0 Journal Article %J Vet J %D 2013 %T Effect of saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus in oral fluid by quantitative reverse transcriptase real-time PCR. %A Decorte, Inge %A Yves Van der Stede %A Nauwynck, Hans %A Nick De Regge %A Ann Brigitte Cay %K Animals %K Excipients %K Porcine respiratory and reproductive syndrome virus %K Real-Time Polymerase Chain Reaction %K Reverse Transcriptase Polymerase Chain Reaction %K RNA, Viral %K Saliva %K specimen handling %K Swine %X

This study evaluated the effect of extraction-amplification methods, storage temperature and saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus (PRRSV) RNA by quantitative reverse transcriptase real-time PCR (qRT-PCR) in porcine oral fluid. The diagnostic performance of different extraction-amplification methods was examined using a dilution series of oral fluid spiked with PRRSV. To determine RNA stability, porcine oral fluid, with or without commercially available saliva stabilisers, was spiked with PRRSV, stored at 4°C or room temperature and tested for the presence of PRRSV RNA by qRT-PCR. PRRSV RNA could be detected in oral fluid using all extraction-amplification combinations, but the limit of detection varied amongst different combinations. Storage temperature and saliva stabilisers had an effect on the stability of PRRSV RNA, which could only be detected for 7 days when PRRSV spiked oral fluid was kept at 4°C or stabilised at room temperature with a commercial mRNA stabiliser.

%B Vet J %V 197 %P 224-8 %8 2013 Aug %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/23489844?dopt=Abstract %R 10.1016/j.tvjl.2013.02.001 %0 Journal Article %J Transbound Emerg Dis %D 2013 %T Large-scale cross-sectional serological survey of Schmallenberg virus in Belgian cattle at the end of the first vector season. %A Estelle Méroc %A Poskin, A %A Van Loo, H %A Quinet, C %A Van Driessche, E %A Delooz, L %A Isabelle Behaeghel %A Riocreux, F %A Hooyberghs, J %A Nick De Regge %A Ann Brigitte Cay %A Thierry van den Berg %A Yves Van der Stede %K Animals %K Belgium %K Bunyaviridae Infections %K Cattle %K Cattle Diseases %K cross-sectional studies %K Enzyme-Linked Immunosorbent Assay %K Female %K Orthobunyavirus %K Seasons %K Seroepidemiologic Studies %K Serologic Tests %X

A cross-sectional survey was conducted in the Belgian cattle population after the first period of infection of the emerging Schmallenberg virus. A total number of 11 635 cattle from 422 herds sampled between 2 January and 7 March 2012 were tested for the presence of Schmallenberg-specific antibodies using an ELISA kit. Between-herd seroprevalence in cattle was estimated at 99.76% (95% CI: 98.34-99.97) and within-herd seroprevalence at 86.3% (95% CI: 84.75-87.71). An Intraclass Correlation Coefficient of 0.3 (P < 0.001) was found, indicating that the correlation between two animals within a herd with respect to their serological status was high. Those results corroborate the conclusion that the Schmallenberg virus was widespread in Belgium during winter 2011. Seroprevalence was shown to be statistically associated to the animal's age (P < 0.0001): with 64.9% (95% CI: 61.34-68.3) estimated for the 6-12 months of age, 86.79% (95% CI: 84.43-88.85) for the 12-24 months of age and 94.4% (95% CI: 93.14-95.44) for the animals older than 24 months. Based on the results of the described serological survey, we can conclude that after the first Schmallenberg virus episode, almost every Belgian cattle has already been in contact with the virus. In consequence, the vast majority of the host animals should have developed post infection protective immunity against the virus.

%B Transbound Emerg Dis %V 60 %P 4-8 %8 2013 Feb %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/23206240?dopt=Abstract %R 10.1111/tbed.12042 %0 Journal Article %J PLoS One %D 2013 %T Validation of a commercially available indirect ELISA using a nucleocapside recombinant protein for detection of Schmallenberg virus antibodies. %A Bréard, Emmanuel %A Lara, Estelle %A Comtet, Loïc %A Viarouge, Cyril %A Doceul, Virginie %A Desprat, Alexandra %A Vitour, Damien %A Pozzi, Nathalie %A Ann Brigitte Cay %A Nick De Regge %A Pourquier, Philippe %A Schirrmeier, Horst %A Hoffmann, Bernd %A Beer, Martin %A Sailleau, Corinne %A Zientara, Stéphan %K Animals %K Antibodies, Neutralizing %K Antibodies, Viral %K Bunyaviridae Infections %K Cattle %K Enzyme-Linked Immunosorbent Assay %K Europe %K Fluorescent Antibody Technique, Indirect %K Gene Expression %K Neutralization Tests %K Nucleocapsid Proteins %K Orthobunyavirus %K Reagent Kits, Diagnostic %K Recombinant Proteins %K Reproducibility of Results %K Roc Curve %K Sheep %X

A newly developed Enzym Like Immuno Sorbant Assay (ELISA) based on the recombinant nucleocapsid protein (N) of Schmallenberg virus (SBV) was evaluated and validated for the detection of SBV-specific IgG antibodies in ruminant sera by three European Reference Laboratories. Validation data sets derived from sheep, goat and bovine sera collected in France and Germany (n = 1515) in 2011 and 2012 were categorized according to the results of a virus neutralization test (VNT) or an indirect immuno-fluorescence assay (IFA). The specificity was evaluated with 1364 sera from sheep, goat and bovine collected in France and Belgium before 2009. Overall agreement between VNT and ELISA was 98.9% and 98.3% between VNT and IFA, indicating a very good concordance between the different techniques. Although cross-reactions with other Orthobunyavirus from the Simbu serogroup viruses might occur, it is a highly sensitive, specific and robust ELISA-test validated to detect anti-SBV antibodies. This test can be applied for SBV sero-diagnostics and disease-surveillance studies in ruminant species in Europe.

%B PLoS One %V 8 %P e53446 %8 2013 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/23335964?dopt=Abstract %R 10.1371/journal.pone.0053446 %0 Journal Article %J Transbound Emerg Dis %D 2012 %T Detection of Schmallenberg virus in different Culicoides spp. by real-time RT-PCR. %A Nick De Regge %A Deblauwe, I %A De Deken, R %A Vantieghem, P %A Madder, M %A Geysen, D %A Smeets, F %A Losson, B %A Thierry van den Berg %A Ann Brigitte Cay %K Animals %K Belgium %K Bunyaviridae Infections %K Cattle %K Cattle Diseases %K Ceratopogonidae %K Insect Vectors %K Orthobunyavirus %K Reverse Transcriptase Polymerase Chain Reaction %K Seasons %X

To identify possible vectors of Schmallenberg virus (SBV), we tested pools containing heads of biting midges (Culicoides) that were caught during the summer and early autumn of 2011 at several places in Belgium by real-time RT-PCR. Pools of heads originating from following species: C. obsoletus complex, C. dewulfi and C. chiopterus were found positive, strongly indicating that these species are relevant vectors for SBV.

%B Transbound Emerg Dis %V 59 %P 471-5 %8 2012 Dec %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/23025501?dopt=Abstract %R 10.1111/tbed.12000 %0 Journal Article %J PLoS One %D 2012 %T DNase SISPA-next generation sequencing confirms Schmallenberg virus in Belgian field samples and identifies genetic variation in Europe. %A Rosseel, Toon %A Scheuch, Matthias %A Höper, Dirk %A Nick De Regge %A Frank Vandenbussche %A Steven Van Borm %K Belgium %K Deoxyribonucleases %K DNA Primers %K Genetic Variation %K High-Throughput Nucleotide Sequencing %K Molecular Sequence Data %K Nucleic Acid Amplification Techniques %K Orthobunyavirus %K RNA, Viral %K Sequence Analysis, RNA %X

In 2011, a novel Orthobunyavirus was identified in cattle and sheep in Germany and The Netherlands. This virus was named Schmallenberg virus (SBV). Later, presence of the virus was confirmed using real time RT-PCR in cases of congenital malformations of bovines and ovines in several European countries, including Belgium. In the absence of specific sequencing protocols for this novel virus we confirmed its presence in RT-qPCR positive field samples using DNase SISPA-next generation sequencing (NGS), a virus discovery method based on random amplification and next generation sequencing. An in vitro transcribed RNA was used to construct a standard curve allowing the quantification of viral RNA in the field samples. Two field samples of aborted lambs containing 7.66 and 7.64 log(10) RNA copies per µL total RNA allowed unambiguous identification of SBV. One sample yielded 192 SBV reads covering about 81% of the L segment, 56% of the M segment and 13% of the S segment. The other sample resulted in 8 reads distributed over the L and M segments. Three weak positive field samples (one from an aborted calf, two from aborted lambs) containing virus quantities equivalent to 4.27-4.89 log(10) RNA copies per µL did not allow identification using DNase SISPA-NGS. This partial sequence information was compared to the whole genome sequence of SBV isolated from bovines in Germany, identifying several sequence differences. The applied viral discovery method allowed the confirmation of SBV in RT-qPCR positive brain samples. However, the failure to confirm SBV in weak PCR-positive samples illustrates the importance of the selection of properly targeted and fresh field samples in any virus discovery method. The partial sequences derived from the field samples showed several differences compared to the sequences from bovines in Germany, indicating sequence divergence within the epidemic.

%B PLoS One %V 7 %P e41967 %8 2012 %G eng %N 7 %1 https://www.ncbi.nlm.nih.gov/pubmed/22848676?dopt=Abstract %R 10.1371/journal.pone.0041967 %0 Journal Article %J Theriogenology %D 2012 %T Impact of a novel inactivated PRRS virus vaccine on virus replication and virus-induced pathology in fetal implantation sites and fetuses upon challenge. %A Karniychuk, U U %A Saha, D %A Vanhee, M %A Geldhof, M %A Cornillie, P %A Ann Brigitte Cay %A Nick De Regge %A Nauwynck, H J %K Animals %K Antibodies, Viral %K Female %K Fetal Diseases %K Fetus %K Gestational Age %K Placenta %K Porcine Reproductive and Respiratory Syndrome %K Porcine respiratory and reproductive syndrome virus %K Pregnancy %K Pregnancy Complications, Infectious %K Sus scrofa %K Swine %K Vaccination %K Vaccines, Inactivated %K Viral Vaccines %K Viremia %X

Preventing congenital infection is important for the control of porcine reproductive and respiratory syndrome (PRRS). Recently, in our laboratory, an inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine has been developed. Promising results in young pigs encouraged us to test the vaccine potency to prevent congenital infection. In the present study, the performance of this experimental inactivated vaccine was investigated in pregnant gilts. An advanced protocol was used to test the PRRSV vaccine efficacy. This protocol is based on recent insights in the pathogenesis of congenital PRRSV infections. Three gilts were vaccinated with an experimental PRRSV 07V63 inactivated vaccine at 27, 55, and 83 days of gestation. Three unvaccinated gilts were included as controls. At 90 days of gestation, all animals were intranasally inoculated with 10(5) tissue culture infectious dose 50 (TCID(50)) of PRRSV 07V63. Twenty days postchallenge animals were euthanized and sampled. The vaccinated gilts quickly developed virus neutralizing (VN) antibodies starting from 3 to 7 days postchallenge (1.0 to 5.0 log2). In contrast, the unvaccinated gilts remained negative for VN antibodies after challenge. The vaccinated gilts had shorter viremia than the control gilts. Gross pathology (mummification) was observed in 8% of the fetuses from vaccinated gilts and in 15% of the fetuses from unvaccinated gilts. The number of fetuses with severe microscopic lesions in the fetal implantation sites (a focal detachment of the trophoblast from the uterine epithelium; a focal, multifocal, or full degeneration of the fetal placenta) was lower in the vaccinated (19%) versus unvaccinated (45%) gilts (P < 0.05). The number of PRRS-positive cells in the fetal placentae was higher in unvaccinated versus vaccinated gilts (P < 0.05). In contrast, the number of PRRS-positive cells in the myometrium/endometrium was higher in vaccinated versus unvaccinated gilts (P < 0.05). Fifty-seven percent of the fetuses from the vaccinated gilts and 75% of the fetuses from the unvaccinated gilts were PRRSV-positive. In conclusion, implementation of the novel experimental inactivated PRRSV vaccine primed the VN antibody response and slightly reduced the duration of viremia in gilts. It also reduced the number of virus-positive fetuses and improved the fetal survival, but was not able to fully prevent congenital PRRSV infection. The reduction of fetal infection and pathology is most probably attributable to the vaccine-mediated decrease of PRRSV transfer from the endometrium to the fetal placenta.

%B Theriogenology %V 78 %P 1527-37 %8 2012 Oct 15 %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/22980086?dopt=Abstract %R 10.1016/j.theriogenology.2012.06.015 %0 Journal Article %D 0 %T Lumpy Skin Disease Virus Genome Sequence Analysis: Putative Spatio-Temporal Epidemiology, Single Gene versus Whole Genome Phylogeny and Genomic Evolution %A Floris C Breman %A Andy Haegeman %A Nina Krešić %A Philips, Wannes %A Nick De Regge %G eng