TY - JOUR T1 - Targeted Whole Genome Sequencing of the Capripoxvirus Genome from Clinical Tissue Samples and Lyophilized Vaccine Batches. JF - Methods Mol Biol Y1 - 2024 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche AB -

Diseases caused by Capripoxviruses (CaPVs) are of great economic importance in sheep, goats, and cattle. Since CaPV strains are serologically indistinguishable and genetically highly homologous, typing of closely related strains can only be achieved by whole-genome sequencing. In this chapter, we describe a robust, cost-effective, and widely applicable protocol for reconstructing (nearly) complete CaPV genomes directly from clinical samples or commercial vaccine batches in less than a week. Taking advantage of the genetic similarity of CaPVs, a set of pan-CaPVs long-range PCRs was developed that covers the entire genome with only a limited number of tiled amplicons. The resulting amplicons can be sequenced on all currently available high-throughput sequencing platforms. As an example, we have included a detailed protocol for performing nanopore sequencing and a pipeline for assembling the resulting tiled amplicon data.

VL - 2732 M3 - 10.1007/978-1-0716-3515-5_12 ER - TY - JOUR T1 - Combined Phylogeographic Analyses and Epidemiologic Contact Tracing to Characterize Atypically Pathogenic Avian Influenza (H3N1) Epidemic, Belgium, 2019. JF - Emerg Infect Dis Y1 - 2023 A1 - Steven Van Borm A1 - Geraldine Boseret A1 - Simon Dellicour A1 - Mieke Steensels A1 - Virginie Roupie A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - Aline Vilain A1 - Michèle Driesen A1 - Marc Dispas A1 - Andy W Delcloo A1 - Philippe Lemey A1 - Ingeborg Mertens A1 - Marius Gilbert A1 - Bénédicte Lambrecht A1 - Thierry van den Berg KW - Animals KW - Belgium KW - Chickens KW - Contact Tracing KW - Epidemics KW - Influenza in Birds KW - Phylogeny KW - Phylogeography KW - Poultry Diseases AB -

The high economic impact and zoonotic potential of avian influenza call for detailed investigations of dispersal dynamics of epidemics. We integrated phylogeographic and epidemiologic analyses to investigate the dynamics of a low pathogenicity avian influenza (H3N1) epidemic that occurred in Belgium during 2019. Virus genomes from 104 clinical samples originating from 85% of affected farms were sequenced. A spatially explicit phylogeographic analysis confirmed a dominating northeast to southwest dispersal direction and a long-distance dispersal event linked to direct live animal transportation between farms. Spatiotemporal clustering, transport, and social contacts strongly correlated with the phylogeographic pattern of the epidemic. We detected only a limited association between wind direction and direction of viral lineage dispersal. Our results highlight the multifactorial nature of avian influenza epidemics and illustrate the use of genomic analyses of virus dispersal to complement epidemiologic and environmental data, improve knowledge of avian influenza epidemiologic dynamics, and enhance control strategies.

VL - 29 CP - 2 M3 - 10.3201/eid2902.220765 ER - TY - JOUR T1 - Combined Phylogeographic Analyses and Epidemiologic Contact Tracing to Characterize Atypically Pathogenic Avian Influenza (H3N1) Epidemic, Belgium, 2019 JF - Emerging Infectious Diseases Y1 - 2023 A1 - Steven Van Borm A1 - Geraldine Boseret A1 - Simon Dellicour A1 - Mieke Steensels A1 - Virginie Roupie A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - Aline Vilain A1 - Michèle Driesen A1 - Marc Dispas A1 - Andy W. Delcloo A1 - Philippe Lemey A1 - Ingeborg Mertens A1 - Marius Gilbert A1 - Bénédicte Lambrecht A1 - Thierry van den Berg VL - 29 CP - 2 M3 - 10.3201/eid2902.220765 ER - TY - JOUR T1 - Complete genome reconstruction of the global and European regional dispersal history of the lumpy skin disease virus. JF - J Virol Y1 - 2023 A1 - Steven Van Borm A1 - Simon Dellicour A1 - Darren P Martin A1 - Philippe Lemey A1 - Agianniotaki, Eirini I A1 - Chondrokouki, Eleni D A1 - Vidanovic, Dejan A1 - Nikola Vaskovic A1 - Tamaš Petroviċ A1 - Sava Laziċ A1 - Xhelil Koleci A1 - Vodica, Ani A1 - Igor Djadjovski A1 - Kiril Krstevski A1 - Frank Vandenbussche A1 - Andy Haegeman A1 - Kris De Clercq A1 - Elisabeth Mathijs AB -

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

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

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

M3 - 10.1128/jvi.01394-23 ER - TY - JOUR T1 - Recombinant LSDV Strains in Asia: Vaccine Spillover or Natural Emergence? JF - Viruses Y1 - 2022 A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - Wannes Philips A1 - Saduakassova, Meruyert A1 - Ilse De Leeuw A1 - Sultanov, Akhmetzhan A1 - Andy Haegeman A1 - Kris De Clercq VL - 14 CP - 7 M3 - 10.3390/v14071429 ER - TY - JOUR T1 - A robust, cost-effective and widely applicable whole-genome sequencing protocol for capripoxviruses JF - Journal of Virological Methods Y1 - 2022 A1 - Elisabeth Mathijs A1 - Andy Haegeman A1 - Kris De Clercq A1 - Steven Van Borm A1 - Frank Vandenbussche M3 - 10.1016/j.jviromet.2022.114464 ER - TY - JOUR T1 - Coding-Complete Sequences of Recombinant Lumpy Skin Disease Viruses Collected in 2020 from Four Outbreaks in Northern Vietnam. JF - Microbiol Resour Announc Y1 - 2021 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Nguyen, Long A1 - Laetitia Aerts A1 - Nguyen, Tho A1 - Ilse De Leeuw A1 - Quang, Minh A1 - Nguyen, Hoang Dang A1 - Wannes Philips A1 - Dam, Thi Vui A1 - Andy Haegeman A1 - Steven Van Borm A1 - Kris De Clercq AB -

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

VL - 10 CP - 48 M3 - 10.1128/MRA.00897-21 ER - TY - RPRT T1 - Key considerations for the implementation of high throughput sequencing based metagenomics (mNGS) in diagnostic clinical, food and veterinary labs : a no-nonsense pointer. the Metastava consortium Y1 - 2021 A1 - Steven Van Borm A1 - Frank Vandenbussche A1 - Kris De Clercq A1 - Andy Haegeman A1 - Sigrid C.J. De Keersmaecker A1 - Kevin Vanneste A1 - Michael Peeters A1 - Steven Van Gucht A1 - Yannick Blanchard A1 - Nicole Pavio A1 - Sandra Martin-Latil A1 - Fabrice Touzain A1 - Isabelle Kempf A1 - Liu, Lihong A1 - Mickhayil Hakhverdyan A1 - Mikael Leijon A1 - Alex Bossers A1 - van der Poel, Wim A1 - Marcel Hulst A1 - Bas Oude Munnink A1 - Miranda De Graaf A1 - Sander van Boheemen A1 - Marion Koopmans A1 - Beer, Martin A1 - Anne Pohlmann A1 - Höper, Dirk ER - TY - JOUR T1 - Metagenomic sequencing determines complete infectious bronchitis virus (avian Gammacoronavirus) vaccine strain genomes and associated viromes in chicken clinical samples. JF - Virus Genes Y1 - 2021 A1 - Steven Van Borm A1 - Mieke Steensels A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Thierry van den Berg A1 - Bénédicte Lambrecht AB -

Infectious bronchitis virus (IBV, genus Gammacoronavirus) causes an economically important and highly contagious disease in chicken. Random primed RNA sequencing was applied to two IBV positive clinical samples and one in ovo-passaged virus. The virome of a cloacal swab pool was dominated by IBV (82% of viral reads) allowing de novo assembly of a GI-13 lineage complete genome with 99.95% nucleotide identity to vaccine strain 793B. In addition, substantial read counts (16% of viral reads) allowed the assembly of a near-complete chicken astrovirus genome, while lower read counts identified the presence of chicken calicivirus and avian leucosis virus. Viral reads in a respiratory/intestinal tissue pool were distributed between IBV (22.53%), Sicinivirus (Picornaviridae, 24%), and avian leucosis virus (37.04%). A complete IBV genome with 99.95% nucleotide identity to vaccine strain H120 (lineage GI-1), as well as a near-complete avian leucosis virus genome and a partial Sicinivirus genome were assembled from the tissue sample data. Lower read counts identified chicken calicivirus, Avibirnavirus (infectious bursal disease virus, assembling to 98.85% of segment A and 69.66% of segment B closely related to D3976/1 from Germany, 2017) and avian orthoreovirus, while three avian orthoavulavirus 1 reads confirmed prior real-time RT-PCR result. IBV sequence variation analysis identified both fixed and minor frequency variations in the tissue sample compared to its in ovo-passaged virus. Metagenomic methods allow the determination of complete coronavirus genomes from clinical chicken samples while providing additional insights in RNA virus sequence diversity and coinfecting viruses potentially contributing to pathogenicity.

VL - 57 CP - 6 M3 - 10.1007/s11262-021-01872-7 ER - TY - JOUR T1 - Nearly Complete Genome Sequences of Two Bluetongue Viruses Isolated during the 2020 Outbreak in the Grand Duchy of Luxembourg. JF - Microbiol Resour Announc Y1 - 2021 A1 - Frank Vandenbussche A1 - Manon Bourg A1 - Elisabeth Mathijs A1 - David Lefebvre A1 - Ilse De Leeuw A1 - Andy Haegeman A1 - Laetitia Aerts A1 - Steven Van Borm A1 - Kris De Clercq AB -

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

VL - 10 CP - 14 M3 - 10.1128/MRA.00210-21 ER - TY - JOUR T1 - WGS- versus ORF5-Based Typing of PRRSV: A Belgian Case Study. JF - Viruses Y1 - 2021 A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - Marylène Tignon A1 - Tamara Vandersmissen A1 - Ann Brigitte Cay AB -

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most widespread and economically devastating diseases in the swine industry. Typing circulating PRRSV strains by means of sequencing is crucial for developing adequate control strategies. Most genetic studies only target the highly variable open reading frame (ORF) 5, for which an extensive database is available. In this study, we performed whole-genome sequencing (WGS) on a collection of 124 PRRSV-1 positive serum samples that were collected over a 5-year period (2015-2019) in Belgium. Our results show that (nearly) complete PRRSV genomes can be obtained directly from serum samples with a high success rate. Analysis of the coding regions confirmed the exceptionally high genetic diversity, even among Belgian PRRSV-1 strains. To gain more insight into the added value of WGS, we performed phylogenetic cluster analyses on separate ORF datasets as well as on a single, concatenated dataset (CDS) containing all ORFs. A comparison between the CDS and ORF clustering schemes revealed numerous discrepancies. To explain these differences, we performed a large-scale recombination analysis, which allowed us to identify a large number of potential recombination events that were scattered across the genome. As PRRSV does not contain typical recombination hot-spots, typing PRRSV strains based on a single ORF is not recommended. Although the typing accuracy can be improved by including multiple regions, our results show that the full genetic diversity among PRRSV strains can only be captured by analysing (nearly) complete genomes. Finally, we also identified several vaccine-derived recombinant strains, which once more raises the question of the safety of these vaccines.

VL - 13 CP - 12 M3 - 10.3390/v13122419 ER - TY - JOUR T1 - Complete Coding Sequence of a Lumpy Skin Disease Virus from an Outbreak in Bulgaria in 2016. JF - Microbiol Resour Announc Y1 - 2020 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Emiliya Ivanova A1 - Andy Haegeman A1 - Laetitia Aerts A1 - Ilse De Leeuw A1 - Steven Van Borm A1 - Kris De Clercq AB -

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

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

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

VL - 9 CP - 4 M3 - 10.1128/MRA.01399-19 ER - TY - JOUR T1 - Evaluation of a commercial exogenous internal process control for diagnostic RNA virus metagenomics from different animal clinical samples JF - Journal of Virological Methods Y1 - 2020 A1 - Steven Van Borm A1 - Fu, Qiang A1 - Raf Winand A1 - Kevin Vanneste A1 - Mikhayil Hakhverdyan A1 - Höper, Dirk A1 - Frank Vandenbussche KW - Animals KW - Feces KW - High-Throughput Nucleotide Sequencing KW - Mengovirus KW - Metagenomics KW - Rna KW - RNA Virus Infections KW - RNA Viruses KW - Swine KW - Swine Diseases KW - Viral Sensitivity and Specificity KW - Virome VL - 283 M3 - 10.1016/j.jviromet.2020.113916 ER - TY - JOUR T1 - Increased viral read counts and metagenomic full genome characterization of porcine astrovirus 4 and Posavirus 1 in sows in a swine farm with unexplained neonatal piglet diarrhea JF - Virus Genes Y1 - 2020 A1 - Steven Van Borm A1 - Kevin Vanneste A1 - Fu, Qiang A1 - Maes, Dominiek A1 - Alexandra Schoos A1 - Eline Vallaey A1 - Frank Vandenbussche VL - 56 CP - 6 M3 - 10.1007/s11262-020-01791-z ER - TY - JOUR T1 - Complete Genome Sequences of Five Foot-and-Mouth Disease Viruses of Serotype A Isolated from Cattle in Nigeria between 2013 and 2015. JF - Genome Announc Y1 - 2018 A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - Hussaini G Ularamu A1 - David O Ehizibolo A1 - Andy Haegeman A1 - David Lefebvre A1 - Annebel De Vleeschauwer A1 - Steven Van Borm A1 - Kris De Clercq AB -

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

VL - 6 CP - 7 M3 - 10.1128/genomeA.00039-18 ER - TY - JOUR T1 - Complete Coding Sequence of Usutu Virus Strain Gracula religiosa/U1609393/Belgium/2016 Obtained from the Brain Tissue of an Infected Captive Common Hill Myna (Gracula religiosa) JF - Genome Announcements Y1 - 2017 A1 - Steven Van Borm A1 - Bénédicte Lambrecht A1 - Frank Vandenbussche A1 - Mieke Steensels VL - 5 CP - 12 ER - TY - JOUR T1 - Complete Coding Sequence of Usutu Virus Strain Gracula religiosa/U1609393/Belgium/2016 Obtained from the Brain Tissue of an Infected Captive Common Hill Myna (). JF - Genome Announc Y1 - 2017 A1 - Steven Van Borm A1 - Bénédicte Lambrecht A1 - Frank Vandenbussche A1 - Mieke Steensels AB -

The complete and annotated coding sequence and partial noncoding sequence of an Usutu virus genome were sequenced from RNA extracted from a clinical brain tissue sample obtained from a common hill myna (), demonstrating close homology with Usutu viruses circulating in Europe.

VL - 5 CP - 12 U1 - https://www.ncbi.nlm.nih.gov/pubmed/28336592?dopt=Abstract M3 - 10.1128/genomeA.00042-17 ER - TY - JOUR T1 - Complete Coding Sequence of Usutu Virus Strain Gracula religiosa/U1609393/Belgium/2016 Obtained from the Brain Tissue of an Infected Captive Common Hill Myna (Gracula religiosa) JF - Genome Announcements Y1 - 2017 A1 - Steven Van Borm A1 - Bénédicte Lambrecht A1 - Frank Vandenbussche A1 - Mieke Steensels VL - 5 CP - 12 M3 - 10.1128/genomeA.00042-17 ER - TY - JOUR T1 - Complete Genome Sequence of the Lumpy Skin Disease Virus Isolated from the First Reported Case in Greece in 2015 JF - Genome Announcements Y1 - 2017 A1 - Agianniotaki, Eirini I. A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Tasioudi, Konstantia E. A1 - Andy Haegeman A1 - Iliadou, Peristera A1 - Chaintoutis, Serafeim C. A1 - Dovas, Chrysostomos I. A1 - Steven Van Borm A1 - Chondrokouki, Eleni D. A1 - Kris De Clercq KW - Illumina KW - LSDV KW - Lumpy skin disease KW - NGS KW - WGS VL - 5 CP - 29 M3 - 10.1128/genomeA.00550-17 ER - TY - JOUR T1 - Complete Genome Sequences of Four Foot-and-Mouth Disease Viruses of Serotype South African Territories 1 (SAT 1), Topotype X, Isolated from Cattle in Nigeria in 2015 JF - Genome Announcements Y1 - 2017 A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - Hussaini G. Ularamu A1 - David O. Ehizibolo A1 - Andy Haegeman A1 - David Lefebvre A1 - David D. Lazarus A1 - Yiltawe S. Wungak A1 - Annebel R. De Vleeschauwer A1 - Steven Van Borm A1 - Kris De Clercq VL - 5 CP - 42 M3 - 10.1128/genomeA.01065-17 ER - TY - JOUR T1 - Correction for Mathijs et al., Complete Genome Sequences of the Neethling-Like Lumpy Skin Disease Virus Strains Obtained Directly from Three Commercial Live Attenuated Vaccines JF - Genome Announcements Y1 - 2017 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Andy Haegeman A1 - King, Alasdair A1 - Nthangeni, Bethuel A1 - Potgieter, Christiaan A1 - Maartens, Louis A1 - Steven Van Borm A1 - Kris De Clercq KW - LSDV KW - Lumpy skin disease KW - NGS KW - PacBio KW - WGS VL - 5 CP - 6 M3 - 10.1128/genomeA.01657-16 ER - TY - JOUR T1 - Laboratory validation of two real-time RT-PCR methods with 5'-tailed primers for an enhanced detection of foot-and-mouth disease virus. JF - J Virol Methods Y1 - 2017 A1 - Frank Vandenbussche A1 - David Lefebvre A1 - Ilse De Leeuw A1 - Steven Van Borm A1 - Kris De Clercq KW - 5' Untranslated Regions KW - Animals KW - DNA Primers KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Reproducibility of Results KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Sensitivity and Specificity KW - Sheep KW - Swine AB -

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

VL - 246 U1 - https://www.ncbi.nlm.nih.gov/pubmed/28457784?dopt=Abstract M3 - 10.1016/j.jviromet.2017.04.014 ER - TY - JOUR T1 - Laboratory validation of two real-time RT-PCR methods with 5′-tailed primers for an enhanced detection of foot-and-mouth disease virus JF - Journal of Virological Methods Y1 - 2017 A1 - Frank Vandenbussche A1 - David Lefebvre A1 - Ilse De Leeuw A1 - Steven Van Borm A1 - Kris De Clercq VL - 246 M3 - 10.1016/j.jviromet.2017.04.014 ER - TY - JOUR T1 - Complete Genome Sequence of Bovine Polyomavirus Type 1 from Aborted Cattle, Isolated in Belgium in 2014. JF - Genome Announc Y1 - 2016 A1 - Steven Van Borm A1 - Rosseel, Toon A1 - Isabelle Behaeghel A1 - Saulmont, Marc A1 - Delooz, Laurent A1 - Petitjean, Thierry A1 - Elisabeth Mathijs A1 - Frank Vandenbussche AB -

The complete and fully annotated genome sequence of a bovine polyomavirus type 1 (BPyV/BEL/1/2014) from aborted cattle was assembled from a metagenomics data set. The 4,697-bp circular dsDNA genome contains 6 protein-coding genes. Bovine polyomavirus is unlikely to be causally related to the abortion cases.

VL - 4 CP - 2 U1 - https://www.ncbi.nlm.nih.gov/pubmed/26941154?dopt=Abstract M3 - 10.1128/genomeA.01646-15 ER - TY - JOUR T1 - Complete Genome Sequence of Capripoxvirus Strain KSGP 0240 from a Commercial Live Attenuated Vaccine JF - Genome Announcements Y1 - 2016 A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - Andy Haegeman A1 - Al-Majali, Ahmad A1 - Steven Van Borm A1 - Kris De Clercq KW - capripoxvirus KW - complete genome KW - KSGP 0240 KW - LSDV KW - Lumpy skin disease VL - 4 CP - 5 M3 - 10.1128/genomeA.01114-16 ER - TY - JOUR T1 - Complete Genome Sequence of Capripoxvirus Strain KSGP 0240 from a Commercial Live Attenuated Vaccine. JF - Genome Announc Y1 - 2016 A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - Andy Haegeman A1 - Al-Majali, Ahmad A1 - Steven Van Borm A1 - Kris De Clercq AB -

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

VL - 4 CP - 5 M3 - 10.1128/genomeA.01114-16 ER - TY - JOUR T1 - Complete Genome Sequence of Pseudorabies Virus Reference Strain NIA3 Using Single-Molecule Real-Time Sequencing. JF - Genome Announc Y1 - 2016 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Verpoest, Sara A1 - Nick De Regge A1 - Steven Van Borm AB -

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.

VL - 4 CP - 3 U1 - https://www.ncbi.nlm.nih.gov/pubmed/27231370?dopt=Abstract M3 - 10.1128/genomeA.00440-16 ER - TY - JOUR T1 - Complete Genome Sequence of the Goatpox Virus Strain Gorgan Obtained Directly from a Commercial Live Attenuated Vaccine JF - Genome Announcements Y1 - 2016 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Andy Haegeman A1 - Al-Majali, Ahmad A1 - Kris De Clercq A1 - Steven Van Borm KW - capripoxvirus KW - complete genome KW - goatpox virus KW - gorgan strain KW - GTPV VL - 4 CP - 5 M3 - 10.1128/genomeA.01113-16 ER - TY - JOUR T1 - Complete Genome Sequences of the Neethling-Like Lumpy Skin Disease Virus Strains Obtained Directly from Three Commercial Live Attenuated Vaccines JF - Genome Announcements Y1 - 2016 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Andy Haegeman A1 - King, Alasdair A1 - Nthangeni, Bethuel A1 - Potgieter, Christiaan A1 - Maartens, Louis A1 - Steven Van Borm A1 - Kris De Clercq KW - capripoxviridae KW - long-range PCR KW - Lumpy skin disease KW - NGS KW - pacbio sequencing KW - SMRT KW - WGS VL - 4 CP - 6 M3 - 10.1128/genomeA.01255-16 ER - TY - JOUR T1 - Complete Genome Sequences of Three African Foot-and-Mouth Disease Viruses from Clinical Samples Isolated in 2009 and 2010. JF - Genome Announc Y1 - 2016 A1 - Steven Van Borm A1 - Rosseel, Toon A1 - Andy Haegeman A1 - Fana, Mpolokang Elliot A1 - Latoya Seoke A1 - Hyera, Joseph A1 - Matlho, George A1 - Frank Vandenbussche A1 - Kris De Clercq AB -

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

VL - 4 CP - 3 U1 - https://www.ncbi.nlm.nih.gov/pubmed/27151795?dopt=Abstract M3 - 10.1128/genomeA.00326-16 ER - TY - JOUR T1 - Investigation of a Possible Link Between Vaccination and the 2010 Sheep Pox Epizootic in Morocco. JF - Transbound Emerg Dis Y1 - 2016 A1 - Andy Haegeman A1 - Zro, K A1 - Sammin, D A1 - Frank Vandenbussche A1 - Ennaji, M M A1 - Kris De Clercq KW - Animals KW - capripoxvirus KW - Disease Outbreaks KW - Genotype KW - Morocco KW - Phylogeny KW - polymerase chain reaction KW - Poxviridae Infections KW - Sheep KW - Sheep Diseases KW - Vaccination KW - Vaccines, Attenuated AB -

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

VL - 63 CP - 6 U1 - https://www.ncbi.nlm.nih.gov/pubmed/25753969?dopt=Abstract M3 - 10.1111/tbed.12342 ER - TY - JOUR T1 - A Tale of Tails: Dissecting the Enhancing Effect of Tailed Primers in Real-Time PCR. JF - PLoS One Y1 - 2016 A1 - Frank Vandenbussche A1 - Elisabeth Mathijs A1 - David Lefebvre A1 - Kris De Clercq A1 - Steven Van Borm KW - Animals KW - DNA Primers KW - Foot-and-Mouth Disease Virus KW - Humans KW - Real-Time Polymerase Chain Reaction AB -

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

VL - 11 CP - 10 U1 - https://www.ncbi.nlm.nih.gov/pubmed/27723800?dopt=Abstract M3 - 10.1371/journal.pone.0164463 ER - TY - JOUR T1 - Using genomics for surveillance of veterinary infectious agents JF - Revue Scientifique et Technique de l'OIE Y1 - 2016 A1 - Elisabeth Mathijs A1 - Frank Vandenbussche A1 - Steven Van Borm KW - Genomics KW - high-throughput sequencing KW - Metagenomics KW - Next-generation sequencing KW - Surveillance KW - typing KW - whole-genome sequencing VL - 35 CP - 1 M3 - 10.20506/rst.issue.35.1.241210.20506/rst.35.1.2424 ER - TY - JOUR T1 - Full-Genome Sequencing of Four Bluetongue Virus Serotype 11 Viruses. JF - Transbound Emerg Dis Y1 - 2015 A1 - Frank Vandenbussche A1 - Sailleau, C A1 - Rosseel, T A1 - Desprat, A A1 - Viarouge, C A1 - Richardson, J A1 - Eschbaumer, M A1 - Hoffmann, B A1 - Kris De Clercq A1 - Bréard, E A1 - Zientara, S KW - Animals KW - Base Sequence KW - Bluetongue KW - Bluetongue virus KW - Europe KW - Genome, Viral KW - Molecular Sequence Data KW - Phylogeny KW - Serogroup KW - Sheep KW - Vaccination KW - Viral Vaccines AB -

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

VL - 62 CP - 5 U1 - https://www.ncbi.nlm.nih.gov/pubmed/24750582?dopt=Abstract M3 - 10.1111/tbed.12178 ER - TY - JOUR T1 - Development and validation of three Capripoxvirus real-time PCRs for parallel testing. JF - J Virol Methods Y1 - 2013 A1 - Andy Haegeman A1 - Zro, K A1 - Frank Vandenbussche A1 - Demeestere, L A1 - Willem Van Campe A1 - Ennaji, M M A1 - Kris De Clercq KW - Animals KW - capripoxvirus KW - Poxviridae Infections KW - Real-Time Polymerase Chain Reaction KW - Sensitivity and Specificity KW - Veterinary Medicine AB -

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

VL - 193 CP - 2 U1 - https://www.ncbi.nlm.nih.gov/pubmed/23850698?dopt=Abstract M3 - 10.1016/j.jviromet.2013.07.010 ER - TY - JOUR T1 - The origin of biased sequence depth in sequence-independent nucleic acid amplification and optimization for efficient massive parallel sequencing. JF - PLoS One Y1 - 2013 A1 - Rosseel, Toon A1 - Steven Van Borm A1 - Frank Vandenbussche A1 - Hoffmann, Bernd A1 - Thierry van den Berg A1 - Beer, Martin A1 - Höper, Dirk KW - Avulavirus KW - bias KW - DNA Primers KW - DNA, Complementary KW - High-Throughput Nucleotide Sequencing KW - Nucleic Acid Amplification Techniques KW - virology AB -

Sequence Independent Single Primer Amplification is one of the most widely used random amplification approaches in virology for sequencing template preparation. This technique relies on oligonucleotides consisting of a 3' random part used to prime complementary DNA synthesis and a 5' defined tag sequence for subsequent amplification. Recently, this amplification method was combined with next generation sequencing to obtain viral sequences. However, these studies showed a biased distribution of the resulting sequence reads over the analyzed genomes. The aim of this study was to elucidate the mechanisms that lead to biased sequence depth when using random amplification. Avian paramyxovirus type 8 was used as a model RNA virus to investigate these mechanisms. We showed, based on in silico analysis of the sequence depth in relation to GC-content, predicted RNA secondary structure and sequence complementarity to the 3' part of the tag sequence, that the tag sequence has the main contribution to the observed bias in sequence depth. We confirmed this finding experimentally using both fragmented and non-fragmented viral RNAs as well as primers differing in random oligomer length (6 or 12 nucleotides) and in the sequence of the amplification tag. The observed oligonucleotide annealing bias can be reduced by extending the random oligomer sequence and by in silico combining sequence data from SISPA experiments using different 5' defined tag sequences. These findings contribute to the optimization of random nucleic acid amplification protocols that are currently required for downstream applications such as viral metagenomics and microarray analysis.

VL - 8 CP - 9 U1 - https://www.ncbi.nlm.nih.gov/pubmed/24086702?dopt=Abstract M3 - 10.1371/journal.pone.0076144 ER - TY - JOUR T1 - Bluetongue sentinel surveillance program and cross-sectional serological survey in cattle in Belgium in 2010-2011. JF - Prev Vet Med Y1 - 2012 A1 - Vangeel, I A1 - Ilse De Leeuw A1 - Estelle Méroc A1 - Frank Vandenbussche A1 - Riocreux, F A1 - Hooyberghs, J A1 - Raemaekers, M A1 - Houdart, P A1 - Y Van der Stede A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - cross-sectional studies KW - Enzyme-Linked Immunosorbent Assay KW - Female KW - Population Surveillance KW - prevalence KW - Reverse Transcriptase Polymerase Chain Reaction KW - Seasons KW - Seroepidemiologic Studies AB -

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

VL - 106 CP - 3-4 U1 - https://www.ncbi.nlm.nih.gov/pubmed/22483650?dopt=Abstract M3 - 10.1016/j.prevetmed.2012.03.011 ER - TY - JOUR T1 - DNase SISPA-next generation sequencing confirms Schmallenberg virus in Belgian field samples and identifies genetic variation in Europe. JF - PLoS One Y1 - 2012 A1 - Rosseel, Toon A1 - Scheuch, Matthias A1 - Höper, Dirk A1 - Nick De Regge A1 - Frank Vandenbussche A1 - Steven Van Borm KW - Belgium KW - Deoxyribonucleases KW - DNA Primers KW - Genetic Variation KW - High-Throughput Nucleotide Sequencing KW - Molecular Sequence Data KW - Nucleic Acid Amplification Techniques KW - Orthobunyavirus KW - RNA, Viral KW - Sequence Analysis, RNA AB -

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.

VL - 7 CP - 7 U1 - https://www.ncbi.nlm.nih.gov/pubmed/22848676?dopt=Abstract M3 - 10.1371/journal.pone.0041967 ER - TY - JOUR T1 - Phylogeographic analysis of avian influenza viruses isolated from Charadriiformes in Belgium confirms intercontinental reassortment in gulls. JF - Arch Virol Y1 - 2012 A1 - Steven Van Borm A1 - Rosseel, Toon A1 - Vangeluwe, Didier A1 - Frank Vandenbussche A1 - Thierry van den Berg A1 - Bénédicte Lambrecht KW - Animals KW - Belgium KW - Charadriiformes KW - Genetic Variation KW - Influenza A virus KW - Influenza in Birds KW - Nucleic Acid Amplification Techniques KW - Phylogeny KW - Phylogeography KW - Sequence Analysis, RNA AB -

Nine influenza viruses isolated from gulls and shorebirds in Belgium (2008-2010), including H3N8, H5N2, H6N1, H11N9, H13N6, H13N8, and H16N3 subtypes, were targeted using random amplification and next-generation sequencing. The gene segments of these viruses segregated into three phylogeographic lineage types: (1) segments circulating in waterfowl in Eurasia with sporadic introduction in other species and in the Americas ("Eurasian avian"), (2) segments circulating in American waterfowl with sporadic introduction to other species and regions ("American avian"), and (3) segments circulating exclusively in gulls and shorebirds and having increased connectivity between the two hemispheres ("Charadriiformes specific"). Notably, an H6N1 and an H5N2 isolated from L. argentatus had mainly Eurasian avian genes but shared a matrix segment of American avian origin (first documentation in European gulls of transhemispheric reassortment). These data support the growing evidence of an important role of Charadriiformes birds in the dynamic nature of avian influenza ecology.

VL - 157 CP - 8 U1 - https://www.ncbi.nlm.nih.gov/pubmed/22580556?dopt=Abstract M3 - 10.1007/s00705-012-1323-x ER - TY - JOUR T1 - Identification and complete genome sequencing of paramyxoviruses in mallard ducks (Anas platyrhynchos) using random access amplification and next generation sequencing technologies. JF - Virol J Y1 - 2011 A1 - Rosseel, Toon A1 - Bénédicte Lambrecht A1 - Frank Vandenbussche A1 - Thierry van den Berg A1 - Steven Van Borm KW - Animals KW - Avulavirus KW - Avulavirus Infections KW - Belgium KW - Ducks KW - Genetic Variation KW - Genome, Viral KW - Genotype KW - High-Throughput Nucleotide Sequencing KW - Molecular Sequence Data KW - RNA, Viral KW - Serotyping AB -

BACKGROUND: During a wildlife screening program for avian influenza A viruses (AIV) and avian paramyxoviruses (APMV) in Belgium, we isolated two hemagglutinating agents from pools of cloacal swabs of wild mallards (Anas platyrhynchos) caught in a single sampling site at two different times. AIV and APMV1 were excluded using hemagglutination inhibition (HI) testing and specific real-time RT-PCR tests.

METHODS: To refine the virological identification of APMV2-10 realized by HI subtyping tests and in lack of validated molecular tests for APMV2-10, random access amplification was used in combination with next generation sequencing for the sequence independent identification of the viruses and the determination of their genomes.

RESULTS: Three different APMVs were identified. From one pooled sample, the complete genome sequence (15054 nucleotides) of an APMV4 was assembled from the random sequences. From the second pooled sample, the nearly complete genome sequence of an APMV6 (genome size of 16236 nucleotides) was determined, as well as a partial sequence for an APMV4. This APMV4 was closely related but not identical to the APMV4 isolated from the first sample. Although a cross-reactivity with other APMV subtypes did not allow formal identification, the HI subtyping revealed APMV4 and APMV6 in the respective pooled samples but failed to identify the co-infecting APMV4 in the APMV6 infected pool.

CONCLUSIONS: These data further contribute to the knowledge about the genetic diversity within the serotypes APMV4 and 6, and confirm the limited sensitivity of the HI subtyping test. Moreover, this study demonstrates the value of a random access nucleic acid amplification method in combination with massive parallel sequencing. Using only a moderate and economical sequencing effort, the characterization and full genome sequencing of APMVs can be obtained, including the identification of viruses in mixed infections.

VL - 8 U1 - https://www.ncbi.nlm.nih.gov/pubmed/21978491?dopt=Abstract M3 - 10.1186/1743-422X-8-463 ER - TY - JOUR T1 - The impact of naturally-occurring, trans-placental bluetongue virus serotype-8 infection on reproductive performance in sheep. JF - Vet J Y1 - 2011 A1 - Saegerman, Claude A1 - Bolkaerts, Benoît A1 - Baricalla, Christine A1 - Raes, Marianne A1 - Wiggers, Laetitia A1 - Ilse De Leeuw A1 - Frank Vandenbussche A1 - Zimmer, Jean-Yves A1 - Haubruge, Eric A1 - Cassart, Dominique A1 - Kris De Clercq A1 - Kirschvink, Nathalie KW - Abortion, Veterinary KW - Animals KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Disease Outbreaks KW - Female KW - Infectious Disease Transmission, Vertical KW - Placenta KW - Pregnancy KW - Pregnancy Complications, Infectious KW - Serotyping KW - Sheep AB -

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

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

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

VL - 58 CP - 6 U1 - https://www.ncbi.nlm.nih.gov/pubmed/21605347?dopt=Abstract M3 - 10.1111/j.1865-1682.2011.01230.x ER - TY - JOUR T1 - Bluetongue virus in wild deer, Belgium, 2005-2008. JF - Emerg Infect Dis Y1 - 2010 A1 - Linden, Annick A1 - Gregoire, Fabien A1 - Nahayo, Adrien A1 - Hanrez, David A1 - Mousset, Benedicte A1 - Massart, Audrey Laurent A1 - Ilse De Leeuw A1 - Vandemeulebroucke, Elise A1 - Frank Vandenbussche A1 - Kris De Clercq KW - Animals KW - Antibodies, Viral KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Deer KW - Disease Reservoirs KW - Geography KW - RNA, Viral KW - Seroepidemiologic Studies KW - Serotyping KW - Sheep KW - Spleen AB -

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

VL - 16 CP - 5 U1 - https://www.ncbi.nlm.nih.gov/pubmed/20409376?dopt=Abstract M3 - 10.3201/eid1605.091217 ER - TY - JOUR T1 - A duplex real-time RT-PCR for the detection of bluetongue virus in bovine semen. JF - J Virol Methods Y1 - 2010 A1 - Vanbinst, Tine A1 - Frank Vandenbussche A1 - Dernelle, Eric A1 - Kris De Clercq KW - Animals KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Reproducibility of Results KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - semen KW - Sensitivity and Specificity KW - virology AB -

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

VL - 169 CP - 1 U1 - https://www.ncbi.nlm.nih.gov/pubmed/20674609?dopt=Abstract M3 - 10.1016/j.jviromet.2010.07.019 ER - TY - JOUR T1 - The most likely time and place of introduction of BTV8 into Belgian ruminants. JF - PLoS One Y1 - 2010 A1 - Saegerman, Claude A1 - Mellor, Philip A1 - Uyttenhoef, Aude A1 - Jean-Baptiste Hanon A1 - Kirschvink, Nathalie A1 - Haubruge, Eric A1 - Delcroix, Pierre A1 - Houtain, Jean-Yves A1 - Pourquier, Philippe A1 - Frank Vandenbussche A1 - Verheyden, Bart A1 - Kris De Clercq A1 - Czaplicki, Guy KW - Animals KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Ceratopogonidae KW - Geography KW - Insect Vectors KW - Population Density KW - Retrospective Studies KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Ruminants KW - Serotyping KW - Sheep KW - Sheep Diseases KW - Time Factors AB -

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

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

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

VL - 5 CP - 2 U1 - https://www.ncbi.nlm.nih.gov/pubmed/20195379?dopt=Abstract M3 - 10.1371/journal.pone.0009405 ER - TY - JOUR T1 - A proposed validation method for automated nucleic acid extraction and RT-qPCR analysis: an example using Bluetongue virus. JF - J Virol Methods Y1 - 2010 A1 - Vandemeulebroucke, Elise A1 - Kris De Clercq A1 - Yves Van der Stede A1 - Frank Vandenbussche KW - Animals KW - Automation KW - Bluetongue virus KW - Cell Line KW - Chick Embryo KW - Clinical Laboratory Techniques KW - Cricetinae KW - Diagnostic Errors KW - nucleic acids KW - Reference Standards KW - Reproducibility of Results KW - Reverse Transcriptase Polymerase Chain Reaction KW - Validation Studies as Topic AB -

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

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

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

VL - 630 U1 - https://www.ncbi.nlm.nih.gov/pubmed/20300993?dopt=Abstract M3 - 10.1007/978-1-60761-629-0_7 ER - TY - JOUR T1 - Bluetongue in Belgium: episode II. JF - Transbound Emerg Dis Y1 - 2009 A1 - Estelle Méroc A1 - Herr, C A1 - Verheyden, B A1 - Hooyberghs, J A1 - Houdart, P A1 - Raemaekers, M A1 - Frank Vandenbussche A1 - Kris De Clercq A1 - Mintiens, K KW - Abortion, Veterinary KW - Animals KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Disease Outbreaks KW - Female KW - Goat Diseases KW - Goats KW - Male KW - Pregnancy KW - Serotyping KW - Sheep AB -

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

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

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

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

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

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

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

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

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

VL - 136 CP - 3-4 U1 - http://www.ncbi.nlm.nih.gov/pubmed/19128895?dopt=Abstract M3 - 10.1016/j.vetmic.2008.11.012 ER - TY - JOUR T1 - Validation of two real-time RT-PCR methods for foot-and-mouth disease diagnosis: RNA-extraction, matrix effect, uncertainty of measurement and precision. JF - J Virol Methods Y1 - 2009 A1 - Goris, Nesya A1 - Frank Vandenbussche A1 - Herr, Cécile A1 - Villers, Jérôme A1 - Yves Van der Stede A1 - Kris De Clercq KW - Animals KW - blood KW - Diagnostic Errors KW - Foot KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - Reverse Transcriptase Polymerase Chain Reaction KW - RNA, Viral KW - Sensitivity and Specificity AB -

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

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

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

VL - 147 CP - 1 U1 - https://www.ncbi.nlm.nih.gov/pubmed/17913251?dopt=Abstract M3 - 10.1016/j.jviromet.2007.08.020 ER - TY - JOUR T1 - Bluetongue in Eurasian lynx. JF - Emerg Infect Dis Y1 - 2008 A1 - Jauniaux, Thierry P A1 - Kris De Clercq A1 - Cassart, Dominique E A1 - Kennedy, Seamus A1 - Frank Vandenbussche A1 - Vandemeulebroucke, Elise L A1 - Vanbinst, Tine M A1 - Verheyden, Bart I A1 - Goris, Nesya E A1 - Coignoul, Freddy L KW - Animals KW - Animals, Zoo KW - Bluetongue KW - Lung KW - Lynx VL - 14 CP - 9 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18760034?dopt=Abstract M3 - 10.3201/eid1409.080434 ER - TY - JOUR T1 - Bluetongue virus antibodies in wild red deer in southern Belgium. JF - Vet Rec Y1 - 2008 A1 - Linden, Annick A1 - Mousset, Benedicte A1 - Gregoire, Fabien A1 - Hanrez, David A1 - Frank Vandenbussche A1 - Vandemeulebroucke, Elise A1 - Vanbinst, Tine A1 - Verheyden, Bart A1 - de Clerck, Kris KW - Animals KW - Antibodies, Viral KW - Belgium KW - Bluetongue KW - Bluetongue virus KW - Deer KW - Sentinel Surveillance KW - Seroepidemiologic Studies KW - Sheep VL - 162 CP - 14 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18390860?dopt=Abstract ER - TY - JOUR T1 - Bluetongue virus serotype 8-associated congenital hydranencephaly in calves. JF - Transbound Emerg Dis Y1 - 2008 A1 - Vercauteren, G A1 - Miry, C A1 - Frank Vandenbussche A1 - Ducatelle, R A1 - Van der Heyden, S A1 - Vandemeulebroucke, E A1 - Ilse De Leeuw A1 - Deprez, P A1 - Chiers, K A1 - Kris De Clercq KW - Animals KW - Animals, Newborn KW - Bluetongue KW - Bluetongue virus KW - Cattle KW - Cattle Diseases KW - Female KW - Hydranencephaly KW - Infectious Disease Transmission, Vertical KW - Male KW - Pregnancy KW - Pregnancy Complications, Infectious KW - RNA, Viral AB -

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

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

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

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

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

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

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

VL - 129 CP - 1-2 U1 - https://www.ncbi.nlm.nih.gov/pubmed/18093753?dopt=Abstract M3 - 10.1016/j.vetmic.2007.10.029 ER - TY - JOUR T1 - Potential of antiviral therapy and prophylaxis for controlling RNA viral infections of livestock. JF - Antiviral Res Y1 - 2008 A1 - Goris, Nesya A1 - Frank Vandenbussche A1 - Kris De Clercq KW - Animals KW - Animals, Domestic KW - Antiviral Agents KW - Cattle KW - Chemoprevention KW - Foot-and-Mouth Disease KW - Foot-and-Mouth Disease Virus KW - RNA Virus Infections KW - RNA Viruses KW - Viral Vaccines AB -

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

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

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

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

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

VL - 55 CP - 8 U1 - http://www.ncbi.nlm.nih.gov/pubmed/18673339?dopt=Abstract M3 - 10.1111/j.1865-1682.2008.01044.x ER -