Equine encephalosis virus: evidence for circulation beyond southern Africa.

Prior to the recent outbreak of equine encephalosis in Israel in 2009, equine encephalosis virus (EEV) had only been isolated from equids in South Africa. In this study we show the first evidence for the circulation of EEV beyond South Africa in Ethiopia, Ghana and The Gambia, indicating that EEV is likely to be freely circulating and endemic in East and West Africa. Sequence analysis revealed that the EEV isolate circulating in The Gambia was closely related to an EEV isolate that was isolated from a horse from Israel during the EEV outbreak in 2009, indicating that the two viruses have a common ancestry. Interestingly horses in Morocco tested negative for EEV antibodies indicating that the Sahara desert may be acting as a geographical barrier to the spread to the virus to North African countries. This evidence for EEV circulation in countries in East and West Africa sheds light on how the virus may have reached Israel to cause the recent outbreak in 2009.

Virological, immunological and pathological findings of transplacentally transmitted bluetongue virus serotype 1 in IFNAR1-blocked mice during early and mid gestation.

Transplacental transmission (TPT) of wild-type Indian BTV-1 had never been experimentally proved. This study was first time investigated TPT of Indian BTV-1 (isolated from aborted and stillborn goat fetal spleens). The sequential pathology, virological and immune cell kinetics (CD4+, CD8+ T-lymphocytes and NK cells in spleen and PBMCs), and apoptosis in IFNAR1-blocked pregnant mice during early (infected on 1 GD) and mid (infected on 8 GD) gestation have been studied. There was higher rate of TPT during mid stage (71.43%) than early (57.14%) stage. In early stage reduced implantation sites, early embryonic deaths, abortions, and necro-haemorrhagic lesions had observed. Mid stage, congenital defects and neurological lesions in foetuses like haemorrhages, diffuse cerebral edema, necrotizing encephalitis and decreased bone size (Alizarin red staining) were noticed. BTV-1 antigen was first time demonstrable in cells of mesometrium, decidua of embryos, placenta, uterus, ovary, and brain of foetuses by immunohistochemistry and quantified by real-time qRT-PCR. BTV-inoculated mice were seroconverted by 7 and 5 dpi, and reached peak levels by 15 and 9 dpi in early and mid gestation, respectively. CD4+ and CD8+ cells were significantly decreased (increased ratio) on 7 dpi but subsequently increased on 15 dpi in early gestation. In mid gestation, increased CD8+ cells (decreased ratio) were observed. Apoptotic cells in PBMCs and tissues increased during peak viral load. This first time TPT of wild-type Indian BTV-1 deserves to be reported for implementation of control strategies. This model will be very suitable for further research into mechanisms of TPT, overwintering, and vaccination strategies.

Comparative Neuropathology of Major Indian Bluetongue Virus Serotypes in a Neonatal BALB/c Mouse Model.

Bluetongue virus (BTV) is neurotropic in nature, especially in ruminant fetuses and in-utero infection results in abortion and congenital brain malformations. The aim of the present study was to compare the neuropathogenicity of major Indian BTV serotypes 1, 2, 10, 16 and 23 by gross and histopathological lesions and virus distribution in experimentally infected neonatal BALB/c mice. Each BTV serotype (20 µl of inoculum containing 1 × 105 tissue culture infectious dose [TCID]50/ml of virus) was inoculated intracerebrally into 3-day-old mice, while a control group was inoculated with mock-infected cell culture medium. Infection with BTV serotypes 1, 2 and 23 led to 65-70% mortality at 7-9 days post infection (dpi) and caused severe necrotizing encephalitis with neurodegenerative changes in neurons, swelling and proliferation of vascular endothelial cells in the cerebral cortex, cerebellum, midbrain and brainstem. In contrast, infection with BTV serotypes 10 and 16 led to 25-30% mortality at 9-11 dpi and caused mild neuropathological lesions. BTV antigen was detected by immunohistochemistry, direct fluorescence antibody technique and confocal microscopy in the cytoplasm of neuronal cells of the hippocampus, grey matter of the cerebral cortex and vascular endothelial cells in the midbrain and brainstem of BTV-1, -2, -10, -16 and -23 infected groups from 3 to 20 dpi. BTV nucleic acid was detected in the infected brain tissues from as early as 24 h up to 20 dpi by VP7 gene segment-based one-step reverse transcriptase polymerase chain reaction. This study of the relative neurovirulence of BTV serotypes is likely to help design suitable vaccination and control strategies for the disease.

Concurrent infection of Bluetongue and Peste-des-petits-ruminants virus in small ruminants in Haryana State of India.

Bluetongue (BT) and peste-des-petits-ruminants (PPR) are major transboundary diseases of small ruminant, which are endemic in India. Testing of bluetongue virus (BTV) and peste-des-petits-ruminants virus (PPRV) from recent outbreaks (2015-2016) in different regions of Haryana State of India revealed that 27.5% of the samples showed the presence of dual infection of BTV and PPRV. Analysis of Seg-2 of BTV (the serotype-determining protein) showed the presence of BTV-12w in several isolates. However, analysis of N gene fragment amplicons showed that viruses belong to lineage IV were most closely related to a pathogenic strain of PPRV from Delhi. This is the first report of co-circulation of PPRV lineage IV and bluetongue virus serotype 12 in the state.

Isolation and evolutionary analysis of Australasian topotype of bluetongue virus serotype 4 from India.

Bluetongue (BT) is a Culicoides-borne disease caused by several serotypes of bluetongue virus (BTV). Similar to other insect-borne viral diseases, distribution of BT is limited to distribution of Culicoides species competent to transmit BTV. In the tropics, vector activity is almost year long, and hence, the disease is endemic, with the circulation of several serotypes of BTV, whereas in temperate areas, seasonal incursions of a limited number of serotypes of BTV from neighbouring tropical areas are observed. Although BTV is endemic in all the three major tropical regions (parts of Africa, America and Asia) of the world, the distribution of serotypes is not alike. Apart from serological diversity, geography-based diversity of BTV genome has been observed, and this is the basis for proposal of topotypes. However, evolution of these topotypes is not well understood. In this study, we report the isolation and characterization of several BTV-4 isolates from India. These isolates are distinct from BTV-4 isolates from other geographical regions. Analysis of available BTV seg-2 sequences indicated that the Australasian BTV-4 diverged from African viruses around 3,500 years ago, whereas the American viruses diverged relatively recently (1,684 CE). Unlike Australasia and America, BTV-4 strains of the Mediterranean area evolved through several independent incursions. We speculate that independent evolution of BTV in different geographical areas over long periods of time might have led to the diversity observed in the current virus population.

Development of Real-Time RT-PCR Assays for Detection and Typing of Epizootic Haemorrhagic Disease Virus.

Epizootic haemorrhagic disease virus (EHDV) is an emerging arboviral pathogen of wild and domestic ruminants worldwide. It is closely related to bluetongue virus (BTV) and is transmitted by adult females of competent Culicoides vector species. The EHDV genome consists of ten linear double-stranded (ds)RNA segments, encoding five non-structural and seven structural proteins. Genome-segment reassortment contributes to a high level of genetic variation in individual virus strains, particularly in the areas where multiple and distinct virus lineages co-circulate. In spite of the relatively close relationship between BTV and EHDV herd-immunity to BTV does not appear to protect against the introduction and infection of animals by EHDV. Although EHDV can cause up to 80% morbidity in affected animals, vaccination with the homologous EHDV serotype is protective. Outer-capsid protein VP2, encoded by Seg-2, is the most variable of the EHDV proteins and determines both the specificity of reactions with neutralizing antibodies and consequently the identity of the eight EHDV serotypes. In contrast, VP6 (the viral helicase), encoded by Seg-9, is highly conserved, representing a virus species/serogroup-specific antigen. We report the development and evaluation of quantitative (q)RT-PCR assays targeting EHDV Seg-9 that can detect all EHDV strains (regardless of geographic origin/topotype/serotype), as well as type-specific assays targeting Seg-2 of the eight EHDV serotypes. The assays were evaluated using orbivirus isolates from the 'Orbivirus reference collection' (ORC) at The Pirbright Institute and were shown to be EHDV pan-reactive or type-specific. They can be used for rapid, sensitive and reliable detection and identification (typing) of EHDV RNA from infected blood, tissue samples, homogenized Culicoides, or tissue culture supernatant. None of the assays detected RNA from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures. The techniques presented could be used for both surveillance and vaccine matching (serotype identification) as part of control strategies for incursions in wild and domestic animal species.

Nutrigenomic evaluation of garlic (Allium sativum) and holy basil (Ocimum sanctum) leaf powder supplementation on growth performance and immune characteristics in broilers.

AIM: In this study, a planned research work was conducted to investigate the nutrigenomic aspects of supplementation of Allium sativum (garlic) and Ocimum sanctum (holy basil) leaf powder on the growth performance and immune characteristics of broilers. MATERIALS AND METHODS: A 6 weeks feeding trial was conducted with 280-day-old Ven Cobb broilers, distributed randomly into seven experimental groups. Each treatment had 4 replicates with 10 birds each. The birds of the control group (T1) were fed a basal diet formulated as per BIS standards. The broilers of treatment groups T2 and T3 were fed basal diet supplemented with the commercially available garlic powder (GP) at levels of 0.5% and 1.0% of the feed, respectively, while broilers in T4 and T5 were fed basal diet supplemented with commercial grade holy basil leaf powder (HBLP) at levels 0.5% and 1.0% of the feed, respectively. Birds in the T6 were fed with 0.5% GP and 0.5% HBLP, whereas T7 was fed with 1.0% GP and 1.0% HBLP. At the end of the feeding trial (6th week), blood samples were collected and analyzed for relative mRNA expression of toll-like receptors (TLR) 2, TLR 4 and TLR 7 using real-time polymerase chain reaction. RESULTS: The mean body weight gain and feed conversion efficiency were improved (p<0.05) in broilers fed the GP and HBLP incorporated diets compared with the control group. The relative mRNA expression levels of TLR 2, TLR 4 and TLR 7 in the peripheral blood of the broilers were found to be increased (p<0.05) in the birds supplemented with graded levels of the GP and HBLP as compared to the untreated group. CONCLUSION: The present work concludes that the inclusion of GP and HBLP could enhance the production performance and immune status of birds by augmenting the T-cell mediated immune response and thereby protects them from disease without decreasing growth traits as a possible substitution to conventional antimicrobials.

Dual Infection with Bluetongue Virus Serotypes and First-Time Isolation of Serotype 5 in India.

Bluetongue is endemic in India and has been reported from most Indian states. Of late, the clinical disease is most frequently seen in the states of Andhra Pradesh, Telangana (erstwhile Andhra Pradesh state), Tamil Nadu and Karnataka. Our analysis of diagnostic samples from bluetongue outbreaks during 2010-2011 from the state of Karnataka identified bluetongue virus (BTV) serotype 5 (BTV-5) for the first time in India. One of the diagnostic samples (CH1) and subsequent virus isolate (IND2010/02) contained both BTV-2 and BTV-5. Segment 2 (seg-2) sequence data (400 bp: nucleotides 2538-2921) for IND2010/02-BTV5, showed 94.3% nucleotide identity to BTV-5 from South Africa (Accession no. AJ585126), confirming the virus serotype and also indicating that Seg-2 was derived from a Western topotype, which is in contrast to serotype 2, that belongs to an Eastern topotype. BTV-5 has been recently reported from Africa, China, French islands and the Americas. Although the exact source of the Indian BTV-5 isolate is still to be confirmed, recent identification of additional exotic serotypes in India is of real concern and might add to the severity of the disease seen in these outbreaks.

Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India.

Bluetongue virus (BTV) infects all ruminants, including cattle, goats and camelids, causing bluetongue disease (BT) that is often severe in naïve deer and sheep. Reverse-transcription-loop-mediated-isothermal-amplification (RT-LAMP) assays were developed to detect eastern or western topotype of BTV strains circulating in India. Each assay uses four primers recognizing six distinct sequences of BTV genome-segment 1 (Seg-1). The eastern (e)RT-LAMP and western (w)RT-LAMP assay detected BTV RNA in all positive isolates that were tested (n=52, including Indian BTV-1, -2, -3, -5, -9, -10, -16, -21 -23, and -24 strains) with high specificity and efficiency. The analytical sensitivity of the RT-LAMP assays is comparable to real-time RT-PCR, but higher than conventional RT-PCR. The accelerated eRT-LAMP and wRT-LAMP assays generated detectable levels of amplified DNA, down to 0.216 fg of BTV RNA template or 108 fg of BTV RNA template within 60-90min respectively. The assays gave negative results with RNA from foot-and-mouth-disease virus (FMDV), peste des petits ruminants virus (PPRV), or DNA from Capripox viruses and Orf virus (n=10), all of which can cause clinical signs similar to BT. Both RT-LAMP assays did not show any cross-reaction among themselves. The assays are rapid, easy to perform, could be adapted as a 'penside' test making them suitable for 'front-line' diagnosis, helping to identify and contain field outbreaks of BTV.

Isolation of Bluetongue Virus 24 from India - An Exotic Serotype to Australasia.

Bluetongue (BT) is a viral disease of ruminants and is caused by different serotypes of bluetongue virus (BTV), which is transmitted by several species of Culicoides midges. The disease is endemic in tropical areas, and incursions have been observed in some of the temperate areas. Twenty-seven recognized serotypes of BTV have been reported so far. Some serotype viruses have been shown to circulate in certain geographical areas. BTV-24 has been reported from Africa, the Mediterranean and the Americas, whereas it is exotic to Australasia. Here, we report isolation of BTV-24 from India and show that it has high sequence homology in genome segment 2 with other Western isolates of BTV-24. Entry of this serotype into Australasian region is a cause of concern.

Molecular epidemiology of bluetongue virus serotype 1 isolated in 2006 from Algeria.

This study reports on an outbreak of disease that occurred in central Algeria during July 2006. Sheep in the affected area presented clinical signs typical of bluetongue (BT) disease. A total of 5245 sheep in the affected region were considered to be susceptible, with 263 cases and thirty-six deaths. Bluetongue virus (BTV) serotype 1 was isolated and identified as the causative agent. Segments 2, 7 and 10 of this virus were sequenced and compared with other isolates from Morocco, Italy, Portugal and France showing that they all belong to a 'western' BTV group/topotype and collectively represent a western Mediterranean lineage of BTV-1.

RNA segment 9 exists as a duplex concatemer in an Australian strain of epizootic haemorrhagic disease virus (EHDV): Genetic analysis and evidence for the presence of concatemers as a normal feature of orbivirus replication.

This paper reports a concatemeric RNA in a strain of epizootic haemorrhagic disease virus (EHDV) serotype 5. Sequencing showed that the concatemeric RNA contains two identical full-length copies of genome segment 9, arranged in series, which has apparently replaced the monomeric form of the segment. In vitro translation demonstrated that the concatemeric RNA can act as a viable template for VP6 translation, but that no double-sized protein is produced. Studies were also performed to assess whether mutations might be easily introduced into the second copy (which might indicate some potential evolutionary significance of a concatemeric RNA segment), however multiple (n=40) passages generated no changes in the sequence of either the upstream or downstream segments. Further, we present results that demonstrate the presence of concatemers or partial gene duplications in multiple segments of different orbiviruses (in tissue culture and purified virus), suggesting their generation is likely to be a normal feature of orbivirus replication.

Genetic and phylogenetic analysis of the non-structural proteins NS1, NS2 and NS3 of epizootic haemorrhagic disease virus (EHDV).

Three unique non-structural (NS) proteins are produced by Epizootic haemorrhagic disease virus (EHDV) during infection of a host cell; NS1, NS2 and NS3. This study presents a complete genetic and phylogenetic analysis of these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each. Accession numbers, gene and protein sizes, ORF positions, G+C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. Unlike the core, or outer-coat proteins, there are no characteristic genetic or phylogenetic traits common to all of the EHDV NS proteins; indicating that each is evolving under different selection pressures. These differences are discussed. Evidence of genetic recombination in genome segment 8 (coding for NS2) is also presented, together with evidence of gene duplication and mutation, suggesting the EHDV genome may have evolved using mechanisms such as these.

Genetic and phylogenetic analysis of the core proteins VP1, VP3, VP4, VP6 and VP7 of epizootic haemorrhagic disease virus (EHDV).

The core proteins of epizootic haemorrhagic disease virus (EHDV) have important roles to perform in maintaining the structure and function of the virus. A complete genetic and phylogenetic analysis was therefore performed on these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each. Accession numbers, gene and protein sizes, ORF positions, G+C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. The inner core proteins (VP1, VP3, VP4 and VP6) were characterised by high levels of sequence conservation, and the ability to topotype isolates very strongly into eastern or western groups. This is particularly evident in genome segment 9 (VP6) which exists as two different sized homologues. VP7 did not topotype, but rather exhibited a more random, radial phylogeny suggestive of genetic drift. With the exception of VP6, all of the core proteins also showed high numbers of synonymous mutations in the third base position, suggesting they have been evolving for a long period of time. Interestingly, VP6 did not show this, and possible reasons for this are discussed.

Genetic and phylogenetic analysis of the outer-coat proteins VP2 and VP5 of epizootic haemorrhagic disease virus (EHDV): comparison of genetic and serological data to characterise the EHDV serogroup.

The outer-coat proteins, VP2 and VP5, of epizootic haemorrhagic disease virus (EHDV) are important for host cell binding during the initiation of infection. They are also known to determine virus serotype. This study presents a complete genetic and phylogenetic analysis of these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each and the correlation of genetic sequence data with serotype. Accession numbers, gene and protein sizes, ORF positions, G+C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. The results show that VP2 is highly variable, is under great pressure to adapt and can be correlated with serotype. While also variable, VP5 appears to be under less adaptive pressure than VP2 but still shows some correlation with serotype. Seven serotypes of EHDV have been defined in this study, although the results do show that some serotypes are extremely closely related--and highlight the benefit of using both molecular and serologic analyses. Analysis of the terminal hexanucleotides showed that the 5' terminus is under greater purifying selection than the 3'. Evidence is also presented that both segments 2 and 6 (coding for VP2 and VP5 respectively) have grown via gene duplication and subsequent mutation.

Emergence of bluetongue serotypes in Europe, part 2: the occurrence of a BTV-11 strain in Belgium.

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.

Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes.

The outer capsid protein VP2 of Bluetongue virus (BTV) is a target for the protective immune response generated by the mammalian host. VP2 contains the majority of epitopes that are recognized by neutralizing antibodies and is therefore also the primary determinant of BTV serotype. Full-length cDNA copies of genome segment 2 (Seg-2, which encodes VP2) from the reference strains of each of the 24 BTV serotypes were synthesized, cloned and sequenced. This represents the first complete set of full-length BTV VP2 genes (from the 24 serotypes) that has been analysed. Each Seg-2 has a single open reading frame, with short inverted repeats adjacent to conserved terminal hexanucleotide sequences. These data demonstrated overall inter-serotype variations in Seg-2 of 29 % (BTV-8 and BTV-18) to 59 % (BTV-16 and BTV-22), while the deduced amino acid sequence of VP2 varied from 22.4 % (BTV-4 and BTV-20) to 73 % (BTV-6 and BTV-22). Ten distinct Seg-2 lineages (nucleotypes) were detected, with greatest sequence similarities between those serotypes that had previously been reported as serologically 'related'. Fewer similarities were observed between different serotypes in regions of VP2 that have been reported as antigenically important, suggesting that they may play a role in the neutralizing antibody response. The data presented form an initial basis for BTV serotype identification by sequence analyses and comparison of Seg-2, and for development of molecular diagnostic assays for individual BTV serotypes (by RT-PCR).

Design of primers and use of RT-PCR assays for typing European bluetongue virus isolates: differentiation of field and vaccine strains.

Bluetongue virus (BTV) is the causative agent of bluetongue, a disease of ruminant livestock that occurs almost worldwide between latitudes 3 degrees S and 5 degrees N. There are 24 serotypes of BTV (currently identified by serum neutralization assays). Since 1998, eight strains of six BTV serotypes (1, 2, 4, 8, 9 and 16) have invaded Europe. The most variable BTV protein is major outer-capsid component VP2, encoded by segment 2 (Seg-2) of the double-stranded RNA virus genome. VP2 represents the major target for neutralizing (and protective) antibodies that are generated in response to BTV infection, and is therefore the primary determinant of virus serotype. RT-PCR primers and assays targeting Seg-2 have been developed for rapid identification (within 24 h) of the six European BTV types. These assays are sensitive, specific and show perfect agreement with the results of conventional virus-neutralization methods. Previous studies have identified sequence variations in individual BTV genome segments that allow different isolates to be grouped on the basis of their geographical origins (topotypes). The assays described in this paper can detect any of the BTV isolates of the homologous serotype that were tested from different geographical origins (different Seg-2 topotypes). Primers were also identified that could be used to distinguish members of these different Seg-2 topotypes, as well as field and vaccine strains of most of the European BTV serotypes. The serotype-specific assays (and primers) showed no cross-amplification when they were evaluated with multiple isolates of the most closely related BTV types or with reference strains of the remaining 24 serotypes. Primers developed in this study will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).

Molecular epidemiology of bluetongue viruses from disease outbreaks in the Mediterranean Basin.

Bluetongue virus (BTV) serotype is primarily controlled by the variable outer coat protein VP2, encoded by genome segment 2. Phylogenetic analyses of segment 2 show that recent Mediterranean isolates of BTV-2 have a similar genetic lineage to those from sub-Saharan Africa and North America but are distinct from Asian strains. In contrast, isolates of BTV-9, from the eastern Mediterranean, are related to a genetic lineage from Asia. BTV-1 from Greece 2001 is also more closely related to Indian isolates, suggesting (in both cases) virus movement from east to west. Recent BTV-4 field isolates from Greece and Turkey are similar to each other, but differ from the Turkish type 4 vaccine strain. These sequencing studies are being used to establish a database for molecular epidemiological studies which is available on the website of the Institute for Animal Health. This resource will support and improve BTV serotype identification methods, by using sequence comparisons (via the Web) rather than by conventional serological techniques that require standardised (and therefore expensive) serological reagents. Phylogenetic trees for BTV genome segment 2 are available on the website.

Development of reverse transcriptase-polymerase chain reaction-based assays and sequencing for typing European strains of bluetongue virus and differential diagnosis of field and vaccine strains.

Bluetongue virus (BTV) is a double-stranded (ds) RNA virus, classified within the genus Orbivirus, family Reoviridae, which causes bluetongue (BT), an infectious, non-contagious disease of ruminants. The virus exists as 24 distinct serotypes, which are currently identified by virus isolation and serum neutralisation assays. The most variable outer capsid protein VP2 (encoded by genome segment 2), is the primary determinant of BTV serotype. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays, based on amplification of segment 2, have been developed for identification of the five European BTV types (BTV-1, BTV-2, BTV-4, BTV-9 and BTV-16). Primer pairs were designed that are specific for each BTV serotype. The resulting RT-PCR assay was both sensitive and specific, providing BTV typing within 24 h. Perfect agreement was recorded between the RT-PCR and virus neutralisation assays. The primers for each serotype could successfully amplify the BTV isolates of that serotype from different regions and showed no cross-amplification of the most closely related BTV serotypes. RT-PCR primers were also developed for the discrimination of field and vaccine strains of BTV serotypes currently circulating in Europe. The primer pairs which could amplify field and vaccine strains of BTV-1, BTV-2, BTV-4 and BTV-9 were validated with several isolates of each serotype from various geographic origins around the world and their type specificity was again tested with the most closely related serotypes. Overall, these RT-PCR assays provide a rapid and reliable method for the identification and differentiation of field and vaccine strains of different BTV types. The primers used in this study are listed on the website of the Institute for Animal Health, Pirbright.