Bluetongue Virus Serotype Research Articles

Defeating Bluetongue Virus: New Approaches in the Development of Multiserotype Vaccines

Bluetongue virus (BTV) is a global threat to domestic and wild ruminants, causing massive economic losses throughout the world. New serotypes of the virus are rapidly emerging in different continents, unfortunately there is little cross-protection between BTV serotypes. The eradication of the virus from a region is particularly complicated in areas where multiple serotypes circulate for a long time. The present review summarizes the actual concerns about the spread of the virus and relevant approaches to develop efficient vaccines against BTV, in particular those focused on a multiserotype design.

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.

HIGH SEROPREVALENCE OF BLUETONGUE IN SHEEP, GOATS AND CATTLE IN ASSIUT GOVERNORATE, EGYPT

A total of 552 serum samples of sheep, goats and cattle from different localities at Assiut governorate, Egypt were screened for qualitative analysis of the BTV antibodies using a commercial competitive ELISA (cELISA) kit. The results showed an overall percentage of BTV positive sheep, goats and cattle serum samples were 41.86%, 24% and 85.42% respectively. A highest percentage of seropositivity was found inArab El- Awamer farm (100%), followed by Abnoub( 63. 64%), Sedfa(11. 43%), veterinary teaching hospital (11.11%), Aboteeg (3.57%) and Bani zeed el akrad (0% ) respectively. From examined sera all over the four years from 2012 to 2015 indicates prevalence of BTV antibodies by ( 62. 69%) during the non hot months and (10%) during the hot months, Female ruminants were more susceptible to the infection (61.29%) than male( 21.67%). The association between sex and prevalence of BT disease among ruminants observed in table (4). The results showed that the prevalence of BT was higher in female (61.29%) than male (21.67%). In conclusion, this study confirms the presence of blue tongue infection in Assiut Governorate, and cELISA were found to be sensitive and effective for screening of BTV group specific antibodies. The disease should be notifying its presence in Egypt since it was free from this disease and more studies should be attempets to estimate the serotype of blue tongue virus

European Bluetongue Serotype 8: Disease Threat Assessment for U.S. Sheep.

Bluetongue virus (BTV) is an orbivirus transmitted by biting midges (Culicoides spp.) that can result in moderate to high morbidity and mortality primarily in sheep and white-tailed deer. Although only 5 serotypes of BTV are considered endemic to the United States, as many as 11 incursive serotypes have been detected in livestock and wildlife in the past 16 years. Introductions of serotypes, with unknown virulence and disease risk, are constant threats to US agriculture. One potential incursive serotype of particular concern is the European strain of BTV-8, which was introduced into Northern Europe in 2006 and caused unprecedented livestock disease and mortality during the 2006-2007 vector seasons. To assess disease risk of BTV-8 in a common white-faced American sheep breed, eight Polled Dorset yearlings were experimentally infected and monitored for clinical signs. Viremia and viral tissue distribution were detected and quantified by real-time qRT-PCR. Overall, clinical disease was moderate with no mortality. Viremia reached as high as 9.7 log10 particles/mL and persisted at 5 logs or higher through the end of the study (28 days). Virus distribution in tissues was extensive with the highest mean titers at the peak of viremia (day 8) in the kidney (8.38 log10 particles/mg) and pancreas (8.37 log10 particles/mg). Virus persisted in tissues of some sheep at 8 logs or higher by day 28. Results of this study suggest that should BTV-8 emerge in the United States, clinical disease in this common sheep breed would likely be similar in form, duration, and severity to what is typically observed in severe outbreaks of endemic serotypes, not the extraordinary disease levels seen in Northern Europe. In addition, a majority of exposed sheep would be expected to survive and act as significant BTV-8 reservoirs with high titer viremias for subsequent transmission to other livestock and wildlife populations.

Complete Genome Sequence of Bluetongue Virus Serotype 8, Which Reemerged in France in August 2015.

We announce here the complete genome sequence (coding and noncoding) of the bluetongue virus (BTV) serotype 8, isolated from a ram in Allier department, France, 2015. Sequence analysis confirms the reemergence of the BTV-8 strain that previously circulated in France until 2009 and other European countries until 2010.

First detection of bluetongue virus serotype 14 in Poland.

Here, we present the first detected cases of bluetongue virus (BTV) in native cattle from Poland. The virus was found in animals located near the Polish-Belarusian and Polish-Lithuanian borders. The positive animals were detected through an official epidemiological surveillance program. A combination of type-specific real-time RT-PCR and phylogenetic tests revealed the presence of BTV serotype 14 (BTV-14). This serotype is highly homologous to the vaccine strain and BTV-14 present in Russia, Lithuania, and Spain (from an animal imported from Lithuania). The most probable route of virus introduction to Poland was transmission through midges. All of the cases were subclinical.

Epidemiology of bluetongue virus in Australasia

Background: Bluetongue is a vector-borne disease of ruminants caused by bluetongue virus (BTV) belonging to Reoviridae. So far, 28 serotypes of BTV have been reported, and distribution of various serotypes is not uniform among different geographical areas. The virus is endemic in tropical areas where vector population is abundant and these areas act as source population for virus spread to the neighbouring temperate sink areas. Australasia is one of the stable source-sink episystems with circulation of different serotypes. During this study, genetic relations of the serotypes circulating in the Australasian region were analysed and the serotypes that are unique to this episystem and those which entered this episystem during recent past were identified. Methods & Materials: Data about BTV serotypes circulating and genome sequence were collected from public databases. Phylogenetic analysis was done using MEGA and BEAST. Results: Majority of the BTV serotypes circulating in Australasia are either unique (BTV-20, -21 and -23) to the region or distinct (BTV-1, -2, -3, -4, -9 and 16) from their counterparts in other source-sink episystems and belong to Eastern topotype. Apart from these, serotypes of western origin are also found to be circulating (BTV-2, -5, -7, -10, -12 and -24), and some (BTV-2 and -10) are indistinguishable from the live attenuated vaccines being used in Africa (BTV-2) and USA (BTV-10). However, these two serotypes are not reported outside India. Conclusion: We could conclude that BTV-20, -21 and -23 are unique to Australasian region and eastern topotype viruses of serotypes BTV-1, -2, -3, -4, -9 and -16 have probably diverged from their western counterparts hundreds of years ago and evolved since then. Among the serotypes of western origin, BTV-5, 7, -12 and -24 seem to have entered this area in the recent past and got established in more than one country whereas vaccine strains of BTV-2 and -10 did not. Identification of circulating serotypes of BTV in this region is important for evaluating virus movement and for vaccine design.

Identification of the Genome Segments of Bluetongue Virus Serotype 26 (Isolate KUW2010/02) that Restrict Replication in a Culicoides sonorensis Cell Line (KC Cells).

Bluetongue virus (BTV) can infect most ruminant species and is usually transmitted by adult, vector-competent biting midges (Culicoides spp.). Infection with BTV can cause severe clinical signs and can be fatal, particularly in naïve sheep and some deer species. Although 24 distinct BTV serotypes were recognized for several decades, additional ‘types’ have recently been identified, including BTV-25 (from Switzerland), BTV-26 (from Kuwait) and BTV-27 from France (Corsica). Although BTV-25 has failed to grow in either insect or mammalian cell cultures, BTV-26 (isolate KUW2010/02), which can be transmitted horizontally between goats in the absence of vector insects, does not replicate in a Culicoides sonorensis cell line (KC cells) but can be propagated in mammalian cells (BSR cells). The BTV genome consists of ten segments of linear dsRNA. Mono-reassortant viruses were generated by reverse-genetics, each one containing a single BTV-26 genome segment in a BTV-1 genetic-background. However, attempts to recover a mono-reassortant containing genome-segment 2 (Seg-2) of BTV-26 (encoding VP2), were unsuccessful but a triple-reassortant was successfully generated containing Seg-2, Seg-6 and Seg-7 (encoding VP5 and VP7 respectively) of BTV-26. Reassortants were recovered and most replicated well in mammalian cells (BSR cells). However, mono-reassortants containing Seg-1 or Seg-3 of BTV-26 (encoding VP1, or VP3 respectively) and the triple reassortant failed to replicate, while a mono-reassortant containing Seg-7 of BTV-26 only replicated slowly in KC cells.

Purification of infective bluetongue virus particles by immuno-affinity chromatography using anti-core antibody.

An immuno-affinity chromatography technique for purification of infective bluetongue virus (BTV) has been descried using anti-core antibodies. BTV anti-core antibodies (prepared in guinea pig) were mixed with cell culture-grown BTV-1 and then the mixture was added to the cyanogens bromide-activated protein-A Sepharose column. Protein A binds to the antibody which in turn binds to the antigen (i.e. BTV). After thorough washing, antigen-antibody and antibody-protein A couplings were dissociated with 4M MgCl2, pH6.5. Antibody molecules were removed by dialysis and virus particles were concentrated by spin column ultrafiltration. Dialyzed and concentrated material was tested positive for BTV antigen by a sandwich ELISA and the infectivity of the chromatography-purified virus was demonstrated in cell culture. This method was applied for selective capture of BTV from a mixture of other viruses. As group-specific antibodies (against BTV core) were used to capture the virus, it is expected that virus of all BTV serotypes could be purified by this method. This method will be helpful for selective capture and enrichment of BTV from concurrently infected blood or tissue samples for efficient isolation in cell culture. Further, this method can be used for small scale purification of BTV avoiding ultracentrifugation.

Epidemiological characteristics and clinicopathological features of bluetongue in sheep and cattle, during the 2014 BTV serotype 4 incursion in Greece.

During 2014, an outbreak of Bluetongue virus (BTV) infections attributed to serotype 4 occurred in Greece and spread to south-eastern Europe. In the present article, the clinical and epidemiological data of 15 sheep flocks and 5 dairy cattle herds affected in Greece are described. In sheep, the most frequent clinical signs observed were fever, hyporexia, and edema of the face. A number of clinically affected sheep had chronic laminitis resulting in chronic lameness. Confirmation of suspect clinical cases was performed using BTV-specific real-time RT-PCR, and serotype 4-specific RT-PCR. The average morbidity of bluetongue in the sheep flocks was estimated to be 15.3% (95% C.I. 6.8-23.8%) and the average mortality and case fatality were 4.5% (95% C.I. 1.5-7.6%) and 32.0% (95% C.I. 18.1-42.9%), respectively. The BTV seroprevalence and the ratio of clinical manifestations-to-infections determined in seven of these flocks, were on average 36.5% (95% C.I. 15.7-57.3%) and 24.6% (95% C.I. 12.8-36.3%). BTV ratio of clinical manifestations-to-infections was higher in the imported western European sheep breeds examined compared to the local ones. In dairy cattle, the average herd prevalence of viremia was 48.8% (95% C.I. 15.3-82.4%) and none had signs associated with bluetongue. The results of this study indicate that the 2014 Greek BTV-4 has significant impact on the health status and the viability of sheep in affected flocks but does not cause clinical signs in cattle, despite the high prevalence of viremia.

Quantification of the increase in the frequency of early calving associated with late exposure to bluetongue virus serotype 8 in dairy cows: implications for syndromic surveillance.

A recent study evaluating whether reproductive data could be used for syndromic surveillance found an increased frequency of early calving (calving occurring a few days earlier than expected) in areas exposed to the Bluetongue virus serotype 8 (BTV-8) in northern Europe. A high proportion of herds infected during the 2006–2009 European outbreak were not reported through the surveillance system. The objectives of this study were (1) to quantify the increase in the frequency of early calving associated with the exposure to BTV-8 in late gestation and (2) to determine whether this association could be found in populations exposed to BTV-8 but without reported clinical signs. Increases in frequency of early calving were quantified for cows in herds located in the 2007 outbreak area in France, reported or not as cases. Increases were detected for cows in both categories of herds with a larger effect in herds reported after clinical signs. Moreover, the largest effect was found for exposures occurring during the latest stage of pregnancy, suggesting that BTV infection could trigger calving in cows in late gestation, a few days earlier than expected. This is the first study quantifying the association between a viral infection and a shortened pregnancy length (still within a normal range). The high magnitude of the increase in frequency of early calving, their occurrence in herds from infected areas but not reported, and the short time interval between exposure and the occurrence of the event confirm the interest of using early calving as an indicator for syndromic surveillance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0296-7) contains supplementary material, which is available to authorized users.

Phylogenetic Analysis of Viral Protein 2 of Bluetongue Virus

Bluetongue is a highly infectious vector born viral disease, and it is a disease of wild and domestic animals (ruminants). Bluetongue is a non-contagious disease of animals and spread by the biting midges (Sperlova. A. and Zendulkova. D. 2011). The name Bluetongue is given by Spreull in 1905 (Spreull, 1905). Bluetongue disease is mild in goats and severe in sheep as sheep is the primary host of bluetongue virus. Cattle act as the reservoir of bluetongue virus (Browne, 1971). The Bluetongue virus is first reported by hutcheon in 1881 During the introduction of European sheep breeds in Southern Africa (Hutcheon, 1902). Later in 1948 it was reported in North America as a sore muzzle disease (Hardy and Price, 1952).). Spare in 1964 reported outbreak of bluetongue disease in India (Spare, 1964). There are several clinical symptoms of Bluetongue disease have been found in ruminants like fever, viraemia, sore muzzle, facial oedema, hyperaemia and congestion, erosion of mucous membrane, haemorrhages, vascular permeability (OIE, 2014). Symptoms are more severe and easily detectable in sheep and these signs are high fever upto 5–7 days, loss of wool, depression and haemorrhages in the coronary band, difficulty in standing and lameness because of painful hoof, excessive salivation, swollen tongue, swelling in nasal and buccal mucosa, pneumonia and death (Tabachnick et.al., 2009). The Severity of clinical signs of bluetongue disease in sheep influenced by the type and strain of infecting virus (Verwoerd & Erasmus, 2004; OIE, 2014). The bluetongue virus is hypervariable in nature therefore, there are 24 serotypes of bluetongue virus are well recognised with two newly proposed serotypes BTV 25 from Switzerland and BTV 26 from Kuwait. In India 22 serotypes have been reported of Bluetongue virus (Prasad et al., 2009; Kumar, 2009). Bluetongue virus belongs to family Reoviridae and genus Orbivirus (Tabachnick et al., 2009). Blue-tongue has a serious economic impact on dairy and wool industry mainly due to high morbidity, mortality and mandatory trade barrier on the movement of BT infected livestock and germ-plasm. BT is evolving into newer challenges and poses ever increasing the threat to associated environment. An Unnatural host like canines have in the past contracted BT infections. Many species of Culicoides have been reported to spread infections. Recently BT has been categorized as multispecies disease by OIE (2014).

Effect of Viral Antigen Levels on the Serological Response and Efficiency of the Binary Ethylenimine-Inactivated Bluetongue Virus Serotype-16 Vaccine

Bluetongue (BT) is a serious hemorrhagic disease of ruminants caused by bluetongue virus (BTV). Inactive BTV vaccines have been successful in field trials in some areas, and inactivated vaccines are considered safer. However, information about the effect of the viral antigen level on the serological response and efficiency of the inactive BTV-16 vaccine is lacking. In the present study, the serological response and efficiency of the viral antigen concentration in the binary ethylenimine-inactivated Chinese BTV serotype-16 vaccine were investigated. The viral antigens in the viral suspension (VS) were quantified using a modified BTV AC-ELISA method. Four batches of vaccine containing 1, 5, 10, and 50 μg/ml of viral antigen were generated from the VS. Four groups of naive Chinese sheep were vaccinated with the different vaccine batches, and the serological response and vaccine efficiency were investigated before and after challenge infection. The vaccines containing 10 and 50 μg/ml of viral antigen induced significant ELISA and neutralizing antibody titers 14 days after vaccination, whereas the vaccines containing 1 and 5 μg/ml of viral antigen did not have these effects. A booster immunization at 21 days enhanced all groups’ antibody titers; however, the increased titer was related to the viral antigen level. In contrast to the serological response, the viral antigen level of the vaccines did not have a significant effect on the vaccine efficiency. With the exception of one sheep from the 5 μg/ml viral antigen group, all vaccinated sheep from the four antigen level groups showed strong resistance to infection based on their clinical symptoms, rectal temperatures and viremia. Collectively, these data suggested that viral antigen levels from 1 to 50 μg/ml had a significant effect on the serological response of the animals but a limited effect on the vaccine efficiency. The BTV-16 vaccine containing 1 μg/ml of viral antigen was sufficient to achieve high efficiency, but only the vaccines with more than 10 μg/ml of antigen induced a significant antibody response. To obtain a better serological response, we suggest the use of vaccines with more than 10 μg/ml of viral antigen. The findings in the study will be useful for BTV vaccine production.

Balance of RNA sequence requirement and NS3/NS3a expression of segment 10 of orbiviruses.

Orbiviruses are insect-transmitted, non-enveloped viruses with a ten-segmented dsRNA genome of which the bluetongue virus (BTV) is the prototype. Viral non-structural protein NS3/NS3a is encoded by genome segment 10 (Seg-10), and is involved in different virus release mechanisms. This protein induces specific release via membrane disruptions and budding in both insect and mammalian cells, but also the cytopathogenic release that is only seen in mammalian cells. NS3/NS3a is not essential for virus replication in vitro with BTV Seg-10 containing RNA elements essential for virus replication, even if protein is not expressed. Recently, new BTV serotypes with distinct NS3/NS3a sequence and cell tropism have been identified. Multiple studies have hinted at the importance of Seg-10 in orbivirus replication, but the exact prerequisites are still unknown. Here, more insight is obtained with regard to the needs for orbivirus Seg-10 and the balance between protein expression and RNA elements. Multiple silent mutations in the BTV NS3a ORF destabilized Seg-10, resulting in deletions and sequences originating from other viral segments being inserted, indicating strong selection at the level of RNA during replication in mammalian cells in vitro. The NS3a ORFs of other orbiviruses were successfully exchanged in BTV1 Seg-10, resulting in viable chimeric viruses. NS3/NS3a proteins in these chimeric viruses were generally functional in mammalian cells, but not in insect cells. NS3/NS3a of the novel BTV serotypes 25 and 26 affected virus release from Culicoides cells, which might be one of the reasons for their distinct cell tropism.

Delineation of the population genetic structure of Culicoides imicola in East and South Africa.

BackgroundCulicoides imicola Kieffer, 1913 is the main vector of bluetongue virus (BTV) and African horse sickness virus (AHSV) in Sub-Saharan Africa. Understanding the population genetic structure of this midge and the nature of barriers to gene flow will lead to a deeper understanding of bluetongue epidemiology and more effective vector control in this region.MethodsA panel of 12 DNA microsatellite markers isolated de novo and mitochondrial DNA were utilized in a study of C. imicola populations from Africa and an outlier population from the Balearic Islands. The DNA microsatellite markers and mitochondrial DNA were also used to examine a population of closely related C. bolitinos Meiswinkel midges.ResultsThe microsatellite data suggest gene flow between Kenya and south-west Indian Ocean Islands exist while a restricted gene flow between Kenya and South Africa C. imicola populations occurs. Genetic distance correlated with geographic distance by Mantel test. The mitochondrial DNA analysis results imply that the C. imicola populations from Kenya and south-west Indian Ocean Islands (Madagascar and Mauritius) shared haplotypes while C. imicola population from South Africa possessed private haplotypes and the highest nucleotide diversity among the African populations. The Bayesian skyline plot suggested a population growth.ConclusionsThe gene flow demonstrated by this study indicates a potential risk of introduction of new BTV serotypes by wind-borne infected Culicoides into the Islands. Genetic similarity between Mauritius and South Africa may be due to translocation as a result of human-induced activities; this could impact negatively on the livestock industry. The microsatellite markers isolated in this study may be utilised to study C. bolitinos, an important vector of BTV and AHSV in Africa and identify sources of future incursions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1277-4) contains supplementary material, which is available to authorized users.

Re-Emergence of Bluetongue Virus Serotype 8 in France, 2015.

At the end of August 2015, a ram located in central France (department of Allier) showed clinical signs suggestive of BTV (Bluetongue virus) infection. However, none of the other animals located in the herd showed any signs of the Bluetongue disease. Laboratory analyses identified the virus as BTV serotype 8. The viro and sero prevalence intraherd were 2.4% and 8.6% in sheep and 18.3% and 42.9% in cattle, respectively. Phylogenetic studies showed that the sequences of this strain are closely related to another BTV-8 strain that has circulated in France in 2006-2008. The origin of the outbreak is unclear but it may be assumed that the BTV-8 has probably circulated at very low prevalence (possibly in livestock or wildlife) since its first emergence in 2007-2008.

Complete genome sequence of the first bluetongue virus serotype 7 isolate from China: evidence for entry of African-lineage strains and reassortment between the introduced and native strains.

Bluetongue virus (BTV) mainly infects sheep but can be transmitted to other domestic and wild ruminants, resulting in a considerable financial burden and trade restriction. Our understanding of the origin, movement, and distribution of BTV has been hindered by the fact that this virus has a segmented genome with the possibility of reassortment, the existence of 27 identified serotypes, and a lack of complete sequences of viruses isolated from different parts of the world. BTV serotype 7 is one of the prevalent BTV serotypes in Asia. Nonetheless, no complete genomic sequence of an Asian isolate of this serotype is available. In an effort to understand the molecular epidemiology of BTV infection in China, for the first time, we report here the complete genome sequence of a BTV serotype 7 strain, GDST008, which was isolated in 2014 in China. This sequence also represents the first complete genome sequence of a BTV serotype 7 from Asia and the third one in the world. Sequence analysis suggests that GDST008 consists of segments from BTV viruses of African lineage as well as those from China. Together, these results improve our understanding of the origin, emergence/re-emergence, and movement of BTV and thus can be applied in the development of vaccines and diagnostics.

Culicoides vector species on three South American camelid farms seropositive for bluetongue virus serotype 8 in Germany 2008/2009

Palearctic species of Culicoides (Diptera, Ceratopogonidae), in particular of the Obsoletus and Pulicaris complexes, were identified as putative vectors of bluetongue virus serotype 8 (BTV-8) on ruminant farms during the epizootic in Germany from 2006 to 2009. BTV may cause severe morbidity and mortality in ruminants and sporadically in South American camelids (SAC). However, the fauna of Culicoides spp. on SAC farms has not been investigated.Therefore, the ceratopogonid fauna was monitored on three farms with BTV-seropositive SAC in Germany. Black–light traps were set up on pastures and in stables from summer 2008 to autumn 2009. Additionally, ceratopogonids were caught in emergence traps mounted on llama dung and dung-free pasture from spring to autumn 2009. After morphological identification, selected Culicoides samples were analysed for BTV-RNA by real-time RT-PCR. The effects of the variables ‘location’, ‘temperature’ and ‘humidity’ on the number of Culicoides caught in black–light traps were modelled using multivariable Poisson regression.In total, 26 species of Culicoides and six other genera of biting midges were identified. The most abundant Culicoides spp. collected both outdoors and indoors with black-light traps belonged to the Obsoletus (77.4%) and Pulicaris (16.0%) complexes. The number of Culicoides peaked in summer, while no biting midges were caught during the winter months. Daily collections of Culicoides were mainly influenced by the location and depended on the interaction of temperature and humidity. In the emergence traps, species of the Obsoletus complex predominated the collections.In summary, the absence of BTV-RNA in any of the analysed Culicoides midges and in the BTV-seropositive SAC on the three farms together with the differences in the pathogenesis of BTV-8 in SAC compared to ruminants suggests a negligible role of SAC in the spread of the virus. Although SAC farms may provide similar suitable habitats for putative Culicoides vectors than ruminant farms, the results suggest that geographic and meteorological factors had a stronger influence on Culicoides abundance than the animal species.

Assessment of population genetic structure in the arbovirus vector midge, Culicoides brevitarsis (Diptera: Ceratopogonidae), using multi-locus DNA microsatellites.

Bluetongue virus (BTV) is a major pathogen of ruminants that is transmitted by biting midges (Culicoides spp.). Australian BTV serotypes have origins in Asia and are distributed across the continent into two distinct episystems, one in the north and another in the east. Culicoides brevitarsis is the major vector of BTV in Australia and is distributed across the entire geographic range of the virus. Here, we describe the isolation and use of DNA microsatellites and gauge their ability to determine population genetic connectivity of C. brevitarsis within Australia and with countries to the north. Eleven DNA microsatellite markers were isolated using a novel genomic enrichment method and identified as useful for genetic analyses of sampled populations in Australia, northern Papua New Guinea (PNG) and Timor-Leste. Significant (P < 0.05) population genetic subdivision was observed between all paired regions, though the highest levels of genetic sub-division involved pair-wise tests with PNG (PNG vs. Australia (FST = 0.120) and PNG vs. Timor-Leste (FST = 0.095)). Analysis of multi-locus allelic distributions using STRUCTURE identified a most probable two-cluster population model, which separated PNG specimens from a cluster containing specimens from Timor-Leste and Australia. The source of incursions of this species in Australia is more likely to be Timor-Leste than PNG. Future incursions of BTV positive C. brevitarsis into Australia may be genetically identified to their source populations using these microsatellite loci. The vector’s panmictic genetic structure within Australia cannot explain the differential geographic distribution of BTV serotypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0250-8) contains supplementary material, which is available to authorized users.

A coiled-coil motif in non-structural protein 3 (NS3) of bluetongue virus forms an oligomer.

Bluetongue, an arthropod-borne non-contagious hemorrhagic disease of small ruminants, is caused by bluetongue virus (BTV). Several structural and non-structural proteins encoded by BTV have been associated with virulence mechanisms. In the present study, the NS3 protein sequences of bluetongue viral serotypes were analyzed for the presence of heptad regions and oligomer formation. Bioinformatic analysis of NS3 sequences of all 26 BTV serotypes revealed the presence of at least three coiled-coil motifs (CCMs). A conserved α-helical heptad sequence was identified at 14-26 aa (CCM-I), 185-198aa (CCM-II), and 94-116 aa (CCM-III). Among these, CCM-I occurs close to the N-terminus of NS3 and was presumed to be involved in oligomerization. Furthermore, the N-terminus of NS3 (1M-R117 aa) was over-expressed as a recombinant fusion protein in a prokaryotic expression system. Biochemical characterization of recombinant NS3Nt protein revealed that it forms SDS-resistant dimers and high-order oligomers (hexamer and/or octamer) under reducing or non-reducing conditions. Coiled-coil motifs are believed to be critical for NS protein oligomerization and have potential roles in the formation of viroporin ring/pore either with six/eight subunits and this is the first study toward characterization of CCMs in NS3 of bluetongue virus.