Strain Of Bluetongue Virus Serotype Research Articles

Genomic Analysis Illustrated a Single Introduction and Evolution of Israeli Bluetongue Serotype 8 Virus Population 2008–2019

Outbreaks of the European Bluetongue virus (BTV) serotype 8 (BTV-8), which are characterized by activity cycles separated by years of inactivity, may be influenced by genetic changes of the virus or by herd immunity. BTV activity in Israel is characterized by similar dynamics, but differs from European countries in its vector population, environmental conditions, and lack of cattle vaccination against this serotype. Comparison of these two geographical systems and characterization of their epidemiological connection is therefore of high interest in-order to better understand the factors influencing BTV-8 evolution. BTV-8, closely related to the European strain, was introduced to Israel in 2008. It was at the center of BT outbreaks in 2010 and 2015–2016 and thereafter was lastly isolated in Israel in 2019. We performed genetic analyses of twelve BTV-8 Israeli strains isolated between 2008 and 2019 and compared them with published sequences of BTV-8 isolated in other countries. The analysis revealed a single introduction of BTV-8 into Israel and thereafter extensive occurrence of genomic drifts and multiple reassortments with local BTV strains. Comparison of the Israeli and Cypriot BTV-8 from 2015 to 2016 suggests transmission of the virus between the two countries and a separate and parallel development from European or other Israeli BTV-8 strains. The parallel development of other BTV-8 strains was demonstrated by the identification of the Israeli BTV-8 ISR-1194/1/19 strain, which exhibited common origin with reassorted Israeli BTV-8 strains from 2010 and additional reassortment of seven segments. In order to reveal the source of BTV-8 introduction into Israel we performed BEAST analysis which showed that a probable common ancestor for both European and Israeli BTV-8 presumably existed in 2003–2004. In 2019, a possible new introduction occurred in Israel, where a novel BTV-8 strain was detected, sharing ~95% identity by segments 2 and 6 with Nigerian BTV-8NIG1982/07 and European–Middle Eastern strains. The results of the study indicate that Israel and neighboring countries consist a separate environmental and evolutionary system, distinct from European ones.

Diversity of Transmission Outcomes Following Co-Infection of Sheep with Strains of Bluetongue Virus Serotype 1 and 8.

Bluetongue virus (BTV) causes an economically important disease, bluetongue (BT), in susceptible ruminants and is transmitted primarily by species of Culicoides biting midges (Diptera: Ceratopogonidae). Since 2006, northern Europe has experienced multiple incursions of BTV through a variety of routes of entry, including major outbreaks of strains of BTV serotype 8 (BTV-8) and BTV serotype 1 (BTV-1), which overlapped in distribution within southern Europe. In this paper, we examined the variation in response to coinfection with strains of BTV-1 and BTV-8 using an in vivo transmission model involving Culicoides sonorensis, low passage virus strains, and sheep sourced in the United Kingdom. In the study, four sheep were simultaneously infected using BTV-8 and BTV-1 intrathoracically inoculated C. sonorensis and co-infections of all sheep with both strains were established. However, there were significant variations in both the initiation and peak levels of virus RNA detected throughout the experiment, as well as in the infection rates in the C. sonorensis that were blood-fed on experimentally infected sheep at peak viremia. This is discussed in relation to the potential for reassortment between these strains in the field and the policy implications for detection of BTV strains.

"Frozen evolution" of an RNA virus suggests accidental release as a potential cause of arbovirus re-emergence.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropod-borne virus of ruminants, emerged in livestock in northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of euros. Although the outbreak was successfully controlled through vaccination by early 2010, puzzlingly, a closely related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this long period without replication, we hypothesise that the second outbreak could have been initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence.

Evidence of reduced viremia, pathogenicity and vector competence in a re-emerging European strain of bluetongue virus serotype 8 in sheep.

SummaryThe outbreak of bluetongue virus (BTV) serotype 8 (BTV‐8) during 2006–2009 in Europe was the most costly epidemic of the virus in recorded history. In 2015, a BTV‐8 strain re‐emerged in France which has continued to circulate since then. To examine anecdotal reports of reduced pathogenicity and transmission efficiency, we investigated the infection kinetics of a 2007 UK BTV‐8 strain alongside the re‐emerging BTV‐8 strain isolated from France in 2017. Two groups of eight BTV‐naïve British mule sheep were inoculated with 5.75 log10TCID50/ml of either BTV‐8 strain. BTV RNA was detected by 2 dpi in both groups with peak viraemia occurring between 5–9 dpi. A significantly greater amount of BTV RNA was detected in sheep infected with the 2007 strain (6.0–8.8 log10 genome copies/ml) than the re‐emerging BTV‐8 strain (2.9–7.9 log10 genome copies/ml). All infected sheep developed BTV‐specific antibodies by 9 dpi. BTV was isolated from 2 dpi to 12 dpi for 2007 BTV‐8‐inoculated sheep and from 5 to 10 dpi for sheep inoculated with the remerging BTV‐8. In Culicoides sonorensis feeding on the sheep over the period 7–12 dpi, vector competence was significantly higher for the 2007 strain than the re‐emerging strain. Both the proportion of animals showing moderate (as opposed to mild or no) clinical disease (6/8 vs. 1/8) and the overall clinical scores (median 5.25 vs. 3) were significantly higher in sheep infected with the 2007 strain, compared to those infected with the re‐emerging strain. However, one sheep infected with the re‐emerging strain was euthanized at 16 dpi having developed severe lameness. This highlights the potential of the re‐emerging BTV‐8 to still cause illness in naïve ruminants with concurrent costs to the livestock industry.

Full-Genome Sequence Analysis of a Reassortant Strain of Bluetongue virus Serotype 16 from Southern India.

The complete genome sequence of a reassortant field strain (IND2014/01) of Bluetongue virus (BTV) serotype 16, isolated from sheep from southern India in 2014, was sequenced. The total genome size was 19,186 bp. Sequence comparisons of all genome segments, except segment 5 (Seg-5), showed that IND2014/01 belonged to the major eastern topotype of BTV.

Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection.

Bluetongue virus (BTV) is an arthropod-borne pathogen that causes an often fatal, hemorrhagic disease in ruminants. Different BTV serotypes occur throughout many temperate and tropical regions of the world. In 2006, BTV serotype 8 (BTV-8) emerged in Central and Northern Europe for the first time. Although this outbreak was eventually controlled using inactivated virus vaccines, the epidemic caused significant economic losses not only from the disease in livestock but also from trade restrictions. To date, BTV vaccines that allow simple serological discrimination of infected and vaccinated animals (DIVA) have not been approved for use in livestock. In this study, we generated recombinant RNA replicon particles based on single-cycle vesicular stomatitis virus (VSV) vectors. Immunization of sheep with infectious VSV replicon particles expressing the outer capsid VP2 protein of BTV-8 resulted in induction of BTV-8 serotype-specific neutralizing antibodies. After challenge with a virulent BTV-8 strain, the vaccinated animals neither developed signs of disease nor showed viremia. In contrast, immunization of sheep with recombinant VP5 - the second outer capsid protein of BTV - did not confer protection. Discrimination of infected from vaccinated animals was readily achieved using an ELISA for detection of antibodies against the VP7 antigen. These data indicate that VSV replicon particles potentially represent a safe and efficacious vaccine platform with which to control future outbreaks by BTV-8 or other serotypes, especially in previously non-endemic regions where discrimination between vaccinated and infected animals is crucial.

Viral replication kinetics and in vitro cytopathogenicity of parental and reassortant strains of bluetongue virus serotype 1, 6 and 8

Bluetongue virus (BTV), a segmented dsRNA virus, is the causative agent of bluetongue (BT), an economically important viral haemorrhagic disease of ruminants. Bluetongue virus can exchange its genome segments in mammalian or insect cells that have been co-infected with more than one strain of the virus. This process, may potentially give rise to the generation of novel reassortant strains that may differ from parental strains in regards to their phenotypic characteristics. To investigate the potential effects of reassortment on the virus’ phenotype, parental as well as reassortant strains of BTV serotype 1, 6, 8, that were derived from attenuated and wild type strains by reverse genetics, were studied in vitro for their virus replication kinetics and cytopathogenicity in mammalian (Vero) cell cultures. The results indicate that genetic reassortment can affect viral replication kinetics, the cytopathogenicity and extent/mechanism of cell death in infected cell cultures. In particular, some reassortants of non-virulent vaccine (BTV-1 and BTV-6) and virulent field origin (BTV-8) demonstrate more pronounced cytopathic effects compared to their parental strains. Some reassortant strains in addition replicated to high titres in vitro despite being composed of genome segments from slow and fast replicating parental strains. The latter result may have implications for the level of viraemia in the mammalian host and subsequent uptake and transmission of reassortant strains (and their genome segments) by Culicoides vectors. Increased rates of CPE induction could further suggest a higher virulence for reassortant strains in vivo. Overall, these findings raise questions in regards to the use of modified-live virus (MLV) vaccines and risk of reassortment in the field. To further address these questions, additional experimental infection studies using insects and/or animal models should be conducted, to determine whether these results have significant implications in vivo.

Genomic diversity among eastern and western topotypes of bluetongue virus serotype 16 based on whole genome sequence analysis

Bluetongue (BT) is a noncontagious, vector-borne disease of domestic and wild ruminants. The causative agent bluetongue virus (BTV) is a double stranded RNA virus belonging to genus Orbivirus within family Reoviridae. Eradication of BTV from endemic regions like India is not an easy task due to the widely distributed Culicoides spp. midge vectors, the ubiquitous distribution of vertebrate hosts and existence of a large number of serotypes of the virus (at least 26 till date). The complete genomes (19,193 base-pairs) of several strains of bluetongue virus serotype 16 (BTV-16) originated from Australia, China, Indian subcontinent, Mediterranean basin, Middle East, Africa (Nigeria) and Europe, were compared. These analysis showed that all ten genome segments of a Nigerian strain are derived from a western lineage, showing only 77% - 84% nt identity with the eastern topotype reference strain 'RSArrrr/16' (and its derivative 'RSAvvvv/16', a vaccine strain) that was originally isolated in Pakistan, 76.4% - 83% with eastern BTV-16 strain from Australia (DPP96) and 77% - 89% with a reassortant strain from India. These detailed comparisons involving global strains showed that there is a very high degree of variation (up to 24%) between BTV-16 strains from eastern and western geographical regions. These data confirm the value of whole genome sequencing for characterization of novel BTV isolates and has helped to identify representative suitable 'reference-strain' of eastern topotype (BTV-16e), western topotype (BTV-16w), as well as 'cross-topotype' reassortant strain (BTV-16r) that are generated in the field for further serological, phylogenetic and molecular epidemiology studies.

No evidence for replication of a field strain of bluetongue virus serotype 1 in the blood of domestic dogs

The potential role of domestic dogs in the long-distance transmission of bluetongue virus (BTV) is currently unproven. This study set out, through an experimental infection study, to investigate whether domestic dogs mount a viraemia post-infection with a field strain of BTV serotype 1. All six experimentally infected dogs seroconverted within 14 days and viral RNA was detected in the blood of the dogs, albeit at significantly lower levels than that seen in domestic ruminants. There was no clear evidence for viral replication in the dogs as no increase in viral RNA was observed in, and it was not possible isolate virus from, the blood of the dogs. There was however evidence for a persistence of viral RNA in the blood of the dogs, which may be evidence for a low level of replication or could be indicative of persistence of the viral inoculum.

Full Genome Sequence of a Western Reference Strain of Bluetongue Virus Serotype 16 from Nigeria

The genome of NIG1982/10, a Nigerian bluetongue virus serotype 16 (BTV-16) strain, was sequenced (19,193 bp). Comparisons to BTV strains from other areas of the world show that all 10 genome segments of NIG1982/10 are derived from a western lineage (w), indicating that it represents a suitable reference strain of BTV-16w.

Complete Genome Sequence Analysis of a Reassortant Strain of Bluetongue Virus Serotype 16 from Italy

The complete genome sequence of a reassortant field strain of bluetongue virus serotype 16 (BTV-16), isolated from cattle in the Apulia region of Italy in 2002, has been determined by Illumina sequencing. Sequence comparisons of segment 1 (Seg-1) to Seg-10, except Seg-5, show that BTV-16 strain ITL2002 belongs to the major eastern topotype of BTV.

Transplacental infection in goats experimentally infected with a European strain of bluetongue virus serotype 8

The capability of the recently emerged European strain of bluetongue virus serotype 8 (BTV-8) to cross the ruminant placenta has been established in experimental and field studies in both sheep and cattle. Seroprevalence rates in goats in North-Western Europe were high during the recent outbreak of BTV-8; however the capability of the virus to infect goats through the transplacental route has not been established.In the present study, four Saanen goats were inoculated with the European strain of BTV-8 at 62days of gestation; this resulted in mild clinical signs, however gross lesions observed post mortem were more severe. Viral RNA was detected by real-time RT-PCR in blood and tissue samples from three fetuses harvested from two goats at 43days post infection. Conventional RT-PCR and genome sequencing targeting viral segment 2 confirmed infection of brain tissue with BTV-8 in two of these fetuses. In total, five of six fetuses demonstrated lesions that may have been associated with transplacental infection with BTV. Infected fetuses did not demonstrate neurological lesions. Low viral RNA concentrations in fetal blood and tissue further suggest that the infected fetuses would probably not have been born viraemic. The implications of these findings with regards to the epidemiology and overwintering of BTV-8 in Europe remains unclear.

Detection of neutralizing antibodies against bluetongue virus serotype 8 by an optimized plasma neutralization test

The neutralization test is used commonly for quantifying neutralizing antibodies and for distinguishing among different virus serotypes (serotyping). Due to the co-circulation of multiple serotypes of Bluetongue virus (BTV), the neutralization test has become an important surveillance method in Europe. However, the existence of different protocols makes test standardization and interpretation of results difficult. The current paper describes the development of a neutralization test using plasma and addresses the factors critical for detection of neutralizing antibodies against BTV serotype 8 (BTV-8), such as virus propagation, stability of virus infectivity and origin of the BTV-8 strain. The results indicated that animals exposed to the Northern European BTV-8 strain developed low neutralizing antibody titers, particularly after vaccination and experimental infection. Although clearly ELISA-positive, these samples often yielded false negative results when tested by the neutralization test using the OIE recommended virus concentration of 100 TCID₅₀/50 μl. The sensitivity of the neutralization test could be improved significantly with retained specificity by using a reduced TCID₅₀ and the homologous European BTV-8 strain instead of the South African reference strain.

Genome Sequence of a Reassortant Strain of Bluetongue Virus Serotype 23 from Western India

The full genome sequence (19,177 bp) of an Indian strain (IND1988/02) of bluetongue virus (BTV) serotype 23 was determined. This virus was isolated from a sheep that had been killed during a severe bluetongue outbreak that occurred in Rahuri, Maharashtra State, western India, in 1988. Phylogenetic analyses of these data demonstrate that most of the genome segments from IND1988/02 belong to the major "eastern" BTV topotype. However, genome segment 5 belongs to the major "western" BTV topotype, demonstrating that IND1988/02 is a reassortant. This may help to explain the increased virulence that was seen during this outbreak in 1988. Genome segment 5 of IND1988/02 shows >99% sequence identity with some other BTV isolates from India (e.g., BTV-3 IND2003/08), providing further evidence of the existence and circulation of reassortant strains on the subcontinent.

The Genome Sequence of a Reassortant Bluetongue Virus Serotype 3 from India

All 10 genome segments (Seg-1 to 10-a total of 19,188 bp) were sequenced from a strain of bluetongue virus serotype 3 (BTV-3) from India (strain IND2003/08). Sequence comparisons showed that nine of the genome segments from this virus group with other eastern topotype strains. Genome Seg-2 and Seg-6 group with eastern BTV-3 strains from Japan. However, Seg-5 (the NS1 gene) from IND2003/08 belongs to a western lineage, demonstrating that IND2003/08 is a reassortant between eastern and western topotype bluetongue viruses. This confirms that western BTV strains have been imported and are circulating within the subcontinent.

The susceptibility of Culicoides imicola and other South African livestock-associated Culicoides species to infection with bluetongue virus serotype 8

In 2006, a strain of bluetongue virus serotype 8 (BTV-8) of sub-Saharan origin was responsible for the first outbreaks in recorded history of clinical bluetongue disease (BT) in northern Europe. In this study, we examine the oral susceptibility of Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae) and other livestock-associated Culicoides species from southern Africa to infection with several strains of BTV-8. Following feeding using an artificial membrane-based method and incubation, virus was found in <1% of C. imicola individuals tested. Higher rates of susceptibility were found, however, for a variety of other South African species, including Culicoides (Avaritia) bolitinos Meiswinkel. Although these results do not preclude the role of C. imicola as a vector of BTV-8, its low susceptibility to BTV indicates that other less abundant Culicoides species may have the potential to play decisive roles in the epidemiology of this virus and should not be excluded from risk assessment studies.

Evaluation of humoral response and protective efficacy of three inactivated vaccines against bluetongue virus serotype 8 one year after vaccination of sheep and cattle

The long-term efficacy of three commercially available inactivated vaccines against bluetongue virus serotype 8 (BTV-8) (BLUEVAC ® 8, Zulvac ® 8, and BTVPUR ® AlSap 8) was evaluated in a seroprevalence study and challenge experiments. Seroprevalences 1 year after vaccination ranged from 75% to 100%. In two infection experiments, groups of vaccinated sheep and cattle selected either randomly or for low antibody levels were challenged with a European BTV-8 strain 12 months after vaccination. With two exceptions, all animals, including those with low antibody levels prior to challenge, were protected from viral replication and clinical disease even at low initial antibody levels. Vaccination of susceptible ruminants in yearly intervals is thus considered an adequate scheme for BTV-8 control in Europe.

Virological and pathological findings in Bluetongue virus serotype 8 infected sheep

Twenty-seven sheep of the four most common Swiss breeds and the English breed Poll Dorset were experimentally infected with a northern European field strain of bluetongue virus serotype 8 (BTV-8). Animals of all breeds developed clinical signs, viremia and pathological lesions, demonstrating that BTV-8 is fully capable of replicating and inducing bluetongue disease (BT) in the investigated sheep. Necropsy performed between 10 and 16 days post-infectionem (d.p.i.) revealed BT-typical hemorrhages, effusions, edema, erosions and activation of lymphatic tissues. Hemorrhages on the base of the Arteria pulmonalis and the left Musculus papillaris subauricularis were frequently present. Histology confirmed the macroscopical findings. Using a score system, clinical manifestation and pathology were found to be significantly related. Furthermore, clinical signs and fever were shown to be indicative for the concurrent presence of high amounts of viral ribonucleic acid (RNA) in blood. Spleen, lung, lymph nodes and tonsils from all animals were analyzed regarding viral RNA loads and infectivity using real-time reverse transcriptase PCR (rRT-PCR) and virus isolation in cell culture, respectively. The highest amount of viral RNA was detected in spleen and lung and rRT-PCR revealed to be a more sensitive method for virus detection compared to virus isolation. A long-term follow-up was performed with three sheep showing that BTV-8 viral RNA in blood was present up to 133 d.p.i. and in certain tissues even on 151 d.p.i. No significant breed-related differences were observed concerning clinicopathological picture and viremia, and the Swiss sheep were as susceptible to BTV-8 infection as Poll Dorset sheep, demonstrating a remarkably high virulence of BTV-8 for indigenous sheep breeds.

Bovine infection with bluetongue virus with special emphasis on European serotype 8

Bluetongue virus (BTV) is an arthropod-borne virus infecting domestic and wild ruminants. Infection in cattle is commonly asymptomatic and characterised by a long viraemia. Associated with the emergence and the recrudescence of BTV serotype 8 (BTV-8) in Northern and Central Europe, remarkable differences have been noticed in the transmission and in the clinical expression of the disease, with cattle showing clinical illness and reproductive disorders such as abortion, stillbirth and fetal abnormalities. Several investigations have already indicated the putative ability of the European BTV-8 strain to cross the bovine placenta and to cause congenital infections. The current epidemiological and pathological findings present an unusual picture of the disease in affected bovines.

Evidence for transplacental and contact transmission of bluetongue virus in cattle

This paper presents evidence that a field strain of bluetongue virus serotype 8 (BTV-8) was transmitted transplacentally and that it was also spread by a direct contact route. Twenty pregnant heifers were imported from the Netherlands into Northern Ireland during the midge-free season. Tests before and after the animals were imported showed that eight of them had antibodies to bluetongue virus, but no viral RNA was detected in any of them by reverse transcriptase-PCR (RT-PCR). Two of the seropositive heifers gave birth to three calves that showed evidence of bluetongue virus infection (RT-PCR-positive), and one of the calves was viraemic. Two further viraemic animals (one newly calved Dutch heifer, and one milking cow originally from Scotland) were also found to have been infected with BTV-8 and evidence is presented that these two animals may have been infected by direct contact, possibly through the ingestion of placentas infected with BTV-8.