Isolation and Cultivation of a New Isolate of BTV-25 and Presumptive Evidence for a Potential Persistent Infection in Healthy Goats.

Recently, several so-called "atypical" Bluetongue virus (BTV) serotypes were discovered, including BTV-25 (Toggenburg virus), in Switzerland. Most "atypical" BTV were identified in small ruminants without clinical signs. In 2018, two goats from a holding in Germany tested positive for BTV-25 genome by RT-qPCR prior to export. After experimental inoculation of the two goats with the BTV-25 positive field blood samples for generation of reference materials, viremia could be observed in one animal. For the first time, the BTV-25-related virus was isolated in cell culture from EDTA-blood and the full genome of isolate "BTV-25-GER2018" could be generated. BTV-25-GER2018 was only incompletely neutralized by ELISA-positive sera. We could monitor the BTV-25 occurrence in the respective affected goat flock of approximately 120 goats over several years. EDTA blood samples were screened with RT-qPCR using a newly developed BTV-25 specific assay. For serological surveillance, serum samples were screened using a commercial cELISA. BTV-25-GER2018 was detected over 4.5 years in the goat flock with intermittent PCR-positivity in some animals, and with or without concomitantly detected antibodies since 2015. We could demonstrate the viral persistence of BTV-25-GER2018 in goats for up to 4.5 years, and the first BTV-25 isolate is now available for further characterization.

A low-passage insect-cell isolate of bluetongue virus uses a macropinocytosis-like entry pathway to infect natural target cells derived from the bovine host.

Bluetongue virus (BTV) causes an economically important disease in domestic and wildlife ruminants and is transmitted by Culicoides biting midges. In ruminants, BTV has a wide cell tropism that includes endothelial cells of vascular and lymphatic vessels as important cell targets for virus replication, and several cell types of the immune system including monocytes, macrophages and dendritic cells. Thus, cell-entry represents a particular challenge for BTV as it infects many different cell types in widely diverse vertebrate and invertebrate hosts. Improved understanding of BTV cell-entry could lead to novel antiviral approaches that can block virus transmission from cell to cell between its invertebrate and vertebrate hosts. Here, we have investigated BTV cell-entry using endothelial cells derived from the natural bovine host (BFA cells) and purified whole virus particles of a low-passage, insect-cell isolate of a virulent strain of BTV-1. Our results show that the main entry pathway for infection of BFA cells is dependent on actin and dynamin, and shares certain characteristics with macropinocytosis. The ability to use a macropinocytosis-like entry route could explain the diverse cell tropism of BTV and contribute to the efficiency of transmission between vertebrate and invertebrate hosts.

The Interaction of Bluetongue Virus VP6 and Genomic RNA Is Essential for Genome Packaging.

The genomes of the Reoviridae, including the animal pathogen bluetongue virus (BTV), are multisegmented double-stranded RNA (dsRNA). During replication, single-stranded (ss) positive-sense RNA segments are packaged into the assembling virus capsid, triggering genomic dsRNA synthesis. However, exactly how this packaging event occurs is not clear. A minor capsid protein, VP6, unique for the orbiviruses, has been proposed to be involved in the RNA-packaging process. In this study, we sought to characterize the RNA binding activity of VP6 and its functional relevance. A novel proteomic approach was utilized to map the ssRNA/dsRNA binding sites of a purified recombinant protein and the genomic dsRNA binding sites of the capsid-associated VP6. The data revealed that each VP6 protein has multiple distinct RNA-binding regions and that only one region is shared between recombinant and capsid-associated VP6. A combination of targeted mutagenesis and reverse genetics identified the RNA-binding region that is essential for virus replication. Using an in vitro RNA-binding competition assay, a unique cell-free assembly assay, and an in vivo single-cycle replication assay, it was possible to identify a motif within the shared binding region that binds BTV ssRNA preferentially in a manner consistent with specific RNA recruitment during capsid assembly. These data highlight the critical roles that this unique protein plays in orbivirus genome packaging and replication.IMPORTANCE Genome packaging is a critical stage during virus replication. For viruses with segmented genomes, the genome segments need to be correctly packaged into a newly formed capsid. However, the detailed mechanism of this packaging is unclear. Here we focus on VP6, a minor viral protein of bluetongue virus, which is critical for genome packaging. We used multiple approaches, including a robust RNA-protein fingerprinting assay, to map the ssRNA binding sites of recombinant VP6 and the genomic dsRNA binding sites of capsid-associated VP6. By these means, together with virological and biochemical methods, we identify the viral RNA-packaging motif of a segmented dsRNA virus for the first time.

Delivering Cargo: Plant-Based Production of Bluetongue Virus Core-Like and Virus-Like Particles Containing Fluorescent Proteins.

This chapter provides a practical guide to the in planta transient production of bluetongue virus-like particles containing a fluorescent cargo protein. Bluetongue virus (BTV) particles are icosahedral, multishelled entities of a relatively large size. Heterologous expression of the four main structural proteins of BTV results in the assembly of empty virus-like particles which resemble the native virus externally, but are devoid of nucleic acid. The space within the particles is sufficient to allow incorporation of relatively large cargo proteins, such as green fluorescent protein (GFP), by genetic fusion to the structural protein VP3. The method described utilizes the pEAQ vectors for high-level transient expression of such particles in Nicotiana benthamiana.

The Culicoides sonorensis inhibitor of apoptosis 1 protein protects mammalian cells from apoptosis induced by infection with African horse sickness virus and bluetongue virus.

African horse sickness virus (AHSV) and bluetongue virus (BTV) are arboviruses of the genus Orbivirus that are transmitted to their vertebrate hosts by Culicoides biting midges. These orbiviruses exhibit lytic infection (apoptosis) in mammalian cells, but cause persistent infection with no cytopathic effects in Culicoides sonorensis cells. Although regulation of apoptosis could thus be integral for establishing persistent virus infection in midge cells, nothing is known about the presence and function of apoptosis pathways in Culicoides midges and their derived cell lines. Here, we report the cloning and functional characterization of an inhibitor of apoptosis protein (IAP), designated CsIAP1, from C. sonorensis cells. The CsIAP1 protein contains two baculoviral IAP repeat (BIR) domains and a RING domain. Silencing of the Cs iap1 gene in C. sonorensis cells caused apoptosis, indicating that CsIAP1 plays a role in cell survival. Stable expression of the CsIAP1 protein in BSR mammalian cells suppressed apoptosis induced by AHSV-4 and BTV-10 infection, and biochemical data indicated that CsIAP1 is an inhibitor of mammalian caspase-9, an initiator caspase in the intrinsic apoptotic pathway. Mutagenesis studies indicated that the BIR2 and RING domains are required for the anti-apoptotic activity of CsIAP1. The results suggest that the mechanism by which CsIAP1 suppresses apoptosis in insect cells may involve inhibition of a Culicoides caspase-9 homologue through a mechanism that requires both the BIR2 and RING domains. This study provides the first evidence that the CsIAP1 protein is a key negative regulator of apoptosis in C. sonorensis cells.

Immunosuppression in sheep induced by cyclophosphamide, bluetongue virus and their combination: Effect on clinical reaction and viremia.

The main purpose of this work was to establish an experimental model for immunosuppression in sheep, and evaluate its possible effects on bluetongue viremia. Animals were allocated in 4 groups: Cy (cyclophosphamide), BT (bluetongue), CyBT (combined Cy and BT) and Co (control), and underwent clinical evaluations, virological testing, peripheral blood immunophenotyping and determination of antiviral humoral immune responses. Intravenous administration of cyclophosphamide (37.5 mg/kg body weight) resulted in immunosuppresion induction, as significant drops were observed in blood leukocytes and lymphocyte subset counts (CD2+, CD4+, CD8+, CD19+), lasting 3-10 days after its administration. Reduction in B-cell (CD19+) counts was more pronounced than in T-/NK-cell (CD2+) counts (92% and 59%, respectively). BTV-9 inoculation resulted in pronounced lymphocytopenia observed from day 1 post-inoculation. Their combined administration resulted in a more intense immunosuppressive effect, as indicated by the greater reduction in lymphocyte, granulocyte, CD4+ and CD8+ cell counts. In group CyBT, earlier initiation of fever by one day (day 6 p.i.) compared to group BT (day 7 p.i.), and delay in antibody responses by one day was observed, compared to group BT. Neutralizing antibodies in both groups (BT, CyBT) were detectable from day 10 p.i., but no significant titer differences were observed. Infectious virus titers were detected from day 4 p.i. in group BT and from day 3 in group CyBT. Statistical significances in virus titers were also observed (greatest mean titer difference: 1.4 log10 CEID50/ml RBCs at day 5 p.i., P < 0.001), indicating possible impact of immunosuppression on virus transmission and epidemiology of bluetongue.

MicroRNA expression profiling of primary sheep testicular cells in response to bluetongue virus infection.

Bluetongue virus (BTV) is a member of the genus Orbivirus within the family Reoviridae and causes a non-contagious, insect-transmitted disease in domestic and wild ruminants, mainly in sheep and occasionally in cattle and some species of deer. Virus infection can trigger the changes of the cellular microRNA (miRNA) expression profile, which play important post-transcriptional regulatory roles in gene expression and can greatly influence viral replication and pathogenesis. Here, we employed deep sequencing technology to determine which cellular miRNAs were differentially expressed in primary sheep testicular (ST) cells infected with BTV. A total of 25 known miRNAs and 240 novel miRNA candidates that were differentially expressed in BTV-infected and uninfected ST cells were identified, and 251 and 8428 predicted target genes were annotated, respectively. Nine differentially expressed miRNAs and their mRNA targets were validated by quantitative reverse transcription-polymerase chain reaction. Targets prediction and functional analysis of these regulated miRNAs revealed significant enrichment for several signaling pathways including MAPK, PI3K-Akt, endocytosis, Hippo, NF-kB, viral carcinogenesis, FoxO, and JAK-STAT signaling pathways. This study provides a valuable basis for further investigation on the roles of miRNAs in BTV replication and pathogenesis.

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.

Pathogenicity study in sheep using reverse-genetics-based reassortant bluetongue viruses.

Bluetongue (BT) disease, caused by the non-enveloped bluetongue virus (BTV) belonging to the Reoviridae family, is an economically important disease that affects a wide range of wild and domestic ruminants. Currently, 26 different serotypes of BTV are recognized in the world, of which BTV-8 has been found to exhibit one of the most virulent manifestations of BT disease in livestock. In recent years incursions of BTV-8 in Europe have resulted in significant morbidity and mortality not only in sheep but also in cattle. The molecular and genetic basis of BTV-8 pathogenesis is not known. To understand the genetic basis of BTV-8 pathogenicity, we generated reassortant viruses by replacing the 3 most variable genes, S2, S6 and S10 of a recent isolate of BTV-8, in different combinations into the backbone of an attenuated strain of BTV-1. The growth profiles of these reassortant viruses were then analyzed in two different ovine cell lines derived from different organs, kidney and thymus. Distinct patterns for each reassortant virus in these two cell lines were observed. To determine the pathogenicity of these reassortant viruses, groups of BTV-susceptible sheep were infected with each of these viruses. The data suggested that the clinical manifestations of these two different serotypes, BTV-1 and BTV-8, were slightly distinct and BTV-1, when comprising all 3 genome segments of BTV-8, behaved differently to BTV-1. Our results also suggested that the molecular basis of BT disease is highly complex.

Reassortment between two serologically unrelated bluetongue virus strains is flexible and can involve any genome segment.

Coinfection of a cell by two different strains of a segmented virus can give rise to a "reassortant" with phenotypic characteristics that might differ from those of the parental strains. Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) segmented virus and the cause of bluetongue, a major infectious disease of livestock. BTV exists as at least 26 different serotypes (BTV-1 to BTV-26). Prompted by the isolation of a field reassortant between BTV-1 and BTV-8, we systematically characterized the process of BTV reassortment. Using a reverse genetics approach, our study clearly indicates that any BTV-1 or BTV-8 genome segment can be rescued in the heterologous "backbone." To assess phenotypic variation as a result of reassortment, we examined viral growth kinetics and plaque sizes in in vitro experiments and virulence in an experimental mouse model of bluetongue disease. The monoreassortants generated had phenotypes that were very similar to those of the parental wild-type strains both in vitro and in vivo. Using a forward genetics approach in cells coinfected with BTV-1 and BTV-8, we have shown that reassortants between BTV-1 and BTV-8 are generated very readily. After only four passages in cell culture, we could not detect wild-type BTV-1 or BTV-8 in any of 140 isolated viral plaques. In addition, most of the isolated reassortants contained heterologous VP2 and VP5 structural proteins, while only 17% had homologous VP2 and VP5 proteins. Our study has shown that reassortment in BTV is very flexible, and there is no fundamental barrier to the reassortment of any genome segment. Given the propensity of BTV to reassort, it is increasingly important to have an alternative classification system for orbiviruses.

Host-seeking activity of bluetongue virus vectors: endo/exophagy and circadian rhythm of Culicoides in Western Europe.

Feeding success of free-living hematophagous insects depends on their ability to be active when hosts are available and to reach places where hosts are accessible. When the hematophagous insect is a vector of pathogens, determining the components of host-seeking behavior is of primary interest for the assessment of transmission risk. Our aim was to describe endo/exophagy and circadian host-seeking activity of Palaearctic Culicoides species, which are major biting pests and arbovirus vectors, using drop traps and suction traps baited with four sheep, as bluetongue virus hosts. Collections were carried out in the field, a largely-open stable and an enclosed stable during six collection periods of 24 hours in April/May, in late June and in September/October 2010 in western France. A total of 986 Culicoides belonging to 13 species, mainly C. brunnicans and C. obsoletus, was collected on animal baits. Culicoides brunnicans was clearly exophagic, whereas C. obsoletus was able to enter stables. Culicoides brunnicans exhibited a bimodal pattern of host-seeking activity with peaks just after sunrise and sunset. Culicoides obsoletus was active before sunset in spring and autumn and after sunset in summer, thus illustrating influence of other parameters than light, especially temperature. Description of host-seeking behaviors allowed us to discuss control strategies for transmission of Culicoides-borne pathogens, such as bluetongue virus. However, practical vector-control recommendations are difficult to provide because of the variation in the degree of endophagy and time of host-seeking activity.

Bluetongue virus (BTV): propagation, quantification, and storage.

As an obligate intracellular parasite, the genome of the Bluetongue virus (BTV) contains ten double-stranded RNA segments which are encapsidated by viral proteins, forming "transport vesicles" that can transmit the viral progeny cell-to-cell efficiently and that can also be transmitted animal-to-animal by a biting midge. BTV is a cytoplasmic virus, and its five major steps of viral infection: attachment, entry, uncoating, assembly, and release, occur only in the cytosol within the infected host cell. Viral replication, suppression of cellular processes, and subsequent pathological damage disrupt many cellular pathways, leading to cellular apoptosis. All of these steps are under very rapid, tight, and efficient control. BTV infects both domestic and wild ruminants, especially sheep, but not humans. BTV is also the prototype in the Orbivirus genus of the Reoviridae family, and has been studied very extensively for the last 25 years. The experimental protocols presented here describe most of the methods that have been used routinely and reproducibly in our lab for our studies of the BTV biosystems.

Rescue of recent virulent and avirulent field strains of bluetongue virus by reverse genetics.

Since 1998, Bluetongue virus (BTV)-serotypes 1, 2, 4, 9, and 16 have invaded European countries around the Mediterranean Basin. In 2006, a huge BT-outbreak started after incursion of BTV-serotype 8 (BTV8) in North-Western Europe. More recently, BTV6 and BTV11 were reported in North-Western Europe in 2008. These latter strains are closely related to live-attenuated vaccine, whereas BTV8 is virulent and can induce severe disease in ruminants, including cattle. In addition, Toggenburg orbivirus (TOV) was detected in 2008 in Swiss goats, which was recognized as a new serotype of BTV (BTV25). The (re-)emergency of known and unknown BTV-serotypes needs a rapid response to supply effective vaccines, and research to study this phenomenon. Recently, orbivirus research achieved an important breakthrough by the establishment of reverse genetics for BTV1. Here, reverse genetics for two recent BTV strains representing virulent BTV8 and avirulent BTV6 was developed. For this purpose, extensive sequencing of full-genomes was performed, resulting in the consensus sequences of BTV8/net07 and BTV6/net08. The recovery of 'synthetic BTV', respectively rgBTV8 and rgBTV6, completely from T7-derived RNA transcripts was confirmed by silent mutations by which these 'synthetic BTVs' could be genetically distinguished from wild type BTV, respectively wtBTV6 and wtBTV8. The in vitro and in vivo properties of rgBTV6 or rgBTV8 were comparable to the properties of their parent strains. The asymptomatic or avirulent properties of rgBTV6 and the virulence of rgBTV8 were confirmed by experimental infection of sheep. Reverse genetics of the vaccine-related BTV6 provides a perfect start to develop new generations of BT-vaccines. Reverse genetics of the virulent BTV8 will accelerate research on the special features of BTV8, like transmission by species of Culicoides in a moderate climate, transplacental transmission, and pathogenesis in cattle.

Spatial analysis of bluetongue cases and vaccination of Swiss cattle in 2008 and 2009.

Bluetongue (BT) is a vector-borne viral disease of ruminants. The infection is widespread globally with major implications for international animal trade and production. In 2006, BT virus serotype 8 (BTV-8) was encountered in Europe for the first time, causing extensive production losses and death in susceptible livestock. Following the appearance of BTV-8 in Switzerland in 2007, a compulsory vaccination programme was launched in the subsequent year. Due to social factors and difficulties to reach animals on high pasture, the regional vaccination coverage varied across the country in both 2008 and 2009. In this study, the effect of vaccination on the spatial occurrence of BTV-8 and the associated relative disease risk in Switzerland in 2008 and 2009 were investigated by a spatial Bayesian hierarchical approach. Bayesian posterior distributions were obtained by integrated nested Laplace approximations, a promising alternative to commonly used Markov chain Monte Carlo methods. The number of observed BTV-8 outbreaks in Switzerland decreased notably from 2008 to 2009. However, only a non-significant association between vaccination coverage and the probability of a spatial unit being infected with BTV-8 was identified using the model developed for this study. The relative disease risk varied significantly across the country, with a higher relative risk of BTV-8 infection in western and north-western Switzerland where environmental conditions are more suitable for vector presence and viral transmission. Examination of the spatial correlation between disease occurrence, control measures and associated ecological factors can be valuable in the evaluation and development of disease control programmes, allowing prioritisation of areas with a high relative risk of disease.

Culicoides vectors of bluetongue virus in Chester Zoo.

On four nights in June 2008, light traps were operated for Culicoides biting midges, the vector species for bluetongue virus (BTV), at five sites in Chester Zoo in north-west England. Over 35,000 Culicoides midges, of 25 species, were captured, including high densities inside animal enclosures. Over 94 per cent of all the Culicoides trapped were females of the Obsoletus group, which is implicated as the vector of BTV serotype 8 in northern Europe. The mean catch of this group per trap per night was over 1500, suggesting a potential risk of BTV transmission if the virus is introduced to Chester Zoo in the animals or midges in the summer.

Two alternative inocula to reproduce bluetongue virus serotype 8 disease in calves.

The aim of this study was to investigate the consequences in calves of two forms of inocula alternative to the use of wild type infectious blood. Two groups of five calves were infected with low cell-passaged virus and infectious blood issued from one animal passage of the same strain. A longitudinal study was implemented and characterised by clinical standardised observations, haematology, BTV RNA detection and viral isolation from blood, detection of serogroup and neutralising antibodies, cytokine expression and post-mortem examination 46 days post-infection (PI). Both tested inocula were able to reproduce clinical expression of the disease, in the bloodstream viral genome was detected until the end of the experiment while virus isolation was possible between days 7 and 31 PI. Humoral immune response developed earlier in calves infected with low cell-passaged virus, while in both groups a massive antibody production was confirmed by the immune balance between IL-4 and IFN-γ expression. Both tested inocula are presented as valid alternative to the use of wild type infectious blood in the study of the pathogenesis of BTV-8 or the efficacy of current and future vaccines.

Interaction of calpactin light chain (S100A10/p11) and a viral NS protein is essential for intracellular trafficking of nonenveloped bluetongue virus.

Bluetongue virus (BTV), a member of the Reoviridae family, is an insect-borne animal pathogen. Virus release from infected cells is predominantly by cell lysis, but some BTV particles are also released from the plasma membrane. The nonstructural protein NS3 has been implicated in this process. Using alternate initiator methionine residues, NS3 is expressed as a full-length protein and as a truncated variant that lacks the initial 13 residues, which, by yeast-two hybrid analyses, have been shown to interact with a cellular trafficking protein S100A10/p11. To understand the physiological significance of this interaction in virus-infected cells, we have used reverse genetics to investigate the roles of NS3 and NS3A in virus replication and localization in both mammalian and insect vector-derived cells. A virus expressing NS3 but not NS3A was able to propagate in and release from mammalian cells efficiently. However, growth of a mutant virus expressing only NS3A was severely attenuated, although protein expression, replication, double-stranded RNA (dsRNA) synthesis, and particle assembly in the cytoplasm were observed. Two of three single-amino-acid substitutions in the N-terminal 13 residues of NS3 showed phenotypically similar effects. Pulldown assay and confocal microscopy demonstrated a lack of interaction between NS3 and S100A10/p11 in mutants with poor replication. The role of NS3/NS3A was also assessed in insect cells where virus grew, albeit with a reduced titer. Notably, however, while wild-type particles were found within cytoplasmic vesicles in insect cells, mutant viruses were scattered throughout the cytoplasm and not confined to vesicles. These results provide support for a role for the extreme amino terminus of NS3 in the late stages of virus growth in mammalian cells, plausibly in egress. However, both NS3 and NS3A were required for efficient BTV growth in insect cells.

A stochastic predictive model for the natural spread of bluetongue.

In recent years the vector-borne diseases (VBD) are (re)-emerging and spreading across the world having a profound impact on human and veterinary health, ecology, socio-economics and disease management. Arguably the best-documented example of veterinary importance is the recent twofold invasion of bluetongue (BT) in Europe. Much attention has been devoted to derive presence-absence habitat distribution models and to model transmission through direct contact. Limited research has focused on the dynamic modelling of wind mediated BT spread. This paper shows the results of a stochastic predictive model used to assess the spread of bluetongue by vectors considering both wind-independent and wind-mediated movement of the vectors. The model was parameterised using epidemiological knowledge from the BTV8 epidemic in 2006/2007 and the BTV1 epidemic in 2008 in South-France. The model correctly reflects the total surface of the infected zone (overall accuracy=0.77; sensitivity=0.94; specificity=0.65) whilst slightly overestimating spatial case density. The model was used operationally in spring 2009 to predict further spread of BTV1. This allowed veterinary officers in Belgium to decide whether there was a risk of introduction of BTV1 from France into Belgium and thus, whether there was a need for vaccination. Given the far distance from the predicted infected zone to the Belgian border, it was decided not to vaccinate against BTV1 in 2009 in Belgium.

Possible means of introduction of bluetongue virus serotype 8 (BTV-8) to Sweden in August 2008: comparison of results from two models for atmospheric transport of the Culicoides vector.

In September 2008, bluetongue virus serotype 8 (BTV-8) infection was detected for the first time in Sweden, in a dairy herd on the west coast. Two different previously published operational atmospheric dispersion models indicate that midges from infected regions in Europe are likely to have reached Sweden by atmospheric transport during an estimated infection window. Both models indicated that the likely dates for the incursion of midges were overnight on August 6 to 7 and August 14 to 15; however, the less constrained model indicated a number of additional possible dates. The distribution of infected herds detected by active surveillance coincides with the regions that were indicated by the models to have been reached by midges from regions in Denmark and Germany with infected herds. It is likely that several points of introduction of infected midges occurred, possibly on different occasions. No alternative routes for introduction of the infection to Sweden were identified, supporting the theory that BTV-8 was introduced by infected midges carried by the wind.

Infectious blood or culture-grown virus: a comparison of bluetongue virus challenge models.

The World Organisation for Animal Health (OIE) currently recommends using infectious ruminant blood as challenge inoculum in bluetongue virus (BTV) vaccination and challenge experiments. The use of virus grown in cultured cells is discouraged because culture passages can lead to changes in virus phenotype, including reduced replication efficiency and virulence in the host, while the OIE considers clinical disease in control animals indispensable evidence of successful infection. In the present study, two groups of five sheep were inoculated with either infectious calf blood lysate or culture-grown bluetongue virus of serotype 8 (BTV-8) (2 × 10(4)TCID(50) and 5 × 10(5)TCID(50), respectively). No pronounced difference in the induction and progression of viraemia as determined by real-time RT-PCR, which is the most objective parameter in the evaluation of vaccine efficacy, was observed. In a second experiment, the virulence of both inocula was confirmed by fatal infection of interferon receptor-deficient mice. The recent availability of highly sensitive molecular methods for the detection of BTV can finally shift the focus away from clinical disease. For the sake of objective and repeatable BTV challenge experiments, the OIE should reconsider its policy on culture-grown virus.