Bluetongue Virus Serotype Research Articles

Isolation of Culicoides- and Mosquito-Borne Orbiviruses in the Southwestern Islands of Japan Between 2014 and 2019.

The circulation of arboviruses in livestock ruminants has often gone unrecognized owing to the fact that a significant percentage of arboviruses probably induce subclinical infections and/or negligible symptoms in infected animals. To determine the current situation of arbovirus circulation in the Yaeyama Islands, attempts to isolate viruses from bovine blood samples collected between 2014 and 2019 have been made. In total, 308 blood samples were collected during the study period, and 43 of them induced cytopathic effects (CPEs) in cell cultures. The identification of the CPE agents was performed by reported RT-PCR assays and a high-throughput analysis with a next-generation sequencing platform. The obtained viruses consisted of an orthobunyavirus (Peaton virus), Culicoides-borne orbiviruses (bluetongue virus serotypes 12 and 16, epizootic hemorrhagic disease virus [EHDV] serotypes 5, 6, and 7, D'Aguilar virus, and Bunyip Creek virus), and potential mosquito-borne orbiviruses (Yunnan orbivirus, Guangxi orbivirus, and Yonaguni orbivirus). Most of the orbiviruses were recovered from washed blood cells with mosquito cell cultures, suggesting that this combination was more efficient than other combinations such as plasma/blood cells and hamster cell lines. This marked the first time that the isolation of EHDV serotypes 5 and 6 and three potential mosquito-borne orbiviruses was recorded in Japan, showing a greater variety of orbiviruses on the islands than previously known. Genetic analysis of the isolated orbiviruses suggested that the Yaeyama Islands and its neighboring regions were epidemiologically related. Some of the viruses, especially the potential mosquito-borne orbiviruses, were isolated during several consecutive years, indicating their establishment on the islands.

Development and Validation of an ELISA for the Detection of Bluetongue Virus Serotype 4-Specific Antibodies.

In this article, we describe the development and evaluation of a double antigen sandwich enzyme-linked immunosorbent assay (ELISA) able to detect serotype 4-specific antibodies from BTV-4 infected or vaccinated animals using a recombinant BTV-4 VP2 protein. The coding sequence of VP2 was inserted into a pVote plasmid by recombination in the Gateway® cloning system. Vaccinia virus (VacV) was used as a vector for the expression of the recombinant VP2. After production in BSR cells, recombinant VP2 was purified by immunoprecipitation using a FLAG tag and then used both as the coated ELISA antigen and as the HRP-tagged conjugate. The performance of the ELISA was evaluated with 1186 samples collected from BTV negative, infected or vaccinated animals. The specificity and sensitivity of the BTV-4 ELISA were above the expected standards for the detection of anti-BTV-4 VP2 antibodies in animals reared in Europe or in the Mediterranean basin. Cross-reactions were observed with reference sera for serotypes 10 and 20, and to a lesser extent with serotypes 12, 17 and 24, due to their genetic proximity to serotype 4. Nevertheless, these serotypes have never been detected in Europe and the Mediterranean area. This ELISA, which requires only the production of a recombinant protein, can be used to detect BTV serotype 4-specific antibodies and is therefore an attractive alternative diagnostic method to serum neutralization.

Environment, vector, or host? Using machine learning to untangle the mechanisms driving arbovirus outbreaks.

Climatic, landscape, and host features are critical components in shaping outbreaks of vector‐borne diseases. However, the relationship between the outbreaks of vector‐borne pathogens and their environmental drivers is typically complicated, nonlinear, and may vary by taxonomic units below the species level (e.g., strain or serotype). Here, we aim to untangle how these complex forces shape the risk of outbreaks of Bluetongue virus (BTV); a vector‐borne pathogen that is continuously emerging and re‐emerging across Europe, with severe economic implications. We tested if the ecological predictors of BTV outbreak risk were serotype‐specific by examining the most prevalent serotypes recorded in Europe (1, 4, and 8). We used a robust machine learning (ML) pipeline and 23 relevant environmental features to fit predictive models to 24,245 outbreaks reported in 25 European countries between 2000 and 2019. Our ML models demonstrated high predictive performance for all BTV serotypes (accuracies > 0.87) and revealed strong nonlinear relationships between BTV outbreak risk and environmental and host features. Serotype‐specific analysis suggests, however, that each of the major serotypes (1, 4, and 8) had a unique outbreak risk profile. For example, temperature and midge abundance were as the most important characteristics shaping serotype 1, whereas for serotype 4 goat density and temperature were more important. We were also able to identify strong interactive effects between environmental and host characteristics that were also serotype specific. Our ML pipeline was able to reveal more in‐depth insights into the complex epidemiology of BTVs and can guide policymakers in intervention strategies to help reduce the economic implications and social cost of this important pathogen.

Safety and efficacy of a Bluetongue inactivated vaccine (serotypes 1 and 4) in sheep

A new inactivated vaccine against Bluetongue virus (BTV) serotypes 1 and 4, was developed from field isolates. Safety and efficacy of the vaccine were evaluated in sheep by serological monitoring and virus nucleic acid detection after experimental infection of vaccinated animals. Seroconversion was observed in vaccinated animals at day 14 post vaccination (pv) with neutralizing antibody titer of 1.9 and 1.8 for serotypes 1 and 4, respectively. The titer increase significantly after the booster reaching 2.7 and persist one year >1.5 for both serotypes. After challenge with virulent isolates, vireamia was recorded in control animals, as evident by q-PCR with threshold cycles (Ct) ranging from 24 to 31 and peaked at day 10 post challenge, while no vireamia was detected in vaccinated animals. Vaccinated sheep were fully protected against the disease and infection.

Serological Cross-Reactions between Expressed VP2 Proteins from Different Bluetongue Virus Serotypes.

Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana, based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three ‘novel’ serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same ‘VP2 nucleotype’.

Detection of arboviruses in Culicoides (Diptera: Ceratopogonidae) collected from animal farms in the border areas of Yunnan Province, China

Biting midges of the genus Culicoides (order Diptera, family Ceratopogonidae) are potential biological vectors for the transmission of certain arboviruses among humans, livestock, and wild animals. This study collected a total of 405 Culicoides individuals from seven animal farms located in five counties in the border areas of Yunnan Province, China, and examined the Culicoides species composition and the major arboviruses carried by the Culicoides species. The collected Culicoides were classified into seven species with variable abundances: Culicoides arakawae (5.43%, 22/405), Culicoides homotomus (1.23%, 5/405), Culicoides obsoletus (19.75%, 80/405), Culicoides orientalis (17.28%, 70/405), Culicoides oxystoma (29.38%, 119/405), Culicoides peregrinus (5.68%, 23/405), and Culicoides nipponensis (21.23%, 86/405). Among the seven species, C. oxystoma and C. nipponensis were distributed in all the five counties with abundances of 13.33–44.87% and 10.00–46.83%, respectively, suggesting that these were the dominant species of Culicoides widespread on animal farms in the border areas. PCR was used to detect major arboviruses in the collected Culicoides specimens, including bluetongue virus (BTV), Japanese encephalitis virus, Dengue virus, Zika virus, African swine fever virus, and African horse sickness virus. Among the tested viruses, only BTV serotype 1 was tested positive in C. oxystoma specimens collected from a buffalo farm. Culicoides oxystoma was the dominant species on animal farms in the sampled areas, but it has not previously been documented as positive for BTV in China. The current results thus suggest that C. oxystoma could be an important vector for BTV transmission in these border areas, which, however, needs to be confirmed by further comprehensive experiments. Overall, the present study provides the first profile of Culicoides species on animal farms in the China, Vietnam, and Myanmar border areas, establishes the prevalence of arboviruses carried by these Culicoides species, and suggests the vector potential of C. oxystoma species for the transmission of BTV.

Long-term determinants of the seroprevalence of the bluetongue virus in deer species in southern Spain

Bluetongue is a vector-borne disease affecting domestic and wild ruminants, with a major socioeconomic impact. Endemic circulation of the bluetongue virus serotype 4 (BTV-4) and BTV-1 have occurred in Spain since 2004 and 2007, respectively. However, epidemiological studies have seldom been approached from a long-term perspective in wild ruminants. A total of 881 deer (red deer and fallow deer) were necropsied from 2005 to 2018 as part of the DNP health-monitoring program. Serum samples were tested for antibodies against BTV with the aims of assessing the temporal trend and to evaluate the modulating factors: individual, populational, environmental, and stochastic. Red deer displayed statistically significant higher seroprevalences of BTV (SBT; 78.6±3.8%) than fallow deer (53.1±4.7%). The detection of BTV-1 and BTV-4 by the serum neutralization test in calves suggested the circulation of both serotypes over the study period. For red deer, wet years together with high densities could provide suitable conditions for vector borne BTV transmission. Moreover, proximity to high suitability habitat for Culicoides, permanent pasturelands, was associated with higher SBT. The differences in the ecology and behaviour of deer species influencing the exposure to the vectors could determine the differences found in the SBT patterns. This study evidences the role that deer species may play in the maintenance of BTV, however, elucidating the epidemiological role of host in different contexts as well as the consequences of climate change on the competent vector populations and its potential effect on the dynamics of BTV infection in hosts communities deserve further research.

Critical parameters of real time reverse transcription polymerase chain reaction (RT-PCR) diagnostics: Sensitivity and specificity for bluetongue virus

A new variant of bluetongue virus serotype 3, BTV3 ITL 2018 (here named: BTV3), was included in serial dilutions in the BT Proficiency Test 2020. Although the OIE-recommended panBTV real time RT-PCR test targeting genome segment 10 (Seg-10) detected this variant, we showed that reverse transcription (RT) at 61 °C instead of 50 °C completely abolished detection. Another Seg-10 panBTV real time RT-PCR test detected BTV3, irrespective of the temperature of RT. In silico validation showed that each of the OIE-recommended PCR primers using IVI-primers contain single mismatches at the -3 position for BTV3. In contrast, WBVR-primers of a second test completely match to the BTV3 variant. Our results suggest that single mismatches caused false negative PCR results for BTV3 at high RT temperature. Indeed, correction of both IVI-primers for BTV3 led to positive results for BTV3 but negative results for all other samples of the BT Proficiency Test 2020. Apparently, variability of the -3 position is sufficient for discriminative PCR detection, although the single mismatch in the IVI-reverse primer was the most important for this phenomenon. Extensive in silico validation showed that targets of both Seg-10 panBTV RT-PCR tests are not completely conserved, and the detailed effect of single mismatches are hard to predict. Therefore, we recommend at least two panBTV RT-PCR tests to minimize the risk of false negatives. Preferably, their PCR targets should be located at completely different and highly conserved regions of the BTV genome to guarantee adequate detection of future BTV infections.

An Early Block in the Replication of the Atypical Bluetongue Virus Serotype 26 in Culicoides Cells Is Determined by Its Capsid Proteins.

Arboviruses such as bluetongue virus (BTV) replicate in arthropod vectors involved in their transmission between susceptible vertebrate-hosts. The “classical” BTV strains infect and replicate effectively in cells of their insect-vectors (Culicoides biting-midges), as well as in those of their mammalian-hosts (ruminants). However, in the last decade, some “atypical” BTV strains, belonging to additional serotypes (e.g., BTV-26), have been found to replicate efficiently only in mammalian cells, while their replication is severely restricted in Culicoides cells. Importantly, there is evidence that these atypical BTV are transmitted by direct-contact between their mammalian hosts. Here, the viral determinants and mechanisms restricting viral replication in Culicoides were investigated using a classical BTV-1, an “atypical” BTV-26 and a BTV-1/BTV-26 reassortant virus, derived by reverse genetics. Viruses containing the capsid of BTV-26 showed a reduced ability to attach to Culicoides cells, blocking early steps of the replication cycle, while attachment and replication in mammalian cells was not restricted. The replication of BTV-26 was also severely reduced in other arthropod cells, derived from mosquitoes or ticks. The data presented identifies mechanisms and potential barriers to infection and transmission by the newly emerged “atypical” BTV strains in Culicoides.

The Bluetongue Disabled Infectious Single Animal (DISA) Vaccine Platform Based on Deletion NS3/NS3a Protein Is Safe and Protective in Cattle and Enables DIVA.

The bluetongue virus (BTV) is transmitted by Culicoides biting midges and causes bluetongue (BT), an OIE-notifiable disease of ruminants. At least 29 BTV serotypes are described as determined by the outer shell proteins VP2 and VP5. Vaccination is the most effective control measure. Inactivated and live-attenuated vaccines (LAVs) are currently available. These vaccines have their specific pros and cons, and both are not DIVA vaccines. The BT Disabled Infectious Single Animal (DISA) vaccine platform is based on LAV without nonessential NS3/NS3a expression and is applicable for many serotypes by the exchange of outer shell proteins. The DISA vaccine is effective and completely safe. Further, transmission of the DISA vaccine by midges is blocked (DISA principle). Finally, the DISA vaccine enables DIVA because of a lack of antibodies against the immunogenic NS3/NS3a protein (DIVA principle). The deletion of 72 amino acids (72aa) in NS3/NS3a is sufficient to block virus propagation in midges. Here, we show that a prototype DISA vaccine based on LAV with the 72aa deletion enables DIVA, is completely safe and induces a long-lasting serotype-specific protection in cattle. In conclusion, the in-frame deletion of 72-aa codons in the BT DISA/DIVA vaccine platform is sufficient to fulfil all the criteria for modern veterinary vaccines.

Novel putative bluetongue virus serotype 29 isolated from inapparently infected goat in Xinjiang of China.

Bluetongue virus (BTV) is the 'type' species of the genus Orbivirus causing bluetongue (BT) in sheep, bovine and other ruminants. Twenty-four serotypes and several atypical serotypes of BTV were identified worldwide. In present study, a novel strain of BTV (V196/XJ/2014) was isolated from an asymptomatic sentinel goat in Yuli County, Xinjiang of China. Serotype identification of this isolate exhibited uniform negative results by serotype-specific conventional RT-PCR and real-time RT-PCR for BTV-1 to BTV-27, and virus neutralization tests using reference sera of BTV-1 to BTV-24. Genomic analysis showed V196/XJ/2014 grouped with atypical serotypes of BTV-25 to BTV-28, BTV-X/XJ1407, BTV-X/ITL2015 and BTV-Y/TUN2017, while segment 2 and VP2 protein of V196/XJ/2014 shared <63.4%/61.4% nucleic acids and amino acids sequence identities with other recognized BTV serotypes and its segment 2 formed a separate 'nucleotype' in phylogenetic tree. These results indicated V196/XJ/2014 does not belong to any reported serotypes of BTV. Further studies of infectivity and pathogenicity showed that goats infected with V196/XJ/2014 did not exhibit observed clinical symptoms, but high level of virus amplification and homologous neutralization antibodies were detected post-infection. Our studies suggested a novel putative serotype of BTV-29 was isolated in Xinjiang of China, which expands our knowledge about the diversity of BTV.

Disease surveillance in England and Wales, April 2021

Disease surveillance in England and Wales, April 2021

Transmission of Bluetongue Virus Serotype 8 by Artificial Insemination with Frozen-Thawed Semen from Naturally Infected Bulls.

Transmission of bluetongue (BT) virus serotype 8 (BTV-8) via artificial insemination of contaminated frozen semen from naturally infected bulls was investigated in two independent experiments. Healthy, BT negative heifers were hormonally synchronized and artificially inseminated at oestrus. In total, six groups of three heifers received semen from four batches derived from three bulls naturally infected with BTV-8. Each experiment included one control heifer that was not inseminated and that remained BT negative throughout. BTV viraemia and seroconversion were determined in 8 out of 18 inseminated heifers, and BTV was isolated from five of these animals. These eight heifers only displayed mild clinical signs of BT, if any at all, but six of them experienced pregnancy loss between weeks four and eight of gestation, and five of them became BT PCR and antibody positive. The other two infected heifers gave birth at term to two healthy and BT negative calves. The BT viral load varied among the semen batches used and this had a significant impact on the infection rate, the time of onset of viraemia post artificial insemination, and the gestational stage at which pregnancy loss occurred. These results, which confirm unusual features of BTV-8 infection, should not be extrapolated to infection with other BTV strains without thorough evaluation. This study also adds weight to the hypothesis that the re-emergence of BTV-8 in France in 2015 may be attributable to the use of contaminated bovine semen.

Nearly Complete Genome Sequences of Two Bluetongue Viruses Isolated during the 2020 Outbreak in the Grand Duchy of Luxembourg.

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

Vaccination With Recombinant Adenoviruses Expressing the Bluetongue Virus Subunits VP7 and VP2 Provides Protection Against Heterologous Virus Challenge.

Bluetongue virus (BTV) is the causative agent of a disease that affects domestic and wild ruminants and leads to critical economic losses. BTV is an arbovirus from the Reoviridae family that is typically transmitted by the bite of infected Culicoides midges. BTV possesses multiple serotypes (up to 28 have been described), and immunity to one serotype offers little cross-protection to other serotypes. The design of vaccines that provide protection across multiple serotypes is therefore highly desirable to control this disease. We previously reported that a recombinant replication-defective human adenovirus serotype 5 (Ad5) that expresses the VP7 inner core protein of BTV serotype 8 (Ad5VP7-8) induced T-cell responses and provided protection. In the present work, we evaluated as BTV vaccine the combination of Ad5VP7-8 with another recombinant Ad5 that expresses the outer core protein VP2 from BTV-1 (Ad5VP2-1). The combination of Ad5VP2-1 and Ad5VP7-8 protected against homologous BTV challenge (BTV-1 and BTV-8) and partially against heterologous BTV-4 in a murine model. Cross-reactive anti-BTV immunoglobulin G (IgG) were detected in immunized animals, but no significant titers of neutralizing antibodies were elicited. The Ad5VP7-8 immunization induced T-cell responses that recognized all three serotypes tested in this study and primed cytotoxic T lymphocytes specific for VP7. This study further confirms that targeting antigenic determinant shared by several BTV serotypes using cellular immunity could help develop multiserotype BTV vaccines.

Within-farm transmission characteristics of bluetongue virus serotype 8 in cattle and sheep in the Netherlands, 2007-2008.

In 2006 and 2007, sheep and cattle farms in the Netherlands were affected by an epidemic of bluetongue virus serotype 8 (BTV-8). In order to obtain insight into the within-farm spread of the virus, five affected cattle and five affected sheep farms were longitudinally monitored between early 2007 and mid or late 2008. The farms were visited between four and seven times to collect blood samples. During each visit, all animals present in the flock or herd were sampled. The samples were analysed for the presence of BTV-8 antibodies (ELISA) and BTV-8 antigen (rRT-PCR). The observed patterns of RT-PCR positives indicate a rapid within-farm virus spread during the vector season. During vector-free periods we observed a complete rRT-PCR positivity decline within a few months. During the vector season a lower bound estimate of the basic reproduction number (R0) ranges from 2.9–6.9 in the cattle herds (one herd not analysed), and from 1.3–3.2 in the sheep flocks in this study.

Potential Acute Renal Injury in Sheep with Bluetongue Serotype 4.

Bluetongue is a vector-borne disease with epidemic potential. Recently, outbreaks of Bluetongue were reported across Greece, caused by the Bluetongue virus (BTV) serotype 4. Regarding its pathogenesis, BTV infection involves various target organs with limited data referring to the kidneys. The objective of this study was to identify the possible impact of BTV infection on kidneys using common renal biomarkers. Urine and blood samples collected from 30 sheep with clinical signs of bluetongue (BTV sheep) and 30 clinically healthy sheep (normal sheep) from the same farms were finally selected and included in the study from an initial population of 47 sheep per group, based on the absence of active urine sediment. Complete urinalysis was performed and urine protein to creatinine ratio (UPC) and urine gamma-glutamyl transferase to creatinine (UGGTC) ratio were determined. Blood urea nitrogen (BUN), creatinine, total proteins, albumin (ALB), and inorganic phosphate (P) were determined in serum samples. UPC and UGGTC were significantly higher (p < 0.05) in BTV sheep compared to normal, whereas urine specific gravity (USG) was significantly lower (p < 0.05). Cylindruria was also detected in BTV sheep, and absence of azotemia in BTV and normal sheep. All these findings are indicative of renal tubular injury and/or dysfunction and suggestive of an association between BTV infection and acute damage of renal tissue.

Biting midge dynamics and bluetongue transmission: a multiscale model linking catch data with climate and disease outbreaks

Bluetongue virus (BTV) serotype 8 has been circulating in Europe since a major outbreak occurred in 2006, causing economic losses to livestock farms. The unpredictability of the biting activity of midges that transmit BTV implies difficulty in computing accurate transmission models. This study uniquely integrates field collections of midges at a range of European latitudes (in Sweden, The Netherlands, and Italy), with a multi-scale modelling approach. We inferred the environmental factors that influence the dynamics of midge catching, and then directly linked predicted midge catches to BTV transmission dynamics. Catch predictions were linked to the observed prevalence amongst sentinel cattle during the 2007 BTV outbreak in The Netherlands using a dynamic transmission model. We were able to directly infer a scaling parameter between daily midge catch predictions and the true biting rate per cow per day. Compared to biting rate per cow per day the scaling parameter was around 50% of 24 h midge catches with traps. Extending the estimated biting rate across Europe, for different seasons and years, indicated that whilst intensity of transmission is expected to vary widely from herd to herd, around 95% of naïve herds in western Europe have been at risk of sustained transmission over the last 15 years.

Pathological and immunological characterization of bluetongue virus serotype 1 infection in type I interferons blocked immunocompetent adult mice

IntroductionWild-type adult mice with intact interferon (IFN) system were neither susceptible to bluetongue virus (BTV) infection nor showed signs of morbidity/mortality. Establishment of immunologically competent wild-type adult mouse model with type I IFNs blockade is necessary to assess the pathogenesis, immune responses and testing of BTV vaccines. ObjectivesPresent study aimed to establish and characterize BTV serotype 1 infection in immunocompetent adult mice with type I IFNs blockade at the time of infection by studying immune responses and sequential pathology. MethodsAdult mice were administered with anti-mouse IFN-α/β receptor subunit-1 (IFNAR1) blocking antibody (Clone: MAR1-5A3) 24 h before and after BTV serotype 1 infection, and sacrificed at various time points. Sequential pathology, BTV localization by immunohistochemistry and quantification by qRT-PCR, immune cell kinetics and apoptosis by flow cytometry, and cytokines estimation by c-ELISA and qRT-PCR were studied. ResultsIFNAR blocked-infected mice developed clinical signs and typical lesions of BT; whereas, isotype-infected control mice did not develop any disease. The IFNAR blocked-infected mice showed enlarged, edematous, and congested lymph nodes (LNs) and spleen, and vascular (congestion and hemorrhage) and pneumonic lesions in lungs. Histopathologically, marked lymphoid depletion with “starry-sky pattern” due to lymphocytes apoptosis was noticed in the LNs and spleen. BTV antigen was detected and quantified in lymphoid organs, lungs, and other organs at various time points. Initial leukopenia (increased CD4+/CD8+ T cells ratio) followed by leukocytosis (decreased CD4+/CD8+ T cells ratio) and significantly increased biochemical values were noticed in IFNAR blocked-infected mice. Increased apoptotic cells in PBMCs and tissues coincided with viral load and levels of different cytokines in blood, spleen and draining LNs and notably varied between time points in IFNAR blocked-infected mice. ConclusionPresent study is first to characterize BTV serotype 1 infection in immunocompetent adult mouse with type I IFNs blockade. The findings will be useful for studying pathogenesis and testing the efficacy of BTV vaccines.

Pathogen detection and disease diagnosis in wildlife: challenges and opportunities.

The availability of rapid, highly sensitive and specific molecular and serologic diagnostic assays, such as competitive enzyme-linked immunosorbent assay (cELISA), has expedited the diagnosis of emerging transboundary animal diseases, including bluetongue (BT) and African horse sickness (AHS), and facilitated more thorough characterisation of their epidemiology. The development of assays based on real-time, reverse-transcription polymerase chain reaction (RT-PCR) to detect and identify the numerous serotypes of BT virus (BTV) and AHS virus (AHSV) has aided in-depth studies of the epidemiology of BTV infection in California and AHSV infection in South Africa. The subsequent evaluation of pan-serotype, real-time, RT-PCR-positive samples through the use of serotype-specific RT-PCR assays allows the rapid identification of virus serotypes, reducing the need for expensive and time-consuming conventional methods, such as virus isolation and serotype-specific virus neutralisation assays. These molecular assays and cELISA platforms provide tools that have enhanced epidemiologic surveillance strategies and improved our understanding of potentially altered Culicoides midge behaviour when infected with BTV. They have also supported the detection of subclinical AHSV infection of vaccinated horses in South Africa. Moreover, in conjunction with whole genome sequence analysis, these tests have clarified that the mechanism behind recent outbreaks of AHS in the AHS-controlled area of South Africa was the result of the reversion to virulence and/or genome reassortment of live attenuated vaccine viruses. This review focuses on the use of contemporary molecular diagnostic assays in the context of recent epidemiologic studies and explores their advantages over historic virus isolation and serologic techniques.