A survey of bluetongue infection in one-humped camels (Camelus Dromedarius); seroprevalence and risk factors analysis.

Bluetongue (BT) is an insect-borne, non-contagious viral disease which affects domestic ruminants including camels and is transmitted by Culicoides spp. Clinical symptoms of BT are typically seen in sheep, although subclinical BT infections are mostly seen in cattle, goats, and camelids. The goal of the present study was to evaluate the sero-prevalence of Bluetongue virus (BTV) in camels from some governorates in Egypt's southern and northern regions, as well as the infection's potential risk factors. During 2020-2021, a cross sectional study was conducted to screen presence of anti-BTV antibodies in 400 serum samples, which were collected randomly from camels, examined using competitive enzyme-linked immunosorbent assay (cELISA). The sera of 102 out of 400 camels tested positive for BTV, representing a frequency of 25.5%. Moreover, the odds of sero-positivity were higher among camels living in Aswan (OR = 5.33, 95%CI: 2.35-12.11), especially in females (OR = 2.63, 95%CI = 1.44-4.09) during summer season (OR = 2.40, 95%CI = 1.20-4.81). Furthermore, the probability of getting BTV infection increased when camels were exposed to the insect vectors (OR = 1.63, 95%CI = 0.87-3.09). The high prevalence of BTV in camels in several Egyptian regions highlights the need for more epidemiological investigations of BTV infection in other ruminant species in order to better control BT disease in these regions.

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.

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.

Spatial Analysis of the 2017 Outbreak of Hemorrhagic Disease and Physiographic Region in the Eastern United States.

Hemorrhagic disease (HD) is considered one of the most significant infectious diseases of white-tailed deer in North America. Investigations into environmental conditions associated with outbreaks suggest drought conditions are strongly correlated with outbreaks in some regions of the United States. However, during 2017, an HD outbreak occurred in the Eastern United States which appeared to be associated with a specific physiographic region, the Appalachian Plateau, and not drought conditions. The objective of this study was to determine if reported HD in white-tailed deer in 2017 was correlated with physiographic region. There were 456 reports of HD from 1605 counties across 26 states and 12 physiographic regions. Of the 93 HD reports confirmed by virus isolation, 76.3% (71/93) were identified as EHDV-2 and 66.2% (47/71) were from the Appalachian Plateau. A report of HD was 4.4 times more likely to occur in the Appalachian Plateau than not in 2017. Autologistic regression models suggested a statistically significant spatial dependence. The underlying factors explaining this correlation are unknown, but may be related to a variety of host, vector, or environmental factors. This unique outbreak and its implications for HD epidemiology highlight the importance for increased surveillance and reporting efforts in the future.

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.

Bluetongue virus in South America: current status based on phylogenetic analysis.

Bluetongue (BT) is an insect-borne disease affecting domestic and wild ruminants. Bluetongue virus (BTV) is the causative agent of the BT disease. BT outbreaks have been widely recorded worldwide. However, in the South American subcontinent, accurate information about the disease and molecular epidemiology is still lacking because little effort has been made to cover the region. This study comprises an exhaustive phylogenetic analysis including all BTV sequences available in databases and reports new Argentinean sequences for Seg 8 and Seg 9. Maximum-likelihood phylogenetic analyses were conducted for Seg 2, Seg 3, Seg 6, Seg 7, Seg 8, Seg 9 and Seg 10. Throughout the study, wide circulation and genetic continuity along the American continent were detected. Also, reassortment events are reported, and the historical virus introduction path into and through South America is suggested.

Detection of bluetongue virus in Culicoides spp. in southern Yunnan Province, China.

BACKGROUND: Culicoides (Diptera: Ceratopogonidae) are vectors for many arboviruses. At least 20 species are considered as vectors or potential vectors of bluetongue virus (BTV) which cause bluetongue disease in ruminants. A BTV prevalence of 30-50% among cattle and goats in tropical southern Yunnan Province, China, prompted an investigation of the potential BTV vectors in this area. METHODS: Culicoides were collected by light trapping at three sites in the tropical region of Yunnan Province. Species were identified based on morphology and DNA sequences of cytochrome c oxidase subunit 1 (cox1). PCR and quantitative PCR following reverse transcription were used to test for the presence of BTV RNA in these specimens. Phylogenetic analysis was used to analyze the cox1 sequences of Culicoides specimens infected with BTV. RESULTS: Approximately 67,000 specimens of Culicoides were collected, of which 748 were tested for the presence of BTV. Five specimens, including two of Culicoides jacobsoni, one of C. tainanus and two of C. imicola, were identified as infected with BTV. No specimens of C. (subgenus Trithecoides) or C. oxystoma tested were positive for BTV infection. CONCLUSIONS: To our knowledge this is the first report of C. jacobsoni as a potential BTV vector and the fourth report of an association between C. tainanus and BTV, as well as the first direct evidence of an association between BTV and C. imicola in Asia. A fourth potential cryptic species within C. tainanus was identified in this study. Further analysis is required to confirm the importance of C. jacobsoni and C. tainanus in BTV epidemiology in Asia.

Production and Easy One-Step Purification of Bluetongue Recombinant VP7 from Infected Sf9 Supernatant for an Immunoenzymatic Assay (ELISA).

Bluetongue (BT) is non-contagious, vector-borne viral disease of domestic and wild ruminants, transmitted by midges (Culicoides spp.) and is caused by Bluetongue virus (BTV). BTV is the type species of the Orbivirus genus within the Reoviridae family and possesses a genome consisting of 10 double-stranded RNA segments encoding 7 structural and 4 nonstructural proteins. Viral Protein 7 (VP7) is the major sera group-specific protein and is a good antigen candidate for immunoenzymatic assays for the BT diagnosis. In our work, BTV-2 recombinant VP7 (BTV-2 recVP7), expressed in Spodoptera frugiperda (Sf9) cells using a baculovirus system, was produced and purified by affinity chromatography from the supernatant of infected cell culture. The use of the supernatant allowed us to obtain a high quantity of recombinant protein with high purity level by an easy one-step procedure, rather than the multistep purification from the pellet. RecVP7-BTV2 was detected using a MAb anti-BTV in Western blot and it was used to develop an immunoenzymatic assay.

Re-emergence of bluetongue virus serotype 4 in Iberian ibex (Capra pyrenaica) and sympatric livestock in Spain, 2018-2019.

Between early October and mid-December 2018, mortalities were detected in Iberian ibex (Capra pyrenaica) populations in southern Spain. In the same region and period, bluetongue virus (BTV) circulation was also reported in sentinel and clinically affected domestic ruminant herds. Molecular analyses confirmed BTV serotype 4 (BTV-4) infection in eight Iberian ibexes from six hunting areas, and in 46 domestic ruminants from seven herds in close proximity to affected hunting estates. Histopathological analyses revealed vascular changes in several organs, pneumonia, lymphoid depletion, inflammatory mononuclear cell infiltrate and fibrosis as the most frequently observed lesions in the affected Iberian ibexes. Epidemiological and laboratory results indicate that BTV-4 was the main aetiological agent involved in outbreaks detected in Iberian ibex populations during the study period. Sequence analyses indicated that the BTV-4 strain detected in Iberian ibex had high homology (99.4%-100%) with strains isolated in livestock during the same period, and with previous isolates (≥98.9%) from Spain and Mediterranean Basin countries. Further studies are warranted to determine the impact of BTV-4 on the health status of Iberian ibex populations after the outbreaks. The inclusion of this species in the surveillance programme may be useful for early detection of BTV, especially in epidemiological scenarios at the wildlife-livestock interface.

Bluetongue virus serotype 12 enters Australia - a further incursion of novel western lineage genome segments.

Bluetongue virus (BTV) is an arbovirus (genus: Orbivirus) that occurs worldwide. It infects domestic and wild ruminant species and can cause disease in livestock, producing high economic impact. Recently, it gained extra prominence throughout Europe, with disease occurring in regions traditionally free of BTV. BTV enters Australia from Southeast Asia via wind-borne infected Culicoides spp. The first Australian isolation was 1975 (BTV-20) and further serotypes were isolated between 1979-86 (BTV-1, -3, -9, -15, -16, -21, -23). Despite increased, more sensitive, monitoring, no more were detected in over two decades, implying a stable BTV episystem of eastern ancestry. Isolations of BTV-2, -7 and -5 then occurred between 2007-15, with the latter two possessing genome segments with high sequence identity to western isolates. We report on the first isolation and genomic characterization of BTV-12, which revealed that three more novel western topotype gene segments have entered northern Australia.

Bayesian optimisation of restriction zones for bluetongue control.

We investigate the restriction of animal movements as a method to control the spread of bluetongue, an infectious disease of livestock that is becoming increasingly prevalent due to the onset of climate change. We derive control policies for the UK that minimise the number of infected farms during an outbreak using Bayesian optimisation and a simulation-based model of BT. Two cases are presented: first, where the region of introduction is randomly selected from England and Wales to find a generalised strategy. This "national" model is shown to be just as effective at subduing the spread of bluetongue as the current strategy of the UK government. Our proposed controls are simpler to implement, affect fewer farms in the process and, in so doing, minimise the potential economic implications. Second, we consider policies that are tailored to the specific region in which the first infection was detected. Seven different regions in the UK were explored and improvements in efficiency from the use of specialised policies presented. As a consequence of the increasing temperatures associated with climate change, efficient control measures for vector-borne diseases such as this are expected to become increasingly important. Our work demonstrates the potential value of using Bayesian optimisation in developing cost-effective disease management strategies.

Type specific seroprevalence of bluetongue virus during 2017-2018 in Andhra Pradesh and Telangana states of India.

Bluetongue (BT) is one of the important viral diseases of domestic and wild ruminants, especially small ruminants such as sheep. Out of the 29 BTV serotypes prevalent in the world, at least 24 of the serotypes are reported in India, either by virus isolation or serology. To better understand the seroprevalence of BTV, we conducted a comprehensive study in the main reservoir hosts of BTV, i.e., cattle and buffaloes of different age groups in Andhra Pradesh and Telangana states of India where the disease is majorly prevalent. A total of 321 blood samples collected from cattle and buffaloes during 2017-2018 were tested for group-specific BTV seroprevalence by c-ELISA, followed by type specific seroprevalence (against BTV-1, 2, 4, 5, 9, 12, 16, and 24) by serum neutralization test. Of the 311 BTV seropositive samples, 112, 98, 102, 127, 2, 113, 160, and 5 samples neutralized BTV-1, 2, 4, 5, 9, 12, 16, and 24, respectively. Twenty-nine samples could not neutralize any of the tested BTV serotypes. Majority of the sera neutralized more than one serotype, up to a maximum of six serotypes. Major finding of the study is detection of BTV serotypes not included in the commercial pentavalent inactivated vaccine. Regular surveillance of circulating serotypes, especially in sentinel reservoir hosts throughout the country can help in designing better multivalent vaccines with suitable vaccine strains, for specific geographic regions.

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.

Co-Circulation of Multiple Serotypes of Bluetongue Virus in Zambia.

Bluetongue (BT) is an arthropod-borne viral disease of ruminants with serious trade and socio-economic implications. Although the disease has been reported in a number of countries in sub-Saharan Africa, there is currently no information on circulating serotypes and disease distribution in Zambia. Following surveillance for BT in domestic and wild ruminants in Zambia, BT virus (BTV) nucleic acid and antibodies were detected in eight of the 10 provinces of the country. About 40% (87/215) of pooled blood samples from cattle and goats were positive for BTV nucleic acid, while one hartebeest pool (1/43) was positive among wildlife samples. Sequence analysis of segment 2 revealed presence of serotypes 3, 5, 7, 12 and 15, with five nucleotypes (B, E, F, G and J) being identified. Segment 10 phylogeny showed Zambian BTV sequences clustering with Western topotype strains from South Africa, intimating likely transboundary spread of BTV in Southern Africa. Interestingly, two Zambian viruses and one isolate from Israel formed a novel clade, which we designated as Western topotype 4. The high seroprevalence (96.2%) in cattle from Lusaka and Central provinces and co-circulation of multiple serotypes showed that BT is widespread, underscoring the need for prevention and control strategies.

Risk-based surveillance for bluetongue virus in cattle on the south coast of England in 2017 and 2018.

BACKGROUND: Bluetongue (BT) is a viral disease of ruminants and camelids which can have a significant impact on animal health and welfare and cause severe economic loss. The UK has been officially free of bluetongue virus (BTV) since 2011. In 2015, BTV-8 re-emerged in France and since then BTV has been spreading throughout Europe. In response to this outbreak, risk-based active surveillance was carried out at the end of the vector seasons in 2017 and 2018 to assess the risk of incursion of BTV into Great Britain. METHOD: Atmospheric dispersion modelling identified counties on the south coast of England at higher risk of an incursion. Blood samples were collected from cattle in five counties based on a sample size designed to detect at least one positive if the prevalence was 5 per cent or greater, with 95 per cent confidence. RESULTS: No virus was detected in the 478 samples collected from 32 farms at the end of the 2017 vector season or in the 646 samples collected from 43 farms at the end of the 2018 vector season, when tested by RT-qPCR. CONCLUSION: The negative results from this risk-based survey provided evidence to support the continuation of the UK's official BTV-free status.

Ovine viperin inhibits bluetongue virus replication.

Viral infections can lead to interferon production, which achieves its antiviral function primarily by activating the JAK/STAT pathway and inducing multiple interferon-stimulated genes (ISGs). Although considerable ISGs have been identified in antiviral researches, little is known about ISGs in bluetongue virus (BTV) infection. Viperin is the most highly induced ISG following BTV infection, which suggests that it may play a critical role in the anti-BTV immune response. The aim of this study was to characterize ovine Viperin (oViperin) and explore whether it can inhibit BTV replication. We cloned the coding sequences (CDS) of sheep Viperin, and the sequence analysis showed that oViperin displayed a high similarity with other species. oViperin has a leucine zipper in the N-terminal, a CxxxCxxC motif in the SAM domain, and a conservative C-terminus. We found that oViperin mRNA expression was significantly up-regulated in a time- and multiplicity of infection (MOI)-dependent manner following BTV infection. oViperin overexpression resulted in a significant inhibition in BTV replication, whereas an oViperin knockdown in MDOK cells increased BTV replication. This study shows for the first time, that oViperin has antiviral activity towards BTV infection and provides important information to research the interaction between BTV and oViperin.

Epizootic Hemorrhagic Disease Virus and Bluetongue Virus Seroprevalence in Wild White-Tailed Deer (Odocoileus virginianus) in Florida, USA.

A wild population of white-tailed deer (Odocoileus virginianus) was surveyed for evidence of past or current epizootic hemorrhagic disease virus (EHDV) and current bluetongue virus (BTV) infections. We collected 121 blood samples from hunter-harvested or live-captured deer from two state-managed properties in northwest Florida, US; live captures were in support of a movement ecology study. Blood samples were tested for antibodies against titers to three EHDV serotypes (EHDV-1, EHDV-2, and EHDV-6), and multiplex quantitative reverse transcription PCR was used to identify the presence of EHDV or BTV viral RNA. Of these samples, 81% (98/121) tested seropositive for at least one of three serotypes of EHDV. Of those testing seropositive, 33% (40/121) contained antibodies for two serotypes, and 19% (24/121) contained antibodies for all three EHDV serotypes. Furthermore, results of generalized linear models indicated that the probability of infection by EHDV serotypes 1 and 6 increased with an animal's age. Our findings indicate that seroprevalence may be high for multiple serotypes in regions where these orbiviruses are endemic. These results could prove useful for managing disease risk in naïve deer populations.

Seroprevalence and Potential Risk Factors of Bluetongue Virus Infection in Domestic Cattle and Goats in Guangxi Province, Southern China.

Bluetongue is one of the most important vector-borne viral diseases that can lead to significant economic losses as a result of reduction of productivity and even death in some susceptible ruminants. However, epidemiological information on bluetongue virus (BTV) infection in cattle and goats is scarce in China. To determine the seropositive rate and risk factors of BTV infection in cattle and goats in Guangxi province, a subtropical region in Southern China, a total of 548 cattle serum samples and 6567 goat serum samples collected from 13 cities across Guangxi province during 2003-2015 were analyzed and found that the seroprevalence is 44.5% (244/548) in cattle and 28.0% (1837/6567) in goats and the main BTV serotypes are BTV-1, -2, -4, and -8. Climatic zone, age, and species are found to be the likely risk factors for BTV infection. To our knowledge, this is the first large-scale serological survey for BTV infection in domestic cattle and goats in Guangxi province, Southern China.

Characterisation of Peste Des Petits Ruminants Disease in Pastoralist Flocks in Ngorongoro District of Northern Tanzania and Bluetongue Virus Co-Infection.

Peste des petits ruminants (PPR) disease was first confirmed in Tanzania in 2008 in sheep and goats in Ngorongoro District, northern Tanzania, and is now endemic in this area. This study aimed to characterise PPR disease in pastoralist small ruminant flocks in Ngorongoro District. During June 2015, 33 PPR-like disease reports were investigated in different parts of the district, using semi-structured interviews, clinical examinations, PPR virus rapid detection test (PPRV-RDT), and laboratory analysis. Ten flocks were confirmed as PPRV infected by PPRV-RDT and/or real-time reverse transcription-polymerase chain reaction (RT-qPCR), and two flocks were co-infected with bluetongue virus (BTV), confirmed by RT-qPCR. Phylogenetic analysis of six partial N gene sequences showed that the PPR viruses clustered with recent lineage III Tanzanian viruses, and grouped with Ugandan, Kenyan and Democratic Republic of Congo isolates. No PPR-like disease was reported in wildlife. There was considerable variation in clinical syndromes between flocks: some showed a full range of PPR signs, while others were predominantly respiratory, diarrhoea, or oro-nasal syndromes, which were associated with different local disease names (olodua-a term for rinderpest, olkipiei-lung disease, oloirobi-fever, enkorotik-diarrhoea). BTV co-infection was associated with severe oro-nasal lesions. This clinical variability makes the field diagnosis of PPR challenging, highlighting the importance of access to pen-side antigen tests and multiplex assays to support improved surveillance and targeting of control activities for PPR eradication.

Origin of Bluetongue Virus Serotype 8 Outbreak in Cyprus, September 2016.

In September 2016, clinical signs, indicative of bluetongue, were observed in sheep in Cyprus. Bluetongue virus serotype 8 (BTV-8) was detected in sheep, indicating the first incursion of this serotype into Cyprus. Following virus propagation, Nextera XT DNA libraries were sequenced on the MiSeq instrument. Full-genome sequences were obtained for five isolates CYP2016/01-05 and the percent of nucleotide sequence (% nt) identity between them ranged from 99.92% to 99.95%, which corresponded to a few (2-5) amino acid changes. Based on the complete coding sequence, the Israeli ISR2008/13 (98.42-98.45%) was recognised as the closest relative to CYP2016/01-05. However, the phylogenetic reconstruction of CYP2016/01-05 revealed that the possibility of reassortment in several segments: 4, 7, 9 and 10. Based on the available sequencing data, the incursion BTV-8 into Cyprus most likely occurred from the neighbouring countries (e.g., Israel, Lebanon, Syria, or Jordan), where multiple BTV serotypes were co-circulating rather than from Europe (e.g., France) where a single BTV-8 serotype was dominant. Supporting this hypothesis, atmospheric dispersion modelling identified wind-transport events during July-September that could have allowed the introduction of BTV-8 infected midges from Lebanon, Syria or Israel coastlines into the Larnaca region of Cyprus.