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African horse sickness (AHS) is an acute, fatal, contagious disease of animals of the family Equidae and is caused by infection with the African horse sickness virus (AHSV). Based on the outer capsid protein VP2, AHSV is classified into nine serotypes (AHSV−1 to −9) with little or no serological cross-reactivity between them. In 2020, AHS outbreaks caused by AHSV−1 were reported in Thailand and Malaysia, marking the first occurrences of AHS in Southeast Asia. However, little is known about the antigenic profile of AHSV−1 VP2. In this study, a recombinant VP2 protein was expressed in Escherichia coli and used as an immunogen, and three monoclonal antibodies (mAbs), designated 7D11, 10A9, and 9E7, against AHSV−1 VP2, were generated. These three mAbs were then successfully used in IFA, WB, and ELISA for the detection of AHSV−1 VP2. Two overlapping linear epitopes, 670NEFDFE675 (E670–675) recognized by 9E7 and 670NEFDF674 (E670–674) recognized by 7D11 and 10A9, were identified through truncation of GST-fused VP2. Amino acid sequence alignment shows that the 670NEFDFE675 motif is completely conserved within AHSV−1 but is highly divergent in other AHSV serotypes. Our studies provide an important tool for basic research into AHSV−1 and for the diagnosis of AHSV−1. [ABSTRACT FROM AUTHOR]

African horse sickness (AHS) is an acute, fatal, contagious disease of animals of the family Equidae and is caused by infection with the African horse sickness virus (AHSV). Based on the outer capsid protein VP2, AHSV is classified into nine serotypes (AHSV−1 to −9) with little or no serological cross-reactivity between them. In 2020, AHS outbreaks caused by AHSV−1 were reported in Thailand and Malaysia, marking the first occurrences of AHS in Southeast Asia. However, little is known about the antigenic profile of AHSV−1 VP2. In this study, a recombinant VP2 protein was expressed in Escherichia coli and used as an immunogen, and three monoclonal antibodies (mAbs), designated 7D11, 10A9, and 9E7, against AHSV−1 VP2, were generated. These three mAbs were then successfully used in IFA, WB, and ELISA for the detection of AHSV−1 VP2. Two overlapping linear epitopes, 670NEFDFE675 (E670–675) recognized by 9E7 and 670NEFDF674 (E670–674) recognized by 7D11 and 10A9, were identified through truncation of GST-fused VP2. Amino acid sequence alignment shows that the 670NEFDFE675 motif is completely conserved within AHSV−1 but is highly divergent in other AHSV serotypes. Our studies provide an important tool for basic research into AHSV−1 and for the diagnosis of AHSV−1.

Simple Summary: The instability of viral RNA, which is susceptible to ubiquitous RNases, poses a significant challenge for its transport and detection in diagnosis. To address this challenge, this study aimed to evaluate the stability and detection limits of various RNA viruses, including the avian influenza virus, Newcastle disease virus, and African horse sickness virus, on Flinders Technology Associates cards. This investigation provides empirical evidence supporting the efficacy of Flinders Technology Associates cards for sample collection and subsequent viral RNA recovery, highlighting their suitability for use in molecular diagnostics. Consequently, based on the demonstrated effectiveness, stability, and safety implications observed in this study, Flinders Technology Associates cards are recommended for virus storage and transport, thus facilitating the molecular detection and identification of RNA viral pathogens. The Flinders Technology Associates (FTA) card, a cotton-based cellulose membrane impregnated with a chaotropic agent, effectively inactivates infectious microorganisms, lyses cellular material, and fixes nucleic acid. The aim of this study is to assess the stability and detection limit of various RNA viruses, especially the avian influenza virus (AIV), Newcastle disease virus (NDV), and African horse sickness virus (AHSV), on the FTA card, which could significantly impact virus storage and transport practices. To achieve this, each virus dilution was inoculated onto an FTA card and stored at room temperature in plastic bags for durations ranging from 1 week to 6 months. Following storage, the target genome was detected using conventional reverse transcription polymerase chain reaction. The present study demonstrated that the detection limit of AIV ranged from 1.17 to 6.17 EID50 values over durations ranging from 1 week to 5 months, while for NDV, it ranged from 2.83 to 5.83 ELD50 over the same duration. Additionally, the detection limit of AHSV was determined as 4.01 PFU for both 1 and 2 weeks, respectively. Based on the demonstrated effectiveness, stability, and safety implications observed in the study, FTA cards are recommended for virus storage and transport, thus facilitating the molecular detection and identification of RNA viral pathogens. [ABSTRACT FROM AUTHOR]

IntroductionCulicoides plays a crucial role as an insect vector in the field of veterinary medicine. The transmission of significant viruses such as bluetongue virus (BTV) and African horse sickness virus (AHSV) by this insect poses a substantial threat, leading to the development of severe diseases in domestic animals. This study aimed to explore the Culicoides species, identify their blood-meal sources, and assess the presence of BTV and AHSV carried by Culicoides in Yuanyang County, Yunnan Province. The aim was to gain insights into the potential vectors of these two viruses and elucidate their potential roles in the transmission of pathogens.MethodsThe midges were collected from cattle (Bos indicus), pig (Sus scrofa), and goat (Capra hircus) pens in Yuanyang County, Yunnan Province in June 2020. Initial identification of midges was conducted through morphological characteristics, followed by molecular identification using the cytochrome C oxidase subunit I (COI) gene. The determination of Culicoides blood-meal sources was accomplished using specific primers targeting the cytochrome b (Cyt b) gene from potential hosts. BTV and AHSV RNA were identified in Culicoides pools through the application of reverse transcriptase PCR and quantitative real-time PCR. Nucleotide homology and phylogenetic analysis were performed using MegAlign (DNAStar) and Mega 6.0 software.ResultsA total of 6,300 Culicoides, consisting of C. oxystoma, C. arakawai, C. imicola, and C. innoxius, were collected from cattle, pigs, and goat pens. The engorgement rates for these species were 30.2%, 54.6%, 75%, and 66.7%, respectively. In the cattle pen, the prevailing species is C. oxystoma (100%). In the pig pen, C. arakawai dominates (70%), with C. oxystoma following at 30%. In the goat pen, C. imicola holds the majority (45.45%), trailed by C. oxystoma (25%), C. innoxius (20.45%), and C. arakawai (9.09%). These Culicoides species were identified as feeding on cattle, pigs, goats, chickens (Gallus gallus), and humans (Homo sapiens). The positivity rates for BTV were 20.00% and 11.54% in blood-fed specimens of C. imicola and C. oxystoma, respectively. Conversely, the positivity rates for BTV in non-blood-fed specimens were 0.00% and 6.67% for C. imicola and C. oxystoma, respectively. BTV was not detected in C. arakawai and C. innoxius. The specimens (YY86) from C. imicola that tested positive for BTV had the closest genetic relationship to YTS-4 isolated from Mangshi, Yunnan Province in 1996. All test results for the nucleic acid of AHSV were negative.ConclusionThe study reveals variations in the species distribution, community composition, blood sucking rate, and blood-feeding sources of Culicoides across different habitats. Notably, C. imicola and C. oxystoma emerge as potential vectors for the transmission of BTV in local animals. Accordingly, this investigation provides crucial insights that can serve as a valuable reference for the prevention and control of BTV in local animals, particularly from the perspective of vector management.

African horse sickness (AHS) is a highly infectious and often fatal disease caused by 9 serotypes of the orbivirus African horse sickness virus (AHSV). In March 2020, an AHS outbreak was reported in Thailand in which AHSV serotype 1 was identified as the causative agent. Trivalent live attenuated vaccines serotype 1, 3, and 4 were used in a targeted vaccination campaign within a 50-km radius surrounding the infected cases, which promptly controlled the spread of the disease. However, AHS-like symptoms in vaccinated horses required laboratory diagnostic methods to differentiate infected horses from vaccinated horses, especially for postvaccination surveillance. We describe a real-time reverse transcription PCR–based assay for rapid characterization of the affecting field strain. The development and validation of this assay should imbue confidence in differentiating AHS-vaccinated horses from nonvaccinated horses. This method should be applied to determining the epidemiology of AHSV in future outbreaks.

Background and Aim: The flinders technology associates (FTA) card is a cotton-based cellulose membrane impregnated with a chaotropic agent that inactivates infectious microorganisms, lyses cellular material, and fixes DNA and/or RNA within the fiber matrix. However, little is known about the effectiveness of these cards for detecting RNA viruses in animals. This study aimed to evaluate the sensitivity of RNA virus detection using conventional reverse-transcription polymerase chain reaction (RT-PCR) on FTA cards. Materials and Methods: A highly virulent Newcastle disease virus (NDV) and an avian influenza virus (AIV) with low pathogenicity were propagated using chicken embryonic eggs. Three days after inoculation, the allantoic fluid was harvested, stored at –80°C, and the stock virus was tested for virus titration. African horse sickness virus (AHSV) was obtained from a live attenuated vaccine that was dissolved and stored at –80°C. For sample preparation, each stock virus was 10-fold serially diluted and each dilution was inoculated onto an FTA card, followed by drying in a Class II safety cabinet. Both the stock virus and infected FTA card were genomically isolated using an extraction kit, FTA purification kit, and extraction kit with Tris-EDTA (TE) buffer. The target genome was then detected by one-step RT-PCR for NDV and AIV, and two-step RT-PCR for African horse sickness, including gel electrophoresis for the detection of specific nucleic acids. Results: The detection limit of stock AIV was compared on FTA cards, using the FTA purification kit, and with TE buffer with an extraction kit. The corresponding results were 1.47, 1.17, and 2.18 log10 EID50, respectively, while for NDV the results were 4.13, 4.83, and 4.84 log10 ELD50. Finally, detection limit of stock AHSV and AHSV on the FTA card extracted using TE buffer with an extraction kit were 4.30 and 4.01 log10 plaque-forming units, respectively. Conclusion: This study demonstrated that the detection limit or sensitivity of all tested RNA viruses on FTA cards did not differ when compared with those of the stock virus and in both methods for RNA isolation on FTA cards. These cards are suitable for collecting and transporting samples infected with RNA viruses, particularly AIV, NDV, and AHSV. Flinders technology associates cards also provide hazard-free samples, a reliable source of RNA for molecular characterization, and sufficient quantity for diagnostic applications based on nucleic acid-based detection.

The Flinders Technology Associates (FTA) card, a cotton-based cellulose membrane impregnated with a chaotropic agent, effectively inactivates infectious microorganisms, lyses cellular material, and fixes nucleic acid. The aim of this study is to assess the stability and detection limit of various RNA viruses, especially the avian influenza virus (AIV), Newcastle disease virus (NDV), and African horse sickness virus (AHSV), on the FTA card, which could significantly impact virus storage and transport practices. To achieve this, each virus dilution was inoculated onto an FTA card and stored at room temperature in plastic bags for durations ranging from 1 week to 6 months. Following storage, the target genome was detected using conventional reverse transcription polymerase chain reaction. The present study demonstrated that the detection limit of AIV ranged from 1.17 to 6.17 EID50 values over durations ranging from 1 week to 5 months, while for NDV, it ranged from 2.83 to 5.83 ELD50 over the same duration. Additionally, the detection limit of AHSV was determined as 4.01 PFU for both 1 and 2 weeks, respectively. Based on the demonstrated effectiveness, stability, and safety implications observed in the study, FTA cards are recommended for virus storage and transport, thus facilitating the molecular detection and identification of RNA viral pathogens.

African horse sickness (AHS) is an acute and subacute infectious disease of equine species caused by the African horse sickness virus (AHSV). The VP7 of AHSV is a group-specific protein conserved in all serotypes and is an excellent candidate for the serological diagnosis and an AHS vaccine component. However, to date, B-cell epitopes on the AHSV VP7 recognized by humoral immune responses remain unclear. This study expressed the recombinant AHSV VP7 soluble in Escherichia coli and purified it for mouse immunization. Four monoclonal antibodies (mAbs) were screened and identified by hybridoma cell fusion, clonal purification, and immunological assays. The B-cell epitopes, recognized by monoclonal antibodies 4B5, 3G10, 3D7, and 4D6, were identified by a series of truncated overlapping peptides expressed as glutathione S-transferase (GST)-fusion proteins. The results revealed that 4B5 recognized the 124VQTGRYAGA132 motif, 3G10 recognized the 140RYYVPQGRT148 motif, while 3D7 and 4D6 recognized the 292QPINPPIFP300 motif. Amino acid sequence alignment indicated that three novel B-cell epitopes were conserved among various AHSV serotypes but unconserved in other orbiviruses, such as the bluetongue and epidemic hemorrhagic disease viruses. This study informs on the antigenic epitopes of AHSV VP7, facilitating future investigations into the serological diagnosis method and epitope-based vaccines against AHSV. • Altering key amino acids at the C-terminus achieved soluble expression of AHSV. • VP7 protein in E. coli for the first time. • Hybridoma technology prepared four monoclonal antibodies specific to the AHSV VP7 protein. • Three new linear B cell epitopes of AHSV VP7 protein were originally mapped using monoclonal antibodies. • This study will facilitate future investigations into diagnosis and epitope-based vaccination against AHSV. [ABSTRACT FROM AUTHOR]

African horse sickness (AHS) is one of the economically most important equid diseases in southern Africa, contributing significantly to equine morbidity and mortality. Annual vaccination with the Onderstepoort Biological Products polyvalent live attenuated vaccine has been the mainstay of prevention in South Africa. The study objectives were to determine if there is a significant relationship between multiple variables (vaccination status, number of AHSV [African horse sickness virus] serotypes contracted, clinical presentation, order of vaccine administration, age, sex and mean Ct value) and case outcome. The study population consisted of samples of AHS cases from South Africa submitted to the Veterinary Genetics Laboratory, University of Pretoria, that were confirmed positive by real-time RT-qPCR from 1 September 2017 to 30 June 2019 with a definitive disease outcome. At a univariable level, unvaccinated horses were 8.7 times more likely to die compared with horses that were vaccinated annually. Vaccination status was not statistically significant at a multivariable level, possibly due to insufficient sample size. Annual vaccination was shown to be protective. The pulmonary form of the disease and a lower Ct value had an increased likelihood of non-survival. Vaccination order was significant at a multivariable level (AHS2 vaccine administered first had a higher likelihood of survival). The study confirmed that increased case fatality was not due to vaccine failure but instead due to multiple variables, with an increased population of unvaccinated horses being one of these. [ABSTRACT FROM AUTHOR]

A unique characteristic of the African horse sickness virus (AHSV) major core protein VP7 is that it is highly insoluble, and spontaneously forms crystalline particles in AHSV-infected cells and when expressed in vitro. The aggregation of AHSV VP7 into these crystals presents many problems in AHSV vaccine development, and it is unclear whether VP7 aggregation affects AHSV assembly or contributes to AHSV pathogenesis. Here, we set out to abolish VP7 self-assembly by targeting candidate amino acid regions on the surface of the VP7 trimer via site-directed mutagenesis. It was found that the substitution of seven amino acids resulted in the complete disruption of AHSV VP7 self-assembly, which abolished the formation of VP7 crystalline particles and converted VP7 to a fully soluble protein still capable of interacting with VP3 to form core-like particles. This work provides further insight into the formation of AHSV VP7 crystalline particles and the successful development of AHSV vaccines. It also paves the way for future research by drawing comparisons with similar viral phenomena observed in human virology. [ABSTRACT FROM AUTHOR]

African horse sickness (AHS) is a debilitating and highly infectious arthropod-borne disease affecting all species of Equidae. The causative agent of AHS is the non-enveloped dsRNA African horse sickness virus (AHSV), belonging in the genus Orbivirus , family Reoviridae. The identification and surveillance of AHSV by simple and reliable diagnostic tools is essential for managing AHS outbreaks. Indirect ELISAs utilising soluble AHSV antigen or recombinant VP7, an immunodominant and serogroup-specific major core structural protein, are commonly used for serological diagnostic assays. Plant production systems are a significant alternative for recombinant protein production, as they are safe, easily scalable, production rates are rapid and upstream processes are more cost-effective than more traditional expression systems. This pilot study reports the successful production of AHSV-5 VP7 quasi-crystals in Nicotiana benthamiana by Agrobacterium tumefacien s-mediated transient expression using the self-replicating pRIC3.0 plant expression vector. After purification by means of density gradient ultracentrifugation, yields of pure VP7 of 2.66 µg/g fresh leaf mass (FLM) were achieved. Purified plant-produced AHSV-5 VP7 detected AHSV-specific antibodies in horse sera in an indirect ELISA and was able to distinguish between AHSV-positive and negative sera. Additionally, plant-produced AHSV-5 VP7 detected AHSV-specific antibodies to the same degree as E. coli- produced VP7. These results justify further investigation into the diagnostic capability of plant-produced AHSV VP7 quasi-crystals. To the best of our knowledge, this is the first report of AHSV VP7 quasi-crystal production in N. benthamiana and the first time that plant-produced VP7's potential as a diagnostic has been assessed. • Transient expression of AHSV-5 VP7 quasi-crystals in Nicotiana benthamiana. • Plant-produced AHSV-5 VP7 detects AHSV-specific antibodies in horse sera. • AHSV-5 VP7 distinguishes between AHSV-positive and negative sera in iELISA. • Plant- and E. coli-produced AHSV-5 VP7 comparably detect AHSV-specific antibodies. [ABSTRACT FROM AUTHOR]

Abstract Background Culicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses and inflict biting nuisance on humans, companion animals and livestock. In temperate regions, transmission of arboviruses is limited by temperature thresholds, in both replication and dissemination of arboviruses within the vector and in the flight activity of adult Culicoides. This study aims to determine the cold-temperature thresholds for flight activity of Culicoides from the UK under laboratory conditions. Methods Over 18,000 Culicoides adults were collected from the field using 4 W down-draught miniature ultraviolet Centers for Disease Control traps. Populations of Culicoides were sampled at three different geographical locations within the UK during the summer months and again in the autumn at one geographical location. Activity at constant temperatures was assessed using a bioassay that detected movement of adult Culicoides towards an ultraviolet light source over a 24-h period. Results The proportion of active adult Culicoides increased with temperature but cold temperature thresholds for activity varied significantly according to collection season and location. Populations dominated by the subgenus Avaritia collected in South East England had a lower activity threshold temperature in the autumn (4 °C) compared with populations collected in the summer (10 °C). Within the subgenus Avaritia, Culicoides scoticus was significantly more active across all temperatures tested than Culicoides obsoletus within the experimental setup. Populations of Culicoides impunctatus collected in the North East of England were only active once temperatures reached 14 °C. Preliminary data suggested flight activity of the subgenus Avaritia does not differ between populations in South East England and those in the Scottish Borders. Conclusions These findings demonstrate seasonal changes in temperature thresholds for flight and across different populations of Culicoides. These data, alongside that defining thresholds for virus replication within Culicoides, provide a primary tool for risk assessment of arbovirus transmission in temperate regions. In addition, the study also provides a comparison with thermal limits derived directly from light-suction trapping data, which is currently used as the main method to define adult Culicoides activity during surveillance.

Background: Culicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses and inflict biting nuisance on humans, companion animals and livestock. In temperate regions, transmission of arboviruses is limited by temperature thresholds, in both replication and dissemination of arboviruses within the vector and in the flight activity of adult Culicoides. This study aims to determine the cold-temperature thresholds for flight activity of Culicoides from the UK under laboratory conditions. Methods: Over 18,000 Culicoides adults were collected from the field using 4 W down-draught miniature ultraviolet Centers for Disease Control traps. Populations of Culicoides were sampled at three different geographical locations within the UK during the summer months and again in the autumn at one geographical location. Activity at constant temperatures was assessed using a bioassay that detected movement of adult Culicoides towards an ultraviolet light source over a 24-h period. Results: The proportion of active adult Culicoides increased with temperature but cold temperature thresholds for activity varied significantly according to collection season and location. Populations dominated by the subgenus Avaritia collected in South East England had a lower activity threshold temperature in the autumn (4 °C) compared with populations collected in the summer (10 °C). Within the subgenus Avaritia, Culicoides scoticus was significantly more active across all temperatures tested than Culicoides obsoletus within the experimental setup. Populations of Culicoides impunctatus collected in the North East of England were only active once temperatures reached 14 °C. Preliminary data suggested flight activity of the subgenus Avaritia does not differ between populations in South East England and those in the Scottish Borders. Conclusions: These findings demonstrate seasonal changes in temperature thresholds for flight and across different populations of Culicoides. These data, alongside that defining thresholds for virus replication within Culicoides, provide a primary tool for risk assessment of arbovirus transmission in temperate regions. In addition, the study also provides a comparison with thermal limits derived directly from light-suction trapping data, which is currently used as the main method to define adult Culicoides activity during surveillance. [ABSTRACT FROM AUTHOR]

• A cocktail of 9 ECRA-AHSV strains elicits an antibody response to all 9 serotypes. • ECRA-AHSV monovalent vaccination induces sustained immunity. • ECRA-AHSV strains trigger balanced Th1 and Th2 responses. African horse sickness virus (AHSV) is an Orbivirus within the Reoviridae family, spread by Culicoides species of midges, which infects equids with high mortality, particularly in horses and has a considerable impact on the equine industry. In order to control the disease, we previously described Entry Competent Replication Abortive (ECRA) virus strains for each of the nine distinct AHSV serotypes and demonstrated their potential as vaccines, first in type I interferon receptor (IFNAR-/-) knockout mice, and then in ponies. In this report we have investigated whether or not a combination ECRA vaccine comprising nine vaccine strains as two different cocktails is as efficient in ponies and the duration of the immunity triggered by ECRA vaccines. In one study, a group of ponies were vaccinated with a cocktail of 4 vaccine strains, followed by a vaccination of the remaining 5 vaccine strains, mimicking the current live attenuated vaccine regimen. In the second study, ponies were vaccinated with a single ECRA-AHSV strain and monitored for 6 months. The first group of ponies developed neutralising antibody responses against all 9 serotypes, indicating that no cross-serotype interference occurred, while the second group developed robust neutralising antibody responses against the single serotype that were sustained at the same level throughout a 6-month study. The results support our previous data and further validate ECRA vaccines as a safe and efficacious replacement of current live vaccines. [ABSTRACT FROM AUTHOR]

Viral metagenomics characterizes known and identifies unknown viruses based on sequence similarities to any previously sequenced viral genomes. A metagenomics approach was used to identify virus sequences in Australian mosquitoes causing cytopathic effects in inoculated mammalian cell cultures. Sequence comparisons revealed strains of Liao Ning virus (Reovirus, Seadornavirus), previously detected only in China, livestock-infecting Stretch Lagoon virus (Reovirus, Orbivirus), two novel dimarhabdoviruses, named Beaumont and North Creek viruses, and two novel orthobunyaviruses, named Murrumbidgee and Salt Ash viruses. The novel virus proteomes diverged by ≥ 50% relative to their closest previously genetically characterized viral relatives. Deep sequencing also generated genomes of Warrego and Wallal viruses, orbiviruses linked to kangaroo blindness, whose genomes had not been fully characterized. This study highlights viral metagenomics in concert with traditional arbovirus surveillance to characterize known and new arboviruses in field-collected mosquitoes. Follow-up epidemiological studies are required to determine whether the novel viruses infect humans.

A unique characteristic of the African horse sickness virus (AHSV) major core protein VP7 is that it is highly insoluble, and spontaneously forms crystalline particles in AHSV-infected cells and when expressed in vitro. The aggregation of AHSV VP7 into these crystals presents many problems in AHSV vaccine development, and it is unclear whether VP7 aggregation affects AHSV assembly or contributes to AHSV pathogenesis. Here, we set out to abolish VP7 self-assembly by targeting candidate amino acid regions on the surface of the VP7 trimer via site-directed mutagenesis. It was found that the substitution of seven amino acids resulted in the complete disruption of AHSV VP7 self-assembly, which abolished the formation of VP7 crystalline particles and converted VP7 to a fully soluble protein still capable of interacting with VP3 to form core-like particles. This work provides further insight into the formation of AHSV VP7 crystalline particles and the successful development of AHSV vaccines. It also paves the way for future research by drawing comparisons with similar viral phenomena observed in human virology.

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The African horse sickness virus (AHSV) belongs to the Genus Orbivirus, family Sedoreoviridae, and nine serotypes of the virus have been described to date. The AHSV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in decreasing size order (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable AHSV protein and the primary target for neutralizing antibodies. Consequently, Seg-2 determines the identity of the virus serotype. An African horse sickness (AHS) outbreak in an AHS-free status country requires identifying the serotype as soon as possible to implement a serotype-specific vaccination program. Considering that nowadays 'polyvalent live attenuated' is the only commercially available vaccination strategy to control the disease, field and vaccine strains of different serotypes could co-circulate. Additionally, in AHS-endemic countries, more than one serotype is often circulating at the same time. Therefore, a strategy to rapidly determine the virus serotype in an AHS-positive sample is strongly recommended in both epidemiological situations. The main objective of this study is to describe the development and validation of three triplex real-time RT-PCR (rRT-PCR) methods for rapid AHSV serotype detection. Samples from recent AHS outbreaks in Kenia (2015-2017), Thailand (2020), and Nigeria (2023), and from the AHS outbreak in Spain (1987-1990), were included in the study for the validation of these methods.

Abstract The outer capsid viral protein 2 (VP2) of African horse sickness virus, encoded by the most variable genome segment 2 (Seg-2), is the primary target for AHSV-specific neutralising antibodies and thus determines the virus serotype. Full length segment 2 sequences from more than 100 AHSVs isolated over the last 80 years were compared and single nucleotide polymorphisms (SNPs) identified between the reference strains and recent field viruses. Regions unique to each individual serotype were identified and primers designed to differentially amplify each of the nine serotypes. The sequences of resulting amplicons contained a significant amount of SNPs to discriminate between field viruses and reference strains or live attenuated viruses. The new serotype specific RT-PCR were subsequently used to determine the prevalence of different AHSV serotypes associated with samples submitted to the Agricultural Research Council - Onderstepoort Veterinary Research Institute during the 2016 / 2017 season. Subsequent sequencing of the PCR products were used to determine if the infections were caused by field or vaccine-derived strains. The serotypes of 70 AHSV positive diagnostic samples submitted to the ARC-OVR were determined. Serotypes 2 and 6 were the most prevalent, while Serotype 1 was the only serotype where sequences identical to the ALV or reference strains were detected in field samples. Based on this study, the incidence of vaccine-derived AHS infections submitted from southern Africa were low. This serotype-specific RT-PCR and sequencing assay could assist with the surveillance and control of equines movement nationally and internationally. It could also provide valuable scientific guidance on the policies and guidelines regulating vaccination and trade of equines in South Africa. [ABSTRACT FROM AUTHOR]

The viral proteins VP1-1, VP2, VP4, VP7 and NS3, of African horse sickness virus serotype 4 (AHSV4), have previously been identified to contain CD8+ T cell epitopes. In this study, overlapping peptides spanning the entire sequences of these AHSV4 proteins were synthesized and used to map epitopes. Peripheral blood mononuclear cells (PBMC) isolated from five horses immunized with an attenuated AHSV4 were stimulated in vitro with the synthesized peptides. Various memory immune assays were used to identify the individual peptides that contain CD8+ T cell epitopes, CD4+ T cell epitopes and linear B cell epitopes. The newly discovered individual peptides of AHSV4 proteins VP1-1, VP4, VP7 and/or NS3 that contain CD8+ T cell, CD4+ T cell or linear B cell epitopes could contribute to the design and development of new generation AHS peptide-based vaccines and therapeutics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Introduction: Culicoides plays a crucial role as an insect vector in the field of veterinary medicine. The transmission of significant viruses such as bluetongue virus (BTV) and African horse sickness virus (AHSV) by this insect poses a substantial threat, leading to the development of severe diseases in domestic animals. This study aimed to explore the Culicoides species, identify their blood-meal sources, and assess the presence of BTV and AHSV carried by Culicoides in Yuanyang County, Yunnan Province. The aim was to gain insights into the potential vectors of these two viruses and elucidate their potential roles in the transmission of pathogens., Methods: The midges were collected from cattle ( Bos indicus ), pig ( Sus scrofa ), and goat ( Capra hircus ) pens in Yuanyang County, Yunnan Province in June 2020. Initial identification of midges was conducted through morphological characteristics, followed by molecular identification using the cytochrome C oxidase subunit I (COI) gene. The determination of Culicoides blood-meal sources was accomplished using specific primers targeting the cytochrome b (Cyt b ) gene from potential hosts. BTV and AHSV RNA were identified in Culicoides pools through the application of reverse transcriptase PCR and quantitative real-time PCR. Nucleotide homology and phylogenetic analysis were performed using MegAlign (DNAStar) and Mega 6.0 software., Results: A total of 6,300 Culicoides , consisting of C. oxystoma, C. arakawai, C. imicola , and C. innoxius , were collected from cattle, pigs, and goat pens. The engorgement rates for these species were 30.2%, 54.6%, 75%, and 66.7%, respectively. In the cattle pen, the prevailing species is C. oxystoma (100%). In the pig pen, C. arakawai dominates (70%), with C. oxystoma following at 30%. In the goat pen, C. imicola holds the majority (45.45%), trailed by C. oxystoma (25%), C. innoxius (20.45%), and C. arakawai (9.09%). These Culicoides species were identified as feeding on cattle, pigs, goats, chickens ( Gallus gallus ), and humans ( Homo sapiens ). The positivity rates for BTV were 20.00% and 11.54% in blood-fed specimens of C. imicola and C. oxystoma , respectively. Conversely, the positivity rates for BTV in non-blood-fed specimens were 0.00% and 6.67% for C. imicola and C. oxystoma , respectively. BTV was not detected in C. arakawai and C. innoxius . The specimens (YY86) from C. imicola that tested positive for BTV had the closest genetic relationship to YTS-4 isolated from Mangshi, Yunnan Province in 1996. All test results for the nucleic acid of AHSV were negative., Conclusion: The study reveals variations in the species distribution, community composition, blood sucking rate, and blood-feeding sources of Culicoides across different habitats. Notably, C. imicola and C. oxystoma emerge as potential vectors for the transmission of BTV in local animals. Accordingly, this investigation provides crucial insights that can serve as a valuable reference for the prevention and control of BTV in local animals, particularly from the perspective of vector management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Meng, He, Wang and Wang.)

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are hematophagous insects, and some species can transmit a plethora of pathogens, e.g., bluetongue virus and African horse sickness virus, that mainly affect animals. The transmission of vector-borne pathogens is strongly temperature dependent, and recent studies pointed to the importance of including microclimatic data when modelling disease spread. However, little is known about the preferred temperature of biting midges. The present study addressed the thermal selection of field-caught Culicoides with two experiments. In a laboratory setup, sugar-fed or blood-fed midges were video tracked for 15 min while moving inside a 60 × 30 × 4 cm setup with a 15-25 °C temperature gradient. Culicoides spent over double the time in the coldest zone of the setup compared to the warmest one. This cold selection was significantly stronger for sugar-fed individuals. Calculated preferred temperatures were 18.3 °C and 18.9 °C for sugar-fed and blood-fed Culicoides, respectively. The effect of temperature on walking speed was significant but weak, indicating that their skewed distribution results from preference and not cold trapping. A second experiment consisted of a two-way-choice-setup, performed in a 90 × 45 × 45 cm net cage, placed outdoors in a sheltered environment. Two UV LED CDC traps were placed inside the setup, and a mean temperature difference of 2.2 °C was created between the two traps. Hundred-fifty Culicoides were released per experiment. Recapture rates were negatively correlated with ambient temperature and were on average three times higher in the cooled trap. The higher prevalence of biting midges in cooler environments influences fitness and ability to transmit pathogens and should be considered in models that predict Culicoides disease transmission., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Abstract Background Biting midges of the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) cause a significant biting nuisance to equines and are responsible for the biological transmission of African horse sickness virus (AHSV). While currently restricted in distribution to sub-Saharan Africa, AHSV has a history of emergence into southern Europe and causes one of the most lethal diseases of horses and other species of Equidae. In the event of an outbreak of AHSV, the use of insecticide treated nets (ITNs) to screen equine accomodation is recommended by competent authorities including the Office International des Épizooties (OIE) in order to reduce vector-host contact. Methods Seven commercially avaliable pyrethroid insecticides and three repellent compounds, all of which are licensed for amateur use, were assessed in modified World Health Organization (WHO) cone bioassay trials in the laboratory using a colony line of Culicoides nubeculosus (Meigen), 1830. Two field trials were subsequently conducted to test the efficiency of treated net screens in preventing entry of Culicoides. Results A formulation of cypermethrin (0.15 % w/w) and pyrethrins (0.2 % w/w) (Tri-Tec 14®, LS Sales (Farnham) Ltd, Bloxham, UK) applied to black polyvinyl-coated polyester insect screen (1.6 mm aperture; 1.6 mm thickness) inflicted 100 % mortality on batches of C. nubeculosus following a three minute exposure in the WHO cone bioassays at 1, 7 and 14 days post-treatment. Tri-Tec 14® outperformed all other treatments tested and was subsequently selected for use in field trials. The first trial demonstrated that treated screens placed around an ultraviolet light-suction trap entirely prevented Culicoides being collected, despite their collection in identical traps with untreated screening or no screening. The second field trial examined entry of Culicoides into stables containing horses and found that while the insecticide treated screens reduced entry substantially, there was still a small risk of exposure to biting. Conclusions Screened stables can be utilised as part of an integrated control program in the event of an AHSV outbreak in order to reduce vector-host contact and may also be applicable to protection of horses from Culicoides during transport.

Orbiviruses are of significant importance to the health of wildlife and domestic animals worldwide; the major orbiviruses transmitted by multiple biting midge (Culicoides) species include bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus. The viruses, insect vectors, and hosts are anticipated to be impacted by global climate change, altering established Orbivirus epidemiology. Changes in global climate have the potential to alter the vector competence and extrinsic incubation period of certain biting midge species, affect local and long-distance dispersal dynamics, lead to range expansion in the geographic distribution of vector species, and increase transmission period duration (earlier spring onset and later fall transmission). If transmission intensity is associated with weather anomalies such as droughts and wind speeds, there may be changes in the number of outbreaks and periods between outbreaks for some regions. Warmer temperatures and changing climates may impact the viral genome by facilitating reassortment and through the emergence of novel viral mutations. As the climate changes, Orbivirus epidemiology will be inextricably altered as has been seen with recent outbreaks of bluetongue, epizootic hemorrhagic disease, and African horse sickness outside of endemic areas, and requires interdisciplinary teams and approaches to assess and mitigate future outbreak threats., (Published by Oxford University Press on behalf of Entomological Society of America 2023.)

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of a range of orbiviruses that cause important veterinary diseases such as bluetongue and African horse sickness. The effective monitoring of Culicoides species diversity and abundance, both at livestock and near potential wildlife hosts, is essential for risk management. The Onderstepoort 220‐V ultraviolet (UV) light trap is extensively used for this purpose. Reducing its power requirements by fitting low‐energy light‐emitting diodes (LEDs) can lead to greater flexibility in monitoring. A comparison of the efficiency of the 220‐V Onderstepoort trap (8‐W fluorescent UV light) with the efficiency of the 220‐V or 12‐V Onderstepoort traps fitted with red, white, blue or green LEDs or a 12‐V fluorescent Onderstepoort trap demonstrated the 220‐V Onderstepoort trap to be the most efficient. All the results showed nulliparous Culicoides imicola Kieffer females to be the dominant grouping. Despite the lower numbers collected, 12‐V traps can be used in field situations to determine the most abundant species. Tests showed that 220‐V fluorescent ultraviolet light traps were more efficient than 220‐V traps fitted with red, white, blue or green LEDs for the collection of livestock‐associated Culicoides species.Similarly, 220‐V fluorescent UV traps collected greater numbers of Culicoides than 12‐V fluorescent traps or 12‐V white, blue or green LED traps.Despite the lower numbers collected, 12‐V traps can be used in field situations to effectively determine Culicoides species composition, population age structure and the most abundant species in an area. [ABSTRACT FROM AUTHOR]

Highlights • Baculovirus-expressed AHS-VP2 and MVA-VP2 vaccines were evaluated in mice. • Clinical protection was complete in mice receiving one or two doses of MVA-VP2. • Clinical protection complete after two doses of baculovirus-expressed VP2. • Significant reduction of viraemia in all vaccinated groups. • Significant levels of immunity were achieved with one dose of either vaccine. Abstract African horse sickness is a severe, often fatal, arboviral disease of equids. The control of African horse sickness virus (AHSV) in endemic countries is based currently on the use of live attenuated vaccines despite some biosafety concerns derived from its biological properties. Thus, experimental vaccination platforms have been developed over the years in order to avoid the biosafety concerns associated with the use of attenuated vaccines. Various studies showed that baculovirus-expressed AHSV-VP2 or modified Vaccinia Ankara virus expressing AHSV-VP2 (MVA-VP2) induced virus neutralising antibodies and protective immunity in small animals and horses. AHSV is an antigenically diverse pathogen and immunity against AHS is serotype-specific. Therefore, AHS vaccines for use in endemic countries need to induce an immune response capable of protecting against all existing serotypes. For this reason, current live attenuated vaccines are administered as polyvalent preparations comprising combinations of AHSV attenuated strains of different serotypes. Previous studies have shown that it is possible to induce cross-reactive virus neutralising antibodies against different serotypes of AHSV by using polyvalent vaccines comprising combinations of either different serotype-specific VP2 proteins, or MVA-VP2 viruses. However, these strategies could be difficult to implement if induction of protective immunity is highly dependent on using a two-dose vaccination regime for each serotype the vaccine intends to protect against. In our study, we have tested the protective capacity of MVA-VP2 and baculovirus-expressed VP2 vaccines when a single dose was used. Groups of interferon alpha receptor knock-out mice were inoculated with either MVA-VP2 or baculovirus-expressed VP2 vaccines using one dose or the standard two-dose vaccination regime. After vaccination, all four vaccinated groups were challenged with AHSV and clinical responses, lethality and viraemia compared between the groups. Our results show that complete clinical protection was achieved after a single vaccination with either MVA-VP2 or baculovirus sub-unit VP2 vaccines. [ABSTRACT FROM AUTHOR]

The attenuated live virus vaccine that is used in South Africa to protect against African horse sickness infection was developed more than 50 years ago. With the selection of the vaccine strains by cell culture passage, a correlation between the size of plaques formed in monolayer Vero cultures and attenuation of virus virulence in horses was found. The large plaque phenotype was used as an indication of cell culture adaptation and strongly correlated with attenuation of virulence in horses. There was never any investigation into the genetic causes of either the variation in plaque size, or the loss of virulence. An understanding of the underlying mechanisms of attenuation would benefit the production of a safer AHSV vaccine. To this end, the genomes of different strains of two African horse sickness isolates, producing varying plaque sizes, were compared and the differences between them identified. This comparison suggested that proteins VP2, VP3, VP5 and NS3 were most likely involved in the determination of the plaque phenotype. Comparison between genome sequences (obtained from GenBank) of low and high passage strains from two additional serotypes indicated that VP2 was the only protein with amino acid substitutions in all four serotypes. The amino acid substitutions all occurred within the same hydrophilic area, resulting in increased hydrophilicity of VP2 in the large plaque strains. [ABSTRACT FROM AUTHOR]

African horse sickness (AHS) is a lethal equine disease transmitted by Culicoides biting midges and caused by African horse sickness virus (AHSV). AHS is endemic to sub-Saharan Africa, but devastating outbreaks have been recorded periodically outside this region. The perceived risk of an AHS outbreak occurring in Europe has increased following the frequent epidemics caused in ruminants by bluetongue virus, closely related to AHSV. Attenuated vaccines for AHS are considered unsuitable for use in non-endemic countries due bio-safety concerns. Further, attenuated and inactivated vaccines are not compatible with DIVA (differentiate infected from vaccinated animals) strategies. All these factors stimulated the development of novel AHS vaccines that are safer, more efficacious and DIVA compatible. We showed previously that recombinant modified Vaccinia Ankara virus (MVA) vaccines encoding the outer capsid protein of AHSV (AHSV-VP2) induced virus neutralising antibodies (VNAb) and protection against AHSV in a mouse model and also in the horse. Passive immunisation studies demonstrated that immunity induced by MVA-VP2 was associated with pre-challenge VNAb titres in the vaccinates. Analyses of the inoculum of these MVA-VP2 experimental vaccines showed that they contained pre-formed AHSV-VP2. We continued studying the influence of pre-formed AHSV-VP2, present in the inoculum of MVA-VP2 vaccines, in the immunogenicity of MVA-VP2 vaccines. Thus, we compared correlates of immunity in challenged mice that were previously vaccinated with: a) MVA-VP2 (live); b) MVA-VP2 (live and sucrose gradient purified); c) MVA-VP2 (UV light inactivated); d) MVA-VP2 (UV light inactivated and diluted); e) MVA-VP2 (heat inactivated); f) MVA-VP2 (UV inactivated) + MVA-VP2 (purified); g) MVA-VP2 (heat inactivated) + MVA-VP2 (purified); and h) wild type-MVA (no insert). The results of these experiments showed that protection was maximal using MVA-VP2 (live) vaccine and that the protection conferred by all other vaccines correlated strongly with the levels of pre-formed AHSV-VP2 in the vaccine inoculum. [ABSTRACT FROM AUTHOR]

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae . Currently, nine serotypes have been defined showing limited cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African Horse Sickness (AHS) in equids with a mortality up to 95% in naïve domestic horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are competent vectors of closely related bluetongue virus. AHS outbreaks cause huge economic losses in developing countries. In the developed world, outbreaks will result in losses in the equestrian industry and will have an enormous emotional impact on owners of pet horses. Live-attenuated vaccine viruses (LAVs) have been developed, however, safety of these LAVs are questionable due to residual virulence, reversion to virulence, and risk on virulent variants by reassortment between LAVs or with field AHSV. Research aims vaccines with improved profiles. Reverse genetics has recently being developed for AHSV and has opened endless possibilities including development of AHS vaccine candidates, such as Disabled Infectious Single Animal (DISA) vaccine. Here, virulent AHSV5 was recovered and its high virulence was confirmed by experimental infection of ponies. ‘Synthetically derived’ virulent AHSV5 with an in-frame deletion of 77 amino acids codons in genome segment 10 encoding NS3/NS3a protein resulted in similar in vitro characteristics as published NS3/NS3a knockout mutants of LAV strain AHSV4LP. In contrast to its highly virulent ancestor virus, this deletion AHSV5 mutant (DISA5) was completely safe for ponies. Two vaccinations with DISA5 as well as two vaccinations with DISA vaccine based on LAV strain AHSV4LP showed protection against lethal homologous AHSV. More research is needed to further improve efficacy, to explore the AHS DISA vaccine platform for all nine serotypes, and to study the vaccine profile in more detail. [ABSTRACT FROM AUTHOR]

Summary: African horse sickness (AHS) is a debilitating and often fatal viral disease affecting horses in much of Africa, caused by the dsRNA orbivirus African horse sickness virus (AHSV). Vaccination remains the single most effective weapon in combatting AHS, as there is no treatment for the disease apart from good animal husbandry. However, the only commercially available vaccine is a live‐attenuated version of the virus (LAV). The threat of outbreaks of the disease outside its endemic region and the fact that the LAV is not licensed for use elsewhere in the world, have spurred attempts to develop an alternative safer, yet cost‐effective recombinant vaccine. Here, we report the plant‐based production of a virus‐like particle (VLP) AHSV serotype five candidate vaccine by Agrobacterium tumefaciens‐mediated transient expression of all four capsid proteins in Nicotiana benthamiana using the cowpea mosaic virus‐based HyperTrans (CPMV‐HT) and associated pEAQ plant expression vector system. The production process is fast and simple, scalable, economically viable, and most importantly, guinea pig antiserum raised against the vaccine was shown to neutralize live virus in cell‐based assays. To our knowledge, this is the first report of AHSV VLPs produced in plants, which has important implications for the containment of, and fight against the spread of, this deadly disease. [ABSTRACT FROM AUTHOR]

Intragenic recombination has been described in various RNA viruses as a mechanism to increase genetic diversity, resulting in increased virulence, expanded host range, or adaptability to a changing environment. Orbiviruses are no exception to this, with intragenic recombination previously detected in the type species, bluetongue virus (BTV). African horse sickness virus (AHSV) is a double-stranded RNA virus belonging to the Oribivirus genus in the family Reoviridae. Genetic recombination through reassortment has been described in AHSV, but not through homologous intragenic recombination. The influence of the latter on the evolution of AHSV was investigated by analyzing the complete genomes of more than 100 viruses to identify evidence of recombination. Segment-1, segment-6, segment-7, and segment-10 showed evidence of intragenic recombination, yet only one (Segment-10) of these events was manifested in subsequent lineages. The other three hybrid segments were as a result of recombination between field isolates and the vaccine derived live attenuated viruses (ALVs).

The ceratopogonid fauna of northern Sardinia (Italy) was investigated using black light traps. The study was carried out in six localities from June to August 2015. A total of 3,879 ceratopogonids were collected and 40 species belonging to 8 genera were identified. The most abundant genus was Culicoides (96%) followed by Forcipomyia (1.5%), Bezzia (1.2%) and Dasyhelea, Atrichopogon, Alluaudomyia, Stilobezzia and Brachypogon (each <1%). Seven new species for Italian fauna were recorded: Forcipomyia bipunctata, Forcipomyia psilonota, Culicoides achrayi, Culicoides cameroni, Culicoides corsicus and Bezzia pygmaea. Stilobezzia sahariensis was reported for the first time in Europe. [ABSTRACT FROM AUTHOR]

Culicoides ( Diptera: Ceratopogonidae) are vectors of pathogens that affect wildlife, livestock and, occasionally, humans. Culicoides imicola ( Kieffer, 1913) is considered to be the main vector of the pathogens that cause bluetongue disease ( BT) and African horse sickness ( AHS) in southern Europe. The study of blood-feeding patterns in Culicoides is an essential step towards understanding the epidemiology of these pathogens. Molecular tools that increase the accuracy and sensitivity of traditional methods have been developed to identify the hosts of potential insect vectors. However, to the present group's knowledge, molecular studies that identify the hosts of C. imicola in Europe are lacking. The present study genetically characterizes the barcoding region of C. imicola trapped on farms in southern Spain and identifies its vertebrate hosts in the area. The report also reviews available information on the blood-feeding patterns of C. imicola worldwide. Culicoides imicola from Spain feed on blood of six mammals that include species known to be hosts of the BT and AHS viruses. This study provides evidence of the importance of livestock as sources of bloodmeals for C. imicola and the relevance of this species in the transmission of BT and AHS viruses in Europe. [ABSTRACT FROM AUTHOR]