Development of two competitive ELISAs based on monoclonal antibodies for the serological detection of Bovine ephemeral fever virus.

Bovine ephemeral fever virus (BEFV) is a member of the genus Ephemerovirus in the family Rhabdoviridae. It is an arthropod-borne virus transmitted by many species of midges and mosquitoes. It can cause severe economic consequences due to losses in milk production and the general condition of cattle and water buffalo. BEF occurs in some tropical, subtropical and warm temperate regions of Africa, Australia, the Middle East and Asia with seasonal outbreaks, but its possible spread to other areas (e.g. Europe) cannot be excluded. Therefore, using and developing rapid diagnostic methods with optimal performance is essential for identifying emerging pathogens and their control. In the present study, we developed two competitive serological ELISAs based on monoclonal antibodies (mAbs), designed by using BEFV inactivated antigen and the BEF recombinant nucleoprotein (N), respectively. A panel of 77 BEF-positive and 338 BEF-negative sera was used to evaluate the two tests. With a diagnostic sensitivity of 97.4 % using the inactivated virus and 98.7 % using the recombinant N, and a diagnostic specificity of 100 % using both antigens, our results suggest that these tests are suitable for the serological diagnosis of BEF.

Immunogenicity of a freeze-dried combined vaccine against Rift Valley fever and bovine ephemeral fever in cattle.

Background: The target of vaccination is to encourage a strong, covering and long-lasting immune response against antigens. For achieving these objectives; effective adjuvant and new vaccine strategies are demanded to make the vaccine sufficiently immunogenic to instigate a powerful immune response. Aim: This study was completed for elaboration and evaluation of freeze-dried combined vaccine against both Rift Valley fever (RVF) and bovine ephemeral fever (BEF) viruses using different stabilizers. Methods: Three formulae were prepared from such vaccine including: formula (1): stabilized with a mixture of 5% Lactalbumin Hydrolysate and 2.5% sucrose, formula (2): stabilized with a mixture of 50% the previous stabilizer and 50% of 1% Carbopol and formula (3): stabilized with 1% Carbopol solution. Samples of the three vaccine formulae were reconstituted on the time of experimental animal vaccination using saponin diluent which acts as an adjuvant for both RVFv and BEFv and as an inactivator BEF virus. The ratio between both viruses in all vaccine formulae was 1:1. Results: All vaccine batches were proved to be free of any foreign contaminants and unharmed for experimentally vaccinated animals. Each of the three groups of calves was vaccinated S/C with 2 ml of a reconstituted vaccine formula and their immune response was evaluated using serum neutralization test. The gained results revealed that the prepared combined freeze-dried vaccine with Carbopol elicited a better humoral immune response than the other two vaccine formulae. Conclusion: It could be recommended to use Carbopol as a stabilizer for the preparation of the aimed vaccine.

Stretching the wings further- susceptibility of Culex pipiens Linnaeus to bovine ephemeral fever virus infection under experimental conditions.

Bovine ephemeral fever (BEF) is a significant viral disease of cattle in the tropical, subtropical, and temperate climatic zones. This disease is also known as three-day sickness due to the spontaneous recovery of the cattle within a short period (usually 3-5 days). Despite its short duration, the disease may have a considerable impact. It can cause heavy economic losses, primarily due to decreased milk production, lowered fertility in bulls, and even fatality in severe cases. The virus is suspected to be transmitted by haematophagous insects (mainly mosquitoes and Culicoides biting midges); however, the identity of a competent vector for BEFV remains a mystery. Here, we investigated whether BEFV may replicate for a short duration in Culex pipiens Linnaeus, 1758, the most prevalent mosquito species in Israel and a potential vector of this virus to Israeli cattle. We applied nested- qPCR to test BEFV abundance in Cx. pipiens every 24 h for 14 consecutive days post-infection. Additionally, we collected eggs laid by BEFV-infected females and investigated BEFV abundance in the different developmental stages of F1 mosquitos. Our results suggest that Cx. pipiens mosquitoes have the potential to act as a vector of BEFV and also indicate that BEFV may be vertically transmitted from Cx. pipiens female parent to her female offspring.

Effectiveness of the strain 919 bovine ephemeral fever virus vaccine in the face of a real-world outbreak: A field study in Israeli dairy herds.

Bovine ephemeral fever virus (BEFV) is a globally spread arthropod-borne RNA virus that has significant economic impacts on the cattle industry. A live attenuated commercial BEF vaccine, based on the Australian BEFV strain 919, is widely used in Israel and other countries. A previous study has suggested the high effectiveness of this vaccine (ULTRAVAC BEF VACCINE™ from Zoetis®), but anecdotal reports of high BEF morbidity among vaccinated dairy herds in Israel casted doubt on these findings. To resolve this uncertainty, a randomized controlled field vaccine effectiveness study was conducted in Israel during a BEF outbreak which occurred in 2021. Eleven dairy herds were enrolled and monitored for BEF-associated morbidity and rumination alteration patterns using electronic monitoring tags (HR Tags, SCR® Dairy, Netanya, Israel). Four of the herds were naturally infected with BEFV during the outbreak, resulting in a total of 120 vaccinated and 311 unvaccinated subjects that were included in the effectiveness study. A mixed-effect Cox proportional hazard regression model was used to calculate the overall hazard ratio between vaccinated and unvaccinated cattle. This analysis demonstrated an average vaccine effectiveness of 60 % (95 % CI = 38 %-77 %) for preventing clinical disease. In addition, a non-statistically significant trend (p = 0.1) towards protection from mortality was observed, with no observation of mortality among the vaccinated groups compared to 2.61 % mortality (7/311) among the unvaccinated subjects. One hundred and thirty vaccinated and unvaccinated calves from affected and non-affected herds and with different status of morbidity were sampled and analysed by serum-neutralization test. The highest titers of BEFV-neutralizing antibodies were found in subjects that were both vaccinated and clinically affected, indicating a booster effect after vaccination. The results of the study provide evidence for the moderate effectiveness of the ULTRAVAC BEF VACCINE™ for the prevention of BEF.

Whole genome characterization and evolutionary analysis of bovine ephemeral fever virus isolated in Iran.

Bovine ephemeral fever virus (BEFV) is an economically important arthropod-borne virus of cattle and water buffaloes which is enzootic in Africa, Australia, and Asia. We characterized the entire length of BEFV BA/RZ/IR strain genome isolated in Iran and compared to the all BEFV full genomes available in the GenBank. The BEFV genomes were phylogenetically classified as 4 lineages including the Middle Eastern, East Asian, Australian, and South African lineages. The Iranian BA/RZ/IR strain, which displayed maximum sequence identity (96.72%) to the Chinese JT02L strain was clustered as a separate branch in the East Asian lineage of the virus. Using Shannon entropy analysis, amino acid variations were detected in the all proteins encoded by BEFV genomes. Particularly, the polymerase L and the accessory proteins Gns, α2 and ß exhibited the highest amino acid variations suggesting their significance in the viral replication efficiency. Our bioinformatics analyses also predict the occurrence of recombination event within the East Asian lineage of BEFV genomes. Our data show that the Chinese Henan 1 may be a hybrid strain constructed of the Chinese JT02L and Iranian BA/RZ/IR BEFV strains as the major and minor parents, respectively. These computational analyses suggest that the homologous recombination may be an evolutionary mechanism for BEFV as a member of the Rhabdoviridae family.

Development and validation of a DIVA ELISA for differentiating BEFV infected from vaccinated animals.

Inactivated vaccine is considered safe and used for prevention of bovine ephemeral fever in several endemic countries. To differentiate between BEFV-infected and vaccinated animals, we developed an ELISA capable of detecting infection-related antibodies against BEFV. Recombinant proteins, including N, P, M, L, GNS, α2, ß and γ, were expressed in E. coli and screened by Western blotting and ELISA. The results showed GNS, α2 and ß specifically reacted with sera from BEFV infected cattle but not sera from vaccinated cattle. A DIVA ELISA based on a C-terminal truncated form of GNS was developed, with 100% sensitivity and 98.0% specificity at a sample to positive-control optical density ratio (S/P) threshold of 0.18. Specificity analysis showed that the assay has no cross-reactivity with antisera of other common bovine viruses. Anti-GNS antibody appears at 3-4 days post infection (dpi) and persists up to 240-300 dpi in the experimentally infected cattle. Sero-epidemiological survey using sera collected from vaccinated cattle in an endemic area in Jiangsu Province revealed sero-positive rate of 2.36% (6/254), indicating that the DIVA ELISA could be used as a reliable diagnostic tool for differentiating BEFV infected from vaccinated animals.

Identification of Cytotoxic T lymphocyte (CTL) Epitope and design of an immunogenic multi-epitope of Bovine Ephemeral Fever Virus (BEFV) Glycoprotein G for Vaccine Development.

Bovine ephemeral fever (BEF), a vector-borne disease of cattle and water buffalo, is enzootic in tropical and subtropical zones of Asia, Australia, and Africa. Since cytotoxic T lymphocytes (CTL) responses may play a key role in the control of bovine ephemeral fever virus (BEFV) infection, it is important to identify and characterize the CTL target epitopes of BEFV antigens. The current study has been designed to identify and characterize the potential CTL epitopes using the Immuno-informatics tools, and it helped find the potent vaccine candidates against BEF. Antigenicity, toxicity, allergenicity, and immunogenicity testing of predicted CTL epitopes was done. Total four CTL epitopes for BEFV G protein, have been identified as potential epitopes. Prediction of the 3D structure of multi-epitope (final structure) was performed using I-TASSER server. Model 1 was selected as the best model with C-Score: -3.71. The modeled G protein structure and multi-epitope structure were validated by the Ramachandran plots Prosa and Verify 3D server. Epitopic regions of 3D protein structure were identified by Chimera UCSF software. Physicochemical properties of the Multi epitope were evaluated using ProtParam server. This is the first report of CTL epitope in the G protein of BEFV. In this manner, they would play an important role in evoking the immune response as well as vaccine development. However, in vitro and in vivo experimental studies are required for suggested epitopes verification. The multi-epitope was designed from regions of the G protein sequence that lacked mutation and genomic diversity. Therefore, it can be introduced as a protein vaccine from all strains of BEFV as a vaccine candidate for design.

Molecular characterization and phylogenetic analysis of bovine ephemeral fever viruses in Khuzestan province of Iran in 2018 and 2020.

BACKGROUND: Bovine ephemeral fever (BEF) is an arthropod-borne viral disease caused by the BEF virus (BEFV). This single-stranded RNA virus that affects cattle and water buffalo is endemic in tropical and subtropical regions including Iran. While BEF is a major disease of cattle in Iran, information regarding its agent, molecular characterization, and circulating viruses are highly limited. The current study aimed to, firstly, determine the genetic and antigenic characteristics of BEFV strains in Khuzestan province in Southwest of Iran in 2018 and 2020 and, secondly, to compare them with strains obtained from other areas. RESULTS: By phylogenetic analysis based on the Glycoprotein gene, BEFV strains were divided into four clusters of Middle East, East Asia, South Africa, and Australia; in which the 2018 and 2020 Iranian BEFV strains were grouped in the Middle East cluster with the Turkish, Indian, and Israeli strains. Depending on the chronology and geographical area, the outbreaks of Turkey (2020), Iran (2018 and 2020), and India (2018 and 2019) are proposed to be related. These BEFVs had the highest identity matrix and the lowest evolutionary distance among the studied strains. Multiple sequence alignment of G1, G2, and G3 antigenic sites showed that these neutralizing epitopes are highly conserved among the strains of the Middle East cluster; however, the strains previously identified in Iran differed in three amino acids placed in G1 and G2 epitopes. CONCLUSION: The findings revealed that BEFVs circulating in the Middle East are closely related phylogenetically and geographically. They also have similar antigenic structures; therefore, developing a vaccine based on these strains can be effective for controlling BEF in the Middle East.

Seroprevalence of Bovine ephemeral fever virus in Gujarat State of India.

Bovine ephemeral fever (BEF) virus (BEFV) is an arthropod borne virus that causes bovine ephemeral fever or three­day sickness in cattle and buffaloes. This is the first report on seroprevalence of BEF in cattle and buffaloes in Gujarat, India. Total of 92 animals, 78 cattle and 14 buffaloes from three regions (districts) of Gujarat state of India, were screened for the presence of anti­BEF antibodies. A total of 27 out of 92 animals were found positive and overall seroprevalence detected was 29.34% (95% CI 20.0­38.6%). A total of 19 out of 78 cattle and 8 out of 14 buffalo's samples were found positive BEFV antibodies. Species­wise seroprevalence in cattle and buffaloes was 24.35% (95% CI 14.8­33.8%) and 57.1% (95% CI 31.2­83.0%), respectively. There was a statistically significant (p < 0.05) species effect based on the seroprevalence. In cattle, location­wise seroprevalence was observed to be 26.82% (95% CI 13.2­40.3%) and 21.62% (95% CI 8.3­34.8%) in Navsari and Banaskantha districts, respectively. The effect of location is not statistically significant (p < 0.05). Cytopathic effect of Vero cells was characterized by rounding, granulation of the cytoplasm within 48­72 hrs of post infection. This was the first report demonstrating the presence of BEFV in Gujarat state.

Short-Term Humoral Immune Response of the pcDNA4-G Plasmid Expressing the Bovine Ephemeral Fever Virus G Gene in BALB/c Mice.

Bovine ephemeral fever (BEF) is an arthropod­borne viral disease characterised by a short­term clinical expression that can lead to significant losses in high­yielding cattle and water buffaloes. In this study, we aimed to generate a recombinant plasmid expressing the glycoprotein (G) of the BEF virus (BEFV) and to stimulate a humoral immune response to this protein in BALB / c mice immunised with the recombinant plasmid. Expression of the encoded protein was demonstrated by western blotting and immunoperoxidase tests. The suitable plasmids were intramuscularly administered to BALB/c mice on days 0, 14 and 21. The antibody response in the immunised mice was measured by a plaque reduction neutralization test (PRNT) and enzyme­linked immunosorbent assay (ELISA). According to BEFV ELISA, only two of the seven animals in these groups exceeded the cut­off value. A significant difference was observed in the mean OD values at 450 nm absorbance in the pcDNA4­G­immunised group when compared with those in the plasmid control group at 30 days (p < 0.05). According to PRNT50 results, a 1:20 (p < 0.05) antibody response was obtained at 30 days in pcDNA4­G (100 µg)­immunised mice, whereas this ratio was 1:80 (p < 0.001) in BEFV­immunised mice (1,000 PFU/0.5 ml). We conclude that the humoral immune response was stimulated in experimental mice immunised with the recombinant plasmid. However, disappointingly, the antibody response was markedly low in pcDNA4­G­immunised mice.

Complete genome sequencing and assessment of mutation-associated protein dynamics of the first Indian bovine ephemeral fever virus (BEFV) isolate.

BACKGROUND: Bovine ephemeral fever (BEF) is a re-emerging disease caused by bovine ephemeral fever virus (BEFV). Although it poses a huge economic threat to the livestock sector, complete viral genome information from any South Asian country, including India, lacks. AIM: Genome characterization of the first Indian BEFV isolate and to evaluate its genetic diversity by characterizing genomic mutations and their associated protein dynamics. MATERIALS AND METHODS: Of the nineteen positive blood samples collected from BEF symptomatic animals during the 2018-19 outbreaks in India, one random sample was used to amplify the entire viral genome by RT-PCR. Utilizing Sanger sequencing and NGS technology, a complete genome was determined. Genome characterization, genetic diversity and phylogenetic analyses were explored by comparing the results with available global isolates. Additionally, unique genomic mutations within the Indian isolate were investigated, followed by in-silico assessment of non-synonymous (NS) mutations impacts on corresponding proteins' secondary structure, solvent accessibility and dynamics. RESULTS: The complete genome of Indian BEFV has 14,903 nucleotides with 33% GC with considerable genetic diversity. Its sequence comparison and phylogenetic analysis revealed a close relatedness to the Middle Eastern lineage. Genome-wide scanning elucidated 30 unique mutations, including 10 NS mutations in the P, L and GNS proteins. The mutational impact evaluation confirmed alterations in protein structure and dynamics, with minimal effect on solvent accessibility. Additionally, alteration in the interatomic interactions was compared against the wild type. CONCLUSION: These findings extend our understanding of the BEFV epidemiological and pathogenic potential, aiding in developing better therapeutic and preventive interventions.

Genome sequence of Bovine Ephemeral fever virus vaccine strain of South African origin.

Bovine Ephemeral fever virus (BEFV) is endemic in South Africa and has a negative economic impact on the meat and dairy industries. Bovine ephemeral fever or three-day stiff-sickness is controlled through annual vaccination with a live attenuated virus manufactured by Onderstepoort Biological Products (South Africa). We announce the genome sequences of two South African Bovine Ephemeral Virus strains; the live attenuated vaccine strain (14 876 nucleotides) and a field strain (14 883 nucleotides). A mutation in the alpha 3 open reading frame rendered the gene non-functional in both genomes. Phylogenetic analysis based on the glycoprotein gene showed that the two strains clustered with the South African lineage.

Isolation and genetic characterization of bovine ephemeral fever virus from epidemic-2020 in Turkey.

Bovine ephemeral fever virus (BEFV) infection occurs seasonally in many tropical and subtropical regions of Africa, Asia (including the Middle East), and Australia while it is exotic in Europe. In this study, the epidemiology of BEFV infection in Turkey that bridges southeastern Europe and Asia, geographically, was investigated according to the comparison of the nucleotide sequences of the virus caused the last epidemic in 2020 with those of the strains previously detected in Turkey as well as BEFV strains from other countries. In the phylogenetic analysis, based on an alignment of full-length G gene sequences, BEFVs from epidemic-2020 were located in Middle Eastern lineage and appear to represent most closely related BEFVs from India-2018 and 2019. The findings will contribute to a better understanding of BEFV epidemiology in Turkey.

Expression and immunogenicity of secreted forms of bovine ephemeral fever virus glycoproteins applied to subunit vaccine development.

AIMS: Vaccines for bovine ephemeral fever virus (BEFV) are available but are difficult to produce, expensive or suffer from genetic instability. Therefore, we designed constructs encoding C-terminally truncated forms (transmembrane anchoring region deleted) of glycoproteins G and GNS such that they were secreted from the cell into the media to achieve high-level antigen expression, correct glycosylation pattern and enable further simple purification with the V5 epitope tag. METHODS AND RESULTS: In this study, synthetic biology was employed to create membrane-bound and secreted forms of G and GNS glycoprotein. Mammalian cell culture was employed as an antigen expression platform, and the secreted forms of G and GNS protein were easily purified from media using a highly effective, single-step method. The V5 epitope tag was genetically fused to the C-termini of the proteins, enabling detection of the antigen through immunoblotting and immunomicroscopy. Our data demonstrated that the C-terminally truncated form of the G glycoprotein was efficiently secreted from cells into the cell media. Moreover the immunogenicity was confirmed in mice test. CONCLUSIONS: The immuno-dot blots showed that the truncated G glycoprotein was present in the total cell extract, and was clearly secreted into the media, consistent with the western blotting data and live-cell images. Our strategy presented the expression of secreted, epitope-tagged, forms of the BEFV glycoproteins such that appropriately glycosylated forms of BEFV G protein was secreted from the BHK-21 cells. This indicates that high-level expression of secreted G glycoprotein is a feasible strategy for large-scale production of vaccines and improving vaccine efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: The antigen expression strategy designed in this study can produce high-quality recombinant protein and reduce the amount of antigen used in the vaccine.

Genetically modified rabies virus vector-based bovine ephemeral fever virus vaccine induces protective immune responses against BEFV and RABV in mice.

Bovine ephemeral fever (BEF), caused by the bovine ephemeral fever virus (BEFV), is associated with an acute febrile infection in cattle and widespread in tropical and subtropical areas, leading to great economic losses to cattle and milk industry. However, no efficacious BEF vaccine is currently available in China. Herein, we generated a recombinant rabies virus (RABV) expressing BEFV glycoprotein (LBNSE-BG), utilizing a reverse genetics system based on the recombinant rabies virus strain LBNSE. It was found that mice immunized with LBNSE-BG produced robust neutralizing antibodies against both BEFV and RABV, and developed complete protection from lethal RABV challenge. Further studies showed that LBNSE-BG activated more dendritic cells (DCs), B cells and T cells in immunized mice than the parent virus LBNSE. Collectively, these findings demonstrate that the recombinant LBNSE-BG described here has the potential to be developed as a cost-effective and efficacious bivalent vaccine for cattle use in endemic areas of BEF and rabies.

Aspirin and 5-Aminoimidazole-4-carboxamide Riboside Attenuate Bovine Ephemeral Fever Virus Replication by Inhibiting BEFV-Induced Autophagy.

Recent study in our laboratory has demonstrated that BEFV-induced autophagy via activation of the PI3K/Akt/NF-κB and Src/JNK pathways and suppression of the PI3K-AKt-mTORC1 pathway is beneficial for virus replication. In the current study, we found that both aspirin and 5-aminoimidazole-4-carboxamide-1-ß-riboside (AICAR) siginificantly attenuated virus replication by inhibiting BEFV-induced autophagy via suppressing the BEFV-activated PI3K/Akt/NF-κB and Src/JNK pathways as well as inducing reversion of the BEFV-suppressed PI3K-Akt-mTORC1 pathway. AICAR reversed the BEFV-activated PI3K/Akt/NF-κB and Src/JNK pathways at the early to late stages of infection and induced reversion of the BEFV-suppressed PI3K-AKt-mTORC1 pathway at the late stage of infection. Our findings reveal that inhibition of BEFV-induced autophagy by AICAR is independent of AMPK. Furthermore, we found that AICAR transcriptionally downregulates the ATG related genes ULK1, Beclin 1, and LC3 and enhances Atg7 degradation by the proteasome pathway. Aspirin suppresses virus replication by inhibiting BEFV-induced autophagy. It directly suppressed the NF-κB pathway and reversed the BEFV-activated Src/JNK pathway at the early stage of infection and reversed the BEFV-suppressed PI3K/Akt/mTOR pathway at the late stage of infection. The current study provides mechanistic insights into the effects of aspirin and AICAR on BEFV replication through suppression of BEFV-induced autophagy.

Investigation of bovine ephemeral fever virus transmission by putative dipteran vectors under experimental conditions.

BACKGROUND: Bovine ephemeral fever virus (Rhabdoviridae: Ephemerovirus) (BEFV) causes bovine ephemeral fever (BEF), an economically important disease of cattle and water buffalo. Outbreaks of BEF in Africa, Australia, Asia and the Middle East are characterized by high rates of morbidity and highly efficient transmission between cattle hosts. Despite this, the vectors of BEFV remain poorly defined. METHODS: Colony lines of biting midges (Culicoides sonorensis) and mosquitoes (Aedes aegypti, Culex pipiens and Culex quinquefasciatus) were infected with a strain of BEFV originating from Israel by feeding on blood-virus suspensions and by intrathoracic inoculation. In addition, in vivo transmission of BEFV was also assessed by allowing C. sonorensis inoculated by the intrathoracic route to feed on male 6 month-old Holstein-Friesian calves. RESULTS: There was no evidence of BEFV replication within mosquitoes fed on blood/virus suspensions for mosquitoes of any species tested for each of the three colony lines. In 170 C. sonorensis fed on the blood/virus suspension, BEFV RNA was detected in the bodies of 13 individuals and in the heads of two individuals, indicative of fully disseminated infections and an oral susceptibility rate of 1.2%. BEFV RNA replication was further demonstrated in all C. sonorensis that were inoculated by the intrathoracic route with virus after 5, 6 or 7 days post-infection. Despite this, transmission of BEFV could not be demonstrated when infected C. sonorensis were allowed to feed on calves. CONCLUSIONS: No evidence for infection or dissemination of BEFV (bovine/Israel/2005-6) in mosquitoes of three different species was found. Evidence was found for infection of C. sonorensis by the oral route. However, attempts to transmit BEFV to calves from infected C. sonorensis failed. These results highlight the challenge of defining the natural vector of BEFV and of establishing an in vivo transmission model. The results are discussed with reference to the translation of laboratory-based studies to inference of vector competence in the field.

Hayes Yard virus: a novel ephemerovirus isolated from a bull with severe clinical signs of bovine ephemeral fever is most closely related to Puchong virus.

Bovine ephemeral fever is a vector-borne disease of ruminants that occurs in tropical and sub-tropical regions of Africa, Asia and Australia. The disease is caused by a rhabdovirus, bovine ephemeral fever virus (BEFV), which occurs as a single serotype globally. Although several other closely related ephemeroviruses have been isolated from cattle and/or arthropods, only kotonkan virus from Nigeria and (tentatively) Mavingoni virus from Mayotte Island in the Indian Ocean have been previously associated with febrile disease. Here, we report the isolation of a novel virus (Hayes Yard virus; HYV) from blood collected in February 2000 from a bull (Bos indicus) in the Northern Territory of Australia. The animal was suffering from a severe ephemeral fever-like illness with neurological involvement, including recumbency and paralysis, and was euthanised. Histological examination of spinal cord and lung tissue identified extensive haemorrhage in the dura mata with moderate perineuronal oedema and extensive emphysema. HYV displayed cone-shaped morphology, typical of rhabdoviruses, and was found to be most closely related antigenically to Puchong virus (PUCV), isolated in 1965 from mosquitoes in Malaysia. Analysis of complete genome sequences of HYV (15 025 nt) and PUCV (14 932 nt) indicated that each has a complex organisation (3' N-P-M-G-GNS-α1-α2-ß-γ-L 5') and expression strategy, similar to that of BEFV. Based on an alignment of complete L protein sequences, HYV and PUCV cluster with other rhabdoviruses in the genus Ephemerovirus and appear to represent two new species. Neutralising antibody to HYV was also detected in a retrospective survey of cattle sera collected in the Northern Territory.

Annexin A2 gene interacting with viral matrix protein to promote bovine ephemeral fever virus release.

Bovine ephemeral fever virus (BEFV) causes bovine ephemeral fever, which can produce considerable economic damage to the cattle industry. However, there is limited experimental evidence regarding the underlying mechanisms of BEFV. Annexin A2 (AnxA2) is a calcium and lipid-conjugated protein that binds phospholipids and the cytoskeleton in a Ca2+-dependent manner, and it participates in various cellular functions, including vesicular trafficking, organization of membrane domains, and virus proliferation. The role of the AnxA2 gene during virus infection has not yet been reported. In this study, we observed that AnxA2 gene expression was up-regulated in BHK-21 cells infected with the virus. Additionally, overexpression of the AnxA2 gene promoted the release of mature virus particles, whereas BEFV replication was remarkably inhibited after reducing AnxA2 gene expression by using the small interfering RNA (siRNA). For viral proteins, overexpression of the Matrix (M) gene promotes the release of mature virus particles. Moreover, the AnxA2 protein interaction with the M protein of BEFV was confirmed by GST pull-down and co-immunoprecipitation assays. Experimental results indicate that the C-terminal domain (268-334 aa) of AxnA2 contributes to this interaction. An additional mechanistic study showed that AnxA2 protein interacts with M protein and mediates the localization of the M protein at the plasma membrane. Furthermore, the absence of the AnxA2-V domain could attenuate the effect of AnxA2 on BEFV replication. These findings can contribute to elucidating the regulation of BEFV replication and may have implications for antiviral strategy development.

South African bovine ephemeral fever virus glycoprotein sequences are phylogenetically distinct from those from the rest of the world.

Bovine ephemeral fever virus (BEFV) is an economically important arbovirus affecting cattle and water buffalo. Currently, isolates can be separated into three phylogenetic groups, differentiated by the place of isolation, namely, East Asia, Australia, and the Middle East. BEFV surface glycoprotein (G) genes from 14 South African field strains collected between 1968 and 1999 were sequenced and compared to 154 published sequences. The BEFV isolates from South Africa were found to be phylogenetically distinct from those from other parts of the world.