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Bovine Ephemeral Fever Virus (BEFV) is a non-contagious virus that commonly infects cattle and water buffalo, reduces milk productivity, decreases the quality of beef, and causes an adverse economic impact on the global livestock industry. However, the evolution of BEFV is unclear, and uncertainty exists regarding its global geodynamics. Consequently, this study aims to comprehend the pattern of viral evolution and gene expression in the BEFV genes G, M, N, and P, including synonymous codons. Additionally, we performed recombination analyses, which exclusively detected recombination signals in the G- and P-genes. Subsequently, a phylogenetic tree was constructed to validate and support these findings. The codon usage bias results showed that the BEFV-selected genes were influenced by both natural and mutation pressure. Furthermore, nucleotide A is more abundant in all the selected genes. The eNC values, ranging from 42.99 to 47.10, revealed the presence of moderate codon usage bias, where gene P exhibited the highest and gene G had the lowest codon usage bias. The neutrality and PR-2 plots, specified codon usage patterns of the genes, are also being shaped by strong selectional pressure. This comprehensive analysis of BEFV genes (G, M, N, and P) sheds light on the molecular evolutionary patterns, co-adaptation, and different genes expression in diverse regions, facilitating the development of preventative programs and insights into viral pathogenesis and vaccine design.Communicated by Ramaswamy H. Sarma.

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The bovine ephemeral fever virus (BEFV) is an enzootic agent that affects millions of bovines and causes major economic losses. Though the virus is seasonally reported with a very high morbidity rate (80-100%) from African, Australian, and Asiatic continents, it remains a neglected pathogen in many of its endemic areas, with no proper therapeutic drugs or vaccines presently available for treatment. The RNA-dependent RNA polymerase (RdRp) catalyzes the viral RNA synthesis and is an appropriate candidate for antiviral drug developments. We utilized integrated computational tools to build the 3D model of BEFV-RdRp and then predicted its probable active binding sites. The virtual screening and optimization against these active sites, using several small-molecule inhibitors from a different category of Life Chemical database and FDA-approved drugs from the ZINC database, was performed. We found nine molecules that have docking scores varying between -6.84 to -10.43 kcal/mol. Furthermore, these complexes were analyzed for their conformational dynamics and thermodynamic stability using molecular dynamics simulations in conjunction with the molecular mechanics generalized Born surface area (MM-GBSA) scheme. The binding free energy calculations depict that the electrostatic interactions play a dominant role in the RdRp-inhibitor binding. The hot spot residues, such as Arg565, Asp631, Glu633, Asp740, and Glu707, were found to control the RdRp-inhibitor interaction. The ADMET analysis strongly suggests favorable pharmacokinetics of these compounds that may prove useful for treating the BEFV ailment. Overall, we anticipate that these findings would help explore and develop a wide range of anti-BEFV therapy.Communicated by Ramaswamy H. Sarma.

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MicroRNAs (miRNAs), as a kind of small noncoding RNAs, have been proved to play a regulatory role in virus infection. However, the role and mechanism of cellular miRNAs in bovine transient fever virus (BEFV) infection are largely unknown. In the present study, we found that bta-miR-101 was significantly up-regulated in the Madin-Darby Bovine Kidney (MDBK) cells upon BEFV infection. Notably, bta-miR-101 mimic dramatically inhibited BEFV replication, while bta-miR-101 inhibitor facilitated BEFV replication, suggesting that bta-miR-101 acted as an anti-viral host factor restraining BEFV replication. Subsequently, NF-κB repressing factor (NKRF) was identified as a target gene of bta-miR-101 by dual luciferase reporter assay, and bta-miR-101 mimic significantly down-regulated expression of NKRF, while bta-miR-101 inhibitor up-regulated its expression, respectively. Furthermore, NKRF could induce apoptosis, and favored the replication of BEFV. Finally, bta-miR-101 inhibited BEFV-induced apoptosis via targeting NKRF to suppress virus replication. In general, our study provides a novel mechanism for bta-miR-101 to exert its antiviral function, which provides a theoretical basis for the development of antiviral strategy.

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Receptors for activated C kinase 1 (RACK1) could competitively combine with mitochondrial antiviral signaling protein (MAVS) to inhibit the type I interferon (IFN) signaling pathway during viral infection in vitro. However, whether RACK1 can degrade MAVS to enhance viral replication is still unknown. In this study, we found that bovine epidemic fever virus (BEFV) infection triggered the expression of RACK1. Overexpression of RACK1 promoted BEFV replication, while knockdown of RACK1 inhibited the replication of BEFV. Further research showed that RACK1 inhibited the type I IFN signaling pathway during BEFV infection by degrading MAVS, and RACK1 degraded MAVS via the ubiquitin-proteasome system. Mechanistically, RACK1 up-regulated the expression of E3 ubiquitin ligase STIP1 homology and U-box containing protein 1 (STUB1), thereby promoting the ubiquitination and degradation of MAVS. In addition, RACK1 degraded MAVS by enhancing the interaction between STUB1 and MAVS but not via its interaction with STUB1. Overall, our study reveals a novel mechanism by which RACK1 inhibits the type I IFN signaling pathway to BEFV infection through degradation of MAVS, thereby promoting viral infection. These findings provide a new perspective for the MAVS degradation regulated by RACK1.

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Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus and causes bovine ephemeral fever of cattle and water buffalo in worldwide. Previous studies have demonstrated that infection with BEFV leads to induction of host cellular apoptosis. However, the role of apoptosis in viral replication and the interaction between viral genes and host genes involved in the process of BEFV-induced apoptosis remains unclear. Herein we investigated the interaction between viral non-structural protein α3 and cellular heterogeneous nuclear ribonucleoprotein K (hnRNP K) in the BEFV-induced apoptosis and its role in virus replication. Overexpression of α3 gene activated caspase 3 and consequently cleaved PARP, ultimately lead to apoptosis. Moreover, virus titer of BHK-21 cells infected with BEFV and then treated respectively by the pan-caspase inhibitor (Z-VAD-FMK) and apoptosis inducer (CCCP) was determined, the results showed that apoptosis promoted viral replication. In addition, knockdown of hnRNP K gene promoted BEFV replication, whereas overexpression of hnRNP K gene had the opposite effects. More importantly, overexpression of hnRNP K inhibited virus-induced apoptosis. Subsequently, it was found that hnRNP K suppressed BEFV replication via degrading viral α3 gene and further inhibited apoptosis induced by α3 gene. Finally, the expression of hnRNP K protein was significantly down-regulated upon BEFV infection, and degradation of hnRNP K protein in BHK-21 cells infected with BEFV was mediated by viral activation of caspase 3. Taken together, these results suggest that apoptosis takes a pivotal role in BEFV replication, and interaction between viral α3 gene and host hnRNP K gene in BEFV-induced apoptosis facilitates BEFV replication.

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BACKGROUND: Bovine ephemeral fever virus (BEFV), the causative agent of bovine ephemeral fever, is an economically important pathogen of cattle and water buffalo. MicroRNAs (miRNAs) are endogenous 21-23 nt small non-coding RNA molecules that binding to a multiple of target mRNAs and functioning in the regulation of viral replication including the miRNA-mediated antiviral defense. However, the reciprocal interaction between bovine ephemeral fever virus replication and host miRNAs still remain poorly understood. The aim of our study herein was to investigate the exact function of miR-3470b and its molecular mechanisms during BEFV infection. RESULTS: In this study, we found a set of microRNAs induced by BEFV infection using small RNA deep sequencing, and further identified BEFV infection could significantly up-regulate the miR-3470b expression in Baby Hamster Syrian Kidney cells (BHK-21) after 24 h and 48 h post-infection (pi) compared to normal BHK-21 cells without BEFV infection. Additionally, the target association between miR-3470b and mitochondrial antiviral signaling protein (MAVS) was predicted by target gene prediction tools and further validated using a dual-luciferase reporter assay, and the expression of MAVS mRNA and protein levels was negatively associated with miR-3470b levels. Furthermore, the miR-3470b mimic transfection significantly contributed to increase the BEFV N mRNA, G protein level and viral titer, respectively, whereas the miR-3470b inhibitor had the opposite effect on BEFV replication. Moreover, the overexpression of MAVS or silencing of miR-3470b by its inhibitors suppressed BEFV replication, and knockdown of MAVS by small interfering RNA also promoted the replication of BEFV. CONCLUSIONS: Our findings is the first to reveal that miR-3470b as a novel host factor regulates BEFV replication via directly targeting the MAVS gene in BHK-21 cells and may provide a potential strategy for developing effective antiviral therapy.

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The aim of this study was to investigate the immunogenicity of a plasmid deoxyribonucleic acid (DNA) vaccine encoding the G1 epitope of bovine ephemeral fever virus (BEFV) G glycoprotein in mice. A plasmid DNA carrying the G1 gene was constructed and designated as pcDNA3.1-G1. The expression of the target gene was confirmed in human embryonic kidney 293 (HEK 293) cells transfected with pcDNA3.1-G1 by indirect immunofluorescent staining. Immunisation experiments were intramuscularly carried out by vaccinating 6-week-old female mice in four groups, including the pcDNA3.1-G1 construct, pcDNA3.1 (+) plasmid alone, BEF-inactivated vaccine and phosphate-buffered saline (PBS) (1X) three times with 2-week intervals. Fourteen days after the last immunisation, the animals were bled and the resulting sera were tested for anti-G1-specific antibodies by immunoblotting analysis, indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralisation (VN) test. Serological assays showed that the pcDNA3.1-G1 construct expressing G1 protein was able to elicit specific antibodies against this antigen. Virus neutralisation test showed that pcDNA3.1-G1 could induce anti-BEFV-neutralising antibodies in mice. Our findings indicated that a new dimension can be added to vaccine studies for bovine ephemeral fever (BEF) using eukaryotic expression plasmids encoding the G1 antigen in the future.

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Bovine ephemeral fever virus (BEFV), identified as the causative pathogen of bovine ephemeral fever (BEF), is responsible for increasing numbers of epidemics/outbreaks and has a significant harmful effect on the livestock industry. Therefore, a rapid detection assay is imperative for BEFV diagnosis. In this study, we described the development of lateral-flow dipstick isothermal recombinase polymerase amplification (LFD-RPA) assays for detection of BEFV. RPA primers and LF probes were designed by targeting the specific G gene, and the amplification product can be visualized on a simple lateral flow dipstick with the naked eyes. The amplification reaction was performed at 38 °C for 20 min and LFD incubation time within 5 min. The detection limit of this assay was 8 copies per reaction, and there was no cross-reactivity with other bovine infectious viruses such as bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine vesicular stomatitis virus. In addition, the assay was performed with total 128 clinical specimens and the diagnostic results were compared with conventional RT-PCR, real-time quantative(q) PCR. The result showed that the coincidence rate of BEFV LFD-RPA and real-time qPCR was 96.09% (123/128), which was higher than conventional RT-PCR. The RPA combined with LFD assay probably provides a rapid and sensitive alternative for diagnosis of BEFV infections outbreak.

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Bovine ephemeral fever (BEF) is caused by the arthropod-borne bovine ephemeral fever virus (BEFV), which is classified in the family Rhabdoviridae and the genus Ephemerovirus. A debilitating and sometimes fatal viral disease, BEF affects cattle and water buffalo. The epizootiology of BEF among cattle in China has not been fully determined. We examined the seroprevalence of the BEFV among cattle in China between January 2012 and June 2014. Among the 2822 serum samples collected from various cattle breeds in 26 provinces in China, the seropositive rate for the BEFV ranged from 0% to 81% between regions and species. Our findings show that BEFV was prevalent in the all of the regions tested in our study and provide the first reliable reference regarding BEF surveillance in China.

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We described the aetiological agents of outbreaks of bovine ephemeral fever (BEF) that occurred in 1985 and 2012 in Turkey, and identify mutations in the viruses from both outbreaks. Outbreaks have emerged periodically every 4-5 years in the same regions in Turkey. Because these regions are located in a subtropical climatic zone, good conditions for vector populations exist. The results of this study show that the BEFVs from outbreaks in Turkey vary significantly. Effective prevention will require a vaccine that contains BEFVs from different genetic clusters.

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The epitope-G1 gene of Bovine ephemeral fever virus (BEFV) glycoprotein was synthesised by PCR and cloned into expression vector pPIC9K to construct recombinant plasmid pPIC9K-G1. Then the pPIC9K-G1 was linearized and transformed into Pichia pastoris GS 115. The recombinant P. pastoris strains were selected by a G418 transformation screen and confirmed by PCR. After being induced with methanol, an expressed protein with 26 kDa molecular weight was obtained, which was much bigger than the predicted size (15.54 kDa). Deglycosylation analysis indicated the recombinant G1 was glycosylated. Western blot and ELISA tests, as well as rabbit immunization and specificity experiments indicated that the target protein had both higher reaction activity and higher immunocompetence and specificity. The recombinant G1 protein could be used as a coating antigen to develop an ELISA kit for bovine ephemeral fever diagnosis.