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.

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.

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.

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.

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.

Genetic characterization of bovine ephemeral fever virus in southern China, 2013-2017.

Bovine ephemeral fever virus (BEFV) can cause bovine ephemeral fever and is an economically important arbovirus of cattle. To expand the knowledge of the molecular epidemiology of BEFV in southern China, the complete surface glycoprotein G gene of BEFV was sequenced from samples collected in five restricted outbreaks from 2013 to 2017, namely 2013ZH, 2014HM, 2015GX, 11082-2016, and qy2017. It was noted that both 2014HM and 11082-2016 were detected in cattle regularly vaccinated with inactivated vaccine. Phylogenetic analysis demonstrated that all five strains grouped into cluster I. However, qy2017 was closer to the BEFV strains identified in Thailand, Japan, and Taiwan after 2000, while 2013ZH, 2014HM, 2015GX, and 11082-2016 were closer to the Chinese strains in 2011 and the Turkey strains in 2012. The analysis of antigenic sites indicated that several amino acid changes occurred between the five strains and the vaccine strain. Importantly, one novel amino acid mutation site was observed in the putative N-linked glycosylation sites of 2013ZH, 2014HM, 2015GX, and 11082-2016. Our study indicated novel genetic characteristics of the newly emerging BEFV strains in southern China and the necessity of updating the component of commercially available inactivated BEFV vaccines in China.

Antigenic variation of bovine ephemeral fever viruses isolated in Iran, 2012-2013.

Bovine ephemeral fever virus (BEFV) is an economic arthropod-borne virus distributed in Africa, Asia, and Australia. Based on the sequence of the gene encoding the surface glycoprotein G, the viral antigenic determinant, BEFV has been phylogenetically classified into three clusters, including Australia, East Asia, and the Middle East. Here, we provide evidence for antigenic variations among the BEFV isolates in Iran during the period of 2012 to 2013 and also the exotic YHL strain, which are all classified into the East Asian cluster of the virus. For this propose, the entire length of the G gene of the viruses were sequenced and phylogenetically compared. The corresponding antigenic sites (G1-G4) were analyzed and antigenic relatedness among these viruses was measured. The two Iranian viruses, which displayed substitutions at residues E503K in the site G1 and E461K in the predicted site G4, were partially neutralized by each other's antisera (R value = 63.23%); however, these two viruses exhibited much lower cross-neutralization that measured by R value as 28.28% and 22.82%, respectively. The crucial substitution at amino acid R218K in the site G3a is believed to be the foremost cause of these declines. The data emphasize the frequent evolution of BEFV in different time periods and geographic regions, in which the new variants can emerge and likely escape from the pre-existing immunities. Thus, continuous monitoring of the circulating viruses is necessary for understanding the viral evolution and evaluation of protective immunity induced by the heterologous viruses.

MiR-3470b promotes bovine ephemeral fever virus replication via directly targeting mitochondrial antiviral signaling protein (MAVS) in baby hamster Syrian kidney cells.

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.

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

BACKGROUND: The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G1 protein with high-affinity and inhibit BEFV replication. METHODS: The purified BEFV G1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G1 was assayed by ELISA. Then the roles of specific G1-binding peptides in the context of BEFV infection were analyzed. RESULTS: The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. CONCLUSION: Two antiviral peptide ligands binding to bovine ephemeral fever virus G1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

Complete genome sequence of a bovine ephemeral fever virus JT02L strain in mainland China.

In this study, we report the complete genome sequence of bovine ephemeral fever virus (BEFV) JT02L, which has been used in our laboratory, in mainland China, for more than a decade. The genome is 14941 nucleotide (nt), comprising a leader sequence of 50 nt, nucleoprotein (N) gene of 1328 nt, phosphoprotein (P) gene of 858 nt, matrix protein (M) gene of 691 nt, glycoprotein (G) gene of 1897 nt, non-structural glycoprotein (GNS) gene of 1785 nt, α1α2 gene of 638 nt, ß gene of 460 nt, γ gene of 400 nt, large multi-functional enzyme (L) gene of 6470 nt and a trailer sequence of 73 nt. Individual genes are separated by intergenic regions (IGRs) of 26, 44, 47, 51, 37, 39, 68 and -21 nt respectively. The overall organization is similar to an Australian BEFV isolate BB7721 but demonstrates some distinctive features including longer α3 and ß open reading frames, intact termination/polyadenylation (TTP) sequence downstream of the ß open reading frame and a longer ß-γ IGR integrated with a 38 nt AT-rich fragment. To our knowledge, this is the first report describing the complete genome of a BEFV strain of East Asian lineage, which may facilitate studies on genomic diversity among geographic strains of BEFV in China and the world.

Characterization of a recombinant Newcastle disease virus expressing the glycoprotein of bovine ephemeral fever virus.

Bovine ephemeral fever (BEF) is caused by the arthropod-borne bovine ephemeral fever virus (BEFV), which is a member of the family Rhabdoviridae and the genus Ephemerovirus. BEFV causes an acute febrile infection in cattle and water buffalo. In this study, a recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of BEFV (rL-BEFV-G) was constructed, and its biological characteristics in vitro and in vivo, pathogenicity, and immune response in mice and cattle were evaluated. BEFV G enabled NDV to spread from cell to cell. rL-BEFV-G remained nonvirulent in poultry and mice compared with vector LaSota virus. rL-BEFV-G triggered a high titer of neutralizing antibodies against BEFV in mice and cattle. These results suggest that rL-BEFV-G might be a suitable candidate vaccine against BEF.

Evolution of bovine ephemeral fever virus in the Australian episystem.

Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that causes a debilitating disease of cattle in Africa, Asia, and Australia; however, its global geodynamics are poorly understood. An evolutionary analysis of G gene (envelope glycoprotein) ectodomain sequences of 97 BEFV isolates collected from Australia during 1956 to 2012 revealed that all have a single common ancestor and are phylogenetically distinct from BEFV sampled in other geographical regions. The age of the Australian clade is estimated to be between 56 and 65 years, suggesting that BEFV has entered the continent on few occasions since it was first reported in 1936 and that the 1955-1956 epizootic was the source of all currently circulating viruses. Notably, the Australian clade has evolved as a single genetic lineage across the continent and at a high evolutionary rate of ∼10(-3) nucleotide substitutions/site/year. Screening of 66 isolates using monoclonal antibodies indicated that neutralizing antigenic sites G1, G2, and G4 have been relatively stable, although variations in site G3a/b defined four antigenic subtypes. A shift in an epitope at site G3a, which occurred in the mid-1970s, was strongly associated with a K218R substitution. Similarly, a shift at site G3b was associated primarily with substitutions at residues 215, 220, and 223, which map to the tip of the spike on the prefusion form of the G protein. Finally, we propose that positive selection on residue 215 was due to cross-reacting neutralizing antibody to Kimberley virus (KIMV). This is the first study of the evolution of BEFV in Australia, showing that the virus has entered the continent only once during the past 50 to 60 years, it is evolving at a relatively constant rate as a single genetic lineage, and although the virus is relatively stable antigenically, mutations have resulted in four antigenic subtypes. Furthermore, the study shows that the evolution of BEFV in Australia appears to be driven, at least in part, by cross-reactive antibodies to KIMV which has a similar distribution and ecology but has not been associated with disease. As BEFV and KIMV are each known to be present in Africa and Asia, this interaction may occur on a broader geographic scale.

Bovine ephemeral fever rhabdovirus α1 protein has viroporin-like properties and binds importin ß1 and importin 7.

Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that is classified as the type species of the genus Ephemerovirus. In addition to the five canonical rhabdovirus structural proteins (N, P, M, G, and L), the large and complex BEFV genome contains several open reading frames (ORFs) between the G and L genes (α1, α2/α3, ß, and γ) encoding proteins of unknown function. We show that the 10.5-kDa BEFV α1 protein is expressed in infected cells and, consistent with previous predictions based on its structure, has the properties of a viroporin. Expression of a BEFV α1-maltose binding protein (MBP) fusion protein in Escherichia coli was observed to inhibit cell growth and increase membrane permeability to hygromycin B. Increased membrane permeability was also observed in BEFV-infected mammalian cells (but not cells infected with an α1-deficient BEFV strain) and in cells expressing a BEFV α1-green fluorescent protein (GFP) fusion protein, which was shown by confocal microscopy to localize to the Golgi complex. Furthermore, the predicted C-terminal cytoplasmic domain of α1, which contains a strong nuclear localization signal (NLS), was translocated to the nucleus when expressed independently, and in an affinity chromatography assay employing a GFP trap, the full-length α1 was observed to interact specifically with importin ß1 and importin 7 but not with importin α3. These data suggest that, in addition to its function as a viroporin, BEFV α1 may modulate components of nuclear trafficking pathways, but the specific role thereof remains unclear. Although rhabdovirus accessory genes occur commonly among arthropod-borne rhabdoviruses, little is known of their functions. Here, we demonstrate that the BEFV α1 ORF encodes a protein which has the structural and functional characteristics of a viroporin. We show that α1 localizes in the Golgi complex and increases cellular permeability. We also show that BEFV α1 binds importin ß1 and importin 7, suggesting that it may have a yet unknown role in modulating nuclear trafficking. This is the first functional analysis of an ephemerovirus accessory protein and of a rhabdovirus viroporin.

Epidemiology and control of bovine ephemeral fever.

Bovine ephemeral fever (or 3-day sickness) is an acute febrile illness of cattle and water buffaloes. Caused by an arthropod-borne rhabdovirus, bovine ephemeral fever virus (BEFV), the disease occurs seasonally over a vast expanse of the globe encompassing much of Africa, the Middle East, Asia and Australia. Although mortality rates are typically low, infection prevalence and morbidity rates during outbreaks are often very high, causing serious economic impacts through loss of milk production, poor cattle condition at sale and loss of traction power at harvest. There are also significant impacts on trade to regions in which the disease does not occur, including the Americas and most of Europe. In recent years, unusually severe outbreaks of bovine ephemeral fever have been reported from several regions in Asia and the Middle East, with mortality rates through disease or culling in excess of 10-20%. There are also concerns that, like other vector-borne diseases of livestock, the geographic distribution of bovine ephemeral fever could expand into regions that have historically been free of the disease. Here, we review current knowledge of the virus, including its molecular and antigenic structure, and the epidemiology of the disease across its entire geographic range. We also discuss the effectiveness of vaccination and other strategies to prevent or control infection.

The geographical distribution and first molecular analysis of Culicoides Latreille (Diptera: Ceratopogonidae) species in the Southern and Southeastern Turkey during the 2012 outbreak of bovine ephemeral fever.

This study investigated the geographical distribution and molecular analysis of Culicoides species in the Southern and Southeastern Turkey during the 2012 outbreak of bovine ephemeral fever (BEF). The midge specimens caught by Onderstepoort-type light traps from livestock farms were tested for molecular evidence of existence of viral genome. Blood specimens were collected from clinically BEF-suspected acute febrile cattle. Total nucleic acid samples obtained from field specimens were checked against the BEF virus G gene and Culicoides internal transcribed spacer 1 (ITS-1) gene. A total of 20,845 Culicoides specimens (20,569 ♀♀, 276 ♂♂) comprising 11 species (Culicoides badooshensis, Culicoides circumscriptus, Culicoides gejgelensis, Culicoides imicola, Culicoides kibunensis, Culicoides longipennis, Culicoides newsteadi, Culicoides nubeculosus, Culicoides odiatus, Culicoides punctatus, Culicoides schultzei, Culicoides spp.) were collected. C. schultzei (18,032) was found as the dominant species and followed by C. imicola (1,857), C. nubeculosus complex (545), and C. circumscriptus (259), respectively. C. kibunensis was identified as new species for this region. PCR positivity of BEF was found 37.14% (13/35) in blood samples whereas no viral genome was obtained from Culicoides specimens. Culicoides spp. ITS-1 gene sequences were analyzed phylogenetically with GenBank ITS-1 sequences. Molecular homology of Culicoides ITS-1 gene was ranged between 62.74 and 71.39%. The results described first molecular detection and phylogenetic analysis of Culicoides ITS-1 gene with reference to the 2012 BEF outbreak in Turkey.

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.

Phylogenetic relationships of the glycoprotein gene of bovine ephemeral fever virus isolated from mainland China, Taiwan, Japan, Turkey, Israel and Australia.

BACKGROUND: The glycoprotein (G) gene sequences of bovine ephemeral fever virus (BEFV) strains derived from mainland China have not been compared with those of the isolates from other countries or areas. Therefore, the G genes of four BEFV isolates obtained from mainland China were amplified and sequenced. A phylogenetic tree was constructed in order to compare and analyze the genetic relationships of the BEFV isolates derived from mainland China and different countries and areas. RESULTS: The complete BEFV G gene was successfully amplified and sequenced from four isolates that originated from mainland China. A total of fifty-one BEFV strains were analyzed based on the G gene sequence and were found to be highly conserved. A phylogenetic tree showed that the isolates were grouped into three distinct lineages depending on their source of origin. The antigenic sites of G1, G2 and G3 are conserved among the isolates, except for several substitutions in a few strains. CONCLUSIONS: The phylogenetic relationships of the BEFV isolates that originated from mainland China, Taiwan, Japan, Turkey, Israel and Australia were closely related to their source of origin, while the antigenic sites G1, G2 and G3 are conserved among the BEFV isolates used in this work.

Finding potential inhibitors against RNA-dependent RNA polymerase (RdRp) of bovine ephemeral fever virus (BEFV): an in-silico study.

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.

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.