Bovine ephemeral fever virus (BEFV) is an overlooked pathogen, recently gaining widespread attention owing to its associated enormous economic impacts affecting the global livestock industries. High endemicity with rapid spread and morbidity greatly impacts bovine species, demanding adequate attention towards BEFV prophylaxis. Currently, a few suboptimum vaccines are prevailing, but were confined to local strains with limited protection. Therefore, we designed a highly efficacious multi-epitope vaccine candidate targeted against the geographically distributed BEFV population. By utilizing immunoinformatics technology, all structural proteins were targeted for B- and T-cell epitope prediction against the entire allele population of BoLA molecules. Prioritized epitopes were adjoined by linkers and adjuvants to effectively induce both cellular and humoral immune responses in bovine. Subsequently, the in silico construct was characterized for its physicochemical parameters, high immunogenicity, least allergenicity, and non-toxicity. The 3D modeling, refinement, and validation of ligand (vaccine construct) and receptor (bovine TLR7) then followed molecular docking and molecular dynamic simulation to validate their stable interactions. Moreover, in silico cloning of codon-optimized vaccine construct in the prokaryotic expression vector (pET28a) was explored. This is the first time HTL epitopes have been predicted using bovine datasets. We anticipate that the designed construct could be an effective prophylactic remedy for the BEF disease that may pave the way for future laboratory experiments.

Bovine ephemeral fever virus (BEFV) is an evolving arbovirus reported across tropical, subtropical and temperate climatic zones globally. This study reveals prominent BEFV outbreaks in India, emerging annually during monsoons in subtropical areas accompanied by a congenial abundance of the vector population. PCR‐based detection of viral genomic RNA in the blood samples collected during outbreaks of 2018–2019 for the first time confirmed the presence of BEFV in India. Phylogenetic analysis based on the glycoprotein gene of BEFV showed the current isolates to have high sequence homology with Middle Eastern lineage with nearly 97%, identity to Turkey (BEFV Ad12/TUR) and Israel (Israel 2006) isolates.

RenalOCT2 substrate Yes No No AV4 AV5 AV6 Physicochemical Properties Formula C18H22N2O6 C9H14Cl2N2O2 C9H11NO3 Molecular weight 362.38 g/mol 253.13 g/mol 181.19 g/mol Molar Refractivity 95.05 63.84 47.19 TPSA 167.10 Ų 89.34 Ų 72.55 Ų Lipophilicity Log Po/w (iLOGP) 1.96 0 1.21 Log Po/w (XLOGP3) -4.02 -0.6 -1.73 Log Po/w (WLOGP) 0.69 1.84 0.46 Log Po/w (MLOGP) -3.99 -1.1 -1.7 Log Po/w (SILICOS-IT) 0.39 0.15 0.54 Consensus Log Po/w -0.99 0.06 -0.24 Water Solubility Log S (SILICOS-IT) -1.29 -1.5 -1.59 Solubility 1.86e+01 mg/ml 7.91e+00 mg/ml 4.68e+00 mg/ml Class Soluble Soluble Soluble Pharmacokinetics GI absorption

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 G NS 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.