Although BEF is one of the most important diseases of cattle in Iran , little is available about its agent and characteristics of circulating viruses. Therefore, in the present study, we characterized the prevalent BEFV strains in Khuzestan province southwest of Iran in 2018 and 2020.
In 2018, the incidence of BEF was sporadic and no report of the disease is available in other regions of the country. Our surveillance program of the BEF showed the absence of the disease in 2019. However, many animals suspected of contracting the three-day sickness were reported in Khuzestan province and other parts of the country in 2020. The RT-nested PCR results confirmed the occurrence of the disease in Khuzestan province as well as other regions of the country. The BEF disease epizootic has been reported in Turkey simultaneous with an outbreak in Iran . The phylogenetic analysis, identity matrix, and distance evolution confirmed the high genetic closeness of prevalent strains in Turkey and Iran in 2020.
According to the phylogenetic analysis of the G gene ectodomain region, the BEFV strains were categorized into four clusters of East Asia, Australia, Middle East, and South Africa. However, previous studies have grouped the BEF strains into the three clusters of East Asia, Middle East, and Australia [20–24]. According to our results and the study of Omar et al , the BEFV strains of South Africa are distinct from those of other regions of the world and fall into the new cluster of South Africa. These strains showed the most distance evolution and the lowest similarity with the identified strains in the present study.
The phylogenetic tree revealed that the IR-2018 and IR-2020 strains are in the Middle East cluster close to Turkish, Indian, and Israeli BEFV strains. However, the viruses related to the outbreaks of 2012 and 2013 in Iran were grouped in East Asia cluster close to the strains from Taiwan, isolated in 2013, and Turkey, isolated in 2012. Most of the strains identified in Turkey belong to the Middle East group; however, in 2012, in some regions of Turkey, some identified BEF viruses were more similar to those placed in the East Asian group [16, 21, 26]. The BEF viruses that were identified in different regions of Iran during 2012 and 2013 were highly similar to those from Turkey . Phylogenetic, identity matrix, and distance evolution analyzes revealed that the BEF viruses previously identified in Iran have a genetic distance from the strains of the present study and may have different sources. It seems that the virus circulating in 2012 and 2013 probably has expanded to the Middle East from East Asia [16, 26], while circulating strains of 2018 and 2020 were domestic strains of the Middle East. Studies of G gene nucleotide and deduced amino acid sequences from 2000 to 2020 indicate the circulation of a domestic strain in the Middle East. However, in 2012 and 2013, another strain that seemed to be exotic was prevalent in Turkey and Iran, but no evidence of the virus has been available since. It remains to be determined whether the structure of the G protein and its epitopes are effective in the compatibility of the virus with competence vectors .
This study demonstrated that BEFVs circulating in the Middle East are closely related phylogenetically and geographically. Pyasi et al reported the outbreaks of BEFV during 2018 and 2019 in India . Their molecular studies have shown that these strains were evolutionarily very close to those of Turkey and Israel, so they strongly suggested that these strains reached India from Israel and Turkey while maintaining their genomic sequence . The findings of the present study also confirm this view. These viruses probably traversed across the countries like Turkey and Israel reaching Iran, and then India. Iran and India do not have a common border, so it is unclear exactly how the virus has been transmitted from Iran to India. The similarity between BEFVs from Iran's and Turkey's 2020-outbreaks (99.4–99.6% nucleotide similarity) was higher than that of IR-2018 and IR-2020 BEFVs (98.4% nucleotide similarity). The simultaneous outbreaks of BEFV in Iran and Turkey and high nucleotide and amino acid similarity indicate the common source of these viruses.
Comparison of G1, G2, and G3 antigenic sites showed that these neutralizing epitopes are highly conserved among the Middle East strains; however, the strains previously identified in Iran differed in three amino acids placed in G1 and G2 epitopes, which two of them were putative attachment site of oligosaccharides. Japanese YHL strain (vaccine strain used in Iran) differed from the Middle East BEFVs, especially IR-2018 and IR-2020 strains in G1 and G2 epitopes. The amino acid substituted in the G1 epitope is a putative N-linked glycosylation site; therefore, it may affect conformation and recognition of the neutralizing epitopes. Consistent with the differences in antigenic sites, Iranian and YHL BEFVs were placed as two distinct clusters. Due to these differences in antigenic structure, the use of heterologous strains as a vaccine may not provide full protective immunity . According to the similarity of circulating strains in the Middle East, the use of these strains to develop vaccines can play an important role in preventing this disease.