BRBV has been identified in many countries, including Japan; however, its genomic information has been limited to reports from only a few countries. Herein, we detected BRBV in cattle from Japan and sequenced the nearly complete viral genomes of two BRBV strains: IBA/2211/2 and LAV/238002. This is the first report detailing the genetic characteristics of BRBV strains in Japan.
According to metagenomic analyses conducted in the USA, Mexico, and Canada, BRBV was detected at high rates in cattle showing respiratory symptoms, and to some extent in cattle not showing respiratory symptoms [4–6]. A study in Japan found that out of 40 nasal swabs from cattle on six farms with respiratory symptoms, 11 (27.5%) from three farms were BRBV-positive, making it the second most detected virus after bovine respiratory syncytial virus [12]. However, the study did not include samples from cattle without respiratory symptoms. Herein, when testing for bovine respiratory pathogens, BRBV was detected in 6/61 samples—which was the highest among the viruses tested. Notably, 4/6 BRBV-positive cattle did not exhibit any respiratory symptoms. Additionally, no respiratory pathogens were exclusively detected in symptomatic cattle among those admitted to Azabu University. BRBV replicates efficiently in the upper respiratory tract but exhibits lower replicative and pathogenic potential in the lungs, typically resulting in asymptomatic infections or mild symptoms in cases of solitary infection [3, 7]. This implies that BRBV spreads undetected, contributing to BRDC, similar to most BRDC pathogens. However, most samples in this study were from cattle admitted to Azabu University for nonrespiratory conditions; therefore, a specific correlation between the detection rate and disease could not be established. The two BRBV genomes identified from this study were from cattle without and with respiratory symptoms in Azabu University and Ibaraki Prefecture, respectively. The inability to isolate these viruses indicates that the relationship between their virologic characteristics and genomic differences could not be determined.
As of December 2023, the GenBank database contains the BRBV genome sequences from seven countries, including the UK, USA, Mexico, Canada, Australia, Sweden, and China. However, the Japanese BRBV genome sequences are not registered. This study obtained two nearly full-length BRBV genomes from Japanese cattle, which shared 84.2% nucleotide and 90.0% amino acid sequence identity. Compared with the 23 complete CDSs of BRBV registered in GenBank, IBA/2211/2 and LAV/238002 exhibited 14 and 24 unique amino acid mutations, respectively. Notably, TRI22LN and R190KS were located at the same positions in both strains, in addition to one identical mutation (TVKN36I), suggesting that these could be characteristic mutations of the Japanese strains. In LAV/238002, 10 mutations were found in VP1, VP2, and VP3 proteins, which form the outer surface of the capsid and contain major neutralizing sites [18–21]. The functions of VP1 and VP2 in BRBV remain unexplored; however, these proteins have been characterized in FMDV and may be related to antigenicity [22]. Certain amino acid mutations in VP1 and VP2 of FMDV reportedly cause immune evasion [22], suggesting that similar mutations in these two proteins of LAV/238002 are involved in immune escape. In IBA/2211/2, 2B protein had an amino acid deletion (1121E) identical to the Chinese strain SC1_LZ02 [11], whereas LAV/238002 exhibited a different deletion (1117DG) nearby. In FMDV, 2B protein, in addition to 2C protein, plays a role in assisting virus maturation by creating a pore in the host cell membrane [23, 24]. These amino acid deletions in 2B protein have not been identified in other Aphthovirus spp., and the biological significance of these mutations requires further investigation.
Advancements in next-generation sequencing have revealed significant genetic diversity in BRBV [8]. However, genomic data are limited in number and geographical distribution, suggesting that additional genome information from unreported countries could enhance our understanding of the genetic diversity of BRBV. Herein, phylogenetic analysis of the polyprotein genes of the two Japanese BRBV strains revealed that they positioned themselves in different branches of the same clade. Analysis of the 12 genes within the polyprotein gene, excluding the leader gene, showed that both Japanese strains belonged to the same clade. However, phylogenetic analysis of leader gene showed that LAV/238002 belonged to a distinct clade, demonstrating a clear evolutionary divergence from other reported BRBV strains. Amino acid sequence analysis supported this divergence, because both Japanese strains, while clustering with viruses from the UK, USA, Canada, and Mexico in gene sequence analysis, diverged significantly in leader protein analysis. The role of leader protein in FMDV suggests involvement in host immune evasion mechanisms [25]. The distinct evolutionary path of leader protein in the two Japanese BRBV strains suggests unique functional characteristics. Functional differences in the leader protein among strains and clades warrant analysis.
Homologous recombination within Aphthovirus spp. such as BRBV is a fundamental evolutionary mechanism [9], and intraspecies recombination in BRBV contributes to its genetic diversity [10, 11]. Herein, homologous recombination events were identified in both the Japanese BRBV strains. The anticipated breakpoints were located from the 3′ end of the VP1 gene to the 5′ end of the 2B gene in IBA/2211/2 and at the 5′ end of leader gene in LAV/238002. Notably, both strains showed high similarity (92.3–93.3%) to a Chinese strain (SC1_LZ02), indicating the involvement of geographically proximal Chinese viruses in the Japanese BRBV evolution. Another possible breakpoint upstream of the VP2 gene was identified in IBA/2211/2. These findings support findings of phylogenetic analysis where the leader gene and protein sequences of IBA/2211/2 and LAV/238002 belong to a different clade compared with other reported BRBV strains, suggesting that the diversity and characteristics of leader genes in the two Japanese strains are because of recombination events.
In conclusion, we report the nearly complete viral genomes of two Japanese BRBV strains—IBA/2211/2 and LAV238002—and elucidate their genetic characteristics. Although these strains show high genetic similarity to known BRBV strains, they exhibit unique mutations and recombination events, indicating a dynamic evolutionary process influenced by regional, environmental, and biological factors. Notably, both Japanese strains display significant divergence in the nucleotide and amino acid sequence of leader compared with other reported BRBV strains, demonstrating remarkable genetic diversity. These findings provide insights into the genetic makeup of the Japanese BRBV strains and enhance our understanding of the genetic diversity and evolutionary mechanisms of BRBV.