Phylogenetic analysis of 3D gene and VP1 gene sequences
Twenty-two representative sequences of partial 3D gene (461nt) were selected randomly for further PCR amplification and sequencing within these positive samples. Sequences isolated from different areas and different species were highly conserved with each other, at the nucleotide level of 92.7%-99.5% and deduced amino acid level of 95%-100%. Maximum-likelihood phylogenetic analysis based on partial 3D regions was generated with representative kobuvirus strains from multiple species, indicating that our sequences belonged toAchivirus A and displayed the closet relatedness with MuKVs. Interestingly, only one strain (YY101) determined during this study showed the closest relationship to the Chinese MuKV strain, GZ85, isolated from Rattus norvegicus (You et al., 2020), while the other 21 sequences formed a major group with MuKV reference strains identified from USA, China and Hungary (Figure 1).
Meanwhile, seven complete VP1 regions (917nt) were successfully determined in the present study, which share 88.7%-99.3% nucleotide identities and 95.1%-100% amino acid identities with one another. In contrast to other references kobuvirus previously reported in Genbank database, they have the highest sequence similarity with the Chinese MuKV strain, MM33 (accession number MN648600.1), at the nucleotide level of 91.3%-93.9%. A phylogenetic tree has been conducted based on the seven VP1 sequences identified in the current study and the available complete nucleotide sequences for MuKV VP1 from three different countries, indicating that our VP1 sequences clustered along with two MuKV strains, GZ80 and GZ84, previously described in Guangdong, China, while other kobuvirus reference strains from China, Hungary and Vietnam formed another groups (Figure 2). The partial 3D and complete VP1 nucleotide sequences of MuKV determined in the present study have been lodged within the Genbank database under the accession numbers MW292451-MW292472 and MW292473-MW292479 , respectively.