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.