Genomic and phylogenetic analyses of complete polyprotein genome
Three complete MuKV polyprotein genome sequences (except the 5’ and 3’UTR) were successfully obtained from three MuKV-positive fecal samples using ten primer pairs in the current study, designating MuKV/XM34/CHN (accession no. MW292482), MuKV/XM86/CHN (accession no. MW292481) and MuKV/YN27/CHN (accession no. MW292480). The obtained genome sequence of MuKV/XM34/CHN was 7714nt long and possessed one complete ORF (7293nt) encoding a potential polyprotein of 2430 amino acids, whereas the acquired 7714-nt-long genome strain of MuKV/XM86/CHN and 7711-nt-long genome strain of MuKV/YN27/CHN contained the complete ORF of 7293nt (2430aa) and 7290nt (2429aa) in length, respectively. Interestingly, the lack of one-amino-acid was found in 3A region of the strain MuKV/YN27/CHN when multiple aligned with the genome of MuKV/XM34/CHN and MuKV/XM86/CHN, and they shared 92.5-97.1% nucleotide identities and 97.1-98.1% amino acid identities with each other. The predicted genomes of our study strains have the same genome organization as identified for other kobuviruses, and the detailed information was shown in Figure 3A.
The genomes identified in the present study showed the highest similarity to the currently published MuKV strain GZ85 (MN648601.1) as the closest match determined by BLASTn search, with 93.2-94.5% nucleotide identities. Moreover, the comparison results indicated our study strains were also closely related to the previously described MuKVs. Consequently, we conducted a comparative sequence analysis for each functional region between our study strains and representative kobuviruses available in the Genbank database. The kobuviruses identified in the present study showed amino acid identities with those of rat (96.0-98.3%), human (80.1-80.7%), canine (80.1-80.5%), and feline kobuvirus reference strains (80.1-80.3%) based upon the complete polyprotein gene. For MuKV/XM34/CHN and MuKV/XM86/CHN, the highest nucleotide identities were presented in P2 region of rat kobuvirus GZ85 strain, with values of 94.6% and 94.7%, respectively, while they also shared the highest amino acid identities (98.7%) to the P2 region of rat08/rAiA/HUN. Regarding MuKV/YN27/CHN, the highest nucleotide (95.4%) and amino acid identities (99.8%) were found in P2 and P1 regions of GZ85, respectively (Table 2). Besides, the similarity plot analysis was generated to further analyze the genetic characteristics, contrasting the complete polyprotein nucleotide gene of our study strains and one feline kobuvirus strain WHJ-1/MF598159.1 (used as an out-group sequence) to the rat kobuvirus reference strain GZ85/MN648601.1 (used as a query sequence). The findings suggested the sequences displayed relatively high similarities to the query sequence in the VP0, VP3, 2A-2C, 3C and 3D regions, whereas different similarities were exhibited in the L, VP1, 3A and 3B regions. Additionally, MuKV/YN27/CHN presented considerably higher similarities in the L, 3A, 3B regions and the sequence connecting 2A and 2B than MuKV/XM34/CHN and MuKV/XM86/CHN (Figure 3B).
Table 3 shows the predicted protease-cleavage sites of our study strains and other reference kobuviruses, including Q/G, P/Q, Q/T, Q/S, Y/V and Q/A. The predicted protease-cleavage sites between L and VP0 (Q/G), 2A and 2B (Q/G), 2B and 2C (Q/G), 2C and 3A (Q/G), and 3C and 3D (Q/S) are conserved among kobuviruses from different species. Besides, the protease-cleavage sites between 3A and 3B is Q/A for the kobuvirus strains, except for porcine kobuvirus (Q/G).
Maximum-likelihood phylogenetic analysis was conducted based on the complete polyprotein nucleotide sequences between our study strains and other representative members of family Picornaviridae , suggesting the strains identified in the present study were located at the same branch as the members of genus Kobuvirus , where the sequences of MuKV are the closest relatives (Figure 4).