METHODS: The RVA G9P[8] genotype from a diarrhea sample was passaged in MA104 cells. The virus was evaluated by TEM, polyacrylamide gel electrophoresis, and indirect immunofluorescence assay. The complete genome of virus was obtained by RT-PCR and sequencing. The genomic and evolutionary characteristics of the virus were evaluated by nucleic acid sequence analysis with MEGA ver. 5.0.5 and DNASTAR software. The neutralizing epitopes of VP7 and VP4 (VP5* and VP8*) were analyzed using BioEdit ver. 7.0.9.0 and PyMOL ver. 2.5.2.
RESULTS: The RVA N4006 (G9P[8] genotype) was adapted in MA104 cells with a high titer (105.5 PFU/mL). Whole-genome sequence analysis showed N4006 to be a reassortant rotavirus of Wa-like G9P[8] RVA and the NSP4 gene of DS-1-like G2P[4] RVA, with the genotype constellation G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1 (G9P[8]-E2). Phylogenetic analysis indicated that N4006 had a common ancestor with Japanese G9P[8]-E2 rotavirus. Neutralizing epitope analysis showed that VP7, VP5*, and VP8* of N4006 had low homology with vaccine viruses of the same genotype and marked differences with vaccine viruses of other genotypes.
CONCLUSION: The RVA G9P[8] genotype with the G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1 (G9P[8]-E2) constellation predominates in China and may originate from reassortment between Japanese G9P[8] with Japanese DS-1-like G2P[4] rotaviruses. The antigenic variation of N4006 with the vaccine virus necessitates an evaluation of the effect of the rotavirus vaccine on G9P[8]-E2 genotype rotavirus.
Methodology: The pangolin P. fungorum (pangolin Pf) genome has a genomic size of approximately 7.7 Mbps with N50 of 69,666 bps. Our study showed that pangolin Pf is a Paraburkholderia fungorum supported by evidence from the core genome SNP-based phylogenetic analysis and the ANI analysis. Functional analysis has shown that the presence of a considerably large number of genes related to stress response, virulence, disease, and defence. Interestingly, we identified different types of secretion systems in the genome of pangolin Pf, which are highly specialized and responsible for a bacterium's response to its environment and in physiological processes such as survival, adhesion, and adaptation. The pangolin Pf also shared some common virulence genes with the known pathogenic member of the Burkholderiales. These genes play important roles in adhesion, motility, and invasion.
Conclusion: This study may provide better insights into the functions, secretion systems and virulence of this pangolin-associated bacterial strain. The addition of this genome sequence is also important for future comparative analysis and functional work of P. fungorum.