Isolation，identification and whole genome analysis of A G1P［7］porcine rotavirus strain

【Objective】To isolate and identify G1P［7］porcine rotavirus（PoRV）and conduct a whole genome analysis，which could provide theoretical basis for the prevention and control of piglet diarrhea and vaccine development. 【Method】Excrement samples from diarrheic piglets were provided by college of Veterinary Medicine，Nanjing Agricul【Objective】To isolate and identify G1P［7］porcine rotavirus（PoRV）and conduct a whole genome analysis，which could provide theoretical basis for the prevention and control of piglet diarrhea and vaccine development. 【Method】Excrement samples from diarrheic piglets were provided by college of Veterinary Medicine，Nanjing Agricultural University. The samples were tested for porcine epidemic diarrhea virus（PEDV），transmissible gastroenteritis virus （TGEV），porcine deltacoronavirus（PDCoV），bovine viral diarrhea virus（BVDV）and PoRV by PCR. Trypsin was added to the supernatant of the PCR-positive samples，which were then inoculated into MA104 cells for virus isolation and purification. The strain was identified by PCR，electron microscopy，indirect immunofluorescence assay，and RNAPAGE. The biological characteristics of the strain were explored by drawing the virus proliferation curve and testing the susceptibility of MA104，Vero，IPEC-J2，HT-29，HRT-18，and Caco-2 cells to the virus. Bioinformatics software was used to analyze the homology of the 11 genes of the isolated strain with various genotypes of rotavirus at home and abroad and to construct a phylogenetic tree of whole genome of the virus.【Result】The PCR results of the excrement samples showed that only PoRV was positive. One strain of PoRV was successfully isolated and named JSNJ2023. The virus could stably replicate and passage on MA104 cells. After three rounds of cloning，a suitable clone was selected. In the third round of cloning，the clone with the highest virus titer was expanded，and a high-titer monoclonal strain was finally obtained. The virus particles had a distinct spherical structure with a clear double-layered capsid feature，with a diameter of approximately 70 nm. The indirect immunofluorescence assay results showed that the JSNJ2023 strain could infect MA104 cells. The RNA-PAGE results indicated that strain JSNJ2023 had the characteristic 11-band electrophoretic pattern of group A rotavirus，arranged as 4：2：3：2. The virus proliferation curve showed that the strain JSNJ2023 reached the peak virus titer in MA104 cells 18 h after inoculation，and the virus titer gradually decreased with the extension of the inoculation time. The susceptibility of the strain JSNJ2023 to cell lines was in the order of MA104，IPEC-J2，HRT-18，HT- 29，Caco2 and Vero cells. The results of gene homology analysis and phylogenetic tree construction showed that the strain JSNJ2023 belonged to group A G1P［7］porcine rotavirus，with the genotype of G1-P［7］-I5.【Conclusion】The PoRV strain JSNJ2023 is successfully isolated and identified as a G1P［7］type rotavirus of group A，with the genotype of G1-P［7］-I5. The strain JSNJ2023 has a high degree of homology with human，porcine and bovine rotaviruses，and its evolution may have been influenced by human，porcine，and bovine rotaviruses，resulting in gene recombination or exchange and the formation of a unique genotype R1-C1-M1-A8-N1-T7-E1-H1）.