Molecular Detection And Genomic Study Of Tibetan Pig Group A Rotavirus In Ganzi Tibetan Autonomous Prefecture,Sichuan Province

Porcine rotavirus group A(PoRVA)is an important pathogen causing diarrhea in piglets,which has caused significant economic losses to the pig industry.Moreover,PoRVA can also be transmitted across species to a variety of mammals such as humans and cattle,which has important public health significance.The virus has been widely circulated in domestic pigs,but there is little epidemiological data on RVA from Tibetan pigs.The purpose of this study is to carry out PoRVA molecular detection on Tibetan pig farms in 7 regions of Ganzi Tibetan Autonomous Prefecture(hereinafter referred to as Ganzi Prefecture)in Sichuan Province in 2021,and to isolate,identify and analyze the successfully typed samples.The results are as follows:1.Detection and genotyping of group A rotavirus in Tibetan pigs in Ganzi Prefecture in 2021From May to June 2021,74 diarrheal fecal samples were collected from 7 Tibetan pig farms in Ganzi Prefecture,including Xiangcheng County,Batang County,Luding County,Derong County,Daocheng County,Yajiang County,and Litang County.202 stool samples.The samples were tested by RT-PCR method that specifically detects PoRVA,and the positive samples were further investigated for the mixed infection of PoRVA and porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),and porcine delta coronavirus(PDCoV).condition.The results showed that the positive rate of PoRVA in diarrhea samples was 24.3%,and the positive rate of PoRVA in non-diarrhea samples was 0.99%;the positive detection rate of Tibetan pig farms was 57.1%;PEDV,TGEV and PDCoV were not detected.In this experiment,2 types of G type and 1 type of P type were identified from PoRVA positive samples,among which G type was G9 type(2/9)and G10(7/9)type;P type was P[13]type(4/9).4);There are 4 strains with both G and P types,2 of which are G9P[13]type and 2 are G10P[13]type.The results of this experiment show that PoRVA is an important pathogen causing diarrhea in Tibetan piglets,and there are at least two genotypes of G9P[13]and G10P[13],which provide a reference for the prevention and control of diarrhea in Tibetan piglets in Ganzi Prefecture.2.Isolation,identification and genomic study of G9P[13]PoRVA from Tibetan pigsMA-104 cells were used to isolate and identify 2 G9P[13]type and 2 G10P[13]type strains,and a cytopathic G9P[13]type strain was successfully isolated.can be separated successfully.After the G9P[13]positive disease material was inoculated with cells,it was blindly passed to the third passage.After 72 hours,the cells appeared cell shedding,shrinking,drawing,and the shed cells gathered.When the cells were continuously passaged to the fifth passage,the cells appeared stable at 36 hours.The lesions were identified as G9P[13]PoRVA by RT-PCR detection,indirect immunofluorescence and electron microscopy.Named RVA/Piglet-tc/CHN/SCLT-FX17/2021/G9P[13],hereinafter referred to as SCLT-FX17 strain.The plaque-purified TCID50 of this strain was 10-5.38/100 μ L.The samples were amplified by RT-PCR,and 11 complete segments of the genome of SCLT-FX17 isolate were successfully obtained,and the genome of the strain was typed by Rota C2.0 gene analysis software,and the genotype was G9-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1.Sequence alignment and phylogenetic tree showed that the VP1,VP2,VP3,VP4,VP6,VP7,NSP4,NSP5 genes of SCLT-FX17 were originated from PoRVA,and the NSP1,NSP2 and NSP3 genes may be derived from human group A rotavirus.It is a reassortment of human-derived strains with porcine-derived strains.Compared with all 33 Chinese G9 porcine-derived virus strains registered in GenBank,SCLT-FX17 strain VP7 gene has 3 unique nucleotide nonsense variants(A270G,T459C,A861G);compared with all 12 registered in GenBank Compared with Chinese P[13]-type swine-derived strains,SCLT-FX17 strain VP4 gene has a total of 18 nucleotide variations,resulting in 7 amino acid variations,which are located in the VP8 region(T120N)and VP5 region(S534A,M551R,K553R,K555T,N558D,L774T),but not in the neutralizing epitope region,the biological significance of these amino acid mutations needs further study.In this experiment,a G9P[13]PoRVA strain of Tibetan pig origin was successfully isolated and the whole genome sequence was obtained,which provided an important reference for in-depth understanding of its molecular characteristics and genetic evolution.Genetic evolution analysis showed that the genome of this strain showed the phenomenon of reassortment between human-derived strains and porcine-derived strains,suggesting that there may be the ability of interspecies transmission,and its public health significance deserves further attention.