The Isolation Of Rotavirus From Different Host Sources In Hubei And The Study On Genetic Variation Of Its Genome

Rotavirus(RV)can infect a variety of young mammals such as pigs,cattle,dogs and cats,and cause diarrhea.It can also cause acute gastroenteritis in children under 5 years of age and infants,which is an important zoonotic pathogen.However,there are few studies on the animal-human transmission of rotavirus.This research takes this as the starting point to collect human,pig,and canine diarrhea related samples in some areas of Hubei.Then rotavirus was detected and isolated.The complete genome sequence was sequenced and the genotypes of its 11 gene segments were analyzed and constructed the phylogenetic tree.We further studied genetic relationship of each gene fragment,as well as the cross-reassortment relationship between human rotavirus,canine rotavirus and porcine rotavirus.The main research results were as follows:1.RV epidemic situation in Hubei province.In this study,human,pig and canine diarrhea related samples and pig intestine tissues were collected from different regions,hospitals,pig farms,and pet hospitals in Wuhan,Xiangyang,and Yichang,Hubei Province from December 2018 to December 2020.A total of 513 samples were collected and detected by RT-PCR.These test results showed that the positive rate of human RV was 21.63%(69/319),porcine RV was 35.29%(6/17),and canine RV was 1.12%(2/177).Then these positive samples were further isolated and identified and the complete genome was sequenced in order to investigate changes in its genotype and genetic characteristics of RV in human,pig and canine.It was expected to provide evidence for the cross-species transmission of RV from different host sources.2.Isolation and identification of RV virusIn this study,firstly the optimal trypsin tolerant concentration of the cells was determined.MA-104 cells were used to isolate all positive samples and RV was activated with a final concentration of 10?g/m L and 5?g/m L trypsin was added to the maintenance solution.The positive samples were inoculated with cells and then subjected to isolate with blind passage.At 48 hours post-infected,the sixth passages culture showed significant cytopathic effect(CPE)such as cell aggregation,shrinkage,shedding,and netting.After ten continuous passages,CPE was stable,and it was identified as RV by RT-PCR and indirect immunofluorescence.Further PCR amplification of VP6 gene and sequencing results showed that this study successfully isolated 11 HRV strains and 2 Po RV strains in Hubei.After further plaque purification,the titers of the 2 Po RV strains named RVA/Po-rcine/CHN/HB-1/2019 and RVA/Porcine/CHN/HB-7/2019 that can be stably passaged were 10^5.63TCID₅₀/ml and 10^6.1TCID₅₀/ml,respectively.Combined with the results of the complete genome sequence of the isolated strains,this genotype of G9P[8]Po RV strains were successfully isolated in China for the first time.It laying a foundation for further research on the biological characteristics of the G9P[8]Po RV strain.It also provided experimental materials and research basis for further analysis of possible genetic reassort-ment.3.Complete genome sequencing and reassortment analysis of RV virusIn order to investigate the genetic evolution of each gene segment of 11 G9P[8]HRV strains and 2 G9P[8]Po RV strains,15 pairs of primers were designed for amplifying genomic sequences by RT-PCR to perform complete genome sequencing analysis.The genetic evolution analysis of the genome showed that 8 G9P[8]strains were belong to Wa-like genotype.Furthermore,they all had the E2 genotype of DS-1-like NSP4.It was suggested that there may be a naturally reassortment result.The genetic evolution analysis of the genomes of two G9P[8]Po RV strains revealed that the 7 gene segments(VP4,VP1,VP2 and NSP2-NSP5)of these pig-derived RV strains were closely related to the human-derived L2448 strain in Wuhan and 11 G9P[8]HRV strains isolated in this study.These strains had the closest genetic relationship.And the VP7 and VP6 genes were closely related to the porcine TM-a strain isolated from Wuhan,suggesting that there may have a phenomenon of reassortment and inter-species transmission between porcine and human rotavirus strains in Hubei,China.