Genomic Characteristics And Interspecies Transmission Of G3P[13] Porcine Group A Rotavirus

Group A rotavirus?RVA?is a major diarrhea-causing pathogen in young animals and children.Porcine Group A rotavirus?PoRVA?is an important diarrhea-causing pathogen in piglet.Moreover,the zoonotic potential of PoRVA has received extensive attention in recent years.The purpose of this study was to isolate RVA strains from fecal samples of piglets,sows,pig breeders and murine in a RVA outbreak swine farm,and to analysis the genomic characteristics of these strains,in order to investigate zoonotic potential of RVA.The results obtained as follows:1.Detection RVA from fecal samples of piglets,sows,pig breeders and murine in a RVA outbreak swine farmIn May 2018,piglets diarrhea outbroke in a swine farm of Sichuan province.23 piglet diarrhea fecal samples,15 clinically healthy sow fecal samples,3 pig breeder fecal samples and 1 murine fecal sample were collected for detecting RVA by a real time RT-PCR,and identifying RVA genotype from RVA-positive samples.The results showed that the RVA detection rate was 95.7%?22/23?in piglet samples,60%?9/15?in sow samples.Other common diarrhea-causing viruses were detected as negative results in these samples from piglets and sows.G3P[13]?n=11?,G9P[13]?n=3?and G3P[23]?n=1?genotype were identified in RVA positive piglet samples;G3P[13]?n=4?genotype was identified in RVA positive sow samples.Interestingly,RVA G3P[13]was detected in three breeder fecal samples and one murine fecal sample.Therefore,the predominant RVA genotype in this swine farm was G3P[13].This study was the first time report that the RVA G3P[13]infected human,it is necessary to further investigate the molecular characteristics and zoonotic potential of the RVA G3P[13].2.Isolation and identification of RVA G3P[13]strainsMA-104 cells was used to isolate RVA G3P[13]strains from positive samples detected by a real time RT-PCR.Cytopathic effect?CPE?characterized as cell shrinking,cell layer splitting,lysis,detachment,and shedding was observed at48 hours post-infection in passage 3.After six continuous passages,CPE was more visible,and the time of occurrence of CPE was stable.RVA strains were identified by RT-PCR and indirect immunofluorescence.Four strains were succ essfully isolated from a piglet fecal sample,a sow fecal sample,a pig breeder fecal sample and a murine fecal sample,named RVA/Piglet-tc/CHN/SCLS-X1/2018/G3P[13],RVA/Pig-tc/CHN/SCLS-3/2018/G3P[13],RVA/Human-tc/CHN/SCL S-R3/2018/G3P[13]and RVA/Murine-tc/CHN/SCLS-M/2018/G3P[13],respectively.After plaque purification of the 4 strains,the TCID₅₀0 was determined as 10⁷.5.5 T CID₅₀/ml,10⁶.8.8 TCID₅₀/ml,10⁶.2.2 TCID₅₀/ml,10⁷.2.2 TCID₅₀/ml,respectively.This r esults laid a foundation for further research the biological characteristics and ge nomic characteristics of isolated RVA strains.3.The interspecies transmission of RVA G3P[13]from pig to humanIn order to investigate the molecular characteristics of RVA G3P[13]strains and the genetic evolution relationship between porcine RVA G3P[13]and human RVA G3P[13],26 pairs of primers were designed for amplifying genomic sequences by RT-PCR.The complete sequence of 11 gene segments of 2 porcine RVA strains and 1human RVA strain were obtained successfully.The constellation of two porcine RVA strains were G3-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1;the human RVA was G3-P[13]-I2-R1-C1-M1-A8-N1-T7-E1-H1.Phylogenetic analysis showed that 10gene segments?VP7,VP4,VP1^VP3 and NSP1^NSP5?in human RVA G3P[13]strain were most closest to two porcine G3P[13]RVA strains in this farm,but the VP6gene was most closest to human RVA strain I321.Moreover,the same recombination event was identified in the VP4 gene of the human RVA G3P[13]strain and the porcine RVA G3P[13]strains,and the breakpoint was located at 1591 bp of VP4genes,and the predicted parents strain were porcine RVA P[13]strain and porcine RVA P[23]strain in this farm.Therefore,the human RVA G3P[13]strain was the porcine RVA G3P[13]strain backbone with the VP6 gene reassortment from human RVA strain.The results in this study showed the interspecies transmissionthe of porcine RVA G3P[13]strain to human from pig.Provided the first case with complete epidemiological link of interspecies transmission of RVA from pig to human,contributing to a greater understanding of the genetic evolution and interspecies transmission of porcine RVA.4.The interspecies transmission of RVA G3P[13]strain from pig to murineIn order to understand the genomic characteristics and the relationship between porcine RVA G3P[13]strains and murine RVA G3P[13]strain,11 complete gene segments of RVA/Murine-tc/CHN/SCLS-M/2018/G3P[13]was obtained successfully.TheconstellationofmurineRVAstrainwas G3-P[13]-I5-R1-C2-M2-A8-N1-T7-E2-H1.Phylogenetic analysis showed that the 7gene segments?VP7,VP4,VP6,NSP1,NSP2,NSP3 and NSP5?were most closest to theRVA/Piglet-tc/CHN/SCLS-X1/2018/G3P[13]and RVA/Pig-tc/CHN/SCLS-3/2018/G3P[13];VP1,VP2 and NSP4 genes most closest to bovine RVA strains;while VP3 gene most closest to caprine RVA strain.The result showed that the genome of murine RVA G3P[13]strain experienced a complex gene reassortment events.Remarkably,different from human RVA G3P[13]strain,recombination event in VP4 gene of the murine RVA strain was not found.The VP4gene of the murine RVA strain was originated from the porcine G3P[13]strain with no recombination event in VP4 gene in this farm.This result indicated that the porcine RVA G3P[13]strain has the potential of interspecies transmission to murine,and murine may play a role in transmission and genetic evolution of PoRVA.