Isolation And Identification Of Porcine Rotavirus G4P[6] Strain And The Establishment And Application Of Novel Diagnostic Methods

Porcine Rotavirus?PoRV?is a common cause of diarrhea and gastroenteritis in suckling piglets and weanling piglets,which can result in diarrhea,vomiting,weight loss and dehydration,even death.PoRV is highly prevalent throughout the world,causing huge economic losses to the pig farms.Due to the frequent occurrence of genetic reassortment among rotaviruses,reassortants containing gene?s?from porcine or human rotaviruses can be detected in human and porcine populations,respectively.PoRV has become a potential public health problem and needs further investigation.To investigate the prevalence of PoRV in China,a total of 2240 serum samples were collected from 45 pig farms in 26 provinces in 2016 and detected for PoRV,transmissible gastroenteritis virus?TGEV?and porcine epidemic diarrhea virus?PEDV?by the ELISA Kits;a total of 191 feces or intestinal contents samples of suspected viral diarrhea were collected from 37 pig farms in 16 provinces in 2016 and detected for PoRV,TGEV and PEDV by the RT-qPCR.Serological examination showed that the positive rates of PoRV,TGEV and PEDV for the pigs were 90.85%,22.99%and 63.10%,respectively;the positive rates of PoRV,TGEV and PEDV for the pig farms were 100%?82.14%and 89.29%,respectively.The co-infections of PoRV+TEGV,PoRV+PEDV,PEDV+TEGV and PoRV+TEGV+PEDV were common.Pathogen detection results showed that the positive ratesof PoRV,TGEV and PEDV for the pigs were 13.09%,0.52%and 59.16%,respectively;the positive rates of PoRV,TGEV and PEDV for the pig farms were 32.43%,2.70%and 70.27%,respectively.The co-infections of PoRV and PEDV were common and serious.The results indicated that PoRV and PEDV were the main pathogens causing the pig diarrhea in China.A strain of PoRV was isolated from clinical samples.The identification and sequence analysis of the virus was performed,the virus was classified as group A PoRV,the genotypes of VP6,VP7 and VP4 were belonged to 15,G4 and P[6],respectively.The virus was designated RVA/Pig/CHN/BJ/2015/G4P[6].Through homology analysis,we found that VP7 and VP4 genes of RVA/Pig/CHN/BJ/2015/G4P[6]are high homologous to human strain R1954 from Chinaand porcine strain 14226₃9 from Vietnam,the homology of VP7 was 96%and 95%,respectively,the homology of VP4 was 95%and 93%,respectively.Taqman MGB Reverse transcription quantitative real-time PCR?RT-qPCR?and Reverse transcription droplet digital PCR?RT-ddPCR?methods were developed for the detection of PoRV.A pair of primers and FAM-labeled TaqMan MGB probe was designed based on the conserved region of NSP3 gene of PoRV.The primers were screened and and the reaction conditions were optimized in RT-qPCR and RT-ddPCR.RT-qPCR results showed that the R2 value of the standard curve was 0.999,the amplification efficiency was 96.498%,the detective limit was 17.5 copies/?L.RT-ddPCR results showed that the R2 value of the standard curve was 0.999,the detective limit was 3.2 copies/?L.The results form detection of 126 clinical samples showed that the sensitivity of RT-ddPCR was higher than RT-qPCR,and the sensitivity of RT-qPCR was higher than RT-PCR.In conclusion,the RT-qPCR and RT-ddPCR methods both have good sensitivity and specificity.To establish the serum diagnosis method of PoRV infection,an indirect ELISA was developed for the detection of PoRV IgA antibody with the VP6 protein of PoRV as coating antigen expressed in the baculovirus expression system.After optimization,125 ng/well of VP6 protein was used to coat the plate,the dilution of clinical serum and HRP Goat anti pig IgA conjugate were 1:100 and 1:20000,respectively.121 clinical serum samples were tested,the sensitivity was 90.5%,the specificity was 86.2%,the positive coincidence rate was 88.4%.These results indicate that the indirect ELISA is a sensitive and specific method for detecting IgA antibodies against PoRV.In summary,this study investigated the prevalence of PoRV in China,isolated a PoRV G4P[6]strain,developed TaqMan MGB RT-qPCR and RT-ddPCR methods for pathogen detection of PoRV,and developed an indirect ELISA method for detection of IgA antibody against PoRV,provided useful technique for detecting PoRV infections and effective prevention and control of PoRV in the pig farms in China.