Establishment Of Multiplex Fluorescent Quantitative RT-PCR Method For Canine Rotavirus And Its Isolation And Identification

Rotaviruses（RV）can infect multiple animal species and have the potential for cross reassortment based on the segmented genome characteristics,so as to realize cross-species transmission.There have been an increasing number of foreign studies on the Across-Species Spreading of RV between different animals or humans and dogs,while the domestic study is still underway.The risk of the Across-Species Spreading of RV in China raises with the increasing population of domestic dogs.Canine rotavirus（CRV）,canine distemper virus（CDV）and canine coronavirus（CCoV）are the main RNA viruses associated with canine diarrhea symptoms.In order to improve the sensitivity and convenience of clinical detection,a multiplex fluorescence quantitative RT-PCR method was established in this study.It can distinguish canine rotavirus,canine distemper virus and canine coronavirus.Simultaneously,the isolation and identification and genetic variation analysis of the CRV had been carried out.Then,the coding regions of 11 segments of the whole genome of the isolated strain were sequenced.Finally,the phylogenetic tree of each gene segment was constructed.Its genetic evolution relationship was analyzed,and the cross reassortment relationship between CRV and HRV or other species was studies.The main study results were as follows:1.The establishment of a multiplex fluorescence quantitative RT-PCR for CRV,CDV and CCoVPrimers and probes were designed for the conserved regions of the VP6 gene of CRV,the N gene of CDV and the M gene of CCoV.A multiplex fluorescence quantitative RT-PCR method was established to simultaneously detect these pathogens.After the factors of the reaction were verified and optimized,the strong specificity of the multiplex fluorescence quantitative RT-PCR was showed in results,which can distinguish and diagnose the infection of three viruses.The sensitivity was good within the detection limits of CRV,CDV and CCoV were 90.80,17.00 and 9.75 copies/μL,respectively.The repeatability was good within the group and the coefficient of variation of repeated trials between groups were all within 5.00%,which can be applied to clinical detection.2.Detection rate of CRV,CDV and CCoVWe had collected 338 samples from domestic dogs in 6 animal hospitals and from homeless stray dogs from October 2019 to May 2021 in Wuhan,Shiyan and Xiangyang,Hubei Province.CRV,CDV and CCoV were detected by the established multiplex fluorescence quantitative RT-PCR method.The results showed that the positive rates of CRV,CDV and CCoV were 20.10%（68/338）,26.00%（88/338）and 18.90%（64/338）respectively.In order to understand the infection,genotype and genetic characteristics of CRV in dogs,and the cross-reassortment relationship with the RV of human and other species,the isolation,identification and genetic evolution of CRV were further analyzed in this study.3.Isolation and identification of CRVFirst,20μg/m L of trypsin was added to the sample supernatant to activate the virus that was used to inoculate MA-104 cells.Then,4μg/m L of trypsin was added to maintain virus growth.A CRV was isolated successfully after 3 blind passages.RT-PCR and IFA was performed to identify the CRV.The CRV strain was named as RVA/Canine-tc/CHN/WH/2020/G3P[3].Obvious cytopathic phenomena such as cell aggregation,netting and abscission were observed after incubation 12 hours post infection.The isolated strain was passaged steadily in MA-104 cells and the viral titer reached 10^7.8 TCID₅₀/m L.4.Whole-genome CDS sequencing and reassortment analysis of CRVThe CDS sequence of the 11 gene segments of CRV were amplified with specific primers and sequenced.The phylogenetic tree was constructed for each gene segment.According to the RCWG,the genomic constellation of the isolated strain based on the 11genes could be described as G3-P[3]-I3-R3-C3-M3-A9-N2-T3-E3-H6,which belonged to an AU-1 like constellation with both AU-1-like and Cat 97-like gene segments.The results showed the closest phylogenic identity of VP7,VP6 and VP4 gene fragments were closely related to HRV（PB1048/Brazil）,HRV（12638/Japan）and RVA（B24-R3/China）from human sewage,respectively.It was suggested that there may be gene reassortment from the isolated strain between HRV and CRV.In summary,this study established a multiplex fluorescence quantitative RT-PCR detection method for CRV,CDV,and CCoV,which laid a foundation for epidemiological investigation and provided technical support for the diagnosis of canine diarrhea.This method was used to investigate the prevalence of canine diarrhea virus in some areas of Hubei Province.Then,a local CRV strain was successfully isolated.Genetic evolution analysis showed that it may be a reassortment strain of AU-1-like and Cat 97-like rotavirus.It was also suggested that RV had a risk of the Across-Species Spreading among human and dogs.