Molecular Detection And Genomic Characteristics Of Canine Coronavirus In Some Areas Of Sichuan

Canine enteric Coronavirus(CCo V)is a single stranded RNA virus with capsule envelope.CCo V is a member of the order Nidovirales,Alphacoronavirus.CCo V is an important pathogen causing viral enteritis in dogs of all ages.CCo V S gene sequence can be divided into different genotypes: CCOV-IIA,CCOV-IIB and CCOV-I.There are many epidemiological investigations on CCo V,but molecular epidemiological data of Sichuan province has not been reported in detail.In this study,CCo V was detected in feces samples of dogs with diarrheal disease from some areas of Sichuan Province,and its genomic characteristics were studied.The results are as follows:1.Molecular epidemiological investigation of CCo V in Sichuan ProvinceIn order to understand the infection of CCo V in some areas of Sichuan Province,a total of 216 stool samples from three areas of Sichuan province(Chengdu,Xichang and Luzhou)from 2019 to 2021 were detected by RT-PCR method reported in literature,and the positive samples were classified.The results showed that 44 samples were positive for CCo V in diarrhea feces,with an average detection rate of 20.4%(44/216).The r T-PCR method for CCo V typing reported in literature showed that the average detection rate of CCOV-IIA,CCOV-IIB and CCOV-I was 41.5%(17/41),34.1%(14/41)and 24.4%(10/41)respectively.Three samples were c COV-IIA and CCOV-IIB mixed infection 7.3%(3/41),three samples were CCOV-I and CCOV-IIA mixed infection 7.3%(3/41),and one sample was CCOV-I and CCOV-IIB mixed infection 2.4%(1/41).The results of this study indicate that CCo V is an important pathogen of canine diarrhea in some areas of Sichuan Province,among which CCOV-I,CCOV-IIA and CCOV-IIB exist simultaneously and different genotypes are infected simultaneously.The results of this study provide reference for the prevention and control of CCo V.2.Genomic characterization of a c COV-IIA strainUsing the sequence of CCOV-IIA Chinese strain B135 JS 2018(MT114544.1)in Gen Bank as a template,22 pairs of amplification primers were designed with Premier 5.0software and Snap Gene software.The amplified sequence fragments were sequentially spliced to obtain the approximately full-length CCOV-IIA genome sequence(28943bp).Megalign and MEGA 7.0 software were used to analyze its similarity and genetic evolution,and the results showed that,The similarity between CCo V/8/China and the Chinese strain B135 JS(MT114544.1)was 78.9%-99.7%,and the homology between CCo V/8/China and The Chinese strain B135 JS(MT114544.1)was 99.4%.The genome obtained in this study was in the same large branch as that of seven Chinese strains including B447 ZJ,and these strains were all strains from after 2018 and formed a new branch.Compared with the complete genome published in Gen Bank,the genetic evolution trend of Chinese strains before and after 2018 was different.There was also a certain genetic distance between c COV-IIA and other foreign c COV-IIA strains,indicating that CCo V strains in China showed a unique trend of genetic evolution in recent years.In this study,114 unique nucleotide sites of CCo V/8/China were changed compared with all the complete 1AB gene sequences of CCOV-IIA,resulting in 51 unique amino acid sites,including 45 amino acid sites on the 1A gene and 6 amino acid sites on the 1B gene.At present,the functions and structures of other non-structural proteins have not been confirmed,and the influence of changes of these sites on the structure and function of the virus is still unclear,which needs further study to confirm.Compared with all 69 CCo V complete S gene sequences,the genome sequences obtained in this study have unique mutations at nucleotide and amino acid sites.A total of10 unique amino acid site variations were caused(QPT169M,A191 V,G451V,A494 P,M582T,D717 E,AGD1055N,SAV1215 L,E1216R,V1217E).Three of them(QPT169M,M582 T,D717E)had the same amino acid site mutation as FCo V.Seven amino acid sites were located in S1 region(QPT169M,A191 V,G451V,A494 P,M582T,D717 E,AGD1055N),and three in S2 region(SAV1215L,E1216 R,V1217E).In addition,the S2 cleavage site of this strain is consistent with that of FCo V,but whether the changes of S1/S2 and S2 ’cleavage sites lead to changes in the pathogenicity of CCo V has not been reported.