RT-PCR Detection And Genomic Characteristics Of Canine Norovirus

Canine norovirus(CNV)belongs to the family Caliciviridae,a member of the genus Norovirus.It is a single-stranded positive-sense RNA virus,which is generally considered to be one of the pathogens that cause canine diarrhea.Currently,CNV strains have been detected in at least 9 countries with a wide geographical distribution,such as Italy,South Korea,the United States,etc.In China,it has only been reported in Hong Kong.CNV,with genetic diversity,can be divided into four genotypes,GIV.2,GVI.1,GVI.2 and GVII based on VP1 gene sequences.To date,there have been no reports of the CNV in mainland of China,and its prevalence and molecular characteristics are not clear.In this study,CNV detection was performed on fecal samples from dogs with diarrhoeal disease from pet hospitals of three provinces and regions in China,and genomic characteristics were studied.The results achieved are as follows:1.The RT-PCR assay for detecting CNV was established successfullyIn this study,three abroad RT-PCR assays for detecting CNV reported were used to detect canine diarrhea fecal samples,but the results were not detected or the detection rate was extremely low.Analysis showed that the nucleotide of the detection target of the existing detection methods had changed.This study aims to establish a more sensitive RT-PCR assay for the detection of CNV.With fully analyzing the commonly used molecular detection target genes of CNV,RdRp gene was selected as the target gene,primers were designed,an RT-PCR assay was established and the reaction system and conditions was optimized.This assay only detected CNV,but could not detect other common canine diarrhea pathogens,with strong specificity;the detection limit of CNV plasmid standard was 4.64×10~3copies/?L with high sensitivity;the inter-batch and intra-batch tests showed that the assay were reproducible.The comparative test showed that the detection rate of CNV in diarrhea samples by the assay from this study was higher than that of the three RT-PCR assays reported abroad.In conclusion,the RT-PCR established in this study provides a more reliable means for the detection and epidemiological investigation of CNV.2.CNV was first proved to exist in dogs in mainland of China with rich genetic diversityCanine diarrhea is a common disease in China.CNV is a diarrhea-causing pathogen in dogs,however,information regard on CNV has not been reported in mainland of China.The purpose of this study was to investigate the prevalence and molecular characteristics of CNV in some provinces in China.In this study,268canine diarrhea fecal samples collected from Sichuan,Chongqing and Henan provinces were detected by RT-PCR assay.The results showed that the detection rate of CNV was 7.8%(21/268),which firstly demonstrated that the presence of CNV in mainland of China.The detection rates of CNV in the samples from Sichuan,Chongqing and Henan were 8.21%(17/207),5%(2/40)and 9.52%(2/21),respectively.Phylogenetic analysis of twenty-one CNV RdRp fragments showed that eighteen of the strains clustered in GVI.2,two clustered in GVI.1 and one clustered in GIV.2,indicating that CNV prevalent in mainland of China has genetic diversity.Analysis the geographic distribution of eighteen CNV GVI.2 positive samples found that these samples were from three provinces,indicating that CNV GVI.2 may be widespread in dogs in China,which is worthy of further investigation and research.All of the CNV-positive diarrhea samples showed co-infection with CDV,CPV,and/or CCo V.The results of this study proved that CNV is common in dogs in mainland of China,and the prevalent CNV strains have unique evolutionary characteristics,which provides an important reference for the prevention and control of canine diarrhea in China.3.Complete genomes of GIV.2,GVI.1 and GVI.2 of CNV were obtained for the first timeIn order to further understand the genome characteristics of the current CNV epidemic strains in China.In this study,based on the detection results of CNV,three sets of primers were designed based on the different genotypes of CNV to amplify the genomes of 21 positive CNV samples,respectively.The complete genomes of strains Dog/C19/19/CH(GIV.2),Dog/Z7/19/CH(GVI.1)and Dog/M9/19/CH(GVI.2)were successfully sequenced,with the length of 7865 bp,7956 bp and 7905 bp,respectively.The 5'-UTR nucleotides of the three strains was identity,and their complete genomes shared 70.8%–76.7%nucleotide identity with each other and shared 57.4%–95.9%nucleotide identity with the known 10 CNV genomes(including 8 nearly full-length genomes).Phylogenetic analysis showed that the three CNV complete genome sequences obtained in this study were clustered in three different branches based on different genotypes.In addition,the complete RdRp,VP1 and VP2 gene sequences of three GVI.2 strains from Sichuan,Chongqing and Henan were obtained in this study.Genetic evolution analysis showed that their molecular characteristics were basically the same without obvious regional differences,indicating that the prevalent CNV strains in mainland of China conserved and had unique co-evolutionary characteristics.Sequence analysis of RdRp,VP1 and VP2 protein showed that strain Dog/C19/19/CH had 5,3 and 7 amino acid mutations on RdRp,VP1 and VP2,respectively;Strain Dog/Z7/19/CH had 5,5 and 8 amino acid mutations on RdRp,VP1 and VP2,respectively;Compared to other GVI.2 strains,four GVI.2 strains(including Dog/M9/19/CH)and the USA GVI.2 strains shared 18 and 12 identical amino acid mutations on VP1 and VP2,respectively.Moreover,the study first reported the complete genomes of GIV.2,GVI.1 and GVI.2 of CNV,and obtained the complete RdRp,VP1 and VP2 sequences of three GVI.2 strains,contributing to understand the molecular characteristics and genetic evolution of CNV.