| As the second largest population of companion animals next only to dogs,cats often share the same living environment with humans,and have frequent contact with humans,domestic animals and poultry.Viral pandemic transmitted by cats show a high incidence trend,and also gradually attract people's attentions.In recent years,with the continuous development of viral metagenomics,the researches on viral communities in wild animals,livestock and poultry had been developed in succession,which not only enriches the genetic diversity of the known viruses,but also greatly expands people' understanding of the unknown viruses.However,the research on viral communities in companion animals started late and the development is not fast enough.At present,there is no systematic study on the viral community in domestic cats in China.Therefore,it is necessary to systematically study the viral community in domestic cats in China,to understand the virus types carried by cats and to master the variation and evolution of the feline viruses.The thorough surveillance on the virus carrying situation in domestic cats is very important for the early warning of the potential feline viral pandemic.In this study,a total of 578 fecal samples were collected from domestic cats from 11 cities in Liaoning,Jilin and Heilongjiang province between 2016 and 2018.We analyzed the composition of fecal virus community in domestic cats by viral metagenomics,and compared the difference on fecal virus community between health and diarrheic cats in the present study.At the same time,the prevalence of the significant carnivores protoparvovirus,feline bocaparvovirus,feline astrovirus and feline kobuvirus was investigated by developing the specific detection methods,and the characteristics of genetic evolution,epidemiology and genome for these viruses were systematically analyzed by cloning the main gene and the complete genome.Furthermore,we first investigated the heterogenous canine bocaparvovirus and gyrovirus in feline feces and analyzed the genetic characteristics of the two viruses.The main results were as follows:(1)Viral metagenomic analyses showed that a total of 13,130,914 contigs were generated and 41.25% contigs were related to viruses from 47 genera in the 25 families.These viral-like contigs can be further classified into vertebrate viruses(25 genera in the 17 families),protozoan viruses(2 genera in one family),phages(13 genera in the 4 families),plant viruses(3 genera in the 3 families)and insect viruses(4 genera in the 3 families).Many newly discovered viruses,such as feline bocaparvovirus,feline kobuvirus,feline astrovirus,canine bocaparvovirus and gyrovirus,were found by viral metagenomic.Furthermore,the viruses belonged to the family Parvoviridae,Astroviridae,Picornaviridae,Coronaviridae and Reoviridae were supposed to the main pathogens causing diarrhea in cats by comparing the difference on fecal virus community between health and diarrheic cats.(2)A PCR-RFLP method for the differential diagnosis of feline parvovirus(FPV)and canine parvovirus(CPV)was successfully developed in this study.The investigation results showed that the nucleotide positive rate of carnivores protoparvovirus was 34.08%(197/578),and a higher positive rate was observed in cats with diarrhea than in health cats.The age and the numbers of cats in the home were estimated to be risk factors,respectively,associated with the nucleotide positive rate of carnivores protoparvovirus.We found that FPV is the preponderant carnivores protoparvovirus in cat population in China,and first provided the molecular evidence of the circulation of CPV-2a and CPV-2b in domestic cats in northeast China.Phylogenetic analysis based on the VP2 gene showed that FPV strains circulated in China had formed a new evolutionary branch,which was the positive selection for the virus evolution.The unique evolution should be highly valued.Moreover,the amino acid variation at the residues 267,324 and 440 may be related to the host specificity of CPV.(3)A multiple polymerase chain reaction(PCR)method for the differential diagnosis of feline bocaparvovirus 1(FBoV1),FBoV2 and FBoV3 was successfully developed in the present study.The investigation results showed that the nucleotide positive rate of FBoV was 22.32%(129/578),and the cats with diarrhea had a higher positive rate than health cats.FBoV1,FBoV2 and FBoV3 are circulated in cat population in China,and FBoV1 is the dominant species.This study first report the circulation of FBoV2 and FBoV3 in China,and found that FBoV3 circulated in China belonged to the new genotype.Furthermore,a stable passage FBoV1 strain was first isolated in the present study.(4)A Taq Man fluorescence quantitative PCR for the specific detection of feline astrovirus(FeAstV)was successfully developed in this study.The investigation results showed that the nucleotide positive rate of FeAstV was 18.17%(105/578),and the prevalence of FeAstV in cats with diarrhea was higher than that in health cats.The season was estimated to be risk factors,associated with the nucleotide positive rate of FeAstV.We first reported the circulation of FeAstV-1 and FeAstV-2 in cat population in China,and FeAstV-1 was the dominant genotype.The prevalence of FeAstV-2 increased year by year,showing a trend of expansion.Genetic analysis showed that FeAstV-1 and FeAstV-2 should be classified into the different species,and FeAstV-1 and FeAstV-2 strains circulated in China had a unique evolutionary trend.(5)A Taq Man fluorescence quantitative PCR for the specific detection of feline kobuvirus(FeKoV)was successfully developed in the present study.The investigation results showed that the nucleotide positive rate of FeKoV was 12.98%,and cats with diarrhea had a higher positive rate than health cats.We first provide the molecular evidence of the circulation of FeKoV in domestic cats in northeast China.Genetic analysis showed FeKoV strains circulated in China formed a unique evolutionary branch.(6)Ten fecal samples were tested to be positive for canine bocaparvovirus(CBoV),12 were positive for gyrovirus(GyV)in 578 feline fecal samples.The analytic results showed that ten CBoV-positive samples belonged to the new genotype of CBoV1.Out of 12 gyrovirus-positive samples,4 were positive for CAV,3 were HGyV/AGV2,3 were GyV3 and 2 were GyV6.We first provide the molecular evidence of the circulation of CBoV1,HGyV/AGV2,GyV3 and GyV6 in domestic cats,suggesting that cats may act as the reservoir for canine bocaparvovirus and various gyroviruses.In conclusion,we investigate the fecal virus community in domestic cats in northeast China using viral metagenomics.The investigation results not only enriched the feline virus database,but also systematically provided the virome database in cats in China.We also found a lot of newly discovered viruses in this study,providing the basic data for the early warning of the potential feline viral pandemic.We developed the specific testing assays for carnivores protoparvovirus,FBoV,FeAstV and FeKoV,and carried out the systemic investigation of these viruses in northeast China in the present study.We first provide the molecular evidence of the circulation of FBoV2,FBoV3,FeAstV-1 and FeAstV-2 in domestic cats in China,and provide the first molecular evidence of CBoV,HGyV/AGV2,GyV3 and GyV6 in cats worldwide,providing the important basic data for the comprehensive understanding of the distribution of the newly discovered feline virus.The scientific phylogenetic analysis also provide the base data for the molecular epidemiological study of the newly discovered feline virus. |
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