Molecular Evolution Characteristics And Pathogenicity Of Canine Parvovirus From Raccoon Dogs

In recent years,with the development of large-scale and intensive farming of raccoon breeding industry,the epidemic disease seriously hindered the healthy development of raccoon breeding industry and caused great economic losses.Canine Parvovirus(CPV)is one of the important pathogens that are harmful to raccoon dogs,and it is prevalent in raccoon dog populations,and its morbidity and mortality are increasing.Therefore,the molecular biological characteristics and pathogenicity of CPV from raccoon dogs were studied.In 2016,some disease materials were collected from three raccoon dogs farms with virus enteritis in Dongping.Three strains of parvovirus were isolated by conventional virus isolation methods,which were named as RDPV-DP1,RDPV-DP2 and RDPV-DP3.respectively.Molecular cloning techniques such as PCR were used to clone and sequence the VP2 gene and NS1 gene of 3 strains of parvovirus.The results showed that there were four amino acid site mutations on VP2 protein,namely S27 T,S297A,N375 D and V562 L.The genotyping of canine parvovirus was determined by several key amino acid sites of VP2 protein.Cloning and sequencing showed that 80 R,93N,103 A,323N,564 S and 568 G of VP2 protein were identical to the corresponding amino acids of CPV-2,so they were identified as CPV-2 type.The 297 site on VP2 mutated from serine to alanine acid,is consistent with the worldwide prevalence of CPV-2a /2b.The 375 site on VP2 mutated from asparagine to aspartic acid,is consistent with the worldwide prevalence of CPV-2a/2b/2c.The mutation of site 562 from valine to leucine is close to sites 564 and 568,which have certain influence on host range.It is worth further study that the mutation from serine to threonine occurred at 27 sites on the VP2 gene of the three strains of parvovirus,which was first discovered in parvovirus.The biological significance of the mutation is still unclear.The VP2 gene sequences of the three parvoviruses isolated were aligned by DNAStar software.The homology of the three parvoviruses at the nucleotide level was 99.9%,and that of the related reference strains at the nucleotide level was 99.1%-99.5%.The amino acidhomology of the VP2 gene of the three parvovirus strains was 100%,and that of the related reference strains was 98.6%-99.5%.From the phylogenetic analysis of the VP2 gene,These 3strains of parvovirus are in the same branch with CPV-2 from 7 raccoons of China and 1 from Chinese fox,and they are not in the same branch as the vaccine strains,indicating that the 3strains of parvovirus are most closely related to CPV-2,which further proves that the 3 strains of parvovirus belong to CPV-2 type.Meanwhile,the comparison of NS1 gene sequences showed that the homology of the three strains of parvovirus at the nucleotide level was99.3%-99.8%,and that of the related reference strains at the nucleotide level was98.9%-100%.The amino acid homology of NS1 gene of 3 strains of parvovirus was99.6%-99.9%,and that of related reference strains was 99.0%-100%.From the phylogenetic analysis of NS1,the three isolated parvoviruses were in the same branch as CPV-2a / 2b /2c.To verify the virulence of the isolated strains,the raccoon dogs were selected as the experimental animal to inoculate RDPV-DP1 by gavage method for the pathogenicity experiment.4-9 days after the challenge,the experimental animals showed loss of appetite or even abolishment,vomiting,depression,lethargy,and diarrhea.The feces became watery and soft at the initial stage,and after that,the feces presented blood stools of coffee color or tomato color,and the blood had a special fishy odor,After the blood was discharged,the experimental animals showed severe dehydration symptoms,the eyeball subsided,the nose was dry,and the skin elasticity decreased significantly.The weight is significantly reduced.The morbidity rate of the inoculated animals was 100%.Virus shedding from the inoculated animals was confirmed by PCR from on days 1 to 12 p.i.Histologic lesions were found in the challenged raccoon dogs euthanized on days 5 and 7 p.i.,including lymphocytopenia in spleen,lymphocytopenia and reticulocytosis in mesenteric lymph nodes,intestinal vilus breakage and inflammatory cell infiltration of the intestinal lamina propria.Except for the euthanized raccoon dogs,all the other challenged raccoon dogs were recovered from the disease.The control animals showed no clinic signs,virus shedding and seroconversion.This study showed that although the three isolated dog parvovirus strains from raccoon dogs were of CPV-2 type,they showed mutations at four amino acid sites on VP2 protein,which was a new type of CPV-2 strain.The pathogenic experiment shows that the virus is highly pathogenic to raccoon dogs and can cause 100% of the disease.It belongs to the strong strainand is not a vaccine strain.This research enriches the evolution data of CPV,which has important theoretical research significance,and provides a theoretical basis for the prevention and control of CPV from raccoon dogs.