Identification And Genotyping Of Canine Parvoviruses In Shanghai From 2014 To 2015

Canine parvovirus disease is caused by canine parvovirus,which is an acute contagious disease and one of the major disease in dogs which is infectious and high morbidity and mortality.Canine parvovirus has found since 1978,it is difficult to prevent and control due to its variation rate and expanding host range,which seriously affected the healthy development of economic animal breeding industry.First,we design a pair of specific primers according to the CPV VP2 sequence,and 30 strains of Shanghai and the surrounding areas were identified and genotyped.The results showed that New CPV-2a is 20 strains accounts for 67%,New CPV-2b is 7 strains accountsfor23% and CPV-2c is3 strain accounted for10%.It can be seen that the Shanghai area is mainly New CPV-2a and New CPV-2b genotypes,and aslo found CPV-2c genotype.Analyzing the VP2 gene sequences found that six nucleotide mutations result in amino acid changes,three strains of CPV-2c mutations occur at the same sites,seven strains of New CPV-2b mutations is also occur at the same sites.We can know that our country canine parvovirus isolates with foreign isolates belong to different branches from phylogenetic tree.Second,in order to explore the gennetic variation and molecular pathogenesis of three different genotypes of CPV,SH14 strain(New CPV-2a),SH1515 strain(New CPV-2b)and SH1516 strain(CPV-2c),the dog intestinal tissues were inoculated into CRFK cells for virus isolation and we used PCR and virology menthods to identify the virus,respectively.The results show that these isolates could grow on CRFK cells and produced cytopathic effect(CPE),including cell rounding,fall of and other stable cytopathic effect,and the specific genome of CPV could beamplified from these cell cultures.Indirect immunofluorescence and western blot experiments have proved that the VP2 protein was expressed in CRFK cells.We could see about CPV particles with the diameter of 20 nm under the electron microscope.Experiment animals inoculated with the isolates exhibited typical symptom of CPV,and CPV genome could be detected from feces after challenged 3 days and continued detoxification for a week.We further determinated the titre of these isolates in CRFK cells,the results showed that the TCID50 of SH14,SH1515 and SH1516 was 10-4.9/0.1mL,10-4.8/0.1mL and 10-4.6/0.1mL,respectively.In order to understand the growth characteristics of the three genotypes CPV,their one-step growth curves on the CRFK were measured,respectively.The results showed that the proliferation dynamics of the three genotype CPVs were similar in CRFK cells,for example,during 0-12 h,it was incubation period;at12h virus particles began releasing,during 36-60 h,it was the rapid proliferation of viruses;at 60 h the virus titers were the highest.Subsequently,virus titers began gradualy decline with cells disrupting and death.Lastly,according to the sequences of the referenced CPV strains from GenBank,we designed five pairs of specific primers for determining the full-length genome of three genotypes CPV.As expected,the whole genome of the three isolates were successfully sequenced the genome length of SH14,SH1515 and SH1516 were 5062 bp,5014bp and 5002 bp.Using DNASTAR software,we also analyzed the genetic relationship among the three CPV isolates and the reference sequences,the results showed that all of the three genotypes CPV have two coding frames: ORF1 and ORF2 which encode NS1,NS2,VP1,VP2 protein,respectively.Among of them,NS1 is 2007 bp,intermediate containing NS2 gene of 498 bp,encoding 699 and 166 amino acids respectively..ORF2 encodes VP1 which is 2184 bp and contains 1755 bp VP2 gene and encoding 728 and 585 amino acids,respectively.However,3’ and 5’ end of their sequences were differences among the three viruses,like other parvovirus,there were two non-coding regions and a Y-shaped hairpin structure flanked the end of CPV genome.Phylogenetic analysis results showed that the homology between SH14 strain(New CPV-2a)and CPV/CN/SD10/2014 strain is the highest(98.8%);the homology between SH1515 strain(New CPV-2b)and CPV/CN/JL6/2013 strain is highest(99.9%);the homology between SH1516 strain(CPV-2c)and CPV-LZ2(2b)strain is 99.2%.Based on the phylogenetic tree,we found that all of the CPVs used in this study could be divided into three branches(SI,SII and SIII),and most of Chinese Canine parvovirus isolates were located in the same branch,indicating that they have has a close genetic relationship.In summary,this study successfully isolated three genotypes CPV which were popular in Shanghai,both of their biology characteristics and whole genome sequences were determined and analyzed.Our results showed that the popular CPV of the Shanghai mainly were New CPV-2a and New CPV-2b.But we also the first time found CPV-2c in Shanghai and Hegfei area,suggesting CPV in the long-term evolution and higher immune pressure has mutated,it may be one of the important reasons of clinically CPV infection gradually increased and the incidence of immune dogs,we should pay more attention on CPV-2c.In a word,this study provided good foundation for further studying on the epidemilogy,pathogenesis of CPV and developing CPV vaccine.