Canine Parvovirus Virus Isolation And Identification And Phylogenetic Analysis

Canine parvovirus (CPV) is a small, non-enveloped virus, containing a single-stranded DNA, belonging to the genus Parvovirus within the family Parvoviridae. CPV is responsible for acute gastroenteritis and leucopenia in puppies, which were first identified in 1978 in many European countries. CPV spread rapidly around the world, causing a virgin-soil pandemic that was marked by high incidence rates and high mortality rates. Retrospective serologic studies have indicated that the apparent immediate ancestor of the virus began infecting dogs in Europe during the early or mid-1970s; this conclusion is based on the finding of antibodies in sera from dogs in Greece, Netherlands, and Belgium in 1974, 1976, and 1977, respectively. The stability of the virus, its efficient fecal-oral transmission, and the near-universal susceptibility of the dog population of the world accounted for this pandemic.In this study, CPC isolation, identification and phylogenetic analysis were conducted. 19 samples were collected since 2009 in Changchun city, among which 17 samples were CPV positive tested by PCR method. The complete VP2 genes of the CPV-positive samples were amplified and sequenced, in order to analysis the epidemic situation of CPV and mutation of VP2 gene of CPV.9 strains of CPV were isolated from the CPV-positive samples, using F81 cell line. Through electron microscopy, hemagglutination assay and PCR method, the CPVs isolated were confirmed. The isolated viruses can multiple in F81 cell line and cause typical CPE, 20-25nm in diameter and have icosahedral symmetry when observed using electron microscopy. The viruses were also confirmed using: direct hemagglutination of swine red blood cells, and polymerase chain reaction assay for the detection of viral DNA.Genetics analysis was conducted, based on the VP2 genes of the CPVs isolation in this study and the CPVs isolated in our laboratory before. Sequences analysis indicated that many site-mutations were presented in the strains isolated in China, and the main predominant antigenic-type CPV is CPV type 2a, while CPV-2b and CPV-2c were also detected in this study. This is the first detection of CPV-2c in China, which has spread around the world. VP2 genes of most of the strains presented some site-mutations, most of which were synonymous-mutation. Among the 17 VP2 genes sequenced, all the deduced VP2 protein presented the Y324I mutation, except for 03/09, 07/09 and 17/09 samples. It has been reported that residue at 324 of VP2 protein is subject to strong positive selection in all parvoviruses of carnivores. Most of the CPVs isolated after 2004 in China contained this mutation.