Isolation, Identification Of Porcine Parvovirus And Phylogeny Analysis Of The NS1and VP2Gene Sequences

Porcine parvovirus (PPV) can cause serious reproductive diseases of swine, The typical clinical signs are embryonic death, mummification, stillbirths and delayed return to oestrus. Besides that, PPV has also been linked to skin lesion and dermatitis occurrence in piglets. PPV is popular in many countries, can affect different age of pigs, the reproductive diseases caused by PPV cause a huge economic losses. This article established a PCR detection method for PPV detection, the positive disease materials were used to isolate PPV by the method of cell culture. Then the biological features of the isolates were studied, the NS1and VP2sequences were sequenced to estimate the evoluation relationship. Specific content as follows:1、Establish PCR detection method and applicationSelect NS1gene sequence as the target gene. We established a PCR detection method of PPV. The sensitivity test results shown that the PCR detection method can detect1.58pg PPV genomic DNA of PPV. From June2010to September2011, we used this PCR method detected312samples which were suspected PPV infection, the results shown only34samples were positive in312samples, the detection rate is10.9%.2. PPV isolation and biological characteristics analysisThe positive samples were cultivated by ST cells, finally eight PPV strains were isolated from different samples. The hemagglutination titer of isolates can reach1:512, and the value of TCID50is about10-5.60/0.1mL. The results of PD50shown that the strain JB, ZD, JZ has the similar antigenicity, DS and ZM has the similar antigenicity, but the antigenicity of the strains GX, SX, HL have a big differences with other isolates.3. Sequencing and analysis of the NS1and VP2gene sequencesThe NS1gene and VP2gene sequences of eight PPV isolates were sequenced, The results of homology analysises shown:the NS1and VP2gene sequences have a high homology between different isolates. Multiple sequence alignment of the PPV NSl and VP2amino acid sequences shown that:several sites are variable, after gene selection pressure analysis, we find that many sites of VP2gene under positive selection pressure, which perhaps are all important for PPV adapting to the change of environment.4. Molecular Evolutionary Genetics Analysis of the PPVsequencesTogether with other date of NSl and VP2gene sequences from the GenBank, phylogenetic trees of NS1and VP2gene deduced amino acid sequences were constructed by N-J method of MEGA5.0separately. The results shown:PPV sequences of China can be divided into four subgroups. Strains SX, JZ, JB, ZD, GX belong to subgroup1, JB and ZD has a near evolutionary distance, SX and JZ has a near evolutionary distance, GX has a far evolutionary distance with other isolates. Strains DS and ZM belong to subgroup2. Another isolate HL belongs to the German subgroup in the VP2tree, perhaps is a new emerging genotype in China.