Isolation, Identification And Study Of Biological Characteristics For Porcine Circovirus Type 2 In Henan

Porcine circovirus (PCV) is a small animal virus with a single-stranded circular DNA genome, which is icosahedral and nonenveloped. PCV contains PCV1 and PCV2, PCV1 was first discovered by Tischer in porcine kidney cell culture PK-15, it is 1759 nucleotides, non-pathogenic, but exists in pigs and porcine cell line widely. However, PCV2 genome consists of 1767 or 1768 nucleotides, which has been associated with postweaning multisystemic wasting syndrome(PMWS). There are two open reading frames (ORF) in PCV2: ORF1 and ORF2. ORF2 codes for structural protein, it is an important sequence to distinguish PCV1 and PCV2. PCV2 was first identified in China in 2000, and the virus have high infection rate in our country's pig farms. The average positive rate of pigs is generally greater than 20%, some more than 50% and even 100%. It can be said that the virus has been prevalent in China at present, and causes significant economic losses every year. China is a country with great quantity of pigs, so there has significance on the study of this disease and its pathogeny.According to the published gene sequences of PCV2 ORF2 in Genbank, a pair of specific primers was designed and systhesized, PCV2 gene was amplified from cases which collected from different areas in Henan, such as Zhengzhou, Linying and Nanyang. Positive cases were treated and inoculated in PK-15, there are 9 strains were isolated. They were detected after blind passaged in PK-15 for 10 generations, and PCV1, PPV, PRV and PRRSV were all negative. Identified by IPMA, specific red-brown reaction was showed up in infected cells.A pair of primers from PCV2 complete sequence was systhesized, and 4 virus strains were amplified, then cloned and sequenced. The 4 strains were named ZZ-PCV2,WZ-PCV2,LY-PCV2 and NY-PCV2. The result showed that NY-PCV2 were 1768 bp, and the others were 1767 bp. The 4 strains and PCV2 complete sequence at home and abroad in GenBank were aligned, it was showed that LY-PCV2 and NY-PCV2 have the nucleotide homology of 98.9%, ZZ-PCV2 and WZ-PCV2 was 99.7%. The 4 strains nucleotide homology were higher than 93% according to other PCV2, while PCV2 ORF2 nucleotide and deduced amino acid homology were higher than 89%. The 4 strains all have specific sequence as TCA/AAC/CCC/CG(T)C in 1479 bp~1468 bp, they all belong to PCV2b.In order to select strains with high titer and cell lines suitable for viruses, PK-15 was cloned and different viruses were inoculated in different cell lines respectively, strains and cell lines were selected by real-time PCR and IFA. LY-PCV2 strain proliferated in McC6 cell line, the TCID50 was 105.1 per milliliter.This study isolated 4 PCV2 strains, and analyzed their homology according to PCV2 strains at home and abroad. These assays established basis for its further study of biological characteristics.