A New Approach For Porcine Circovirus Type 2 Isolation And The Full Length Genome Analysis Of Guangdong Trains

Porcine circovirus type 2 (PCV2) could cause PMWS (Post-weaning pigs multisystemic wasting syndrome),PDNS(Porcine dermatitis and nephropathy syndrome),PNP(Proliferative and necrotizing pneumonia), PRDC( Porcine respiratory disease complex) and CT(congenital tremors) and reproductive disturbance. In 1991, PMWS was first reported in Canada.From then on, PCV2 had widely spread in the world. PCV2 was first found in 2000 by Lang Hongwu , from then on , more and more reports certificated that PCV2 has widely infection in our country and has caused great economic lose in the pig industry. PCV2 are usually mixed infection with other pathogens in clinic, such as PRRSV (Porcine reproductive and respiratory syndrome virus), PRV (Pseudorabies virus), PPV (Porcine Parvovirus), PCV1 and CSFV (Classical swine fever virus); and PCV2, PCV1, CSFV and PPV virus could not show siginifican cytopathic effect (CPE), which bringing some difficulty to virus isolation, Swine hyperpyrexia epidemic situation in 2006, not only PRRSV, but alsoPCV2, PPV, PRV and CSFV mixed infection cases had been found. PK-15cell serial subcultivations usually use to PCV2 isolation and purification, and identify the effect through specific detection of specific virus. There are not better ways to remove other unknown virus. Thus through identifying PCV2 virus in highly pathogenic porcine reproductive and respiratory syndrome cases, this study using infection of PCV2 whole gene establishes a set of isolation ways of PCV2 virus. The most research works were as following:1. According to porcine circovirus type2 (PCV2) gene sequence in GenBank, chose only site on the genome and designed a pair of primer. 29 highly pathogenic porcine reproductive and respiratory syndrome cases which collecting on 4 areas in Guangdong from May to November 2008 amplified by PCR, 4 cases were found to be positive and cloned them to pMD18-T carrier, screened out positive recombinant plasmids pMD-PCV2 and sequenced, and compared and analyzed between sequencing results and reference virus strains having been reported. The results show that: 4 PCV2 whole gene are all 1767 bp, and are named GDYFJ,GDSHJ,GDZQJ,GDBLJ. Homology between the 4 gene strains and other isolated strains in our country has big difference, and has not obvious orderliness; but has near relationship with 3 virus strains from Guangdong and Guangxi province. So, we believe that PCV2 has not variation, and the factors of the epidemic situation are more pigs'immunosuppression which arising by PCV2.2. Prepared abundant pMD-PCV2 positive plasmids, picked out PCV2 whole genome by Sacâ…¡, and then DNA was cyclized with T4-ligase. Furtherly the full length DNA was used to transfect the PK-15, and copied whole virus by the infection of the whole genome. Through IFA and PCR proved that transfection cells have plenty of viral particle which having duplication activity. Picked out cells which having higher virus by fluorescent quantitative PCR, replicated virus to improve virus content and titer, and isolated pure PCV2 virus strain. All the work established bases for studying on PCV2 infection, virus immunity and pathogenic mechanism, and molecular epidemiology.