Epidemiologic Investigation Of Mixed Infection By PRRSV And PCV2

Porcine Reproductive and Respiratory Syndromevirus(PRRSV)and Porcine Circovirus type 2(PCV2)are the causative agent of postweaning multisystemic wasting syndrome(PMWS)that has become an emerging and important disease in swine.The PCV2 co-infection increases the severity of PRRSV-induced interstitial pneumonia in pigs and induces the lymphoid depletion, granulomatous inflammation, and necrotizing hepatitis characteristic of PMWS. According to epidemiological survey, the disease occurs in China, and has a prevalent tendency in recent years.The samples of sick pigs were collected and detected by multiplex PCR, which has been constructed through PRRSV and PCV2, the result showed the mixed infection of PRRSV and PCV2 was quite serious. The NSP2 of PRRSV and whole gene of PCV2 were cloned, sequenced and analyzed to find out the genetic relationship and evolution regularity between this prevalent strain and other strains, to make some basic researches for the epidemiological investigation and pathogenic mechanism of PRRSV and PCV2. The contents as follows:1.Establishment and application of a multiplex PCR assay for simultaneous detection of PCV2 and PRRSVAccording to the gene sequences of PCV2 and PRRSV in GenBank, two pairs of specific primers were designed for amplifying the two specific fragments of PCV2 ORF2 and PRRSV NSP2 respectively. After optimization of annealing temperature and primers concent rations, multiplex PCR (mPCR) was established for simultaneous detection of the two viruses. The results showed that the mPCR was highly sensitive in that as little as 1 pg of the respective virus, 1.66pg(PCV2), 1.6 pg ( PRRSV) respectively. No band was amplified from classical swine fever virus (CSFV), porcine parvovirus (PPV), Japanese encephalitis virus(JEV), and salmonella. 67 samples from sick pigs were detected using multiple PCR , 49 were positive for PRRSV and 37 were positive for PCV2 , 31 were mixed infection with PRRSV and PCV2, the rate of mixed infection was 46.27%, it is very serious among the pigs with mixed infection of PRRSV and PCV2. 2.Molecular epidemiology investigation of mixed infection of PRRSV and PCV2We have cloned, sequenced, and analyzed partial NSP2 of PRRSV and whole genes of PCV2 by PCR amplification, and identified the genetic relationship and evolution regularity between this strain and others. The sequencing analysis of partial NSP2 of PRRSV showed that the JL08(187) has the homology over 97% blasted with domestic prevalent strains such as JAX1, YN02, JLYS, HUNH2, HQ6, JX5 and so on, which demonstrated the accuracy and reliability of the RT-PCR. The homology were under 80% compared to the avirulent vaccine strains including CH-1A,CH-1R and VR2332. It has a poor homology compared to the classic european PRRSV strain. Sequence analysis of PCV2 whole genome and its main ORF1 and ORF2 showed the genome of PCV2 was 1767bp consisted with reports before. The nucleotide sequence homology was 95% compared to other regions. 3. Diagnosis of typical mixed infection cases of porcine reproductive and respiratory syndrome and Porcine circovirus type2PRRSV and PCV2 have been most important pathogens for swine recently, and being widely spreaded in the swine herds. In this test, we used PCR to detect PRRSV and PCV2 from some pathogenetic tissue. The results showed that PRRSV and PCV2 were positive, and the main fatality cause of the pigs were mixed infection by PRRSV and PCV2.