Detection Of PCV2, PPV, PRV, PRRSV And CSFV In Cases Of High Swine Fever In Jiangsu Province During 2005 And 2006 And Some Vegions Of Nucleotide Sequencing Of PRRSV Isolates Collected In 2005 And 2006

Two multiplex PCR assays were developed and subsequently evaluated for their effectiveness as means to simultaneously detect multiple viral infections of swine. Specific primers for each of three DNA viruses, namely, porcine parvovirus, porcine circovirus type 2 and pseudorabies virus, were synthesized and used. 511bp, 404bp and 277bp were amplified in a single reaction for each one, respectively. At the same time, specific primers for each of two RNA viruses, namely, porcine reproductive and respiratory syndrome virus and classical swine fever virus, were synthesised and used in another reaction, 876bp and 465bp were amplified for each one, respectively. The efficiency and sensitivity of the two multiplex PCRs seemed to meet their potential application for routine molecular diagnostic purposes.211 suspicious samples from cases of high swine fever collected from Jiangsu area were detected for the five viruses by two multiplex PCRs, and the results showed that 93 cases were PCV2 positive, with the positive rate 44.1%; 8 cases were PPV positive, 5 cases were PRV positive, 108 cases were PRRSV positive, 22 cases were CSFV positive, and the positive rates were 3.9%, 2.4%, 51.2% and 10.4% respectively. The positive rate of PCV2/PRRSV/HCV mixed infection was 5.4% (11 of 211), PCV2/PRRSV/PRV mixed infection rate was 0.5% (1 of 211), PCV2/PRRSV mixed infection rate was 25.6% (54 of 211), PRRSV/HCV mixed infection rate was 2.4.% (5 of 211), PCV2/HCV mixed infection rate was 1.9%(4 of 211), PCV2/PPV mixed infection rate was 3.4%(7 of 211), PCV2/PRV mixed infection rate was 1.9%(2 of 211), HCV/PRV mixed infection rate was 0.5%(1 of 211). The positive rate of PCV2 infection in 2005 was 21.2%, which rised up to 73.2% in 2006. The positive rate of PRRSV in 2005 was 38.1%, and reached 67.7% in 2006. The results suggested that the positive rates of PCV2 and PRRSV infection rised up obviously during the two years, while the positive rates of PPV, PRV and HCV infection almost remained the same, PRRSV and PCV2 seemed to become epidemic in Jiangsu mentioned regions.The open reading frame 5(ORF5) of ten PRRSV isolates and partial Nsp2 gene of six isolates were amplified by RT-PCR. The PCR products were cloned into pGEM-T easy vector and sequenced. The results showed that all of the ORF5 genes were 603bp in length. The sequences were compared with sequences of PRRSV ORF5 published in GenBank, and the results indicated that ten PRRSV isolates were closely related to each other, exihibited 96.9% to 99.8% nucleotide sequence identities. There was 84.5% to 92.2% nucleotide identity with other American type PRRSV in GenBank. While the sequences of the Nsp2 genes were different, especially PRRSVs isolated in 2006. There was a continuous deletion of 87 bases in Nsp2 gene compared with that of other PRRSVs isolated before. The PRRSVs isolated in 2006 exihibited 97.9% to 98.9% nucleotide sequence identities each other, while remained parts of Nsp2 genes of Nsp nucleotide sequence identities of PRRSV isolated in 2005 exhibited 96.5% to 98.5%, and no deletions were found. The PRRSVs isolated in Jiangsu between 2005 and 2006 exihibited 94.4% to 96.1% nucleotide sequence identities in Nsp2 gene except a deletion of 87 bases observed in isolates collected in 2006.