Detection Of PCV2,PRRSV,CSFV,PPV,PRV,JEV,EMCV,PEV And PTV In Cases Of High Swine Fever In Jiangsu Province In 2007 And Evaluation Of TaqMan Real-time Fluorescent Quantitative RT-PCR To Differentiate Virulent CSFV Strains From Avirulent CSFV

Multiplex PCR assay was developed and subsequently evaluated for their effectiveness as means to simultaneously detect multiple viral infections of swine. At first, specific primers for each of two RNA viruses, namely, porcine reproductive and respiratory syndrome virus (PRSSV) and classical swine fever virus (CSFV), were synthesised and used in the reaction, 743 bp and 288 bp fragments were amplified for each one, respectively. It is emphasized that specific primers for porcine reproductive and respiratory syndrome virus can differentiate the PRRSV with a thirty amino acids deletion in Nsp2 region from the classical ones. The efficiency and sensitivity of the multiplex RT-PCR seemed to meet their potential application for routine molecular diagnostic purposes.Secondly, specific primers for each of two RNA viruses, namely, encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV), were synthesised and used in another reaction, 284 bp and 1057 bp fragments were amplified for each one, respectively. Thirdly, a nested RT-PCR protocol is described now suited to detect all known porcine enterovirus and porcine teschovirus serotypes using three sets of primers, and the sizes of three fragments were 158 bp,221 bp and 313 bp, respectively. Three species were porcine teschoviruses (PTV) PTV-1～PTV-11; porcine enterovirus A (PEVA); porcine enterovirus B (PEVB). The efficiency and sensitivity of these RT-PCRs also seemed to meet their potential application for routine molecular diagnostic purposes.141 suspicious samples from cases of high swine fever collected from Jiangsu area were detected for the nine viruses by the developed multiplex RT-PCR mentioned above and multiplex PCR for detection of porcine circovirus (PCV), porcine parvovirus (PPV) and pseudorabies virus (PRV) established previously in our laboratory, and the results showed that 61 cases were PCV2 positive, with the positive rate 43.3%; 96 cases were PRRSV positive, with the positive rate 68.1%; 69 cases were CSFV positive, the positive rate was 48.9%; 67 cases were PTV positive, the positive rate was 47.5%; 34 cases were PEVB positive, the positive rate was 24.1%; 9 cases were EMCV positive, the positive rate was 6.6%, the positive rates of PPV, PRV, JEV, PEVA were 0%. The positive rate of PCV2/PRRSV mixed infection was 39.6% (52 of 141), PCV2/PRRSV/CSFV mixed infection rate was 8.5% (12 of 141), PCV2/PRRSV/PTV mixed infection rate was 5.7% (8 of 141). PRRSV/CSFV mixed infection rate was 29% (41 of 141), PRRSV/PTV mixed infection rate was 24.8% (35 of 141).The results suggested that PRRSV and PCV2 seemed to become epidemic in Jiangsu regions, and PRRS Nsp2 variant strains were predominant consistantly in 2007. The results also showed that classical swine fever was also severe in the cases with high swine fever from this region, and the synergy between classical swine fever and high swine fever in the cases mentioned above was obvious. It is emphasized that epidemiology of Teschen disease and porcine enterovirus infection of pigs was rarely studied in the past in our country, and the positive rate for PTV and PEV were 47.8% and 24.1%, respectively, among cases with high swine fever from Jiangsu region.In this study, we are going to develop a TaqMan real-time fluorescent quantitative RT-PCR to differentiate virulent CSFV strains from avirulent ones. CSFV specific primers and two differently labeled TaqMan probes for the differentiation of wild-type CSFV strains from C-srtain virus were designed in the 5'-UTR of the viral genome of CSFV. The results showed that the virulent CSFV strain Shimen and C-strain vaccine virus could not be differentiated by TaqMan RT-PCR developed in this study, and more efforts based on this technique are bing taken.