Isolation, Identification Of The Highly And Lowly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus (PRRSV) And Establishment A Duplex RT-PCR Technology To Differentiate The Highly And Lowly PRRSV

Porcine reproductive and respiratory syndrome virus(PRRSV) is causative agent of porcine reproductive and respiratory syndrome(PRRS).The PRRSV could be divided into highly and lowly pathogenic PRRSV basing on its pathogenic ability to pig.Since July 2007,the highly pathogenic PRRSV(HP-PRRSV) marked by 3 the 30 amino-acid deletion in nonstructural protein 2(NSP2) has been epidemic in China.In order to rapid differentiate the HP-PRRSV and LP-PRRSV,first,some HP-PRRSV strains were isalated,and then basing on analysis the NSP2 gene of the HP-PRRSV and HP-PRRSV strains,the duplex RT-PCR method for detection the highly and lowly pathogenic porcine reproductive arid respiratory syndrome virus was established and applied on clinic.Some PRRSV strains were isolated from the pigs infected "High fever disease",and the probable co-infected and continued pathogenies with PRRSV in the PRRS cases were isolated and identified.The pathogenic character of an isolated HP-PRRSV strain(nominated HN-LY,NSP2 GenBank No.:AB359239) was evaluated by virus challenge assay in pig.A common lower primer and two specific upper primers of the duplex RT-PCR technology for detection HP-PRRSV and LP-PRRSV were designed by anlysis the molecular variation character of Nsp2.The sensitivity, specificity,application range and repetition assay of the duplex PCR were tested,and clinic suspicious PRRSV infected samples were detected by the established duplex PCR assay in contrast to the routine virus isolation and identifying method and other PCR assay which could be only used to detection the HP-PRRSV.The application assay of the duplex RT-PCR method was carried out by detection 9376 clinic suspicious PRRSV infected samples since January 1,2007, and the RT-PCR results were also analyzed.The result indicated total eight PRRSV strains were sececessfully isolated from the "high-fever swines".The virus challenge test revealed the HN-LY PRRSV strain was high-pathgenic to pig,and the pathogenic character of which was consisitent with the HP-PRRSV. All the isolated HP-PRRSV strains existing a unique molecular hallmark,which was a discontinuous deletion of 30 amino acids in NSP2.The duplex RT-PCR assay being used to differentiate the HP-PRRSV and LP-PRRSV was successfully established.The specificity and the sensitivity test of the duplex RT-PCR revealed that the detection threshold of the duplex PCR was 100 copy of HP-PRRSV or LP-PRRSV,and no products were amplified from the nucleic acids of the other 8 kinds of pathogenic microorganism acting as the negative control.The 4 times repetition test indicated that the duplex RT-PCR was reproducible,and the application ranges test demonstrated that the samples including the serum,whole blood,organ samples,nasal swabs etc could be successfully amplified by the established duplex RT-PCR assay,whereas the positive ratio of serum samples were a little more higher than the others.The results of 82 clinic suspicious samples detecting by the established duplex RT-PCR assay were consistent with that detecting by the routine virus isolation and identifying method and other PCR assay only for detection of HP-PRRSV.The detection results of 9376 suspicious PRRSV samples indicated the total positive ratios of PRRSV was 43.04%,and among which the positive ratio of HP-PRRSV,LP-PRRSV as well as the duplex positive ratis of HP-PRRSV and LP-PRRSV was 27.28%,9.01%and 7.2%, respectively.Our study revealed that the established duplex PCR method was highly specific and sensitive,and was fit for rapid clinic diagnosing of differentiating the HP-PRRSV and LP-PRRSV.