Identification Of A Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus And Molecular Epidemiology Of The Virus

A highly pathogenic disease, characterized by high and continued fever high-mortality, occurred inthe pigs in main pig-producing provinces in China in 2006. To investigate the causative agent of thepandemic disease, we selected 8 clinical samples of infected pigs from Hunan province. The result ofamplification using porcine reproductive and respiratory syndrome virus (PRRSV) ORF7 and ORF5specific primers indicated that seven out of eight samples were PRRSV positive. PRRSVs were thenisolated successfully on MARC-145 cells with an obvious cytopathologic effect, characterized by cellcongregationand contraction. As to the isolates, specific fragments could be amplified by RT-PCR withPRRSV ORF7 and ORF5 specific primers, while no fragments could be amplified by RT-PCR or PCRwith CSFV, PCV2 or PRV specific primers. The results of IFA detection were positive with anti-PRRSVGP5, M and N McAb. These experiments showed that the viruses isolated in this study were PRRSVsand the HUN4 isolate was selected for the whole genome sequencing. The sequences analysis showedthat the length of the complete genome sequence was 15352bp. The most interesting feature was that itlacked 30 amino acids in NSP2 protein compared with the classical strains like CH-1a or VR-2332. Theartificial infection test showed that all five infected pigs died at 7, 8, 12, 16 and 21 days post inoculation,respectively. The clinical and pathological observations of pigs were similar to those appeared in fieldinvestigation. In conclusion, the emerged PRRSV featured by deletions in NSP2 is highly pathogenic topigs and is possibly the predominant causative agent of the so-called"high fever"disease in China in2006.According to the complete genome sequences of PRRSV in GenBank, two pairs of specific primerswere designed to amplify the complete GP5 gene and the partial NSP2 gene, respectively, and then wereused to detect the 339 samples from 16 provinces in China. The results showed that 146 samples werepositive and the positive rate was 43.1%. 64 positive samples were sent to company for sequencing. Thesequencing results showed that 56 isolates were highly pathogenic PRRSV variants with twodiscontinuous deletions in NSP2 protein. Another 8 isolates did not have deletions like that, indicatingthey were not highly pathogenic PRRSV variants. The Analysis of GP5 of PRRSV variants showed thatthe number and positions of N-glycosylated sites changed. The primary neutralizing epitope in position39 changed from L to I and the virulence related sites in position 13 and 151 retained the features (R13and R151) like very virulent strains. The PRRSVs in our country could be classified into four mainsubgroups according to the results of phylogenetic trees. All the highly pathogenic PRRSVs in the treeclustered in one branch, which was called subgroup I and 8 non-PRRSV variants were mainlydistributed in subgroup II. The subgroup I had low homology with classical strains and vaccine strains,while, had highest homology with subgroup II and had lowest homology with subgroup IV.A pair of primers was designed based on the NSP2 gene sequences of PRRSV strains available inGenBank and the highly pathogenic PRRSV isolates identified in our laboratory. A reversetranscription-polymerase chain reaction (RT-PCR) was developed to differentiate the classical PRRRSVs and highly pathogenic PRRSVs. For the classical PRRRSVs and highly pathogenic PRRSVs,DNA fragments of sizes 230bp and 320bp were amplified, respectively, indicating two types of isolatescould be differentiated by RT-PCR method. The specificity test showed that there was no co-reactionwith other viruses, such as PCV2, PRV and CSFV. The sensitivity of detection was equal to 10TCID50for PRRSVs. The detections of 56 known clinical samples and 5 artificial infected samples wererepeated for 3 times. The results were the same with the detection results before, which indicated thismethod had good repeatability and highly coincided with other detection methods. The differentiatingresults were identified by sequencing methods, which indicated this method could differentiate highlypathogenic PRRSVs from classical PRRSVs. Application of the method for detecting the clinicalsamples demonstrated that the RT-PCR is rapid, simple and specific for detecting PRRSV and can beused in differential diagnosis of the highly pathogenic PRRSV from the classical PRRSV in the furtherepidemiological investigation.