Isolation, Identification And Biological Characteristics Of Porcine Reproductive And Respiratory Syndrome Virus Anhui Strains

A severe pig disease designated as"high fever disease'',which spread more than 20 provinces in China in 2006, caused great economic losses. The infringed pigs experienced the common clinical symptoms such as reddening of the skin or a purple color in the ears, edema of the eyelids, conjunctivitis and extensive hemorrhage in pre-weaning piglets with high incidence and mortality. In this study, the epidemic strains of porcine reproductive and respiratory syndrome virus (PRRSV) were isolated and identified. The sequence analysis and animal regression test suggested that 5 strains of isolates fell into the variant strains of PRRSV with very virulence.60 suspect samples of lung, spleen and lymph node collected from the infected herds in Anhui were used for virus isolation identification and PCR amplification; five suspect strains of PRRSV were isolated from the PCR positive samples. The isolates grew well and induced specific cytopathogenic effect (CPE) on Marc-145 cells after a serial of passages. Electronic microscopy showed the virion is enveloped with the diameter of about 50-80nm. All virus isolates can reacted specifically with the positive serum against standard strain of PRRSV VR-2332 by indirect immunofluorescence assay (IFA) and specific fragments for variant of PRRSV were amplified from infected cell culture with the isolated strains by RT-PCR. Our data supported that the isolates belong to variant strains of PRRSV, which designated as SCH07, DY07, FX07, SZH08 and HSH08 respectively.PRRSVs with different geographic origins are classified into 2 major genotypes, the European type and North American type. Nonstructural protein 2 (Nsp2) and glycoprotein 5 (encoded by ORF5) are regarded as 2 regions of high heterogeneity that are involved in the pathogenicity of PRRSV strains. Partial Nsp2 and ORF5 genes of five isolate strains were amplified by RT-PCR and cloned into the pMD18-T vector for sequencing. The results of sequence analysis demonstrated that 5 PRRSV isolates shared 97.1%～98.7% sequence homology of Nsp2 and 96.9 %～98.7 % sequence homology of ORF5, shared more than 96.9 %～99.7 % homology with HuB1, HuN4 and JXA1 strains, which suggested that they belong to North American type. Sequence analysis showed that all the PRRSVs isolated from the infected pigs in this outbreak of"high fever disease"share a discontinuous deletion of 30 amino acid in Nsp2 gene, which contributes to the virulence of PRRSV in pigs. To evaluate the pathogenicity of PRRSV isolates, animal regression test was conducted. Four 40-day-old pigs with antibody negative of PRRSV divided into 2 groups, 2 pigs in test group were challenged with SCH07 isolate strain with TCID50 of 10-4.7/0.1ml, and another 2 pigs were design as control. At 3-4 days post-challenge, the inoculated pigs experienced serious signs of''high fever disease''with high body temperatures, anorexia, erythematous blanching rash in ears, and nervous symptom and died at day 15 post-challenged and The characteristic gross lesion of extensive hemorrhage in lung, spleen, lymph node were found in the challenged pigs with PRRSV isolate SCH07. The results of pathological histology showed typical interstitial pulmonitis, lymphocyte sparseness and reticulocyte hyperplasia of groin, which was consistent with that of variant strain of PRRSV HuB1, HuN4 and JXA1 strains.Our data indicate that identical variants of PRRSV are responsible for the epidemic of"high fever disease"in Anhui province.