Molecular Epidemiology Of Porcine Reproductive And Respiratory Syndrome Viruses From Swine Farms In Eastern China

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRSV with an enveloped single-stranded RNA virus in the family of Arteriviridae, is characterized by respiratory stress and reproductive failure. There are two genotypes of PRRSV, European genotype and American genotype, based on the high variability of the whole genome among isolates from different areas, especially the ORF5 gene. This study was aimed to establish a RT-PCR assay to discriminate two genotypes of PRRSV from tissues and to evaluate the molecular epidemiology of PRRSV based on the divergence of ORF5 within isolates from eastern China.Firstly, three paired of primers were designed based on the conserved region franking N gene of strains VR-2332 and Lelystad. The results showed that patial N gene of American genotype (382bp) or the whole N gene of European genotype (576bp) could be amplified specifically from respective reference strains by RT-PCR. Moreover, it could also detect both genotypes simultaneously. One hundred and sixty-eight samples from sick pigs were detected by the assay. There were 43 positive samples of American genotype,6 positive for European genotype and 1 sample with both genotypes.To get insights into the molecular epidemiology of PRRSV in eastern China, two pairs of primers for ORF5 were designed to amplify the whole genes of American genotype (723bp) or European genotype (702bp) from PRRSV positive samples. The sequencing results indicated that the homology of 23 sequenced American genotype PRRSV isolates was 90.4%-99.5% at nucleotide level and 88.1%-99.5% at amino acid level to each other, while it was only 87.1%-88.7% nucleotide sequence similarity to VR2332, indicating that the GP5 gene was highly variable among isolates of American genotype prevainling in eastern China. On the other hand, there were 5 European type PRRSV isolates with 98.3%-98.5% nucleotide sequence homology and 96.0%-96.5% amino acid identity to each other. However, they were only 92.7%-93.2% (nt) and 91.1%-91.6 (aa) homologous to strain Lelystad, indicating that they might be the mutants from European type vaccines. However, the other two European type isolates of 87.6% and 90.1% nucleotide identities to vaccine strain PRRSV-EU, and 87.3% (nt)和88.6% (aa) homologous to Lelystad, suggesting that these two strains might be the wild PRRSV isolates of European genotype from unknown sources.Phylogenetic ananylsis conducted with ORF5 gene of PRRSV-US showed that the isolates in 2006 clustered as a separate group from those in 2004 and 2005, shifting away from the vaccine strains or old isolates in late 1990s. Another finding was that the sequences of European type isolates in 2006 were much further away from the vaccine strains such as PRRSV-EU.In conclusion, the differential RT-PCR assay for PRRSV of the US and EU genotypes, elucidation of the genetic variations of the PRRSV isolates over the years, preliminary revealing of the possibility of PRRSV EU genotypy in the region would help enrich molecular epidemiological data of PRRSV in China and provide some basis for proper control of the disease.