| Porcine Reproductive and Respiratory Syndrome (PRRS) caused by porcine reproductive and respiratory syndrome (PRRSV) is an infectious disease that threats global swine industry. At present,there is a complex of PRRSV strains circulating in pig populations in China, which involves the American strains including classical strains, highly pathogenic strains and NADC30-like strains, as well as the European strains. Such complicated situation poses great pressure to the prevention and control system of PRRS in China.In order to grasp the trend of PPRSV distribution and genomic evolution, the whole sequences of 61 PRRSV isolated in China during 2008 to 2015 were determined, and then compared with representative PRRSV strains. Genetic variation of important genes Nsp2, GP3 and GP5 were analyzed.The results shows that within 61 PRRSV isolates, 5 isolates belongs to EU-PRRSVs isolated and 56 belongs to American PRRSVs.Within the 56 American PRRSVs, there were two NADC30-like PRRSV and 54 HP-PRRSVs. The 54 HP-PRRSV isolates show 95.2%～99.9% homology, and they share 97.2%～99,3% homology comparing with representative strain of HP-PRRSV (JXA1). The homology between two NADC30-like strains was 99.9%, and their genome shares 95.4% similarity comparing with NADC30 PRRSV representative strain (NADC30) . The 5 EU-PRRS isolates are of 84.4% to 99.7%homology, and comparing with the representative strain of EU PRRSV LV strain, they share 87.2%～90.9% homology. Gene sequence analysis of important viral proteins shows that PRRSV is changing constantly. The non-structural proteins Nsp2 and structural protein GP5 have the most significant mutations. Nsp2 sequence analysis indicated that deletion of gene segment in the Nsp2 site with various sizes ranging from 1 to l50 aa occurred in all 61 isolates. In the important structural protein GP5, most of the isolates show significant changes of GP5 gene, especially of the location and number of glycosylation sites. Structural protein GP3 also has some changes: in EU-PRRSV isolates, there are multiple deletions and mutations at GP3 hypervariable region. We also report four isolates of PRRSV with new gene deletions, and such deletion with certain number and location of nucleotide or amino acid has not yet been reported in China. Our results show that since 2008, the current circulating PRRS strains in China are mainly HP-PRRSVs,the emergence of EU-PRRSV and NADC-30 like PRRSVs has made the situation a complexity, an improved and strengthened surveillance system in China is essential and in urgent need.There are various strains of PRRSV circulating in pigs in China, and their genomes of these viruses are changing constantly. Considering these characters, in order to improve current universal RT-PCR test of PRRSV used in surveillance, we established the detection method of PRRSV universal fluorescent quantitative RT-PCR. According to the published PRRSV European type and American type gene sequence, specific probes and primers were designed, selected and test optimized. This method has good specificity, sensitivity and repeatability, and can specifically detect PRRSV America and European strains, with no cross reaction with other virus detection of other swine viruses. It is can detect 20 copies/μL for American strains and 22 copies/μL for European strains. Particularly, it shows better sensitivity when detecting NA classical strains and EU PRRSV strains. The efficiency of variation was within 2.5% for in repeat trails within the same batch and between different batches. A number of 812 animal samples and clinical samples were tested, and the coincidence rate was 94.2%. The results show that the detection method of PRRSV universal real-time RT-PCR can be applied in diagnosis and surveillance of PRRSV infection.In summary, this study has elucidated the distribution and genetic evolution of PRRSV in China,and established a PRRSV universal RT-PCR method, which is important for the prevention, control and eradication of PRRS. |
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