Biological Characteristics And Genomic Sequencing Of Three PRRSV Isolates And Construction Of PRRSV/JSyx/2006 Genomic Infectious Clone

High mutated PRRSV isolates appeared in China in the summer, 2006. They are characterized by a 90-bp deletion in the gene encoding protein Nsp2, and isolates from different regions of China share with the common deletion. The PRRS viruses with this feature are considered as the cause of a great loss in pig industry since 2006 in China.To evaluate the pathogenicity of these isolates with specific genomic characteristics, PRRSV/Changzhou/2005, PRRSV/JSyx/2006, PRRSV/Yangzhou/2008 isolates were selected, and furthur studied.The similar cytopathic effect caused by three isolates was observed after infecting the Marc-145 cells. RT-PCR with the specific primers for Nsp2 gene showed that PRRSV/Changzhou/2005 was non-deleted strain while the other two were isolates with 90 bp deleted in Nsp2 region. Animal challenge experiment revealed that PRRSV/JSyx/2006 and PRRSV/Yangzhou/2008 strain caused challenged piglets to present some typical PRRS symptoms and lessions: skin cyanopathy and pore bleeding, the body temperature was elevated in pigs challenged with PRRSV/JSyx/2006 and PRRSV/Yangzhou/2008 than that of pigs challenged with PRRSV/Changzhou/2005.No challenged pigs died during 22 days post-challenge.Based on sequences of PRRSVs deposited in GenBank, pairs of primers were designed to amplify full genome of these three isolates for sequencing.The region encoding Nsp2 with a 90 bp discontinuous deletion both in PRRSV/JSyx/2006 and PRRSV/Yangzhou/2008, including with a deletion of 3 bases (encoding phenylalanine) at 2947-3033nt position which is the characteristics of PRRSV/Changzhou/2005.Isolates in China were divided to 2 brenchs through aligning. CH-1a, HB-1, PRRSV/Changzhou/2005, PRRSV/JSyx/2006 and PRRSV/Yangzhou/2008 belonged to the same brench. PRRSV/JSyx/2006 and PRRSV/Yangzhou/2008 had high homology with other isolates from different areas of China during the same period.Based on the whole sequence of PRRSV/JSyx/2006, eight segments covering PRRSV/JSyx/2006 full genome were amplifed by RT-PCR using pairs of primers. Full cDNA were cloned into pCR(?)2.1 vector firstly, then the infectious cDNA was clone into pCMVmc1 vector, and the rescue virus pCMV-PRRSV/JSyx/2006 was constructed.