Genetic Characteriation And Isolation Of Porcine Reproductive And Respiratory Syndrome Virus Prevailing In Southeastern China

Porcine reproductive and respiratory syndrome (PRRS) is characterized by severe reproductive failure in sows (late-term abortions, dead, weak and mummified piglets), respiratory disease and increased preweaning mortality. The PRRSV possesses four characteristics that may contribute to difficulties in prevention and treatment of the disease, including production of effective vaccines. These include tropism for macrophages or macrophage-lineage cells, remarkable antigenic variations among PRRSV field isolates, antibody-dependent enhancement of the virus infection (ADE), and ability to establish persistent infection. The main objectives of the present study are (1) to analyze the variations of the GP5 genes of PRRSV isolates from some pig farms in southeastern China, (2) to express and purify the GP5 protein for ELISA coating and preparation of polyclonal antibodies to GP5 for indirect immunofluorescent detection of PRRSV, and (3) to identify and recover a field PRRSV strain from infected tissues.Reverse transcription PCR was used to amplify the GP5 gene from the tissue samples of pigs suspected of having PRRSV in southeastern Chinese provinces, and gene fragments were cloned and sent for sequencing. Phylogenetic analysis indicates that all isolates from the region from 2004 to 2007 belong to subgroup 1 of North American type. The isolates from this study shared identities of 89.6-100% at the nucleotide level and 90.7-99.5% at the amino acid level. Their nucleotide identity was 88.1-90.2% and amino acid similarity, 85.8-89.6% to the classical North American isolate VR2332.The gene encoding truncated GP5 was cloned into the expression vector pET-30a, the recombinant protein was efficiently expressed in E. coli Rosetta in the form of inclusion bodies after induction with IPTG as revealed by SDS-PAGE, and the protein yield after purification was 87.5mg per liter culture of the recombinant bacteria. Western blotting and ELISA indicated that this recombinant protein could react with PRRSV positive serum. The recombinant was used to generate the anti-GP5 polyclonal antibody in rabbit with titer as high as 1:32768. which was used for identification of PRRSV by indirect immunofluorescent assay.The tissues samples that were PRRSV positive by RT-PCR were collected and used to inoculate onto porcine alveolar macrophage (PAM) monolayers. Replication of PRRSV in the cells was confirmed by RT-PCR fragment targeting GP5, indirect immunofluorescent assay and transmission electron microscopy. The virus had deletion of 29 amino acids in NSP-2 and could induce the cytopathic effect after subculturing in Marc-145 cell line with TCID50 of about 104.59 after 10 passages.The findings in this study provide some basis for further research into the genetic variations, pathogenesis and immune intervention.