Effect Of Porcine Serum Amyloid A3 On Replication Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS)caused by porcine reproductive and respiratory syndrome virus(PRRSV)is a viral infection disease that has important impact on pig industry.PRRS is emerged in the late 1980 s and causes huge economic losses in China as well as worldwide.Serum amyloid protein 3(SAA3)is an interferon-inducible protein and serves as a biomarker of infection.The dynamics of porcine SAA3 during PRRSV infection and the effect of porcine SAA3 on PRRSV replication were unknown.Therefore,the current studies analyzed the changes of porcine SAA3 at mRNA and protein levels during PRRSV infection and further determined the effect of porcine SAA3 on PRRSV replication.The coding region of porcine SAA3 was cloned by PCR based on the genome sequence of porcine SAA3 and inserted into Flag7.1 vector to generate a recombinant plasmid expressing porcine SAA3.The sequence of the cloned porcine SAA3 was aligned with human and mouse counterparts to analyze the antigenic epitopes.Rabbits were immunized with the peptides synthesized based on the antigenic epitopes of porcine SAA3 to generate antiserum specific to porcine SAA3.The generated antiserum was characterized by ELISA,western-bot and immunofluorescence assay,and showed a specificity and sensitivity to porcine SAA3.The changes of porcine SAA3 in PRRSV-infected PAMs as well as in tissues of PRRSV-infected pigs were determined at both mRNA and protein levels.Porcine alveolar macrophages(PAMs)infected with PRRSV were harvested at different time points and subjected to real-time PCR analysis.The tissues collected from 30 days-old specific-pathogen-free pigs infected with PRRSV were subjected to real-time PCR and western blot for analysis of the levels of porcine SAA3 m RNA and protein,respectively.The level of porcine SAA3 mRNA in PRRSV-infected PAMs was significantly higher than that in mock-infected cells from 12 h post-infection.Porcine SAA3 mRNA in the tissues of PRRSV-infected pigs was up-regulated at different levels with the top of lung,in which the m RNA level was200 times higher than that in mock-infected pig.The levels of porcine SAA3 protein in PRRSV-infected tissues,such as lung and kidney,were remarkably higher than that in mock-infected pigs,as detected by western blot analysis.These data indicated that PRRSV infection up-regulated porcine SAA3 expression at different levels.The effect of porcine SAA3 on PRRSV infection was determined by exogenously expressing porcine SAA3 in PRRSV-infected cells or by treating PRRSV-infected cells with SAA protein.The level of PRRSV m RNA in cells expressing porcine SAA3 was significantly increased compared with control cells,as detected by real-time PCR.PRRSV titers in cells expressing porcine SAA3 were higher than control cells,suggesting that porcine SAA3 facilitated PRRSV replication.Further studies demonstrated that SAA assisted in PRRSV adsorption to host cells.In conclusion,porcine SAA3 gene was cloned and expressed,and the antiserum specific to porcine SAA3 was generated in the current studies.PRRSV infection up-regulated porcine SAA3 expression at both mRNA and protein levels.Porcine SAA3 was able to facilitate PRRSV replication and to assist inPRRSV adsorption to host cells.