The Studies Of Complement System Response After HP-PRRSV Infecting Pigs

Porcine reproductive and respiratory syndrome virus(PRRSV) is considered as a significant contributor to porcine reproductive and respiratory syndrome(PRRS).And PRRSV persistent infections can cause immune suppression and secondary infection of bacteria or viruses, which increases the difficulty of treatment. The complement system plays a crucial role in the innate defense against common pathogens. It serves as a first line of defense against invading pathogens and play important role in anti-viral. But complement system response and interaction with pigs after infection of PRRSV are rarely reported and only known to Xiao qi(Xiao et al., 2010) and Zhou Ping(Zhou et al., 2011) involved in transcription level of complement gene expression information.The former chip results showed in Yorkshire and Landrace hybridization pigs, 4 and 7 days after the north American PRRSV infection in lung tissue samples, complement activation factor was upregulated and complement inhibitor is downregulated;While the latter results showed that the Tongcheng pigs HP- PRRSV infection after 5 days, complement inhibiting factor in the alveolar macrophages CLU rise significantly, the key factor of the complement activation CFP and expression of C3 was downregulated and the complement system is restrained.Therefore, in order to study the pig complement response and function of the system after PRRSV infection, in the early stage of the research of this study has been carried out through the Tongcheng pigs and Large white pigs.According to artificial infection test materials and transcriptome analysis data before and after infection, we have screened the complement system differentially expressed genes after infection of Highly Pathogenic blue-ear virus(Highly Pathogenic PRRS, HP-PRRSV).According to the study in Tongcheng pigs and Large White pigs after artificial infection of HP-PRRSV and transcriptome analysis data, this research content is as follows:(1) Genetic analysis of the complement pathway: Here we employed the Illumina Hi Seq TM2000 platform to perform a Digital Gene Expression analysis of the porcine lung transcriptome response to HP-PRRSV infection and we used RNA- Seq Tophat 2.0 to map the sequencing reads output and perform statistics of differentially expressed genes and transcription. Then complement differentially expressed genes were screened after infection of HP-PRRSV by using threshold a Fold Change 2 or 2 or less or more, at the same time P 0.01 or less.Our results suggested Tongcheng pig C3 AR complement of genes, C4 A, C5 associated with serine protease 1(the MBL- associated serine protease, MASP- 1) gene is raised, will promote the activation of complement lectin way; upregulated CFH and downregulated CFP gene can produce inhibition to complement the bypass way;Complement inhibitor CD55, CD59, CLU and C4 BPB were upregulated and they will inhibit complement invertase and membrane complex formation, which have inhibit the whole of the complement system.In Large white pigs, complement lectin MASP- 1 gene was upregulated, and classic way C1 R raised and C4 A C3AR raised and conducive to the complement system activation. upregulated CFH and downregulated CFP gene can produce inhibition to complement the bypass way;Complement inhibitor CD55, CD59, CLU and C4 BPB were upregulated and they will inhibit complement invertase and membrane complex formation, which have inhibit the whole of the complement system.In conclusion, according to the transcriptome data, complement system is activated and suppressed. Therefore we deduced Tongcheng pig complement activation degree is higher than large white some system.(2) RNA level quantitative validation: After infection of HP-PRRSV, differentially expressed genes of complement system were detected by Real-Time PCR in alveolar macrophage. Quantitative results showed that complement gene C3 AR, C4 A, CFH, CD55, CD59 and CLU are upregulated and CFP gene is downregulated in Tongcheng pigs and Large White pigs.(3) C3 d deposition was detected by Immunohistochemistry in the lung tissues after infection of HP-PRRSV: Immunohistochemical results showed that the the C3 d was detected and the complement system was activated in Tongcheng and Large White pigs.(4) Serum C3 changes of complement system after HP- PRRSV infection: After infection of HP-PRRSV in Tongcheng pigs and Large White pigs, serum complement factor C3 was detected by ELISA.The results showed that: in Tongcheng pigs C3 level of were significantly higher on 5 day, 7 day than 0 day; In Large White pigs C3 level of was stable. Tongcheng pigs 5 days and 7 days C3 level were significantly higher than Large White pigs suggesting that HP- PRRSV infection after city activation degree is higher than large white pig.The complement system response of Tongcheng pigs and Large White pigs was explored from the transcription level and protein level after HP- PRRSV infection.From transcription data, complement system is activated and then is inhibited by complement inhibitor C3 d deposition was detected in the lung tissue by Immunohistochemistry and showed that the complement system is activated. Determination of C3 level in serum is different between Tongcheng and Large White pigs: In Tongcheng pigs serum C3 protein is rising significantly and in Large White C3 level tends to be stable. Tongcheng pigs 5 days and 7 days C3 level were significantly higher than Large White pigs. I hope these results will provide certain reference for the future study of PRRSV disease-resistant breeding.