RNA-Seq Analysis And Determination Of Restriction Factors Against Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS), caused by porcine reproductive and respiratory syndrome virus(Porcine reproductive and respiratory syndrome virus, PRRSV), is an acute and highly contagious viral infection disease with huge economic losses to the pig industry in the world. In 2006, an outbreak of highly pathogenic porcine reproductive and respiratory syndrome virus(HPPRRSV) occurred in China, which has the stronger appeal and pathogenicity bringing greater harm to pig industry. HP-PRRSV predominantly infects and replicates in Porcine alveolar macrophages(PAMs). Type I IFN can inhibit PRRSV replication, whereas IFN exerts its antiviral activity via inducing ISGs(Interferon stimuted genes, ISGs) to encode the limiting factor. The studies about looking for host restriction factors which can efficiently inhibit HP-PRRSV have recently become popular. But it’s a huge project to screen the IFN stimulated restriction factors because there are many kinds of ISGs. As we all know, RNA-Seq technology is the next-generation sequencing technology with the advantages of high throughput, high efficiency and good sensitivity. In this study, host restriction factors against HPPRRSV in PAMs, which were pretreated with porcine IFN-ɑ, were high-throughput screened using RNA-Seq technology.In this experiment, ISG15, an anti-PRRSV limiting factor, acted as the indication. The sampling time points were determined according to the expression of ISG15 in PAMs infected with HP-PRRSV. 12 hours after inoculation was finally chosen. PAMs were divided into four different treatment groups, respectively IFN, IFN+PRRSV, PRRSV and C(control group) group. Changes in global gene expression in IFN-ɑ-pretreated PAMs in response to HP-PRRSV was detected by RNA-Seq analysis. 346 differentially expressed genes(DEGs) were transcriptionally up-regulated by IFN-α. Among these up-regulated DEGs, 93 showed significantly attenuated expression level in response HP-PRRSV infection. These attenuated DEGs were remarkably enriched in immune-response-related terms, such as the "RIG-I-like receptor signaling pathway", "response to virus", "response to the stimulus ". 16 antiviral DEGs were significantly up-related in response to IFN-ɑ, including IRG6(RSAD2), IFI44, ISG20, PKR, LOC100738990(TRIM5), BST2, OAS1, IFIT1(ISG56), IRF7, IFIT2, ISG15, LOC100627004(IFITM1), ISG12(A)(IFI27), IFITM3, IFIT5, among which 15 were attenuated in the IFN+PRRSV group.We examined whether HP-PRRSV was inhibited by 16 antiviral DGEs through siRNA-traeted PAMs. Then the PAMs were infected with HP-PRRSV and the replication of HP-PRRSV was detected. The results showed PKR, OAS1, IFIT1(ISG56), ISG15, IFI44, ISG20, BST2, IRF7, IFIT2, LOC100627004(IFITM1), IFITM3, IFIT5 and GBP1 could significantly inhibit the replication of PRRSV.GLRX1 is a protein with multifunction, such as oxidation, signal transduction, anti-apoptosis. RNA-Seq test results showed that IFN significantly up-regulated the expression of GLRX1, but HPPRRSV infection significantly inhibited the expression of GLRX1. The expression was down-regulated by siRNA interference in PAMs. Results showed that HP-PRRSV replication was significantly increased, indicating GLRX1 could inhibit the replication of HP-PRRSV. We compared the expression of GLRX1 in different tissues of pigs infected PRRSV and health ones. The expression of sick pigs GLRX1 in inguinal lymph nodes, heart, lymph nodes hilar were significantly decreased, while the expression in the small intestine, lung and muscle were significantly increased. It was suggested HPPRRSV could affect GLRX1 distribution in various tissues.In the study, host restriction factors against HP-PRRSV were screening and determined by RNASeq. Our results provided the basic data for further exploring the mechanisms of immune response and immune invasion by HP-PRRSV.