Regulation Of Heme Oxygenase-1on Porcine Reproductive And Respiratory Syndrome Infection

Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductiveand respiratory syndrome virus (PRRSV), is characterized by reproductive failure inbreeding animals and respiratory distress in all ages of pigs. Since antigenic variability,macrophage tropism, antibody-dependent enhancement (ADE), persistent infection and othercharacteristics of PRRSV infection, PRRSV often mixed infection with other pathogensclinically, and currently pathogenesis and immune mechanism of the disease remains unclear,and there is no really effective control measures. PRRS has become one of the mostchallenging issues in veterinary immunology research field.At present, PRRS has prevailed inthe main worldwide pig-raising country and districts and caused pig-raising industry largeeconomic losses. So it becomes one of the hot research areas.According to our previous study,high-throughput sequencing technology were used to determine transcriptome changes in thelung tissue of piglets after PRRSV infection. We found that heme oxygenase1gene wassignificantly upregulated after PRRSV infection. In vitro studies also showed HO-1gene wassignificantly up-regulated after PRRSV infection in Marc-145cells, implying that HO-1mayplay an important regulatory role in PRRSV infection. Based on previous study, the role ofHO-1during PRRSV infection in vitro were determined.1.PRRSV infection significantly affect HO-1expression in Marc145and PK-15CD163celllines.HO-1expression at different time points after highly pathogenic PRRSV SD-16andclassical PRRSV CH-1a infectionin Marc145and PK-15CD163cells were determined by Realtime PCR and Western blot.Highly pathogenic PRRSV and classical PRRSV infection cansignificantly up-regulated HO-1expression in Marc145cells.Both of the two strains ofPRRSV infection down-regulate HO-1expression in PK-15CD163cells.2. HO-1induction significantly inhibited PRRSV infection on Marc145and PK-15CD163cellsAfter treatment of Marc145and PK-15CD163cells withspecific HO-1inducer CoPP, Realtime PCR and Western blot was used to detect the mRNA and protein expression levels ofHO-1gene and PRRSV N gene.The results showed that, CoPP concentration dependent induce HO-1mRNA and protein expression. Simultaneously,PRRSV infection wassignificantly inhibited on Marc145and PK-15CD163cells.The effect of HO-1induction ondifferent PRRSV strains infection on Marc145cells were further determined by IFA, FACSand TCID50Results showed that, CoPP concentration dependent inhibitedboth PRRSV straininfection on Marc145cells. These results indicate that inhibition of PRRSV infection by HO-1inductionwas PRRSV strain independent.3. Adenovirus-mediatedHO-1over-expression significantly inhibited PRRSV infetion onMarc145and PK-15CD163cellsEffect of HO-1over-expression on PRRSV infection were determined by usingrecombinant adenovirus.HO-1and PRRSV N gene mRNA and protein expression levels andculture supernatant progeny virus titers were determined by Real time PCR, Western blot andTCID50.Adenovirus-mediated HO-1over-expression in Marc145and PK-15CD163cellssignificantly inhibited the synthesis of PRRSV N protein and supernatant and progeny virustiters.4. Knockdown of basal expression of HO-1by siRNA promoted PRRSV infection onPK-15CD163cellsTo investigate whether basal level of HO-1affected PRRSV infection, HO-1specificsiRNA were used to knockdown the expression of HO-1in normal PK-15CD163cells and theeffect of HO-1knockdown on PRRSV infection were determined.The results showed that,HO-1specific siRNA significantly knockdown HO-1expression in normal PK-15CD163cells,and simultaneously, PRRSV infection increased significantly. These results indicated thatbasal level of HO-1can affect PRRSV infection.5. HO-1knockdown partially reversed the inhibitory effect of HO-1on PRRSVinfectionIn order to verify the specificity of HO-1-mediated anti-PRRSV infection effect, HO-1specific siRNA were co-incubated with CoPP and Adv-HO-1. PRRSV infection on PK-15CD163were determined by Real time PCR, Western blot and TCID50.The results showed that,HO-1specific siRNA partially reversed the inhibition effect of HO-1induction and HO-1overexpression on PRRSV infection.In conclusion, HO-1showed a pronounced antiviral activity in PRRSV infection. Thesefindings suggest that up-regulation of HO-1exerts as a host cellular defense mechanismagainst PRRSV infection, and HO-1induction or overexpression may potentially be a usefulprevention and treatment strategy against PRRSV infection.