Effects Of Host Protein ZAP And IL-7 On The Replication And Immunization Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus(PRRSV)causes reproductive failure in sows and respiratory diseases in all pigs.PRRSV has became the most economically important pathogen impacting the swine industry of our country.The antiviral effector molecules which are involved in host protective responses to PRRSV infection are not fully understood.Study the antiviral factors against PRRSV infection is important to understand the mechanism of host immune response against PRRSV,and will be helpful for development of novel antiviral therapies in future.Current vaccination strategies against PRRSV only provide limited protection,it is necessary to explore the new vaccines and adjuvants to control PRRS.Based on the above background,this paper mainly includes the following aspects:1.Transcriptome analysis of MAVS transfected Marc-145 cellsIn this study,we found that PRRSV infection reduced the endogenous MAVS expression in Marc-145 cells,and over-expression of MAVS inhibited PRRSV replication in vitro.These results indicate that MAVS is involved in the interaction between PRRSV and host immune response,and MAVS mediates a powerful anti-PRRSV process.To study the MAVS mediated anti-PRRSV response,transcriptome analysis of MAVS transfected Marc-145 cells was performed.The results showed that the antiviral immune responses were significantly up-regulated by MAVS,and the type I interferon and a plenty of antiviral factors were induced.These date provide a basis for further understanding of MAVS mediated immune response and screening of novel anti-PRRSV cellular restriction factors.2.ZAP inhibits PRRSV replication and interacts with viral Nsp9In this study,a zinc finger antiviral protein,termed ZAP,was screened from MAVS induced antiviral genes by transcriptome sequencing.We found ZAP is an interferon stimulates gene,and ZAP suppresses PRRSV replication at the early stage of PRRSV infection.We also found that the viral protein Nsp9,a RNA-dependent RNA polymerase(RdRp)interacts with ZAP.The interacting locations were mapped to the zinc finger domain of ZAP and the N-terminal amino acids 150-160 of Nsp9.These findings suggest that ZAP is an effective antiviral factor for suppressing PRRSV infection,and shed light on virus-host interaction.3.PRRSV Nsp4 antagonises the antiviral activity of ZAPOur previous results showed that the host protein ZAP can significantly inhibit the replication of PRRSV,but the effect of PRRSV on ZAP and the mechanism of PRRSV escaping the antiviral effect of ZAP remain unknown.In this study,We found that PRRSV infection significantly down-regulated the endogenous ZAP expression in Marc-145 cells.We confirmed PRRSV nonstructural protein 4(Nsp4),a 3C-like proteinase,is responsible for ZAP reduction.Nsp4 reduces ZAP expression at the protein level by cleaving ZAP,and the cleavage depending on the protease activity of Nsp4.Meanwhile,the anti-PRRSV activity of ZAP could be antagonized by Nsp4 in Marc-145 cells.In addition,we found the 180 amino acid of Nsp4 also plays an important role in the degradation of ZAP,and recombinant PRRSV containing Nsp4 mutant decreased the degradation of ZAP.Taken together,those findings reveal a manner of PRRSV Nsp4 antagonizing the antiviral activity of ZAP,and shed light on a new strategy evolved by PRRSV to escape the host defense.4.IL-7 enhanced the immune effect of PRRSV GP5 proteinPRRSV-GP5 protein is the most important protective antigen,but the ability of GP5 to induce neutralizing antibodies is weak,due to the glycosylation modification and decoy epitope of GP5 protein.In order to enhance the immune effect of GP5,we optimized the gene sequence of GP5(named as GP5m),and choose porcine IL-7 as molecular adjuvant.The GP5m protein,IL-7-GP5m fusion protein and IL-7/GP5m co-expressed protein were prepared by baculovirus expression system.Piglets were vaccinated with the three subunit vaccines,respectively.The control group treated with same amount of PBS.All piglets were challenged with PRRSV at six weeks post vaccination.The results showed that IL-7/GP5m vaccinated pigs had a higher antibody levels and a better lymphocyte proliferation response.When challenged with virulent PRRSV,IL-7/GP5m vaccinated pigs had significantly lower levels of viremia,lower viral RNA load,and a higher protection rate.This study suggest that porcine IL-7 could act as a molecular adjuvant,which could improve the immune effect of PRRSV GP5 protein.This study provides a reference for the development of new subunit vaccine of PRRSV.