Examination Of The Functional Protein Genes Related To Immunoregulation Of PRRSV And Immunicity Of Recombinant PRRSV With NGS Deletion In GP5

Porcine reproductive and respiratory syndrome virus(PRRSV)was an important pathogen that caused huge economical losses to the world pig industry,especially the Chinese pig industry.The PRRSV was a positive strand RNA virus that encoding at least 14 nonstructural proteins and 8 structural proteins,which is easy to mutate.The infection of PRRSV could cause immunosuppression of the host,and with lots of researches on the pathogenic and immune mechanism of PRRSV,the genes that lead to symptoms and immunosuppression remains unclear.Currently,vaccines against PRRSV were mainly divided into two types:live attenuated vaccines and inactivated vaccines,but neither was an ideal option.Live attenuated vaccines offer protections only between homologous strains,and there were risks of reversion in virulence and recombination with wild strains.Comparing to the live attenuated vaccines,the inactivated vaccines were much safer,but their protection was weaker than former.This research deeply studied the questions mention above by using reverse genetic techniques,which mainly includes:1.Attenuation of PRRSV NT0801 by serially passaged and analysis of full-length genomes of viruses in different passagesThe virulence of PRRSV could be attenuated after serially passage in vitro.Meanwhile,during the process,mutations or deletions could occur in the PRRSV genome,which may be involved the PRRSV virulence.In this study,PRRSV strain was isolated in our lab and NT0801-F80 was derived from its parental isolate NT0801 by 80 passages in Marc-145 cells.Experimental infection of piglets clearly demonstrated that strain NT0801－F80 is less virulent than NT0801.However,whole genome sequencing showed that the genomes of the parental and attenuated strains are highly conserved compared with those of four other pairs of virulent parental/attenuated vaccine strains(VR2332 and RespPRRS MLV,JA142 and Ingelvac?ATP MLV,CH-la and CH-1R,and JXA1 and JXAR).The attenuated strain NT0801-F80 has only 21 nucleotide changes,producing only 14 amino acid(aa)changes in Nsp2,GP2,GP3,and GP5,compared with those aa sequences of the virulent parental strain.These mutated aa in the attenuated virus may be involved in virulence.These data provide valuable information on the attenuation mechanism of PRRSV that should be useful in future research.2.Analysis of critical amino acids in PRRSV N protein inducing IL-10 expressionPRRSV infection could caxuse immune suppression,which has not been understood clearly.IL-10 is an important immune suppressive cytokine.Several studies have demonstrated that PRRSV infection could cause the upregulation of IL-10.However,the functional proteins of PRRSV in inducing the IL-10 are not clear.In this study,we obtained the 1705bp IL-10 gene promoter sequence by genome walking kit,cloned into pGL3-Basic plasmid and obtained pIL-10 to establish corresponding dual luciferase reporter gene detection system.Meanwhile,the pVAX-N eukaryotic expression plasmid was obtained by cloning PRRSV N protein gene into pVAXl vector.The fact that PRRSV N protein could upregulate IL-10 promoter activity was confirmed by the system mentioned above.On this basis,the key domains in N protein gene regulating IL-10 promoter activity were explored by constructing N protein mutants.Moreover,alanine substitution mutation analysis revealed that the N protein residues 33-37,65-68,and 112-123 were related to the upregulation of IL-10 promoter activity.In order to study the role of potential residues in regulating the IL-10 induction on PRRSV,we first construct the infectious.cDNA clone of NT0801 strain(pNT/wt).Then,recombinant PRRSV with mutations at residues 33-37 in the N protein(rQ33-5A and rS36A),recovered from corresponding infectious cDNA clones,induced significantly lower levels of IL-10 production in infected monocyte-derived dendritic cells,as compared to their revertants rQ33-5A(R)and rS36A(R),and the wild-type recombinant PRRSV strain rNT/wt.These data indicate that type 2 PRRSV N protein plays an important role in IL-10 induction and the N-N non-covalent domain is associated with this activity.These findings provide an important example of rationally designed modifications to abolish the host innate response without alterations to the replication potential of the virus.3.Role of PRRSV Nsp2 gene in inducing inflammatory responses and analysis of its critical determinantsThe inflammatory responses induced by variable PRRSV strains were different.Non-structural protein 2(Nsp2)gene,especially its central hypervariable region(HV2),is recognized as the most variable region within the PRRSV genome,such as insertions or deletions.Compared with Nsp2 of VR-2332,the genes in strains BB0907,Em2007,MN184C and TJM-F92 has 30aa,68aa,111aa and 120aa deletions,respectively.The role of those regions of Nsp2 gene in inducing inflammatory responses is not clear.In this study,we first constructed the infectious cDNA clone of HP-PRRSV BB0907 strain(pBB).Base on the infectious cDNA clone pBB,four recombinant PRRSV strains,rBB/+30aa(30 aa addition in HV2 as those in VR2332),rBB/A68aa(68aa deletion as Em2007),rBB/Δ111aa(deletion of aa323-433 as MN184C)and rBB/A120aa(deletion of aa628-747 as TJM-F92),were individually rescued by using a highly pathogenic type 2 PRRSV cDNA infectious clone.They all displayed similar growth characteristics in pulmonary alveolar macrophages(PAM)cultures compared to the parent virus rBB.The results of real-time PCR and ELISA for inflammatory cytokines showed that the transcriptional and post-transcriptional levels of IL-P,IL-2,IL-6 and TNF-a in rBB/Δ11 laa and rBB/Δ120aa infection groups were significant lower than those in rBB,while the levels of these inflammatory cytokines had no significant differences between rBB/+30aa,rBB/A68aa and rBB groups.Meanwhile,IκB phosphorylation level was remarkably decreased in rBB/Δ111aa and rBB/Δ120aa,compared with those in rBB/+30aa,rBB/A68aa and rBB groups.It indicated that the region of aa323-433 and aa628-747 in HV2 of Nsp2 could modulate inflammatory cytokines production in PAM in vitro.It should be helpful for us to understand the different pathogenicity of PRRSV strains with genetic variations in Nsp2.4.Construction and immunogenicity recombinant PRRSV with N-Linked glycans deletion in GP5Vaccination is one of effective methods to prevent and control PRRSV.However,the current vaccine is not perfect.PRRSV infection could cause the delayed neutralization antibody response and cellular response.It has been suggested that N-Linked glycans on PRRSV GPs interferes with the neutralizing antibody response.In the present study,we systematically tested the influence of glycosylation sites in GP5 on PRRSV infectivity using the infectious cDNA clone.Our results demonstrated that mutants harboring single mutation on NGS at N30,N35,N44 or N51 in GP5 could be successfully rescued.However,only a part of mutants carrying multiple mutations at NGSs in GP5 could be recovered.In addition,the mutations on NGSs were stable in most of the mutant viruses by passage 10 times on MARC-145 cells,whereas,the mutation on NGS at N30 was reverted in rN30/44S and rN30/44/51.The glycosylation assay demonstrated that N35,N44 and N51 were the NGSs and the N30 was not the NGS.Then,we developed the mutant viruses carrying deglycosylation in GP5,and then the mutant viruses were subsequently inactivated and mixed with adjuvant.The results in mice immunization showed that 35,44 and 51 NGS play an effect on induction of neutralization antibody.Then,we developed three mutant viruses carrying deglycosylation in GP5(rN35S,rN44K and rN35/44S),and then the mutant viruses were subsequently inactivated and mixed with adjuvant.We tested whether pigs administrated with inactivated vaccine could well generate the protective immune response against PRRSV challenge.The efficacy of the inactivated mutant viruses were compared with that of the inactivated wild-type PRRSV.It was found that pigs in the groups administered the inactivated rN35/44S could induce higher serum neutralizing(SN)antibodies at six weeks post-vaccination.Following challenge with PRRSV,pigs in groups inoculated with inactivated rN35/44S showed lightest clinical signs,lowest viremia and pathological lesion of lungs,as compared to those of the inactivated viruses group and challenge control groups.These findings provide a foundation to develop the new type of vaccine.