Evaluation Of Function Proteins And Key Amino Acid Sites For Tregs And Neutralizing Antibody Inductions Of Procine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus(PRRSV)has now been one of the most important diseases in pigs,leading to significant economic losses in swine industry worldwide.The virus genome had a great variant.PRRSV negatively modulates host immune responses,resulting in persistent infection.Adaptive immune responses of the host act as an important source of selective pressure in the evolutionary process of the virus.However,the mechanism of immunosuppression and immune escape of PRRSV is unclear yet.In this study,a novel PRRSV isolate with deletion in GP3 gene was found in mid-eastern China.It was found that highly pathogenic PRRSV(HP-PRRSV)strain BB0907 could induce Tregs in the co-culture system of monocyte-derived dendritic cells(MoDCs)and peripheral blood mononuclear cells(PBMCs).And the 15N and 46R amino acid(aa)residues of N protein were critical for induction of Tregs.Meanwhile,the resistant strains to the neutralization antibody(NAb)were generated under NAb pressure.It was found that the aa residues 102 and 104 in GP5 and 70 in M were the key sites in NAb against PRRSV.All these data should be useful for understanding the mechanism of immunity to PRRSV and monitoring the antigen variant strains in the future.The contents of this research contain four parts as following:1.A novel isolate with deletion in GP3 gene of PRRSV from ChinaThe PRRSV strain SH1211 was isolated from a large-scale pig farm with PRRSV infected in Shanghai in 2012.The full-length genome of the SH1211 strain was sequenced and analyzed.The SH1211 genome was 15,313 nt in size,excluding the polyadenylated sequences,and shared 94.9%nucleotide sequence identity with the HP-PRRSV strain,JXA1.A deletion at aa positions 68 and 69 was identified in the predicted GP3 protein of SH1211,compared with the GP3 of North American PRRSV isolates.The predicted GP2 and GP5 proteins of the SH1211 strain shared only 91.5%and 85.1%aa sequence identities with those of the JXA1 strain,respectively.Phylogenetic analysis showed that the complete genome of SH1211 is most closely related to the HP-PRRSV strains isolated in China.However,based on the coding sequences of the GP2 and GP5 proteins showed that SH1211 is most closely related to the QYYZ strain.A recombination analysis indicated that the SH1211 strain might have been generated through recombination events between the JXA1 and QYYZ genomes,in which the GP2 and GP5 coding sequences were exchanged.Thus,SH1211 is a novel PRRSV strain with significant variation.Our genetic analysis of the SH1211 strain enriches the virus epidemiological data.2.Examination of the function protein of PRRSV and the key aa sites related toinduction of TregsIn this study,we constructed the culture method of MoDCs and the detection method of CD4+CD25+Foxp3+ Tregs.It was found that HP-PRRSV strain BB0907 induced more Tregs than classical PRRSV strains S1 by using the co-culture system of MoDCs and PBMCs.PRRSV-induced Tregs had a significant suppressive effect on proliferation of PHA-stimulated lymphocytes.In order to definite the Tregs epitopes of PRRSV,we expressed the recombinant GP5,M and N proteins of HP-PRRSV by using baculovirus expression systems.It was found that only N protein could induce Tregs.Synthetic peptides covering the whole length of N protein showed that three aa regions,15-21,42-48 and 88-94,in N protein played an important role in Treg induction.Recombinant PRRSVs rescued from HP-PRRSV infectious cDNA clones showed that the 15N and 46R residues of N protein were critical for induction of Tregs.Moreover,the phenotype of induced Tregs closely resembled that of TGF-?-secreting Th3 Tregs in swine.Our results should be useful for understanding the mechanism of immunity to PRRSV in the future.3.Examination of the key aa sites in GP5 and M proteins regulate the induction of neutralizing antibody against PRRSVIn this study,HP-PRRSV strain BB0907 was continuously passaged under the pressure of porcine NAb serum in vitro and five resistant variant strains BB5s,BB10s,BB20s,BB30s and BB34s were obtained.The results of neutralization assays showed that the NA titers of BB34s were significantly drcreased when compared with that of parent strain BB.Sequencing results showed that nine different aa substitutions occurred in the GP2(G198V),GP3(P69S,K71R,and S226P),GP4(D43N and F44S),GP5(Y102C and G104R)and M(R70K)proteins in BB34s when compared with those in BB strain.To determine the effect of the aa substitutions on neutralization,14 recombinant PRRSV strains containing these mutations were generated by site-directed mutagenesis using BB34s and BB infectious cDNA clones.The neutralization assay results revealed that the aa residues 102 and 104 in GP5 and 70 in M were the key sites in NAbs against HP-PRRSV.Alignment of GP5 and M sequences revealed that the variant aa residues were frequent among American type PRRSV strains.It may be helpful for monitoring the antigen variant strains in the field and developing new vaccine against PRRSV in the future.4.Pathogenesis of HP-PRRSV in China Tibetan swineTibetan pig is a special swine type.It has not been reported wheather it has susceptibility to HP-PRRSV.In this study,fifteen 4-week-old Tibetan piglets were divided into three groups each with 5 in the highland area.Group 1 was inoculated intranasally with HP-PRRSV strain BB0907.Group 2 was placed in contact with Group 2 two days after challenge.Group 3 was mock-infected and used as controls.All groups were isolately feeded and observed for 14 days after challeng.The results showed that 80%and 40%piglets in the inoculated and contacted groups had high temperatures(over 40.0°C)after 3-5 days,and showed clinical signs,which including depression,anorexia,lethargy,eye sticky secretions and hind limb paralysis.The viremia appeared on 4-14 days after infection.The main lesion was interstitial pneumonia.The TNF-?,IL-1? IL-6 and IL-10 expression in the serums,BALFs and lymph node tissues of the infected piglets were significantly increased compared with those in mock-infected control group.It indicated that HP-PRRSV had a high pathogenic to Tibetan pigs.It is necessary to detect and prevent PRRSV infection in this high-land region in China in the further.