Efficacy Evaluation Of Two MLV Vaccines Against Emerging PRRSV-2 And Development Of PRRSV-2 NSP1β Mutant Vaccine Strain And Blocking Monoclonal Antibody

Porcine reproductive and respiratory syndrome(PRRS)caused by PRRSV is an important disease.The first PRRSV named CH-1a was isolated in 1996 in China.In 2006,a highly pathogenic PRRSV(HP-PRRSV)characterized by unique discontinuous 30 aa deletion in NSP2 protein emerged causing heavy economic loss to pig industry.Seven commercial modified live virus vaccines are widely used to control PRRS in China with their corresponding virus strains including VR2332 MLV,R98,JXA1-R,HuN4-F112,GDr180,TJM-F92 and CH-1R,respectively.All the vaccine strains were generated by attenuation of parental strains.Since 2006,the highly pathogenic PRRS has been effectively controlled with the widespread use of MLV vaccines.However,NADC30-like PRRSV-2 strains were reported in 2013 in China.The epidemic situation of PRRSV-2 became complicated.1.Genomic,cell replication characteristics and pathogenicity analysis of emerging PRRSV-2In this study,42 out of 132 tissue samples collected from pig farms in central and east of China between 2016 and 2018,were confinned to be PRRSV-2 positive by RT-PCR.Base on phylogenetic analysis of ORF5 and ORF6,the proportion of lineage 8 was 18.38%.Three isolates closely related to QYYZ strain belonged to lineage 3,while three other strains were clustered into lineage 1 represented by NADC30.Four PRRSV-2 strains named JS18-3,ZJnb16-2,SDbzl6-2 and HNXX16 were successfully isolated and sequenced.The results of complete genome evolution analysis indicated that JS18-3 belonged to classical lineage 8.ZJnb16-2 belongs to lineage 8 which is closely related to HP-PRRSV,while SDbz16-2 and HNXX16 belong to lineage 1.The recombination analysis results showed that ZJnb16-2 was a recombinant virus between lineage 8(JXA1-like)and lineage 3(QYYZ-like),SDbz16-2 was a recombinant virus among lineage 1(NADC30-like)and lineage 8(JXA1-like).JS18-3 was a triple recombinant strain from lineage 8,1 and 3(CH-la-like,NADC30-like,GDsg-like).There is no recombination signal for HNXX16.Cell tropism results showed that ZJnb16-2,HNXX16 and SDbz16-2 could not replicate in Marc-145 cells,while JS18-3 could.In addition,4871-6635 of JS18-3 shared the highest 99.3%identity with HP-PRRSV representative strain JXA1 in nucleotide sequence indicating that it is evolving towards HP-PRRSV.Compared with the original CH-la-like strain JX07,replication enhancement and severe cytopathic effects were observed in JS18-3 infected Marc-145 cells and PAMs.The pathogenicity of ZJnb16-2,SDbz16-2 and JS18-3 was also evaluated.The results showed that ZJnb16-2 had relatively strong virulence compared with SDbz16-2,and the pathogenicity of JS18-3 strain was significantly stronger than that of the parental strain CH-la.Pathogenicity results indicated that piglets inoculated with ZJnb16-2,SDbz16-2 and JS18-3 presented persistent fever,dyspnoea,serious microscopic lung lesions and lymph node congestion.This study confirmed that the emerging lineage 1 and 3 tend to recombine with other lineages.Recombination led to the emergence of new pathogenic PRRSV-2 in China,which may evade the protective immunity induced by conventional vaccines.Therefore,it is urgent to update the vaccine strains and develop a universal PRRSV vaccine.2.Clinical protective effect of two MLV vaccines against ZJnb16-2 and efficacy evaluation of two MLV vaccines against emerging PRRSV-2ZJnb16-2 was a recombinant PRRSV-2 with lineage 3 genome features.In this study,efficacy evaluation of two representative MLV vaccines named HuN4-F112 and VR2332 MLV against ZJnbl6-2 was performed.The results showed that the viremia of both vaccine virus was detectable at 7,14,21 days post vaccination.The results of cross-protective efficacy showed that piglets in HuN4-F 112/ZJnb 16-2 group presented lower body temperature,higher average daily weight gain and milder clinical symptoms as compared to VR2332-MLV/ZJnb16-2 and Mock/ZJnb16-2.Viremia reduction was observed in serum from immunized piglets at 21 days after challenge.However,all the vaccinated piglets exhibited different degrees of lung and lymph node lesions.Microscopic lesion including collapsed alveoli with infiltration of numerous inflammatory cells in alveolar spaces and exfoliated epithelial cells in the bronchiole could be observed in all challenged piglets under microscopic examination.In coclusion,both VR2332 MLV and HuN4-F112 could not provide fully protection against ZJnb16-2 infection.In addition,viral persistence in clinical environment greatly increases the possibility of recombination with epidemic isolates leading to possible reversion to virulence.The overall efficacy of current MLV vaccines against emerging PRRSV-2 was conducted in terms of cross-reactivity of neutralizing antibody and cellular immunity.Hyperimmune serum with neutralization titer between 1:16-1:64 were prepared from immunized pigs with vaccine strains HuN4-F112(lineage 8)and JK-100(lineage 5).Neutralization experiments based on PAMs showed that both HuN4-F112 and JK-100 derived neutralization serum could not neutralize ZJnb16-2,SDbz16-2.JS18-3 could replicate on Marc-145 cells.Subsequently,the cross neutralization response to JS18-3 was evaluated using the neutralization serum against HuN4-F112.Results showed that the neutralization titer against JS18-3 was only 1:6-1:8.These results indicated that neutralizing antibodies produced by vaccine immunization could only neutralize closely related strains.No cross reactivity against emerging lineage 1 and 3 PRRSV-2 was observed.IFN-y secreting cells(SC)targeting HuN4-F112,VR2332 MLV,ZJnb16-2,JS18-3,HNXX16 were detected in blood from 21 and 28 days post vaccination.The number of IFN-y-SC was extremely variable(20-100 IFN-y-SC per 106 PBMC).This also confirmed the existence of cellular immunity cross reactivity against different lineages of PRRSV-2 in China.In summary,aberrant and delayed cell-mediated immune responses after vaccine immunization led to the insufficient cross-protection effect of MLV vaccine against emerging virus.3.Rescue of HuN4-F112 vaccine viruses with either NSP1β point mutation or NSP1 random mutationType I interferon is essential for regulating adaptive immune response.However,MLV vaccines could also suppress type Ⅰ IFN production which is a tremendous benefit for the vaccine virus to survive in the host.Therefore,the development of a vaccine that its IFN suppression function was attenuated or eliminated and could stimulates the interferon response will improve its cross-protection efficiency.NSP1βis an importante type I IFN viral antagonists encoded by PRRSV.Its 121-135aa is closely related to the IFN suppression function.Therefore,we selected NSP1β as the research target.In this study,an infectious cDNA clone of PRRSV-2 vaccine strain HuN4-F112 was generated.The virus was successfully rescued through transfecting recombinant plasmid to Marc-145 cells.The mutant strains(vRR128129AA,vKR124128AA,vL126A and vL135A)in the coding regions of NSP1β proteins were successfully rescued based on the infectious clone of HuN4-F112.Infection test results showed that the growth characteristics of the mutant strains were similar to the parent strain HuN4-F112 in Marc-145 cells.However,the mutant strains of vRR128129AA and vKR124128AA grew slowly and were eliminated at 48 hours post infection in PAMs.In contrast,HuN4-F112,vL126A and vL135A replicates fast over time.qPCR results showed that vRR128129AA and vKR124128AA up-regulated expression of IFN-α,IFN-β and ISG15.Hence,the introduction of the mutation allows the vaccine virus to be rapidly eliminated by macrophages,which leads to transient infection.The inhibitory effect for innate immunity response was weakened and mutant virus could trigger a transient immune response.The likelihood of possible reversion to virulence for vRR128129AA and vKR124128AA was solved.The triggered interferon response will effectively modulate the adaptive response.Mutant virus could be applied as new vaccine strains candidate for PRRS prevention and control in the future.On this basis,we introduced random mutations into NSP1 coding gene using the error-prone PCR in order to identify more interferon-sensitive mutations.The randomly mutated NSP1 encoding sequences was subcloned and sequenced.Sequencing results showed that each clone contained 6-9 point mutations in nucleotide sequence at different locations.As mentioned aboved,infectious clone of PRRSV-2 HuN4-F112 containing NSP1 gene with random mutations gene was successfully constructed.The proportion of mutant plasmids in the positive plasmid was 20%.Partially constructed recombinant plasmids were successfully transfected into Marc-145 cells to save random mutation virus.This lays a solid foundation for the subsequent discovery of multiple interferon-sensitive sites on the PRRSV genome by perforrming interferon pressure to the mutant virus combined with high-throughput sequencing technology,and provides a new method for the development of PRRSV interferon-sensitive vaccine.4.Preparation of monoclonal antibody which could block PRRSV-2 from infecting PAMsPRRSV-2 utilizes the same receptor when infecting target cells.Hence,monoclonal antibody against receptors could potentially block the infection of different lineages PRRSV-2.pCD163 is the core receptor for PRRSV infecting target cells.In this study,the recombinant plasmid pFAST-HTB-pCD163-SRCR5-9 was successfully constructed.pCD163-SRCR5-9 protein was expressed in the supernatant of cells after transfecting bacmid into sf9 cells.In vitro virus combination test results showed that purified pCD163-SRCR5-9 protein could bind to ZJnb17-1 and HNXX16 strains,thereby blocking the virus from binding to cell surface receptors.Sera from pCD163-SRCR5-9 protein immunized mice were able to prevent viral infection in PAMs.12 monoclonal antibodies against pCD163-SRCR5-9 were developed using traditional monoclonal antibody technology combined with IFA.Monoclonal antibodies 8H2 and 4H7 had inhibitory effect on PRRSV invasion.The titer of the infected virus reduced 100-1000 fold on average and the virus copy number decreased about 104.The epitope identificated by 8H2 and 4H7 were located in the PSTI and SRCR6 region of pCD163 protein and the epitopes were located in the 1-15 and 1-30 amino acid,respectively.The broad spectrum blocking monoclonal antibody could be used to treat infected sows of high economic value.Identification of key areas related to PRRSV infection greatly expanded the application of gene editing technology in PRRS resistant pig breeding.