| Porcine reproductive and respiratory syndrome（PRRS）is an acute,highly contagious viral major infectious disease caused by porcine reproductive and respiratory syndrome virus（PRRSV）that causes reproductive disorders and respiratory symptoms as typical clinical signs in pigs.Sub-lineage 1,Sub-lineage 5 and Sub-lineage 8 PRRSV strains are the popular strains in China in recent years.NADC30-like PRRSV strains belong to Sub-lineage 1 and their genomes are highly susceptible to recombination.The NADC30-like PRRSV strain has evolved endemically since its introduction to China,and no targeted vaccine is available for this strain.In this study,we conducted a molecular epidemiological survey,isolation and identification of prevalent strains and pathogenicity analysis of PRRSV in Sichuan Province,and constructed a recombinant pseudorabies virus co-expressing NADC30-like PRRSV GP5 and M proteins,investigated the biological properties and target delivery characteristics of recombinant pseudorabies virus,and evaluated the safety and immunogenicity of recombinant pseudorabies virus by inoculation with mice and pigs,and provided reference for the development and application of vaccines against NADC30-like PRRSV strains.The main studies are as follows:1.Molecular epidemiological survey of PRRSV in Sichuan and isolation and identification of epidemic strains and pathogenicity analysisThe 282 PRRSV nucleic acid-positive samples were detected from 539 samples collected in 13 cities in the Sichuan region from 2016 to 2020,and PRRSV-1 positive samples were detected for the first time in Sichuan.Phylogenetic analysis combined with the PRRSV-2 ORF5 gene sequences obtained from Sichuan region in NCBI revealed that there were five subtypes of PRRSV-2 strains in Sichuan region,which were Sub-lineage8.7（60.71%）,Sub-lineage 5.1（11.31%）,Sub-lineage 1.8（18.45%）,Sub-lineage 1.5（2.97%）,Sub-lineage 3.5（6.55%）.A total of 16 prevalent PRRSV strains were isolated in this study,and homologous recombination analysis revealed genetic recombination in four NADC30-like PRRSV strains.The results of pathogenicity analysis of NADC30-like PRRSV strain proved that CHSCDJY-2019 was a virulent strain,capable of causing typical clinical signs of PRRSV and high levels of viremia,but no mortality occurred in infected piglets.2.Construction and biological characterization of recombinant pseudorabies virus co-expressing NADC30-like PRRSV GP5 and M proteinsIn this study,the ORF5 and ORF6 gene fragments of PRRSV CHSCDJY-2019 strain were amplified by RT-PCR,and the target gene fragments were inserted into the p EGFP-g I/28K vector by homologous recombination technique to construct the transfer vector p EGFP-2A-NC-OFR5-6 after the introduction of cleavage peptides.The recombinant virus was obtained by homologous recombination,Crispr/cas9 technique,and the pure recombinant virus rPRV-NC56 was obtained after empty spot purification.The results of Western Blot and IFA identification showed that rPRV-NC56 was able to express the exogenous protein efficiently.Transmission electron microscopy of rPRV-NC56-infected cells showed that exogenous gene insertion and expression did not affect the assembly and morphology of PRV virus particles,while virus-like particles（VLPs）of approximately 40～60 nm in diameter,similar in size and morphology to PRRSV virus particles,were observed in the cytoplasmic vesicles of infected cells.The biological characteristics of rPRV-NC56 were not significantly different from the parental strain with a virus titer of 108.52TCID50/m L.Continuous cell passaging assays demonstrated that the rPRV-NC56 strain was genetically stable with stable exogenous gene insertions and virulence gene deletions.3.Safety and immunogenicity evaluation of rPRV-NC56 in miceThis study evaluated the safety and immunogenicity evaluation of recombinant pseudorabies virus rPRV-NC56 in mice.The results showed that there was no clinical manifestation and death in mice after rPRV-NC56 inoculation,no significant upregulation of IL-6 and TNF-αin serum,and no significant pathological changes in brain tissue.rPRV-NC56 inoculation of mice induced high levels of PRV and NADC30-like PRRSV-specific immune responses,which protected mice against lethal doses of PRV wild virus.The potency of neutralizing antibodies against NADC30-like PRRSV in the sera of rPRV-NC56-immunized mice（1:13.64）was significantly higher than that of HP-PRRSV（1:6.82）4 weeks after booster immunization.The proliferation index（SI value）of the NADC30-like PRRSV inactivating antigen-stimulated group of rPRV-NC56-immunized mouse spleen cells was extremely significantly higher than that of the HP-PRRSV inactivating antigen-stimulated group.4.Mechanism of rPRV-NC56 induced early mucosal immune responseIn this study,two immunization routes,intranasal and intramuscular,were designed,and immunohistochemistry（IHC）,q PCR and immunofluorescence were used to analyze the in vivo colonization,targeted antigen delivery,and induction of immune-related cell differentiation and migration in mucosal tissues of mice with different immunization routes of recombinant virus.The results showed that rPRV-NC56 had an earlier colonization time and higher viral load in mucosal tissues such as lung and intestine after intraintranasal immunization than intramuscular injection immunization,whereas intramuscular injection immunization had an earlier colonization time and higher viral load in the spleen and kidney.rPRV-NC56 targets antigen delivery to APCs such as epithelial cells,DCs,and macrophages in the lung and intestine.Intranasal immunization induced a significant upregulation of MCP-1 expression and a significant increase in the number of DCs,monocytes,macrophages,CD4+T lymphocytes,and CD45+leukocytes in the lung and intestine compared with intramuscular immunization,and induced a significant increase in the number of s-Ig A-secreting cells in the lung and intestine.Intranasal immunization with PRV-NC56 induced higher levels of PRV and NADC30-like PRRSV-specific humoral and cellular immunity after booster immunization than intramuscular immunization.5.Evaluation of the safety and immunization effect of rPRV-NC56 in pigsSafety of rPRV-NC56 was evaluated by vaccinating newborn piglets and gestating sows.No clinical manifestations and normal growth after intramuscular and intranasal inoculation of rPRV-NC56 with 108TCID50/m L in newborn piglets.Gestating sows inoculated with 108TCID50/m L of rPRV-NC56 showed no clinical manifestations,and the number of litters and healthy piglets born in the inoculated sows were not significantly different from the control group.Sentinel piglets and sows tested negative for antibodies to g B and GP5 proteins.The immunization effect of rPRV-NC56 was evaluated by intramuscular and intranasal immunization of weaned piglets in terms of humoral immunity,cellular immunity and attack protection.Intramuscular and intranasal immunization with PRV-NC56 induced high levels of PRV-specific neutralizing antibodies（1:70.30 and 1:91.38）and PRRSV-specific neutralizing antibodies（1:18.31 and 1:20.7）in piglets 2 weeks after booster immunization,with negative results forPRV g E and PRRSV N protein antibodies in all groups.2 weeks after booster immunization,piglets in each group were attacked with PRV-XJ strain and PRRSV CHSCDJY-2019 strain,respectively.100%protection against PRV attack after immunization with rPRV-NC56.Immunization with rPRV-NC56 greatly reduced the clinical manifestations of NADC30-like PRRSV infection and viremia caused by NADC30-like PRRSV,and reduced the viral load of PRRSV in various tissues,and there were no significant pathological changes in the lungs and hilar lymph nodes of the immunized piglets after the attack. |
