Construction And Immunized Piglets Evaluation Study Of Recombinant Ndv Vector Vaccines Candidates Of NADC30-like PRRSV And HP-PRRSV

Since porcine reproductive and respiratory syndrome(PRRS)was first reported in China in1995,it has caused great losses to the domestic pig industry.PRRS is caused by the PRRSV virus,which belongs to the family Arteriviridae and genus Arterivirus.The original prevalent PRRSV strain in China was type Ⅱ.In 2006,PRSSV was prevalent in China,with high fever and high mortality after infection,which caused devastating damage to the pig industry.It was named highly pathogenic PRRSV(HP-PRRSV),and the representative strain was JXA1.Due to the characteristics of rapid mutation of PRRSV,the emergence of multi-lineage variants has increased the difficulty of prevention and control,and the variants are mainly Lineage 1,3,5 and 8.The NADC30-like strain was imported from other countries in 2013,which further complicated the current situation of epidemic prevention in China.The dominant strains were JXA1-like and NADC30-like lineages.However,there is a risk of homologous recombination with wild strains,the emergence of new variants,and the repeated infection of pig farms with PRRSV and even the occurrence of new epidemics.Therefore,there is an urgent need for a novel vaccine against HPPRRSV and NADC30-like.SD-A19(NADC30-like)-ORF5 and YN-LQ(JXA1-like HP-PRRSV)-ORF5 sequences were inserted into the NDV vector plasmid,and monovalent or bivalent vaccines expressing GP5 protein were obtained by reverse genetics.The recombinant virus stably expressed the foreign protein,which was verified by PCR,restriction enzyme digestion,Western blot and indirect immunofluorescence.After confirming that the recombinant virus did not enhance the virulence,the mouse immune experiment was carried out.Specific antibodies against HP-PRRSV and NADC30-like were produced in mice immunized with r L-link-GP5 at 35 days after the first immunization.Flow cytometry analysis of CD3,CD4 and CD8 cells in splenic lymphocytes showed a large number of differentiation of CD3+CD4+ T cell subsets and increased intracellular cytokine secretion of IL-4 and IFN-γ at 35 days after the first immunization.The results of T lymphocyte proliferation assay showed that the SI value of r L-link-GP5 was 3.14,1.16 times that of r L-A19-GP5 and 1.12 times that of r L-LQGP5 after specific stimulation of PRRSV,indicating that RL-LINK-GP5 could induce a certain degree of cellular immunity in mice.The recombinant vaccine candidate has certain immune effect in mice.The results of piglet immune experiments showed that the specific antibody OD value of r Llink-GP5 recombinant candidate vaccine against HP-PRRSV was 2.33,and the NADC30-like antibody OD value was 2.03.The r L-link-GP5 and NADC30-like vaccines could achieve 1:28and 1:45 neutralizing antibodies against HP-PRRSV and HP-PRRSV,respectively.The results of cytokine ELISA showed that the recombinant vaccine candidates mainly stimulated IFN-γproduction,and only r L-A19-GP5 and r L-link-GP5 recombinant vaccine candidates achieved better stimulation of IFN-γ secretion.Flow cytometry analysis of CD3,CD4 and CD8 cells in spleen lymphocytes showed that only r L-link-GP5 recombinant candidate vaccine could induce differentiation to some extent,while all three recombinant candidate vaccines could induce cellular immune responses 42 days after the first immunization.These results indicated that r LLQ-GP5,r L-A19-GP5 and r L-link-GP5 could improve the immunity and cellular immunity of piglets to a certain extent.In the piglet challenge protection experiment,the body temperature of piglets in the three recombinant vaccine groups did not significantly increase,and the organ viral load was reduced to varying degrees,which effectively protected piglets.r L-link-GP5 recombinant vaccine had a better protective effect than the other two recombinant vaccine candidates.In this study,the recombinant NDV with stable expression of HP-PRRSV and/ or NADC30-like GP5 proteins was successfully rescued,and the immune experiments in mice and piglets were conducted to verify the immune effect of the prepared vaccine,which provides a new idea for the prevention and control of PRRSV.