Isolation And Identification Of PRRSV,Cnstruction And Immungenicity Of Recombinant Adenovirus Vaccine For PRRSV

Porcine reproductive and respiratory syndrome(PRRS)is one of the most economically devastating diseases for the pig industry worldwide.The disease can cause reproductive problems in sows(stillbirth,mummified fetuses,miscarriage,and resulting in high mortality of newborn piglets)and growth disorders of piglets(mainly caused by respiratory diseases,characterized by interstitial pneumonia).It has resulted in huge economic losses in the world every year,and has been reported can increase the host's susceptibility to other pathogens.In addition to the direct loss of productivity,more resources and manpower are required to prevent and monitor PRRSV pathogens.Recently,PRRSV has broken the regional restrictions in the world,the new imported cases were constantly appearing in our country in recent years,which greatly increases the difficulty of PRRS prevention and control in China.To investigate the epidemiological characteristics of the disease,clinical samples of lung,spleen,and lymph nodes were collected and analyzed in different suspected farms,including Heilongjiang,Jilin,Liaoning,Inner Mongolia,and Guangxi.In the study,corresponding recombinant adenovirus candidate vaccines were also constructed to provide a theoretical basis for the design of different subtypes of PRRSV vaccines.To analyze the molecular epidemiological characteristics of PRRSV in our country from2016 to 2020,2,773 suspected clinical samples were collected in nine provinces including Heilongjiang,Jilin,Liaoning,Inner Mongolia,and Guangxi.After RT-PCR detection and sequencing,a total of 19 PRRSV full-length gene sequences and 99 GP5 gene sequences were obtained.Phylogenetic Analysis of GP5 gene of 63 HP-PRRSV strains showed that 21 were NADC30-Like strains,7 were NADC34-Like strains,and 7 were QYYZ-Like strains,one strain was LV-Like strain.At present,HP-PRRSV-like strains as the main epidemic strains are still prevalent in our country,and the percentage cases of NADC30-Like strain is on the rise.NADC34-Like strain is an imported strain has been first reported in Heilongjiang and Jilin provinces.According to the epidemiological survey results,PRRSV in China is the genetic diversity,the result provides a theoretical basis for clarifying the epidemic evolution rule of PRRSV,as well as the deployment of the epidemic prevention and control in the futureSubsequently,the PRRSV-ZDXYL-China-2018-1 strain was isolated and identified,and its pathogenicity was verified through infectivity experiment.The results of gene sequence analysis showed that PRRSV-ZDXYL-China-2018-1 strain belonged to NADC34-Like in type2 lineage,and was closely related to US strain 1-7-4 based on GP5 gene analysis,it was sharing96.7% and 97.1% nucleotide identity to NADC34 and US-ISU10 strains,respectively.The homology with the NADC30-Like strain in China is 85.6%.The results of the pigs infection experiment showed that the isolated strain can cause fever,general dyspnea and other clinical manifestations in piglets,and lead to moderate interstitial pneumonia according to the histopathological analysis.In addition,recombinant adenovirus vaccine candidates against the newly emerged NADC34-Like strain were constructed in the study,the immunogenicity of the vaccine was investigated in mice and piglets.The results of Western Blot and indirect immunofluorescence showed that the recombinant adenovirus vaccines pac-Ad5-34-GP3,pac-Ad5-34-GP5 and pac-Ad5-34-GP35 were successfully constructed.During mice immunization test,the mice could produce specific antibodies,neutralizing antibodies,and cytokines IFN-? and IL-4 when mice were vaccinated with recombinant adenovirus vaccine.After 35 days of immunization,the levels of IFN-? cytokine expression in the pac-Ad5-34-GP3,pac-Ad5-34-GP5 and pac-Ad5-34-GP35 groups were significantly higher(P<0.05)than those of in the PBS control and the adenovirus control group.pac-Ad5-34-GP3,pac-Ad5-34-GP5 and pac-Ad5-34-GP35 experimental groups The expression level of IL-4cytokine was 1.37 times,1.49 times,1.36 times and 1.48 times higher than those of the PBS and the adenovirus group,respectively.Furthermore,all recombinant adenovirus vaccine showed high levels of lymphocytes proliferation in the experimental groups compared to the PBS and the adenovirus groups,indicated that the vaccines can cause corresponding Th1 and Th2 immune responses,and produce humoral and cellular immune responses against PRRSV.After inoculation with PRRSV recombinant adenovirus in pigs,specific antibodies,neutralizing antibodies,and cytokines IFN-? and IL-4 were detected in experiment pigs.35 days after immunization,the levels of IFN-? cytokine in the pac-Ad5-34-GP3,pac-Ad5-34-GP5 and pac-Ad5-34-GP35 experimental groups were significantly higher(P<0.05)than those of the PBS and the adenovirus group.The expression levels of IL-4 cytokines in the groups pac-Ad5-34-GP3,pac-Ad5-34-GP5 and pac-Ad5-34-GP35 were 1.52 times,1.59 times,1.30 times,1.60 times,1.92 times,and 1.36 times higher than those of the PBS control group and the adenovirus group,respectively.Recombinant adenovirus Vaccines of pac-Ad5-34-GP3,pac-Ad5-34-GP5 and pac-Ad5-34-GP35 showed significantly higher(P<0.05)stimulation index compared to the PBS and the adenovirus control groups.All vaccines can cause corresponding Th1 and Th2 immune responses based on animal experiment.After the challenge,no obvious clinical symptoms were observed in the immune groups compared with the control group,vaccinated animals could reduce the occurrence of viremia,and the occurrence of viremia was alleviated,with no obvious pathological changes in the lungs,indicating that recombinant adenovirus vaccine could provide a good protective immunity and produce a good humoral and cellular immune response at the same time.