Pathogenicity Of A Jxa1-like Strain Of PRRSV And The Immunogenicity Of Recombinant Adenovirus Vaccines

Porcine Reproductive and Respiratory Syndrome(PRRS)is one of the highly contagious infectious diseases caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)that seriously harm the pig industry.PRRSV is an important pathogen that can cause pig disease worldwide.Pigs of different genders,ages and breeds can be infected.Clinically,it can cause respiratory diseases and premature birth of pregnant sows,miscarriage and mummified fetus.Currently,the protective effects of current commercial inactivated PRRSV vaccines and live attenuated vaccines are very limited,and neutralizing antibodies and cell-mediated immune responses are weak.There is a risk that the vaccine strains are likely to spread in the field and have strong virulence.It is unable to provide sufficient conditions for the prevention and control of PRRSV.In this study,a PRRSV named 17-1 0-PI was isolated from a pig farm with PRRS syndrome from the Guangxi province.The successful isolation of the virus was verified by means of RT-PCR,CPE,IFA,etc.Ten pairs of primers were designed according to the PRRSV strain,and the whole genome sequence of the 17-10-PI was amplified and sequenced.To express the 17-10-PI strain and explore the source of its genome,the Nsp2 and GP5 genes was compared with the characteristic strains from China and the other countries.The four weeks old piglets confirmed to be free of PRRSV were inoculated with a JXA1-Like 17-10-PI strain.Explore the pathogenicity of 17-10-PI strain by observing the clinical syndrome,temperature,viremia,cytokines,and pathological changes of the test piglets.According to the PRRS epidemic prevention and control situation of China in recent years.The recombinant adenovirus candidate vaccine was constructed successfully.The evaluation of its immunogenicity and protection through animal experiments provides a reference for the early warning of the development of new vaccines.1.Whore genome sequencing and phylogenetic analysis of a JXA1-like strain 17-10-PI Porcine reproductive and respiratory syndrome virusThe obvious CPE has appeared after 17-10-PI strain was inoculated into Marc-145 cells.Meanwhile,the cell isolate 17-10-PI was identified by RT-PCR,IFA,and Western-Blot.The whole genome of the isolate is 15 320 bp in length(excluding the polyA),including a 5’-UTR(189 bp)and a 3’-UTR(149 bp).The results showed that the PRRSV 17-10-PI strain has closer relationship with JXA1-like stains,and the whole gene homology is 98.8%.The homology with the Lelystad virus(LV)strain and VR-2332 strain is 57.8%and 88.9%,respectively.2.Pathogenicity of 17-10-PI strain of porcine reproductive and respiratory syndrome virusPigs infected with the 17-10-PI strain,showed typical symptoms of viral infection and a febrile response starting from 2 days post-infection(dpi).The pigs infected with 1 7-10-PI had an early febrile peak at 6 dpi and a second peak at 9 dpi,they had average rectal temperatures that rose above 40℃ within 6-10 dpi.A piglet has died at 15 dpi.The average daily gain of piglets in the 17-10-PI group and the PBS group was 0.266 kg and 0.423 kg,respectively,and there was a highly significant difference between the two groups(P<0.01).Test results showed that the isolate could replicate in pigs and cause viremia,and cytokine concentrations of serum of 17-10-PI infected piglets increased at different degrees.The increase of IFN-α and IL-2 was relatively obvious.Necropsy and pathological examination showed that the 17-10-PI strain can cause obvious pathological damage such as stromal proliferative pneumonia and massive infiltration of inflammatory cells.3.Construction and immune effect in mice of recombinant adenovirus candidate vaccinesA synthetic gene,designated con-major,the encoding defined epitopes was used to generate a recombinant adenovirus designed pac-Ad5-con-major.The chimeric antigen included T-cell epitopes of the NADC30-like and JXA1-like lineage strains.Meanwhile,the NADC30-like lineage vaccine(pac-Ad5-30-GP5)has been successfully constructed using recombinant adenovirus as a vector.The immunologic test results of mice showed that the pac-Ad5-con-major and pac-Ad5-30-GP5 test groups can highly significantly increase the level of GP5 specific antibodies in the serum(P<0.05)at 35 days after immunization,compared with the commercial vaccine immunization group.The recombinant adenovirus immune group(pac-Ad5-con-major:85.12 pg/mL;pac-Ad5-30-GP5:86.61 pg/mL)can significantly increase the expression of IL-4 at 35 days after immunization(P<0.05)compared with the PBS group(60.13 pg/mL).The pac-Ads-con-major immunization group can induce a relatively significantly high level of IFN-y(506.49 pg/mL)better than the commercial vaccine immunization group(330.00 pg/mL),there is a significant difference between immunization groups(P<0.05).The pac-Ad5-con-major immunization group(2.17)can significantly increase the SI index compared with the other immunization groups(pac-Ad5-30-GP5:1.82;commercial vaccine group:1.78)(P<0.05),after PRRSV specific stimulation.4.Evaluation of the immune effect of recombinant adenovirus candidate vaccines in pigsThe immunologic test results of pig showed that the specific antibody level of the pac-Ad5-con-major immunization group reached a peak at 35 days after immunization,which was higher than that of the other immunization groups,and continued to be significantly higher than other immunized groups from 14 to 35 days after immunization(P<0.05).The specific neutralizing antibody titer of recombinant adenovirus immunization groups reached the peak(pac-Ad5-con-major:17.78±6.96;pac-Ad5-30-GP5:16.10±2.23)at 35 days after immunization.The expression level of IL-4 in the recombinant adenovirus immunization groups(pac-Ad5-con-major:52.97 pg/mL;pac-Ad5-30-GP5:55.49 pg/mL)are higher than the commercial vaccine group(50.29 pg/mL)at 35 days after immunization.Compared with the commercial vaccine group(214.60 pg/mL),the recombinant adenovirus group can significantly induce a higher level of IFN-γ(pac-Ad5-con-major:253.33 pg/mL;pac-Ad5-30-GP5:254.69 pg/mL)(P<0.05).After PRRSV specific stimulation,the recombinant adenovirus pac-Ad5-con-major immunization group(2.60)can extremely significantly increase the SI index(P<0.001),compared with the PBS group(1.42).Meanwhile,the recombinant adenovirus pac-Ad5-con-major immunization group was higher than the other immunization groups(pac-Ad5-30-GP5:2.11;commercial vaccine group:2.45).5.Challenge test of recombinant adenovirus candidate vaccines in pigsAfter the NADC30-like strain was challenged,the body temperature of the PBS group reached the peak(40.64℃)at 5 dpi,and the body temperature was higher than 40℃ within 2～9 dpi.In addition,the body temperature of each immunization group began to decrease after 5 dpi,and it was normal that the body temperature on each day was lower than 40.0℃,gradually.The recombinant adenovirus vaccines can highly significantly reduce the blood viral load of the experimental piglets(pac-Ad5-con-major:24.95 times,P<0.01;pac-Ad5-30-GP5:16.36 times,P<0.01),compared with the PBS group.The viral genome copies in the lung(5.88 times)and liver(5.94 times)of the commercial vaccine group were significantly higher than that in the pac-Ad5-con-major immunization group(P<0.05).Apart from,the viral genome copies of the recombinant adenovirus groups had a lower level than the commercial vaccine group in inguinal lymph nodes and other tissues.There are also varying degrees of reduction in the number.The pathological section results showed that in the PBS control group,there was more lymphocyte infiltration around the bronchus,a small amount of cell necrosis,obvious pulmonary interstitial hyperplasia,and a large number of alveolar cavity expansion.In the pac-Ad5-con-major,pac-Ad5-30-GP5 and commercial vaccine test groups,lung pathological sections showed local alveolar wall thickening,accompanied by a small amount of lymphocyte infiltration.In conclusion,a PRRSV JXA1-like lineage strain(17-10-PI)was successfully isolated,which can cause severe clinical symptoms and pathological changes in experimental piglets,and belongs to highly pathogenic PRRSV;The recombinant adenovirus vaccines of pac-Ad5-30-GP5 and pca-Ad5-con-major were successfully constructed;The recombinant adenovirus vaccines can effectively induce the humoral and cellular immune responses of experimental mice and pigs.Some immune indicators of recombinant adenovirus vaccine are better than the commercial vaccine,which can protect pigs from PRRSV to a certain extent.