Construction And Vaccine Studies On A Pseudorabies Virus With Deletions In GE And GI And A Pseudotyped Virus For Avian Influenza Virus

1 Study on a gE^-/gI^-mutant stainPseudorabies(Aujeszky's disease) is one of the main diseases threatening the pig industry.Eradicating PRV in the United States and Europe has shown great progress,but PR is still an endemic problem in China and many other countries.In the process of controlling PRV,the ability to detect gE antibodies in infected pigs is crucial.In this study, we report the generation of a recombinant PRV virus with gI and gE gene deletions and the testing of its immunogenicity and protective immunity in vaccinated Balb/c mice and piglets.The main research was described an follows.1.1 Construction of PRV Ea gE^-/gI^-mutant stainPseudorabies virus(PRV) belongs toαherpesvirus family with the characteristics of neurotropism and strong latency,gE and gI are main virulent genes of pseudorabies virus. They all play important roles in the growth of PRV in nerve tissues.Transfer plasmid pIESE was generated by digesting the plasmid pIECMV with Stuâ… and BstEâ…¡.Sequence analysis dedicated there was about 1247bp deletion between Stuâ… site of gI gene and BstEâ…¡site of gE gene.Genomic DNA of PRV Ea strain was co-transfected into IBRS-2 cells with transfer plasmid pIESE.After obvious cytopathic effect(CPE) was observed on day 2 post-transfection,the infected cells containing recombinant pseudorabies PRV Ea gI^-/gE^-viruses were harvested,frozen and thawed three times and virus isolated by plaque formation on IBRS-2 cells.The recombinant virus was confirmed by Southern blotting and indirect immunofluorescence assay(IFA),and results showed the ORF of gI and gE genes had already been deleted by the way of homologous recombination.1.2 Analysis of immunization and virus challenge1.2.1 Immunization and virus challenge in miceFour groups of seven 4-week-old female Balb/c mice were each inoculated at footpad with either 10⁵ TCID₅₀ of recombinant PRV gI^-/gE^-vaccine,or 105 TCID₅₀ of PRV gG^-/LacZ⁺(Ea strain) vaccine,or 10⁵ TCID₅₀ of a commercial PRV gE^-(Bartha strain) inactivated vaccine or 100μL of DMEM.Mice were boosted once 28 days later with the same dose of inoculum respectively.Sera were collected at 0,21,28,35,42,49 and 56 days post vaccination for immunoassays.Mice then were challenged intramuscularly with 2×10⁶ TCID₅₀ of PRV Ea.After vaccination of mice,PRV ELISA and neutralizing antibodies were detected as early as 14 days after a single immunization in the sera of mice immunized with all of 3 of inactive vaccines.Neutralizing antibody titers increased significantly after the boost,and came to their peak value at day 42,then decreased between 42-49 days.No anti-gE antibodies were detected in mice immunized with PRV gI^-/gE^-or with commercial PRV gE^-at any of time point tested.No Antibodies to PRV and PRV gE were detected in control mice that received DMEM.The data showed that 10⁶TCID₅₀ PRV Ea gE^-/gI^-was effectively at protection 2×10⁶ TCID₅₀PRV Ea challenge. 1.2.2 Immunization and virus challenge in pigsTwenty 14-day-old piglets tested negative for PRV antibodies were randomly divided into four groups of five.Each group of piglets was inoculated intramuscularly with either 10⁵TCID₅₀ of PRV gI^-/gE^-vaccine or 10⁵TCID₅₀ of PRV gG^-/LacZ⁺ vaccine,or 10⁵ TCID₅₀ of PRV gE^-(Bartha strain) inactivated vaccine or 2ml of DMEM,and boosted once by the same dose and the same route 28 days later.Sera were collected at 0,21,28, 35 and 42 days post vaccination for immunoassays.All the pigs then were challenged intramuscularly with 5×10⁶ TCID₅₀ of PRV Ea strain.PRV ELISA,PRV gE-ELISA and microneutralizing assay were performed to assess antibodies against PRV.Antibodies to PRV could be detected by PRV ELISA and microneutralizing assay 14 days after vaccination.Anti-gE antibodies were not detected in 96-well microtiter plates coated with PRV gE monoclonal antibody in the PRV gE^-groups.In the virus challenge study,both vaccinated groups and control group were challenged with 5×10⁶ TCID₅₀ of the virulent PRV Ea strain.No clinical sign of PRV infection was observed with pigs vaccinated with recombinant virus PRV gI^-/gE^-and PRV gG^-/LacZ⁺,and all pigs of this group survived.In the other group vaccinated with commercial PRV gE^-,two pigs displayed obvious clinical symptoms(fervescence,diarrhea,disgorging,incoordination etc) on day 5 following challenge,and died on day 10 after challenge.Pigs in the DMEM control group showed neurological symptoms of PRV infection on day 2 following challenge and all died on day 7 after challenge.The HA protein is involved with receptor binding and eliciting immune responses, and also represents the best understood model system for cell entry.In this study,we incorporated hemagglutinin(HA) protein of H5N1 into HIV particles,and generated H5 hemagglutinin pseudotyped virus(HIV/H5-HA),and evaluated its protection by virulent wild-type avian influenza virus(AIV) challenged in mice.1.2.3 The packaging and detecting of HIV/H5-HAHA gene of avian Influenza Virus(AIV) H5N1 subtype was amplified by RT-PCR and cloned into pcDNA3.1⁽⁺⁾ vector,then were cotransfected with retroviral vectors HIV-1 gag/pol encoded,pCMVâ–³S.2 andthe HIV packagable vector encoding aβ-galactosidase (β-gal) reporter gene,pHR'-CMVLacZ into 293T cells.The HA protein expression could be verified by Western blot.Three main bands were seen which is consistent with the expected molecular weight for the full-length HA0 and the cleaved subunits HA1 and HA2.Further,HA protein expression was comfirmed by FACS,EM and cell-cell fusions. Infection activity of HIV/H5-HA were confirmed by LacZ staining and infecting 293Tcells.To test whether the AIV/H5-HA was infectious and displayed the similar host range as AIV,we infected 293T,BHK,Vero,PK-15,IBRS-2 and MDCK cell strains with HIV/H5-HA.The results showed that HA pseudotyping particles have wide cell tropism.1.2.4 Reproducing entering host cell mechanism of AIVIn the experiments,Hemagglutination and treatment with NH₄Cl tests were taken to assess the pathway of HIV/H5-HA entering host cell.For hemagglutination test, HIV/H5-HA was detected to make chicken erythrocyte agglutinate,and HA titer≧1:32. We treated the Vero cells with various concentration of ammonium chloride(0-50mM) before infection.The results displayed that treatment with 40mM NH₄Cl could cause complete inhibition of infection by HIV/H5-HA.There existed linear correlation between OD₄₂₀ value and the concentration of NH₄Cl for both of HIV/H5-HA(y=-0.028x+1.473, R²=0.960).It showed that HIV/H5-HA entering the host cells perform a pH-dependant manner.All of above results revealwe developed a pseudotyped virus system for studying AIV entering host cell mechanism and immunologic response between AIV and host cells.1.2.5 Immunization and virus challenge studies in miceFive groups of six 6-week-old female Balb/c mice were injected intramuscularly,the first 3 groups injected with various HIV/H5-HA,while IA group injected with inactivated AIV and control group with 100μl PBS.HI was performed to detect antibodies in sera. HI antibodies could be detect in HIV/H5-HA inoculated groups but not found in both inactivated AIV group and PBS inoculated group on 7-day post-vaccination,and compared with IA and control groups,HI titers induced by HIV/H5-HA increased 2-4 fold.The mean HI titers increased significantly after immunization for all vaccinated groups in the following week,and reached a maximum were on 7 days after challenge.In the challenge experiments,mice in the control group showed severe signs of illness (apathy,paralysis),and lost approximately 26.22%of their body weights,and died within 6 days post-challenge.All of the mice in vaccinated groups appeared slight sick within 7 days post-challenge.Body weights of mice in vaccinated groups lost approximately12.78%-21.07%within 7 days post-challenge,but regain their body weights to the pre-challenge level in the following week.HIV/H5-HA in this report were effective at protect mice against a lethal challenge by 10⁷ EID₅₀ of HPAIV H5N1 virus.