Construction Of Recombinant Fowl Adenovirus Expressing VP2 Protein Of Infectious Bursal Disease Virus

Fowl adenovirus serotype 4(FAdV-4)CH/HNJZ/2015 strain is a highly pathogenic epidemic strain isolated from Henan,China in 2015,belonging to species C in genus aviadenovirus,Adenoviridae.It is a nonenveloped double-stranded DNA virus,which is the main pathogen of Hepatitis-hydropericardium syndrome(HHS),but its pathogenesis is still unclear.The function of many unknown genes remains to be seen.Therefore,it is necessary to establish a reverse genetic manipulation system for the FAdV-4 CH/HNJZ/2015 strain to help resolve these issues.Adenovirus has the advantages of relatively stable virions,low pathogenicity,wide host range,easy genetic manipulation,high virus titer,easy concentration and storage,and ability to accommodate larger exogenous genes.It has been widely studied and applied in basic research,vaccine preparation,and genetics treatment and is considered to be one of the most promising vectors for gene transfer.Infectious bursal disease(IBD),also known as"Gumboro",is a high mortality and contagious diseases caused by infectious bursal disease virus(IBDV).It mainly infected 3-6 weeks old chicks.After infection,the bursa of the chickens is seriously damaged,causing immunosuppression and high mortality.Since the discovery of IBD in chickens in 1957,it has been prevalent in many countries and regions around the world.It is also listed as one of the three major infectious diseases that harm global poultry health including Newcastle disease and chicken Marek's disease.Its high mortality has brought the poultry industry huge economic losses.The main method to prevent and control IBD is to vaccinate chickens to obtain active or passive immune protection.Although all countries have better commercial vaccines,the virulence of IBDV strains is increasing during the epidemic process,so that the antigenicity of commercial vaccines cannot be completely matched with the epidemic strains,resulting in clinical immune failures.The prevention and treatment of IBDV poses a serious challenge,so it is urgent to develop a new vaccine that matches the antigenic structure of the epidemic strain.VP2 protein is the main host protective antigen of IBDV and the main structural protein of IBDV.With the rapid development of molecular biology technology,the novel viral vector vaccine with viral VP2 gene as target gene has become the main trend in development of IBD vaccine research.In this study,a full-length genomic infectious clone of the FAdV-4 CIH/HNJZ/2015 strain was constructed by ExoCET direct cloning.Recombinant fowl adenovirus was constructed by inserting the antigenic gene VP2 of infectious bursal disease virus into the foreign gene insertion site of CH/HNJZ/2015 by seamless site-directed mutagenesis technology.The research results are as follows:1.Establishment of reverse genetic system of FAdV-4 CH/HNJZ/2015 strainExoCET direct cloning technology,which combines exonuclease-mediated in vitro homologous recombination with RecET-mediated intracellular homologous recombination,allow us to directly and efficiently clone large fragments of DNA.The genome of the highly pathogenic strain of FAdV-4 CH/HNJZ/2015 was directly cloned into the p15A vector by this method.The infectious clone containing the complete viral genome was obtained,and the virus rescue was successfully carried out.The reverse genetic system of fowl adenovirus type 4 CH/HNJZ/2015 strain is convenient for genetic modification and transformation,which provides a powerful tool for the study of gene function and pathogenesis of FAdV-4 new strain.2.Inserting the antigen gene of IBDV to construct recombinant fowl adenovirus plasmidAdenovirus is a potential viral vector with the advantages of susceptibility,low pathogenicity,wide host range,good stability,easy DNA manipulation,high virus titer.easy concentration and storage,and ability to accommodate larger gene fragments.By seamless site-directed mutagenesis technology,we have inserted the antigenic gene VP2 of chicken infectious bursal disease virus into the exogenous gene insertion site of plasmid p15A-cm-HNJZ.3.Rescue of recombinant fowl adenovirusThe constructed recombinant virus plasmid was linearized by restriction enzyme digestion and transfected into chicken liver cancer cells(LMH).After 6 days post-transfection,the cells showed obvious cytopathic effect,and the specific primers were Lused in PCR toamplify the VP2 gene in the recombinant virus.PCR results indicated that the recombinant FAdV-4,rHNJZ-1966-IBDV-VP2 was rescued successfully.4.Expression verification of foreign genes in recombinant FAdV-4The expressionof VP2 protein by the recombinant virus rHNJZ-1966-IBDV-VP2 was verified by Western blotting,and a band with the expected size as the positive control was obtained,indicating that VP2 can be expressed normally in the rHNJZ-1966-IBDV-VP2.The position of the VP2 protein expressed by rHNJZ-1966-IBDV-VP2 in the cells was confirmed by indirect immunofluorescence assay,and the results showed that the target protein was mainly in the cytoplasm.5.Replication ability of recombinant FAdV-4 in vitroThe parental FAdV-4 strain CH/HNJZ/2015 and the recombinant virus rHNJZ-1966-IBDV-VP2 were respectively infected into LMH cells,and the virus-infected cells were harvested at different time points after infection,and the TCID50 of the virus harvested at each time point was measured,then the growth curve of the virus was drawn.There was no significant difference in the proliferation dynamics of the recombinant virus rHNJZ-1966-IBDV-VP2 and FAdV4 parental strain CH/HNJZ/2015,indicating that the recombinant virus rHNJZ-1966-IBDV-VP2 has good replication ability in vitro.6.Genetic stability test of rHNJZ-1966-IBDV-VP2In order to evaluate the genetic stability of the recombinant virus rHNJZ-1966-IBDV-VP2,the recombinant virus was serially passaged 40 times,and a PCR test was performed on the VP2 gene in the recombinant virus genome every 10 generations.The results showed that the VP2 gene in each generation of recombinant virus can be amplified.A band of uniform gene size indicates that the VP2 gene is stably present during replication of the recombinant virus rHNJZ-1966-IBDV-VP2.In this study,the reverse genetic manipulation system of FAdV-4 highly pathogenic strain CH/HNJZ/2015 was established by ExoCET direct cloning technology.It provides an effective platform for further research on the function of unknown virus genes,molecular pathogenesis and the interaction mechanism with the host.VP2 gene was inserted into the foreign gene insertion site of FAdV-4 CH/HNJZ/2015 strain by seamless site-directed mutagenesis technology.The adenoviral plasmid was successfully rescued by the virus,and the rescued virus was able to stably replicate and inherit in vitro,laying a foundation for inactivated recombinant viral vector vaccine research.