The Construction Of Enteric Fowl Adenovirus Serotype 4 (FADV-4) Vector System As Vehicle For High-efficent Preventive Addtives

With the continuous development of animal husbandry,the intensification and scale of livestock and poultry breeding are constantly increasing,and the pressure on disease prevention and control is also increasing.The traditional group immunity of poultry is achieved through injection,which requires a dual process with the breeding process,which is time-consuming and labor-intensive.Functional feed additives refer to additives that enter the body of animals through feed addition and perform certain biological functions.According to material properties,it can be divided into nutritional additives and non nutritional additives.Among them,non nutritional additives mainly include substitutes for antibiotic and immune enhancer products,but there are few reports on non nutritional additives that can prevent and control major diseases in livestock and poultry breeding(referred to as preventive functional additives).The existing preventive functional additives mostly use bacteriophages,yeast,and probiotics as carriers to deliver disease protection antigens.Due to the limited post processing of antigens,their delivery efficiency for virus antigens,especially envelope virus antigens,is mostly not ideal.The viral vector that can achieve mammalian post processing of viral antigens and efficiently deliver antigens in the intestine has become the preferred choice.Entergenic adenoviruses as vehicle for antigen delivery have multi-advantages than other viral vectors in aspects of safty,stability,immunogenicity,host range,insertion capacity and repeated oral administration.Comparing with commercial replication-defective adenovirus,IIIa gene deleted single-cycle adenovirus(SC-Ad)has the similar biosafety,but the genome copy number and expression level of forein interest increased dramatically.Due to abovementioned advantages,this study focused on the model of fowl entergenic adenovirus serotype four(FAd V-4)which caused the hepatitis-hydropericardium syndrome(HHS)in poultry,and established FAd V-4 infectious clones(wild type FAd V-4 and SCFAd V4,respectively)and rescue cell line to explore the feasibility of FAd V-4 as antigen delivery vehicle for preventive functional additives.The main research contents are as follows:1.Wild-type FAd V-4 isolation and identification.In this study,a large number of liver tissues were collected from the chicken suspected to be infected with HHS.After physical and appropriate amount of antibiotics treatment,the supernatant was extracted and inoculated in LMH cell line for virus proliferation.Cytopathogenic effect was appeared 36 hours post inoculation.Then the viral genome DNA was isolated and identified by PCR.The PCR and phylogenetic analysis confirmed the viral isolate was 100% homology with FAd V-4.Subsequently,the animal reinfection experiment was conducted by injecting non-immune broilers with isolated FAd V-4,and it was found that the chickens in the experimental group had hemorrhagic spots on the liver surface,massive pericardial effusion in the pericardium,and renal enlargement.The symptom was in consistent with FAd V-4,which indicated a strain of FAd V-4 was successfully isolated and the virus was highly pathogenic.2.Wild-type FAd V-4 infectious clone construction and verification.The FAd V-4 genome was approximately 46 kb in size.In this study,the FAd V-4infectous clone was constructed using Gibson Assembly technology in vitro.Steps were described as follows.First,the vector backbone p Shuttle-FAVITR-FR was amplified using PCR for FAd V-4 infectious clone with a template from porcine adenovirus serotype 3infectious clone.Second,the vector backbone of PCR amplification was assembled with FAd V-4 genome DNA according Gibson Assembly procedure and the mixture was transformed into EZ10 competent cells.Third,the positive infectious clones were verified by nucleic acid gel electrophoresis,restriction enzyme digestion,PCR amplification and virion rescue in LMH cells.The result showed that 15 from 18 seclected clones could rescue viron particles and the viral replification level was similar to wild-type FAd V-4,which indicated that FAd V-4 infectious clone was susscessfully obtained.3.SC-FAd V4 infectious clone construction.To further construct the infectious clone of SC-FAd V4,the IIIa gene need to be deleted from wild-type FAd V-4 genome and a recombinant cell line stable expressing IIIa gene LMH-IIIa need to be established as well.First,the targeting fragment including ccd BCm expression box flanking PmeⅠ restriction site and left/right homologous arms for IIIa deletion was synthetized.The thermosensitive p KD46 expression λ-red recombineases was pretransformed into DB3.1 competent cells with FAd V-4 infectious clone p Shuttle-FAd V4.Second,the targeting fragment with ccd B-Cm expression cassette were transformed into DB3.1competent cells(p KD46 & p Shuttle-FAd V4/DB3.1)for IIIa replacement by ccd B/Cm under λ-red recombination,the result genome plasmid was named p Shuttle-FAd V4(ΔIIIa::ccd B-Cm).Third,the cells(p KD46 & p Shuttle-FAd V4/DB3.1)was inoculated at42℃ for p KD46 vector remove,and the ccd B-Cm expression cassette was removed by PmeⅠ enzyme digestion and self-ligation.The result genome plasmid was named p ShuttleFAd V-4(ΔIIIa::Pme I).4.LMH-IIIa cells constrction based on IIIa gene targeting insertion.Besides construction IIIa deleted SC-FAd V4 vector of p Shuttle-FAd V-4(ΔIIIa::Pme I),t he cells line for IIIa gene expression is another pivotal factor.In order to realize the site-spec ific insertion of IIIa gene,we co-transfected CRISPRE-Cas9 in-one vector(p X330-U6-Apoe1 sg RNA-CBh-h Sp Cas9/p X330-U6-Apoe2 sg RNA-CBh-h Sp Cas9)with homologous recom bination donor vector(donor-sg RNA1/ donor-sg RNA2)into LMH cells to establish IIIa stab le expression cell line(LMH-IIIa).Under the selection with puromycin,several cells clones were formed and replicated.Meanwhile,the transgene was successfully expressed by Wester n blot indentification.In conclusion,this study successfully isolated one strain of FAd V-4 in Shaanxi.Based on the isolated FAd V-4 genome,the infectious clone of wt FAd V-4 was futher constructed.To prepare the recombinant genome for SC-FAd V4,the IIIa in wt FAd V-4 genome was deleted and the cell line stable expression IIIa was established as well to verify the rescue of SC-FAd V4 virions after genome transfection.This study provides necessary materials and technical support for SC-FAd V4 system establishment,and for the study of FAd V4 molecular pathogenesis and application as preventive additives based on SC-FAd V4.