Isolation And Identification Of FAdV-8b And Development Of Bivalent Vaccine(FAdV-4+FAdV-8b)

Fowl adenoviruses(FAdVs)belongs to the genus Adenovirus in the family of Adenoviridae.FAdVs are associated with a wide range of avian diseases,including inclusion body hepatitis(IBH),hepatitis hydropericardium syndrome(HHS)and gizzard erosion ulceration(GEU).Fowl adenovirus serotype 4(FAdV-4)and FAdV-8b are causative agents of HHS and IBH.Epidemiological data show that the outbreaks of HHS and IBH in China has been increasing since 2015,and HHS and IBH co-infections were often reported in clinical.Currently there are no commercially available bivalent vaccines for the prevention and control of both HHS and IBH caused by FAdV-4 and-8b.Therefore,it is of great significance to develop an efficient vaccine for prevention and control of HHS and IBH.In this study,a FAdV-8b strain was isolated from liver samples of suspected IBH-infected chickens from a farm in Shandong Province.The whole genome sequence of FAdV-8b strain was obtained by high-throughput sequencing,and the genetic evolution analysis was carried out.The pathogenicity of FAdV-8b in SPF chicken embryos and chickens was studied.At present,there are no commercially available bivalent vaccines for prevention and control of both HHS and IBH in China.In this study,the Fiber1 of FAdV-4 was replaced by the Fiber of FAdV-8b through reverse genetic technology to construct a recombinant chimeric virus r FAdV-4-Fiber/8b expressing the Fiber protein of FAdV-8b.The immunogenicity and immune efficacy of inactivated vaccine based on r FAdV-4-Fiber/8b were comprehensively evaluated.This study laid the foundation for the control and prevention of HHS and IBH and bivalent vaccine development.1.Isolation,identification and genetic evolution analysis of fowl adenovirus serotype 8b strainIn 2021,a suspected outbreak of inclusion body hepatitis(IBH)occurred in a farm in Shandong Province.The diseased chickens exhibited typical clinical symptoms including depression and fluffy feathers,and post-mortem examination of diseased chickens indicated swollen livers.The mortality of chickens is 20%.In order to clarify the pathogen of the disease,the liver tissue of chickens was collected in this study.The pathogen was determined to be FAdV-8b by PCR detection.The virus was isolated using Leghorn male hepatocellular(LMH)cells.A Chinese epidemic strain of FAdV-8b was successfully isolated and named CH/SDQD/2021.Subsequently,high-throughput sequencing of the viral genome was performed.Homology and genetic evolution analysis were performed using MEGA7.0 and Meg Align software.The results showed that the full-length genome of the isolate was 44000 bp,the G+C content was 58%,and 32 open reading frames were encoded.The homology between the FAdV-8b isolate CH/SDQD/2021 and the FAdV-E strain was 93.4%-98.9%,and the highest homology 98.9% was found with the Chinese strain He B20 isolated in2020 The results showed that the strain CH/SDQD/2021 had the same origin as the epidemic strains of FAdV-8b in China,but there was diversity.The isolation of this strain provided a basis for the investigation of IBH prevalence and related vaccine development in China.2.The in vitro proliferation and pathogenicity study of FAdV-8b isolate CH/SDQD/2021In order to explore the proliferation of FAdV-8b strain CH/SDQD/2021 on LMH cells and its pathogenicity to susceptible chickens,the FAdV-8b isolate CH/SDQD/2021 was cultured on LMH cells,and the peak viral titer was105.4TCID50/100 μL.The pathogenicity of the isolate was evaluated by inoculating SPF chickens of different ages and chicken embryos with different doses of the virus.The survival rate of 10-day-old chicken embryos inoculated with 106 TCID50 of virus through the chorioallantoic membrane was 50%,and the survival rate of chicken embryos inoculated with 107 TCID50 virus was 30%.The survival rate of chicken embryos inoculated with 107 TCID50 virus through the allantoic cavity was 20%.Clinical symptoms such as depression and reduced feed intake began to appear in 1to 2-week-old chickens 24 hours after infection with the virus.The survival rate of1-week-old SPF chickens inoculated with two different doses of FAdV-8b virus by intramuscular injection was zero.The survival rate of 2-week-old SPF chickens inoculated with 106 TCID50 of the virus was 90%.The survival rate of chickens inoculated with 107 TCID50 of the virus was 30%.Typical lesions of IBH were observed in both infected chickens and chicken embryos,such as fragile and enlarged liver,jaundice,white necrosis on the surface,and enlarged kidney with urate deposition.The histopathological results showed that the liver cells had nuclear fragmentation,vacuolar degeneration,glomerular swelling and hemorrhage,and renal tubular edema.No obvious histological changes were found in the blank control group.3.Construction and identification of recombinant FAdV-4 expressing FAdV-8b Fiber proteinAs there is no available bivalent vaccine for the prevention and control of HHS caused by FAdV-4 and IBH caused by FAdV-8b in China,this study used reverse genetic technology to replace Fiber1 of FAdV-4 with Fiber of FAdV-8b to construct a recombinant chimeric virus r FAdV-4-Fiber/8b.The results showed that the recombinant virus could stably express FAdV-8b Fiber.The growth kinetics of the chimeric virus r FAdV-4-Fiber/8b was similar to the parental virus on LMH cells.The pathogenicity study of r FAdV-4-Fiber/8b showed that replacing Fiber 1 with FAdV-8b Fiber did not change the pathogenicity of the virus.At present,there is a lack of research on the infection mechanism of FAdV-8b.The results of this study show that replacing Fiber 1 of FAdV-4 with FAdV-8b Fiber has little effect on the infection of recombinant virus in vitro and in vivo,suggesting that FAdV-8b may infect host cells in a similar way as FAdV-4,and FAdV-8b Fiber may play the same role as FAdV-4 Fiber1 in the initiation of infection.Our results support further studies to elucidate the infection mechanism of different serotypes of FAdV,thereby improving the prevention and control strategies of HHS and IBH.4.Evaluation of immunogenicity and immune efficacy of bivalent inactivated vaccine based on r FAdV-4-Fiber/8bIn order to evaluate the protective effect of bivalent inactivated vaccine based on r FAdV-4-Fiber/8b on chickens infected with FAdV-4 and FAdV-8b,the inactivated r FAdV-4-Fiber/8b vaccine was used for single dose immunization.The immunogenicity evaluation results showed that the vaccine could induce the body to produce neutralizing antibodies against FAdV-4 and FAdV-8b.A single dose of vaccine immunization can provide clinical protection against FAdV-4 and FAdV-8b challenge.The immunized chickens did not show any clinical symptoms during the entire test period,and there was no visual autopsy lesion.The viral load in various organs and tissues of immunized chickens was significantly lower than that of non-immunized challenged chickens,while the viral load of FAdV-4 or FAdV-8b in various organs and tissues of immunized chickens was similar to that of non-challenged control chickens.The results of this study prove that cross-protection against different FAdV can be provided by modifying or replacing the Fiber gene of FAdV.This will lay the foundation for the development of gene therapy vectors and multivalent candidate vaccines.