Isolation And Identification Of Fowl Adenovirus 11 And Preliminary Study Of Trivalent Vaccine

Since 2015,hepatitis-hydropericardium syndrome（HHS）caused by fowl adenovirus 4（FAdV-4）and inclusion body hepatitis（IBH）associated with fowl adenovirus8b（FAdV-8b）and fowl adenovirus 11（FAdV-11）have been prevalent in chickens in China,causing serious economic losses to Chinese poultry industry.Epidemiological investigation indicated that FAdV-4,FAdV-8b and FAdV-11 were the main serotypes infected by chickens in China,and concurrent infection was very common in poultry farms,yet there was no trivalent vaccine against these three serotypes.In this study,a FAdV-11 FJSW/2021 strain was isolated from the clinical samples collected from a chicken farm in Fujian Province.The whole genome sequence was determined and the genetic evolution analysis was carried out to study the biological characteristics of the isolated strain.In the previous study,the Fiber of FAdV-8b was used to replace the Fiber-1 of FAdV-4 to construct r FAdV-4-fiber/8b,and a bivalent inactivated vaccine based on r FAdV-4-fiber/8b was developed.In this study,the fiber gene of FAdV-11 was inserted into the 1966-bp deletion region in the genome of the recombinant virus r FAdV-4-fiber/8b to construct a recombinant virus r FAdV-4-fiber/8b+11 that simultaneously expresses FAdV-8b and FAdV-11 Fiber proteins.FAdV-4-fiber/8b+11 has similar in vitro replication capacity and pathogenicity to susceptible SPF chickens as FAdV-4.A single dose of inactivated r FAdV-4-fiber/8b + 11 vaccine could produce specific antibody immune responses to FAdV-4,FAdV-8b and FAdV-11,and provided effective clinical protection against FAdV-4,FAdV-8b and FAdV-11 infection.This study laid a foundation for the development of a trivalent vaccine against HHS and IBH.The study includes the following four aspects:1.Isolation,identification and phylogenetic analysis of FAdV-11 Chinese epidemic strainsIn order to study the genetic variation of FAdV-11,a FAdV-11 strain was isolated from the liver of chickens suspected to have inclusion body hepatitis.The whole genome sequence of FJSW/2021 isolate was determined.The results showed that the genome of the virus is 44154 bp in length,and the genome composition is highly consistent with the reported FAdV-11.The phylogenetic analysis showed that the FJSW/2021 isolate shares97.68%-99.97% homology with the published FAdV-11 isolates at home and abroad,indicating FJSW/2021 belongs to FAdV-11.There is an increase in the number of amino acids of the p TP protein of FJSW/2021 isolate;FJSW/2021 is 99.97% identical to the the Canadian non-pathogenic strain ON NP2,but FJSW/2021 has more amino acids in the100 K and 33 K coding regions.The amino acid sequence alignment analysis of the main structural proteins Fiber,Hexon and Penton showed that the FJSW/2021 strain is identical to the domestic strain HBQ12 and the non-pathogenic strain ON NP2.However,the amino acid sequences encoding the three proteins are quite different from those of Australian isolates.In this study,a FAdV-11 strain was successfully isolated and its whole genome sequence was determined.The results suggested that the Chinese FAdV-11 strains may have the same origin and evolve continuously,which enriched the knowledge of molecular epidemiology and genetic diversity of Chinese FAdV-11 isolates and laid a foundation for the development of related vaccines.2.The in vitro proliferation and pathogenicity study of the FAdV-11 isolate FJSW/2021In order to study the in vitro proliferation characteristics of FAdV-11 Chinese isolate FJSW/2021,the virus titer was determined on Leghorn male hepatocellular cell（LMH）.The results showed that FJSW/2021 could proliferate in LMH and cause cytopathic effects.The virus titer began to rise at 12 h post infection,and reached a peak titer of 10^5.3TCIID₅₀/100μL at 96 h post infection,and then entered a plateau period.One-week-old and two-week-old SPF chickens were intramuscularly infected with 10⁶ and 10⁷ TCID₅₀ of FAdV-11 Chinese isolate FJSW/2021,respectively.The mortality of one-week-old chicken was 100%,and of the two-week-old chicken was 40%-50%.The dead chickens showed similar IBH symptoms,suggesting that the pathogenicity of the virus in susceptible chickens of different ages was different.This study provided guidance for the establishment of animal models for FAdV-11 infection and the evaluation of the immune efficacy of the related vaccines.3.Construction and identification of recombinant r FAdV-4-fiber/8b+11 expressing FAdV-8b/-11 fiber proteinsEpidemiological studies have shown that FAdV-4,FAdV-8b and FAdV-11 are the main serotypes causing HHS and IBH in China.In order to develop a trivalent vaccine for effective prevention and control of HHS and IBH,the FAdV-11 Fiber gene was inserted into the 1966-bp natural deletion region of the r FAdV-4-fiber/8b genome using the FAdV-4reverse genetics system.The infectious clone was constructed and the recombinant virus r FAdV-4-fiber/8b+11 was successfully rescued.The r FAdV-4-fiber/8b+11 was identified by PCR and Western Blot.The results showed that FAdV-11 Fiber was successfully inserted and expressed,and the recombinant virus had good stability.The recombinant virus and the parental virus had similar proliferation characteristics on LMH.The recombinant virus and the parental virus FAdV-4 were inoculated to two-week-old SPF chickens.Chickens infected with the recombinant virus showed typical HHS necropsy changes,and the mortality rate was lower than that of the FAdV-4 infected chickens.The viral load of each organ was significantly higher than that of the uninfected group.In this study,the recombinant r FAdV-4-fiber/8b+11 expressing FAdV-8b/-11 fiber protein was successfully constructed which laid a foundation for the development of a trivalent vaccine against HHS and IBH.4.Evaluation of immunogenicity and immune efficacy of inactivated vaccine based on r FAdV-4-fiber/8b+11In order to evaluate the immunogenicity and immune efficacy of the inactivated r FAdV-4-fiber/8b+11vaccine,one-week-old chickens were immunized by intramuscular injection.The level of anti-FAdV-4/-8b/-11 antibody in chickens after immunization was determined by ELISA every week.The results showed that the inactivated vaccine could induce specific antibodies against FAdV-4/-8b/-11.Two weeks after immunization,FAdV-4,FAdV-8b and FAdV-11 isolates were used for challenge test.The results showed that r FAdV-4-fiber/8b +11 as an inactivated vaccine could provide full protection against FAdV-4/-8b/-11 infection.The results suggest that r FAdV-4-fiber/8b+11 can be used as an efficient trivalent vaccine to prevent FAdV-4,FAdV-8b and FAdV-11 infection.