| Since May 2015,hepatitis-hydropericardium syndrome(HHS)caused by a new genotype of fowl adenovirus serotype 4(FAd V-4)has been reported in Shandong,Jiangsu,Anhui,Henan,Hebei,Liaoning,Jilin,Zhejiang,Hubei,and Shanxi,China,causing significant economic losses to Chinese poultry industry.FAdV-4 infects mainly 3-to 5-week-old broiler chickens.The disease is characterized by 20 and 80%mortality,accumulation of clear,straw-colored fluid in the pericardial sac,enlarged and discolored liver with foci of hemorrhage and/or necrosis.It is of great significance to develop new and highly effective vaccine against FAdV-4 epidemic strain for prevention and control the HHS in China.In this thesis,a recombinant Newcastle disease virus LaSota vaccine strain expressing the fowl adenovirus serotype 4 fiber2 gene,rLaSota-Fiber2 was constructed by using reverse genetic technology.The immunogenicity and immune efficacy of rLaSota-Fiber2 was evaluated.An indirect ELISA for detecting FAdV-4-specific antibody was established using purified fiber2 as the coating antigen.Anti-fiber2 monoclonal antibody was also developed.The thesis consists of the following sections:1.Construction and identification of recombinant Newcastle disease virus expressing the Fiber2 gene of fowl adenovirus serotype 4 Chinese epidemic strainThe aim of this study is to construct a recombinant Newcastle disease virus(NDV)expressing the Fiber2 gene of fowl adenovirus serotype 4(FAdV-4)Chinese epidemic strain,and lay the foundation for developing safe,cheap and highly effective vaccine against NDV and FAdV-4 infections.The full-length Fiber2 gene from FAdV-4 Chinese epidemic strain was inserted between the phosphoprotein(P)and matrix protein(M)in the genome of NDV by using the reverse genetic system of NDV LaSota vaccine strain.A recombinant NDV of rLaSota-Fiber2 was rescued and confirmed by RT-PCR,sequencing,and Western blot.The results showed that the Fiber2 gene was inserted at the correct site and in correct direction,and the Fiber2 protein was expressed as soluble form rather than as an incorporated protein into the NDV vector particles.The results of standard pathogenicity index tests and growth properties of the 10th passage of the rLaSota-Fiber2 indicated that rLaSota-Fiber2 retained low pathogenicity and high titer growth capacity in embryonated chicken egg as the parental virus of LaSota strain with the highest 50%embryo infectious dose(EID50)of 108.5/100 μL,the mean death time(MDT)of 120 h,and both the intracerebral pathogenicity index(ICPI)and the intravenous pathogenicity index(IVPI)of zero.rLaSota-Fiber2 and its parent LaSota strain grew to similar titers at same time points with no significant difference in growth dynamics.The recombinant rLaSota-Fiber2 will provide safe,cheap and highly effective candidate vaccine against NDV and FAdV-4 infections.2.Prokaryotic expression of the Chinese fowl adenovirus serotype 4 Fiber2 protein and development of an indirect ELISA for antibody detectionTo establish a method for detecting specific antibodies against FAdV-4,the codon-optimized Fiber2 gene was cloned into the pET-32a(+)vector,and the expression of Fiber2 protein was induced in E.coli BL21(DE3).After optimization of a series of conditions,an indirect ELISA for detecting FAdV-4-specific antibodies was established by using the purified Fiber2 protein as a coating antigen.There was no cross-reaction between FAdV-4 antigen and antibodies against the Newcastle disease virus,infectious bursal disease virus,infectious bronchititis virus,egg-dropping syndrome virus,H9 and H5 subtype AIV,indicating that the established ELISA had a good specificity.The ELISA has good sensitivity and repeatability.Anti-FAdV-4 positive serum with 1:3200 dilution was still ELISA positive.The Intra-and inter-assay coefficient of variation was 1.5%-5.2%and 1.1%-5.6%,respectively.Seroepidemiological prevalence of FAdV-4 infection in partial chicken farms in Henan province and surrounded area was investigated by using the eatablished ELISA.The results indicated that all the tested chicken farms were anti-FAdV-4 antibody positive with average positive rate of 59.53%.The results showed that the established ELISA in this study can be used for the dection of antibodies against FAdV-4,and provides a method for the monitoring and epidemiological investigation of FAdV-4.3.Immunogenicity and protective efficacy of recombinant Newcastle disease virus expressing the Fiber2 gene of fowl adenovirus serotype 4 Chinese epidemic strainIn this section,the immunogenicity and protective efficacy of the rLaSota-Fiber2 was evaluated by intramuscular vaccination of 2-week-old SPF White Leghorn chicks with the live or inactivated rLaSota-fiber2in SPF chickens.The anti-FAdV-4 antibody was detected by using an indirect ELISA,and the anti-NDV antibody was detected by using hemagglutination inhibition assay after immunization.The results showed that both live and inactivated rLaSota-Fiber2 can induce anti-FAdV-4 and anti-NDV specific antibody.The challenge experiment results demonstrated that single-dose intramuscular vaccination of 2-week-old SPF White Leghorn chicks with the live or inactivated rLaSota-fiber2 provided complete protection against virulent NDV challenge.However,single-dose intramuscular vaccination with the live rLaSota-fiber2 vaccine provided better protection against virulent FAdV-4 challenge.These results indicate that the rLaSota-Fiber2 is a promising bivalent vaccine candidate to control both HHS and ND. |
