Construction of a recombinant duck enteritis virus (DEV) expressing hemagglutinin of H5N1 avian influenza virus based on an infectious clone of DEV vaccine strain and evaluation of its efficacy in ducks and chickens.
BACKGROUND
Highly pathogenic avian influenza virus (AIV) subtype H5N1 remains a threat to poultry. Duck enteritis virus (DEV)-vectored vaccines expressing AIV H5N1 hemagglutinin (HA) may be viable AIV and DEV vaccine candidates.


METHODS
To facilitate the generation and further improvement of DEV-vectored HA(H5) vaccines, we first constructed an infectious clone of DEV Chinese vaccine strain C-KCE (DEV(C-KCE)). Then, we generated a DEV-vectored HA(H5) vaccine (DEV-H5(UL55)) based on the bacterial artificial chromosome (BAC) by inserting a synthesized HA(H5) expression cassette with a pMCMV IE promoter and a consensus HA sequence into the noncoding area between UL55 and LORF11. The immunogenicity and protective efficacy of the resulting recombinant vaccine against DEV and AIV H5N1 were evaluated in both ducks and chickens.


RESULTS
The successful construction of DEV BAC and DEV-H5(UL55) was verified by restriction fragment length polymorphism analysis. Recovered virus from the BAC or mutants showed similar growth kinetics to their parental viruses. The robust expression of HA in chicken embryo fibroblasts infected with the DEV-vectored vaccine was confirmed by indirect immunofluorescence and western blotting analyses. A single dose of 10(6) TCID50 DEV-vectored vaccine provided 100 % protection against duck viral enteritis in ducks, and the hemagglutination inhibition (HI) antibody titer of AIV H5N1 with a peak of 8.2 log2 was detected in 3-week-old layer chickens. In contrast, only very weak HI titers were observed in ducks immunized with 10(7) TCID50 DEV-vectored vaccine. A mortality rate of 60 % (6/10) was observed in 1-week-old specific pathogen free chickens inoculated with 10(6) TCID50 DEV-vectored vaccine.


CONCLUSIONS
We demonstrate the following in this study. (i) The constructed BAC is a whole genome clone of DEV(C-KCE). (ii) The insertion of an HA expression cassette sequence into the noncoding area between UL55 and LORF11 of DEV(C-KCE) affects neither the growth kinetics of the virus nor its protection against DEV. (iii) DEV-H5(UL55) can generate a strong humoral immune response in 3-week-old chickens, despite the virulence of this virus observed in 1-week-old chickens. (iv) DEV-H5(UL55) induces a weak HI titer in ducks. An increase in the HI titers induced by DEV-vectored HA(H5) will be required prior to its wide application.
Jiangsu Academy of Agricultural Sciences/National Research Center of Veterinary Biologicals Engineering and Technology, Nanjing, 210014, China. jcwang@263.net.
Jichun
J
Wang
Shandong Vocational Animal Science and Veterinary College, Weifang, 261061, China. sdmxgam@163.com.
Aimin
A
Ge
Jiangsu Academy of Agricultural Sciences/National Research Center of Veterinary Biologicals Engineering and Technology, Nanjing, 210014, China. xumengwei1746109@aliyun.com.
Mengwei
M
Xu
Jiangsu Academy of Agricultural Sciences/National Research Center of Veterinary Biologicals Engineering and Technology, Nanjing, 210014, China. zhisheng.wang@163.com.
Zhisheng
Z
Wang
Jiangsu Academy of Agricultural Sciences/National Research Center of Veterinary Biologicals Engineering and Technology, Nanjing, 210014, China. 985654669@qq.com.
Yongfeng
Y
Qiao
Jiangsu Academy of Agricultural Sciences/National Research Center of Veterinary Biologicals Engineering and Technology, Nanjing, 210014, China. 617651353@qq.com.|College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China. 617651353@qq.com.
Yiqi
Y
Gu
Jiangsu Academy of Agricultural Sciences/National Research Center of Veterinary Biologicals Engineering and Technology, Nanjing, 210014, China. 523264193@qq.com.|College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China. 523264193@qq.com.
Chang
C
Liu
Jiangsu Academy of Agricultural Sciences/National Research Center of Veterinary Biologicals Engineering and Technology, Nanjing, 210014, China. lym613@hotmail.com.
Yamei
Y
Liu
Jiangsu Academy of Agricultural Sciences/National Research Center of Veterinary Biologicals Engineering and Technology, Nanjing, 210014, China. houjibo@jaas.ac.cn.
Jibo
J
Hou
D000818:Animals; D002645:Chickens; D004372:Ducks; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics* / Q000276:immunology*; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics* / Q000276:immunology*; D007252:Influenza Vaccines / Q000235:genetics* / Q000276:immunology*; D005585:Influenza in Birds / Q000276:immunology / Q000517:prevention & control; D025922:Mardivirus / Q000235:genetics*; D009154:Mutation; D014779:Virus Replication
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines