Abstract
BackgroundHighly 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.MethodsTo 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 (DEVC-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.ResultsThe 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 106 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 107 TCID50 DEV-vectored vaccine. A mortality rate of 60 % (6/10) was observed in 1-week-old specific pathogen free chickens inoculated with 106 TCID50 DEV-vectored vaccine.ConclusionsWe demonstrate the following in this study. (i) The constructed BAC is a whole genome clone of DEVC-KCE. (ii) The insertion of an HA expression cassette sequence into the noncoding area between UL55 and LORF11 of DEVC-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.
Highlights
Pathogenic avian influenza virus (AIV) subtype H5N1 remains a threat to poultry
Three days after the co-transfection of DNA from DEVC-KCE and the bacterial artificial chromosome (BAC) transfer vector plasmid pDEVgc-pHA2, a recombinant DEVC-KCE harboring mini-F plasmid sequences was successfully generated as indicated by its production of green fluorescence under UV light (488 nm; Fig. 1)
A total of seven colonies with chloramphenicol resistance were obtained after electroporation of DEVC-KCE-mini-F DNA into Escherichia coli DH10B competent cells, one of which, termed pDEVC-KCE, was selected for further restriction fragment length polymorphism (RFLP) analysis
Summary
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. A DEV-vectored vaccine harboring the hemagglutinin (HA) of the highly pathogenic avian influenza virus (AIV) subtype H5N1 was generated based on this BAC, and robust expression of HA was confirmed in the infected cells [9]. The safety of this vaccine remains questionable owing to its development from a virulent parental strain.
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