Generation And Identification Of A Duck Enteritis Virus(DEV)Expressing Hemagglutinin Of High Pathogenic Avian Influenza Virus(H5N1)

H5N1 avian influenza virus as a highly pathogenic influenza virus has attracted great corncerning of the world.The inactivated vaccine has been used widely as the primary means of prevention and control of avian influenza currently in China.However,due to poor immune effect of inactivated vaccine,such as less T cell immunity,and that it is not a DIVA vaccine that can differentiate infection of the virus in field condition.The risk of spreading of live viruses is another disadvantage during the production of the vaccine.Therefore,development of a safe,efficient,high quality new vaccines has become a priority for effective prevention and control of avian influenza.Live viral vectored vaccine can express efficiently foreign protective antigens of infection pathogens.Moreover,the induction of both humoral immunity and cellular immunity,can achieve the purpose of sterilized immunity.So viral vected vaccine has become one of the focuses of new vaccines.Duck enteritis viral(DEV),as a member of the herpes virus,is with a genome of approximately 158Kbp,and contains many growing non-essential genes that do not affect the growth characteristics of DEV after deletion.Because of these characteristi-cs,DEV has the potential to serve as an expression of exogenous genes.To facilitate the generation and further improvement of DEV vectored HA(H5)vaccine,we constructed an infectious clone of DEV of the Chinese vaccine strain C-KCE.Based on the bacterial artificial chromosome(BAC),we generated efficiently a DEV vectored HA(H5)vaccine(DEV-H5(UL55))by insertion of a synthesized HA(H5)expression cassette with pMCMV IE promoter and a consensus HA sequences into the non-coding area between the UL5 5 and LORF11.Restriction fragment length polymorphism(RFLP)of DEV BAC or mutants showed the bands as suspected with slight differences.Recovered virus from the BAC or mutants showed similar growth kinetics as parental virus(no significant difference).The results of indirect immunofluorescence(IIF)and western blotting indicated the robustexpression of HA after infection of the vectored vaccine in chicken embryo fibroblasts(CEFs).It was concluded that we successfully constructed a vectored duck enteritis virus(DEV)stably expressing hemagglutinin of H5N1 avian influenza virus based on an infectious clone of DEV vaccine strain.Through this study,we can draw the following conclusions:(1)in this experim-ent the constructed DEV BAC contains the whole genome of DEV vaccine strain C-KCE;(2)HA gene inserted between the UL55 and LORF11 DEVC-KCE dose not affect the growth characteristics of DEV;(3)Although for a week-old DEV-H5(UL55)chicks have a certain virulences,it can also produce in three-week-old chickens a strong humoral response;(4)DEV-H5(UL55)can induce a low HI antibody in commercial ducks.