| H5N1subtypeavian influenza virus(AIV) is a single-stranded, negative-sense, enveloped RNA virus which belongs to the Orthomyxoviridae family.This pathogen isperceived as a serious threat to the development of the poultry industry and a significant emerging pandemic threat. The structural protein of AIV could recognize and binding the host cell surface receptors to infect cells. Thus, as the most important protective antigen, the HA protein has been the target protein of various influenza vaccine studies.We have been successfully constructed the recombinant duck enteritis virus(DEV) r DEVus78 Ha, which expressing the hemagglutinin(HA) gene of H5N1 subtype AIV HB/49 strain. Vaccination with r DEVus78 Ha recombinant virus in ducks could induce rapid and strong immune protection, but the immunologic mechanism of this recombinant virus is still unknown. During the process of highly pathogenic avian influenza virus(HPAIV) infecting cells, the signal peptide and the transmembrane domain of HA protein have an effect on itsthe activity and expression. In order to evaluate the protective efficacy of antigens that presented in different ways,weconstructed and rescued two duck enteritis viruses(DEV) recombinant viruses by using of Red E/T homologous recombination and Gateway cloning technology, andinsertingthe signal peptide deleted(r DEV-PK) and transmembrane region deleted(r DEV-PSM) HA gene of the H5N1 subtype AIV HB/49 strain between the US7 and US8 genes of the DEV genome, respectively. Both recombinant viruseswere successfully constructed.Three recombinant virus r DEVus78 Ha 、 r DEV-PK and r DEV-PSMwere then used to immune three-week-old specific-pathogen-free(SPF) grade ducks and evaluate protective efficacy after 3 and 7 days vaccination by intranasal infection with HB/49 virus.Simultaneously for further study of rapid protection provided by r DEVus78 Ha, it was used to immunize three-week-old SPF grade ducks, the inactivated avian influenza vaccine Re-5 strain immunized group and PBS group were also set as controls, accordingly. Three ducks in each group were euthanized at 1,3,5,7 and 14 days post-inoculation, and the spleen, bursa of fabricius, windpipe fluid, saliva and tear were collected,the PBMC, splenic lymphocytes and blood serum were also separated. The fluorescent quatititive PCR, flow cytometry and indirect ELISA were used to evaluate the innate and adaptive immunity.The identified results indicatedthat both recombinant virus were successfully constructed, the signal peptide deleted HA protein(expressed from r DEV-PK)cannot transferred to the cells membrane but gathered in the infected cells; the transmembrane region deleted HA protein(expressed from r DEV-PSM) were secreted to extra cells. Neither signal peptide nor transmembrane region missing appeared to affect the replication of recombinant virus, but neither of them can provide rapid protection against homologous HPAIV. But the recombinant virus r DEVus78 Ha strain expressing complete signal peptide and transmembrane region of HA gene could up-regulated expressing of IFN-γand Granzyme-A at transcriptional level, enhanced the stimulating proliferation abilityof lymphocyte and induced more CD4+T-lymphocytes, evoked high cellular immune responses against the infections.This study indicate that the signal peptide and transmembrane region are both indispensable partof HA protein that could produce rapid specific immune responses; and the enhanced cellular immune responses may be the critical factor that r DEVus78 Ha virus possesses the rapid specific protective immunity. Our resultsprovided the foundationoffurther studies inrapid specific immunologic mechanism and rapid immune vaccines. |
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