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Construction of recombinant Marek's disease virus (rMDV) co-expressing AIV-H9N2-NA and NDV-F genes under control of MDV's own bi-directional promoter.

ABSTRACT: To qualitatively analyze and evaluate a bi-directional promoter transcriptional function in both transient and transgenic systems, several different plasmids were constructed and recombinant MDV type 1 strain GX0101 was developed to co-express a Neuraminidase (NA) gene from Avian Influenza Virus H9N2 strain and a Fusion (F) gene from the Newcastle disease virus (NDV). The two foreign genes, NDV-F gene and AIV-NA gene, were inserted in the plasmid driven in each direction by the bi-directional promoter. To test whether the expression of pp38/pp24 heterodimers are the required activators for the expression of the foreign genes, the recombinant plasmid pPpp38-NA/1.8kb-F containing expression cassette for the two foreign genes was co-transfected with a pp38/pp24 expression plasmid, pBud-pp38-pp24, in chicken embryo fibroblast (CEF) cells. Alternatively, plasmid pPpp38-NA/1.8kb-F was transfected in GX0101-infected CEFs where the viral endogenous pp38/pp24 were expressed via virus infection. The expression of both foreign genes was activated by pp38/pp24 dimers either via virus infection, or co-expression. The CEFs transfected with pPpp38-NA/1.8kb-F alone had no expression. We chose to insert the expression cassette of Ppp38-NA/1.8kb-F in the non-essential region of GX0101?Meq US2 gene, and formed a new rMDV named MZC13NA/F through homologous recombination. Indirect fluorescence antibody (IFA) test, ELISA and Western blot analyses indicated that F and NA genes were expressed simultaneously under control of the bi-directional promoter, but in opposite directions. The data also indicated the activity of the promoter in the 1.8-kb mRNA transcript direction was higher than that in the direction for the pp38 gene. The expression of pp38/pp24 dimers either via co-tranfection of the pBud-pp38-pp24 plasmid, or by GX0101 virus infection were critical to activate the bi-directional promoter for expression of two foreign genes in both directions. Therefore, the confirmed function of the bi-directional promoter provides better feasibilities to insert multiple foreign genes in MDV genome based vectors.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC3944216 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Construction of recombinant Marek's disease virus (rMDV) co-expressing AIV-H9N2-NA and NDV-F genes under control of MDV's own bi-directional promoter.

Zhang Zhenjie Z Ma Chengtai C Zhao Peng P Duan Luntao L Chen Wenqing W Zhang Fushou F Cui Zhizhong Z

PloS one 20140305 3

To qualitatively analyze and evaluate a bi-directional promoter transcriptional function in both transient and transgenic systems, several different plasmids were constructed and recombinant MDV type 1 strain GX0101 was developed to co-express a Neuraminidase (NA) gene from Avian Influenza Virus H9N2 strain and a Fusion (F) gene from the Newcastle disease virus (NDV). The two foreign genes, NDV-F gene and AIV-NA gene, were inserted in the plasmid driven in each direction by the bi-directional pr ...[more]

PMID: 24599338

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Project description:Chlamydia psittaci (C. psittaci) is an obligate intracellular zoonotic pathogen that can be transmitted to humans from birds. No efficacious commercial vaccine is available for clearing chlamydial infection due to lack of potential vaccine candidates and effective delivery vehicles. Herpesvirus of turkeys (HVT) is an efficacious commercially available vaccine against Marek's Disease virus (MDV). In this study, a recombinant HVT-delivered vaccine against C. psittaci and Marek's disease was developed and examined. The 5'-terminus of pmpD gene (pmpD-N) encoding the N-terminal fragment of polymorphic membrane protein D of C. psittaci was inserted into a nonessential region of HVT genome using reverse genetics based on an infectious bacterial artificial chromosome (BAC) clone of HVT. The recombinant virus (rHVT-pmpD-N) was recovered from primary chicken embryo fibroblast (CEF) cells by transfection of modified HVT BAC DNA containing the pmpD-N gene. The rHVT-pmpD-N construct was confirmed to express PmpD-N by immunoblot and immunofluorescence. The rHVT-pmpD-N was stable during 20 passages in vitro. The growth kinetics of rHVT-pmpD-N was comparable to that of parental HVT in vitro and in vivo. One-day-old SPF chickens inoculated subcutaneously with rHVT-pmpD-N displayed increased PmpD-specific antibody levels and a vigorous PmpD-specific lymphocyte proliferation response using HVT vector or CEF cells as control. Furthermore, the percentage of CD4+ cells was significantly elevated in rHVT-pmpD-N-immunized birds as compared to the parental HVT. All chickens vaccinated with rHVT-pmpD-N or parental HVT were protected completely against challenge with a very virulent strain of Marek's Disease virus (MDV) RB-1B. Post challenge with C. psittaci CB7 strain, a significant decrease in respiratory distress, lesions and Chlamydia load was found in the rHVT-pmpD-N-vaccinated group compared to the parental HVT. In conclusion, our study suggests that the rHVT-pmpD-N live vaccine may be viable as a candidate dual vaccine that provides protection against both very virulent MDV and C. psittaci.

Project description:BackgroundAs a low pathogenic influenza virus, avian influenza virus subtype H9N2 (H9N2 AIV) often induces high morbidity in association with secondary bacterial infections in chickens or mammals. To explore this phenomenon, the relationship between intestinal microflora changes and bacterial translocations was studied post H9N2 AIV challenge and post AIV infection plus Ageratum-liquid treatment.MethodsIllumina sequencing, histological examination and Neongreen-tagged bacteria were used in this study to research the microbiota composition, intestinal barrier, and bacterial translocation in six weeks of BALB/c mice.ResultsH9N2 AIV infection caused intestinal dysbacteriosis and mucosal barrier damages. Notably, the villus length was significantly reduced (p?<?0.01) at 12 dpi and the crypt depth was significantly increased (p?<?0.01) at 5 dpi and 12 dpi with infection, resulting in the mucosal regular villus-length/crypt-depth (V/C) was significantly reduced (p?<?0.01) at 5 dpi and 12 dpi. Moreover, degeneration and dissolution of the mucosal epithelial cells, loose of the connective tissue and partial glandular atrophy were found in infection group, indicating that intestinal barrier function was weakened. Eventually, intestinal microbiota (Staphylococcus, E. coli, etc.) overrun the intestinal barrier and migrated to liver and lung tissues of the mice at 5 and 12 dpi. Furthermore, the bacteria transferred in mesentery tissue sites from intestine at 36?h through tracking the Neongreen-tagged bacteria. Then the Neongreen-tagged bacteria were isolated from liver at 48?h post intragastrical administration. Simultaneously, Ageratum-liquid could inhibit the intestinal microbiota disorder post H9N2 AIV challenge via the respiratory tract. In addition, this study also illustrated that Ageratum-liquid could effectively prevent intestinal bacterial translocation post H9N2 AIV infection in mice.ConclusionIn this study, we report the discovery that H9N2 AIV infection could damage the ileal mucosal barrier and induce the disturbance of the intestinal flora in BALB/c mice resulting in translocation of intestinal bacteria. In addition, this study indicated that Ageratum-liquid can effectively prevent bacterial translocation following H9N2 infection. These findings are of important theoretical and practical significance in prevention and control of H9N2 AIV infection.

Dataset Information

Construction of recombinant Marek's disease virus (rMDV) co-expressing AIV-H9N2-NA and NDV-F genes under control of MDV's own bi-directional promoter.

Publications

Construction of recombinant Marek's disease virus (rMDV) co-expressing AIV-H9N2-NA and NDV-F genes under control of MDV's own bi-directional promoter.

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