Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Sep 13;12(9):1047.
doi: 10.3390/vaccines12091047.

Construction of Recombinant Marek's Disease Virus Co-Expressing VP1 and VP2 of Chicken Infectious Anemia Virus

Kai Li  1   2   3 , Yongzhen Liu  1 , Changjun Liu  1 , Yanping Zhang  1 , Hongyu Cui  1 , Xiaole Qi  1 , Jiayong Zhang  4 , Jia Xu  4 , Suyan Wang  1 , Yuntong Chen  1 , Yulu Duan  1 , Yulong Gao  1 , Xiaomei Wang  1   5
Affiliations

Construction of Recombinant Marek's Disease Virus Co-Expressing VP1 and VP2 of Chicken Infectious Anemia Virus

Kai Li et al. Vaccines (Basel). .

Abstract

The chicken infectious anemia virus (CIAV) has been reported in major poultry-producing countries and poses a significant threat to the poultry industry worldwide. In this study, two Marek's disease virus (MDV) recombinants, rMDV-CIAV-1 and rMDV-CIAV-2, were generated by inserting the CIAV VP1 and VP2 genes into the MDV vaccine strain 814 at the US2 site using the fosmid-based rescue system. For rMDV-CIAV-1, an internal ribosome entry site was inserted between VP1 and VP2, so that both proteins were produced from a single open reading frame. In rMDV-CIAV-2, VP1 and VP2 were cloned into different open reading frames and inserted into the MDV genome. The recombinant viruses simultaneously expressed VP1 and VP2 in infected chicken embryo fibroblasts and exhibited growth kinetics similar to those of the parent MDV. The two recombinant viruses induced antibodies against CIAV in chickens. A single dose of the recombinant viruses provided strong protection against CIAV-induced anemia in chickens. These recombinant VP1- and VP2-expressing MDVs are potential vaccines against CIAV in chickens.

Keywords: Marek’s disease virus; VP1; VP2; chicken infectious anemia virus; vaccine.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Construction of fosmids containing CIAV VP1 and VP2 genes. (A) The genomic structure of the MDV vaccine strain 814. (B) A schematic diagram of the recombinant fosmid 814E-CIAV-1 with the VP1-IRES-VP2 cassette inserted within the US2 gene in the MDV genome. (C) A schematic diagram of the recombinant fosmid 814E-CIAV-2 with the VP1 and VP2 expression cassette inserted within the US2 gene in the MDV genome.
Figure 2
Figure 2
The cytopathic effect (CPE) induced by the recombinant Marek’s disease virus (MDV) containing the VP1 and VP2 genes in chicken embryo fibroblasts (CEFs). The recombinant MDVs were inoculated in CEFs for 4–5 days before the CPE was checked. Bar length, 200 μm.
Figure 3
Figure 3
Detection of VP1 and VP2 expression by the recombinant viruses. The rescued viruses (rMDV-CIAV-1, rMDV-CIAV-2) or the parental virus (rMDV) chicken embryo fibroblasts (CEFs) in six-well tissue culture plates were infected for 4 days, and the expression of VP1 and VP2 was detected in an indirect immunofluorescence assay with anti-VP1 and anti-VP2 antibodies (1:1000). Bar length, 200 μm.
Figure 4
Figure 4
Growth kinetics and genetic stability of the recombinant Marek’s disease viruses (MDVs) in chicken embryo fibroblasts (CEFs). (A) Comparison of the replication kinetics of the recombinant MDVs and the parental virus (rMDV) in CEFs. (B) PCR detection of the VP1 and VP2 genes inserted in the recombinant MDVs passaged 10 (P10) and 20 (P20) times in CEFs. (C) Confirmation of VP1 and VP2 expression by the recombinant MDVs passaged 20 times in CEFs with immunofluorescence assay. Bar length, 400 μm.
Figure 5
Figure 5
Antibody responses and protective efficacy induced by the recombinant Marek’s disease viruses (MDVs) in chickens. Groups of 10 chickens were inoculated with 2000 PFU of the recombinant MDVs and challenged with the chicken infectious anemia virus (CIAV) at 21 days post-vaccination. (A) The sera samples were collected 3 weeks post-vaccination and 2 weeks post-challenge to detect antibodies against CIAV using ELISA. (B) Hematocrit (HCT) of chickens in each group after 2 weeks post-challenge with CIAV. (C) The thymus index of chickens in each group after 2 weeks post-challenge with CIAV. *, p < 0.05; ***, p < 0.001; ns, no significant difference.
See this image and copyright information in PMC

References

    1. Jeurissen S.H., Wagenaar F., Pol J.M., van der Eb A.J., Noteborn M.H. Chicken anemia virus causes apoptosis of thymocytes after in vivo infection and of cell lines after in vitro infection. J. Virol. 1992;66:7383–7388. doi: 10.1128/jvi.66.12.7383-7388.1992. - DOI - PMC - PubMed
    1. Renshaw R.W., Soine C., Weinkle T., O’Connell P.H., Ohashi K., Watson S., Lucio B., Harrington S., Schat K.A. A hypervariable region in VP1 of chicken infectious anemia virus mediates rate of spread and cell tropism in tissue culture. J. Virol. 1996;70:8872–8878. doi: 10.1128/jvi.70.12.8872-8878.1996. - DOI - PMC - PubMed
    1. Peters M.A., Jackson D.C., Crabb B.S., Browning G.F. Chicken anemia virus VP2 is a novel dual specificity protein phosphatase. J. Biol. Chem. 2002;277:39566–39573. doi: 10.1074/jbc.M201752200. - DOI - PubMed
    1. Zhuang S.M., Shvarts A., van Ormondt H., Jochemsen A.G., van der Eb A.J., Noteborn M.H. Apoptin, a protein derived from chicken anemia virus, induces p53-independent apoptosis in human osteosarcoma cells. Cancer Res. 1995;55:486–489. - PubMed
    1. Danen-Van Oorschot A.A., Fischer D.F., Grimbergen J.M., Klein B., Zhuang S., Falkenburg J.H., Backendorf C., Quax P.H., Van der Eb A.J., Noteborn M.H. Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells. Proc. Natl. Acad. Sci. USA. 1997;94:5843–5847. doi: 10.1073/pnas.94.11.5843. - DOI - PMC - PubMed

LinkOut - more resources