Biological Characteristics Of A Meq-deleted Marek's Disease Virus And Its Immune Effect

Genetically engineered vaccine and live vector vaccine of Marek's disease virus (MDV) with good immunogenicity and high-level biological safety provide a new method for effective prevention of Marek's disease (MD) , avian influenza (AI) and other avian diseases. On the basis of the related studies, the following research work was carried out to study the MDV (GX0101 strain) and its meq gene by the latest bacterial artificial chromosome (BAC) recombination technique in this paper.1. Construction of a meq-deleted strain (GX0101△meq) and the study of its biological Characteristics: In this study, the meq gene was knocked out from a pathogenic MDV strain GX0101 through BAC technique. The infectious clone GX0101Δmeq BAC was purified and used to transfect chicken embryo fibroblasts (CEF). Indirect immunofluorescence assay (IFA) results showed that the meq genes of GX0101 were knockout successfully. The rescued GX0101Δmeq could replicate effectively in CEF. Comparison of pathogenicity between GX0101 and GX0101Δmeq to 1-day old SPF chickens showed that deletion of meq gene led to the loss of the pathogenicity and oncogenicity of GX0101, indicating that the GX0101Δmeq could be a good candidate vaccine strain.2. Study of the relationship between meq gene and MDV-caused immunosuppression: 1-day old SPF chickens were infected with GX0101Δmeq and GX0101, respectively. Immune system variables included relative lymphoid organ weight, blood lymphocytes and antibody production following vaccination against AIV and NDV, and the dynamic changes of T lymphocytes (CD4+/CD8+). The results showed that the immunosuppression caused by GX0101 was significantly decreased due to the deletion of meq gene.3. Evaluation of immune effects of GX0101Δmeq: 1-day old chickens were inoculated intra-abdominally (i.a.) with 2000 PFU of GX0101Δmeq or CVI988/Rispens, respectively. All chickens were challenged i.a. with a very virulent (vv) MDV strain rMd5 after five days. The organs of chickens with MD-specific symptoms were collected during the following 90-d period and after 90 days the samples of heart, liver and spleen of live chickens were all collected. Histopathological evaluation showed that the protection index of GX0101Δmeq or CVI988/Rispens for chickens were 100% and 89% respectively, indicating that GX0101Δmeq is able to induce better immune protection against vv MDV challenge.4. The immuno-protective effect of GX0101Δmeq-BAC: 1-day old chickens were immunized with 10μg of GX0101Δmeq-BAC intramuscularly (i.m.) or 2000 PFU of CVI988/Rispens i.a. Challenge was performed (i.a.) using 500PFU of very virulent rMd5 at day 5 and 12 post-immunization (p.i.), respectively. The organs of MD-specific lesions during 90-d period and the samples of heart, liver and spleen of live chickens after challenge were collected. Histopathological evaluation showed that the protection index of GX0101Δmeq-BAC or CVI988/Rispens for chickens were 33% and 87% on 5 day post challenge, and the protection index of GX0101Δmeq-BAC was 53% on 12 day post challenge. These results indicated that GX0101Δmeq-BAC could provide some immune protection for chickens.5. Construction of a recombinant MDV-GX0101Δmeq-H9-HA expressing AIV H9- HA gene: A recombinant pcDNA3.1 was constructed, which could highly express the HA of H9N2 subtype AIV in CEF. Moreover, a recombinant MDV (rGX0101Δmeq-H9-HA) was constructed successfully by inserting the AIV H9-HA gene cassette to replace the meq gene in the GX0101 genome using"Red / ET"recombination technology.In a word, the series of studies in this paper provided new ideas for the development of novel and efficiently genetic engineering vaccine for MDV and AIV in the future.