Construction of Recombinant Marek's Disease Virus Expressing the NDV-F gene and its Replication in Chickens and in Vitro / 病毒学报

We used a meq-deleted attenuated MDV-I strain GX0101Δmeq as a vector to construct a recombinant virus expressing the exogenous gene NDV-F. The ORF of exogenous gene NDV-F was inserted into the eukaryotic expression vector pcDNA3.1(-). Then, the expression cassette of NDV-F which contains the CMV promoter was amplified. Simultaneously, we amplified the selected gene Kan+ expression cassette and inserted them into the PMD18-T vector. Tandem expression cassettes were amplified using primers containing the 50-bp homologous arm of MDV-US2. The PCR product was electroporated into EL250 host bacteria containing GX0101Δmeq. Then, the Kan+ expression cassette was deleted from the recombinant virus genome using 1% arabinose. The plasmid of the positive clone which the Kan+ expression cassette was deleted was extracted and transfected into CEFs to rescue the recombinant virus. The recombinant virus was injected into chickens to observe its growth and replication. The recombinant virus rMDV-F containing the exogenous gene NDV-F was rescued successfully. The recombinant virus could duplicate and express well in CEFs, and grow and replicate well in chickens. Using GX0101Δmeq as a vector, combined with a recombinant system of Red E/T and FLP/FRT, we constructed a recombinant virus that expressed the exogenous gene NDV-F. This study could lay the foundation for further study of recombinant viruses.

Molecular characterization of duck enteritis virus CHv strain UL49.5 protein and its colocalization with glycoprotein M

The UL49.5 gene of most herpesviruses is conserved and encodes glycoprotein N. However, the UL49.5 protein of duck enteritis virus (DEV) (pUL49.5) has not been reported. In the current study, the DEV pUL49.5 gene was first subjected to molecular characterization. To verify the predicted intracellular localization of gene expression, the recombinant plasmid pEGFP-C1/pUL49.5 was constructed and used to transfect duck embryo fibroblasts. Next, the recombinant plasmid pDsRed1-N1/glycoprotein M (gM) was produced and used for co-transfection with the pEGFP-C1/pUL49.5 plasmid to determine whether DEV pUL49.5 and gM (a conserved protein in herpesviruses) colocalize. DEV pUL49.5 was thought to be an envelope glycoprotein with a signal peptide and two transmembrane domains. This protein was also predicted to localize in the cytoplasm and endoplasmic reticulum with a probability of 66.7%. Images taken by a fluorescence microscope at different time points revealed that the DEV pUL49.5 and gM proteins were both expressed in the cytoplasm. Overlap of the two different fluorescence signals appeared 12 h after transfection and continued to persist until the end of the experiment. These data indicate a possible interaction between DEV pUL49.5 and gM.

Evaluation of the pathogenicity of a field isolate of Marek's disease virus integrated with retroviral long terminal repeat sequence / 病毒学报

The pathogenicity of a field isolate of Marek's disease virus (MDV) named GXY2 integrated with retroviral long terminal repeat (LTR) sequence from a chicken with MD tumors was evaluated. Experimental chickens were divided into group A, B, C, D and E. The later four groups were vaccinated on one-day-old with CVI988/Rispens for group B and D, with HVT for group C and E, while group A was taken as no-vaccinated control. On 8-day-old, group A, B and C were challenged with GXY2 by intra-abdominal injection, group D and E were kept as un-challenged control. All the birds were raised routinely until 82 days post-challenge (PC), died birds during the experiment and the slaughtered birds at the end of the experiment were necropsied and examined for gross lesions of MD and further confirmed by a developed polymerase chain reaction (PCR) based differential diagnosis technique for avian neoplastic diseases. The results showed that time of onset of MD death of group A, B and C were PC 25, 77 and 29 days with the incidences of visible MD visceral tumors. On PC 82 days, tumor incidences and mortalities of group A, B and C were 72%, 34.8% and 50%, 84%, 21.7% and 20%, respectively. The vaccination protection of CVI988/Rispense and HVT were 51.67% and 30.56% respectively. Among all the visceral organs, heart had the highest tumor incidences (23.5%), and then followed by liver (14.7%) and gizzard (10.3%). The weight-gain of unvaccinated birds was significantly depressed and severe dystrophy of thymus and bursa of Fabricius were also found. The results of the study demonstrated that isolate GXY2 possessed the ability of causing acute tumors and overcoming the protection of the vaccinations of either CVI988/Rispense or HVT.

Construction of Marek's disease virus serotype 814 strain as an infectious bacterial artificial chromosome / 生物工程学报

The aim of this study was to construct the complete genome of Marek's disease virus serotype 814 strain as an infectious bacterial artificial chromosome (BAC). Using self-designed selection marker Eco-gpt (1.3 kb) and BAC vector pBeloBAC11 (7.5 kb), we constructed the transfer plasmid pUAB-gpt-BAC11. The plasmid pUAB-gpt-BAC11 and MDV total-DNA were cotransfected into secondary CEFs; we put the virus-containing cells in selection medium for eight rounds and obtained purified recombinant viruses. Recombinant viral genomes were extracted and electroporated into E. coli, BAC clones were identified by restriction enzyme digestion and PCR analysis. Finally, we obtained 38 BAC clones, DNA from various MDV-1 BACs was transfected into CEFs, and recombinant virus was reconstituted by transfection of MDV-BAC2 DNA. We successfully cloned the complete genome of MDV-1814 strain as an infectious bacterial artificial chromosome. With these cloned genomes, a revolutionary MDV-DNA engineering platform utilizing RED/ET recombination system was constructed successfully, which can help the understanding of MDV gene functions and promote the using of MDV as a vector for expressing foreign genes. In addition, it opens the possibility to generate novel MDV-1 vaccines based on the BACs.

Construction and immunological characterization of recombinant Marek's disease virus expressing IBDV VP2 fusion protein / 生物工程学报

A transfer plasmid vector pUC18-US10-VP2 was first constructed by inserting the gene of the enhancer green fluorescent protein(eGFP) fused to the VP2 gene of very virulent Infectious bursal disease virus (IBDV) JS strain into the US10 fragment of the Marek's disease virus (MDV) CV1988/Rispens. The recombinant virus, designated as rMDV, was developed by co-transfecting CEF with the transfer plasmid vector and simultaneously infecting with the CVI988/Rispens virus. The PCR and IFA results indicated that the rMDV is stable after 31 passages. Chickens vaccinated with rMDV were protected from challenge with 100LD50 of IBDV JS. The protection ratio of the chickens vaccinated with the 1000PFU, 2000PFU, 5000PFU of the rMDV were 50%, 60%, and 80% respectively. It is interesting that the average histopathology BF lesion scores of chicken group immunized with 5000PFU of rMDV by one-time vaccination was close to that of chicken group vaccinated with IBDV live vaccine NF8 strain for twice (2.0/1.5). There is no difference in protection between the groups (P > 0.05) but significent difference between groups immunized with 5000 PFU of rMDV and with normal MDV. This demonstrated that rMDV expressing VP2 fusion protein was effective vaccine against IBDV in SPF chickens.

Polymerase chain reaction for the detection and differentiation of Marek's disease virus strains MDV-1 and HVT

Marek's disease [MD] is a lymphoproliferative disease of chickens characterized by lymphocytic infiltration of various organs. The present study was an attempt to use polymerase chain reaction [PCR] to optimize a rapid and reliable assay for detection of MDV genome. Detection of serotype I of MDV [MDV-l] was confirmed by presence of a 200 bp DNA fragment as a PCR product. Differentiation of MDV- I and herpesvirus of turkeys [HVT] was also conducted using specific primers from the glycoprotein A [gA] gene and a 388 bp DNA fragment was amplified from HVT genome. The specificity of the test was confirmed by sequencing of PCR products. Results indicate that MDV-l can be diagnosed in clinical samples and inoculated cell cultures which is used for virus isolation. In addition, differentiation between MDV- I and HVT viruses was confirmed based on the size of PCR products. The test proved to be rapid and reliable and be performed as a robust diagnostic test in veterinary diagnostic laboratories