Codon pair bias deoptimization of the major oncogene meq of a very virulent Marek's disease virus.

Codon pair bias deoptimization (CPBD) has been successfully used to attenuate several RNA viruses. CPBD involves recoding a viral protein-coding sequence to maximize the number of codon pairs that are statistically underrepresented in the host, which presumably slows protein translation and, hence, causes virus attenuation. However, since recoding preserves the amino acid composition and codon bias, attenuated and parental viruses are antigenically identical. To determine if Marek's disease virus (MDV), a highly oncogenic herpesvirus of the chicken with a large double-stranded DNA genome, can be attenuated by CPBD of its major oncogene meq, we recoded the gene to minimize (meq-D), maximize (meq-O), or preserve (meq-R) the level of codon pairs that are overrepresented in the chicken protein-coding sequences. Unexpectedly, mutants carrying recoded genes produced comparable or increased levels of Meq in the context of viral infection in cultured cells. In addition, parental virus and mutant viruses carrying recoded meq genes replicated with comparable kinetics in vitro and in vivo, and were equally virulent in susceptible chickens. In summary, CPBD of meq failed to produce any quantifiable attenuation of MDV and confirms differences in the complexity of applying CPBD to large DNA viruses versus RNA viruses.

Turkey herpesvirus with an insertion in the UL3-4 region displays an appropriate balance between growth activity and antibody-eliciting capacity and is suitable for the establishment of a recombinant vaccine.

We constructed turkey herpesvirus (HVT) vector vaccines in which the VP2 gene of infectious bursal disease virus (IBDV) was inserted into the HVT genome in the following regions: UL3-4, UL22-23, UL45-46, and US10-SORF3. We then evaluated the relationship between the gene insertion site and the capacity of the virus to elicit antibodies. rHVT/IBD (US10) showed good growth activity in vitro, with growth comparable to that of the parent HVT. On the other hand, rHVT/IBD (UL3-4), rHVT/IBD (UL22-23), and rHVT/IBD (UL45-46) exhibited decreased growth activity in chicken embryo fibroblast (CEF) cells compared to the parent HVT. However, the rHVT/IBD (US10) elicited lower levels of virus-neutralizing (VN) antibodies compared to the other constructs. rHVT/IBD (UL3-4) and rHVT/IBD (UL45-46) appeared to be similar in their ability to elicit VN antibodies. Based on the results of in vitro and in vivo assays, rHVT/IBD (UL3-4) was selected for further testing. In a challenge assay, rHVT/IBD (UL3-4) protected chickens from challenge with virulent Marek's disease virus serotype 1 and IBDV. In conclusion, the site of gene insertion may have a strong effect on the growth of the vector virus in vitro and its antibody-eliciting capacity. Insertions in the UL3-4 region permitted a balance between growth activity and VN-antibody-eliciting capacity, and this region might therefore be an appropriate insertion site for IBDV VP2.

Marek's disease virus morphogenesis.

Marek's disease virus (MDV) is a highly contagious virus that induces T-lymphoma in chicken. This viral infection still circulates in poultry flocks despite the use of vaccines. With the emergence of new virulent strains in the field over time, MDV remains a serious threat to the poultry industry. More than 40 yr after MDV identification as a herpesvirus, the visualization and purification of fully enveloped infectious particles remain a challenge for biologists. The various strategies used to detect such hidden particles by electron microscopy are reviewed herein. It is now generally accepted that the production of cell-free virions only occurs in the feather follicle epithelium and is associated with viral, cellular, or both molecular determinants expressed in this tissue. This tissue is considered the only source of efficient virus shedding into the environment and therefore the origin of successful transmission in birds. In other avian tissues or permissive cell cultures, MDV replication only leads to a very low number of intracellular enveloped virions. In the absence of detectable extracellular enveloped virions in cell culture, the nature of the transmitted infectious material and its mechanisms of spread from cell to cell remain to be deciphered. An attempt is made to bring together the current knowledge on MDV morphogenesis and spread, and new approaches that could help understand MDV morphogenesis are discussed.

Efficacy and safety of cell associated vaccines against Marek's disease virus grown in a continuous cell line from chickens.

The Marek's disease virus (MDV) vaccine strains CVI 988 and herpes virus of turkeys (HVT) strain FC126, usually are grown in primary chicken embryo fibroblasts (CEF). We found that the strains could be grown also in the so-called JBJ-1 cell line to titres in the same range as when chicken embryo fibroblasts were used. The JBJ-1 cell line is a fibroblast-like continuous chicken cell line, which can be grown in flat bottom tissue culture flasks, roller bottles and on micro carriers. We investigated the efficacy of experimental CVI 988 vaccines grown in JBJ-1 cells and the efficacy of combinations of CVI 988 grown in JBJ-1 cells with HVT FC 126 also grown in JBJ-1 cells. The study was performed in accordance with European Pharmacopoeia monograph 0589 for live MDV disease vaccines. Groups of 1-day-old SPF chicks were vaccinated subcutaneously or intramuscularly, with 10(2.5) TCID50 per dose of CVI 988 alone or in combination with 500PFU per dose of HVT. As a control a group vaccinated with CVI 988 grown in CEF was included. One group was not vaccinated. Five days after vaccination all chickens were challenged with the very virulent MDV strain RB1B. After challenge the chickens were observed for a period of 70 days for signs of Marek's disease (MD). The protection induced by CVI 988 grown in JBJ-1 cells and the combination of CVI 988 and HVT-FC126 both grown in JBJ-1 cells, amply complied with the requirements of the European Pharmacopoeia which prescribes that the protection index should be at least 80%. The safety of the vaccines grown in JBJ-1 cells was tested in a field study in commercial layer chickens. No signs of MD were noticed during the study and no other signs attributable to the vaccine. It is concluded that the JBJ-1 cell line is a suitable substrate for the current vaccines against MD.

Antiviral properties of combination peptides of HIV-1 Rev NLS and NES.

Nuclear translocation signal has been identified as a mediator of protein shuttling between nuclear and cytoplasm. Here we report that the combination of peptides from nuclear localization signal (NLS) and nuclear export signal (NES) of HIV-1 Rev have an antiviral activity against the Herpes virus of turkey and Marek's disease virus serotype 1.