Marek's disease herpesvirus: a model of vaccine-dependent adaptation.

Marek's disease (MD) virus (MDV) is an alphaherpesvirus that causes a rapid-onset T-cell lymphoma in chickens. In order to preserve the viability of poultry industry, non sterilizing vaccines have been used since fifty years, preventing lymphoma development but leading to an imperfect control of MD. Vaccination has been accompanied with the increase in virulence of MDV forcing the development of new vaccine formulations. Several loci of MDV genome are variable and have evolved in link with virulence of MDV strains. It has been shown that MDV is in fact constituted by a dynamic population of genetic variants with a distribution linked to viral strain phenotype. In this context, we have shown that CVI988/Rispens vaccine, still the most efficient one against hypervirulent MDV strains, is composed of twenty variants, variable from one batch to another, evolving likely as RNA virus quasispecies.

Vaccine and oncogenic strains of gallid herpesvirus 2 contain specific subtype variations in the 5' region of the latency-associated transcript that evolve in vitro and in vivo.

Gallid herpesvirus 2 (GaHV-2) is the alphaherpesvirus responsible for Marek's disease (MD), a T-cell lymphoma of chickens. The virulence of the GaHV-2 field strain is steadily increasing, but MD is still controlled by the CVI988/Rispens vaccine. We tried to determine distinguishing traits of the CVI988/Rispens vaccine by focusing on the 5' end region of the latency-associated transcript (5'LAT). It includes a variable number of 60-bp tandem repeats depending on the GaHV-2 strain. By analyzing six batches of vaccine, we showed that CVI988/Rispens consisted of a population of 5'LAT molecular subtypes, all with deletions and lacking 60-bp tandem repeat motifs, with two major subtypes that probably constitute CVI988/Rispens markers. Serial passages in cell culture led to a substantial change in the frequency of CVI988/Rispens 5'LAT subtypes, with non-deleted subtypes harboring up to four 60-bp repeats emerging during the last few passages. Dynamic changes in the distribution of 5'LAT-deleted subtypes were also detected after infection of chickens. By contrast, the 5'LAT region of the oncogenic clonal RB-1B strain, which was investigated at every step from the isolation of the clonal bacmid RB-1B DNA to the isolation of the ovarian lymphoma cell line, consisted of non-deleted 5'LAT subtypes harboring at least two 60-bp repeats. Thus, vaccine and oncogenic GaHV-2 strains consist of specific populations of viral genomes that are constantly evolving in vivo and in vitro and providing potential markers for epidemiological surveys.

Investigation of the hepatotoxicity profile of chemical entities using Liverbeads and WIF-B9 in vitro models.

The cytotoxicity profile of various chemical entities was evaluated using two in vitro hepatocyte models. Liverbeads is a cryopreserved model consisting of primary hepatocytes entrapped in alginate beads. WIF-B9 is a hybrid cell line obtained by fusion of rat hepatoma (Fao) and human fibroblasts (WI38). Various reference hepatotoxicants were tested and ranked according to their equivalent concentration 50 (EC50) for various biochemical endpoints (lactate dehydrogenase (LDH) release, 3-(4,5 dimethylthiazol 2yl)-2,5-diphenyl-2H tetrazolium bromure (MTT) activity, adenosine triphosphate (ATP) and glutathione (GSH) levels). The ranking obtained was comparable in both models and consistent with previously published results on hepatocyte monolayers. Ketoconazole, erythromycin estolate, retinoic acid, telithromycin and alpha-naphthyl-isothiocyanate were among the most toxic chemicals in both models, with an EC50 < 200 microM. Troleandomycin, spiramycin, erythromycin, diclofenac, taurodeoxycholate, warfarin, galactosamine, valproic acid and isoniazid were found to be less toxic. Few marked differences, potentially linked to metabolism pathways, were observed between EC50s in the two models for compounds such as cyclosporine A (10 and > 831 microM) and warfarin (5904 and 1489 microM) in WIF-B9 and Liverbeads, respectively. The results obtained indicate that Liverbeads and WIF-B9 cells are reliable in vitro models to evaluate the hepatotoxic potential of a wide range of chemicals, irrespective of structure and pharmaceutical class.

Cytochrome P450 expression-induction profile and chemically mediated alterations of the WIF-B9 cell line.

BACKGROUND INFORMATION: WIF-B9 is a hybrid cell line obtained by fusion of rat hepatoma cells (Fao) and human fibroblasts (WI38). It exhibits the structural and functional characteristics of differentiated hepatocytes, including active bile canaliculi. The aim of the present study was to characterize the WIF-B9 cell line as a model for analysing drug-induced hepatic effects. The drug metabolism potential of WIF-B9 cells was identified by studying the rat and human CYP (cytochrome P450) mRNA constitutive expression profile and induction potential after exposure to reference inducers. The morphological alterations provoked by chemical entities were also characterized. RESULTS: Competitive reverse transcriptase-PCR revealed that four rat (1A1, 2B1/2, 2E1 and 4A1) and four human (1A1, 2Cs, 2D6 and 2E1) CYP mRNA isoforms were constitutively expressed in WIF-B9 cells. The rat CYP forms were expressed at levels 2-4 orders of magnitude higher than the human forms. Exposure for 20-72 h to increasing concentrations of CYP reference inducers (beta-naphthoflavone, 3-methyl cholanthrene, dexamethasone, phenobarbital, clofibrate and pregnenolone 16alpha-carbonitrile) revealed that the rat CYP 1A1, 1A2, 3A1, 3A2 and 4A1 and human CYP 1A1 and 2Cs mRNAs were inducible. Rat CYP 1A1 and 1A2 were the most inducible isoforms since they were overexpressed up to 100-fold after 20-48 h of treatment with beta-naphthoflavone. Human CYP 1A1 and 2Cs mRNAs were induced 3-fold after 48 h of treatment with phenobarbital. Other mechanisms involved in hepatotoxicity were explored using microscopy and immunofluorescence. The WIF-B9 cell line exhibited fragmentation and dilatation of bile canaliculi upon exposure to erythromycin, and to isoniazid and cytochalasins, respectively. Monensin promoted cell depolarization and cytoplasmic granulation. Ethionine promoted cytoplasmic vacuolation and dilatation of the Golgi structures. CONCLUSIONS: These results indicate that the CYP expression and induction profiles and the morphological features of WIF-B9 cells allow prediction in vitro of the induction and hepatotoxicity profiles of chemical entities.