Differential quantification of cloned CVI988 vaccine strain and virulent RB-1B strain of Marek's disease viruses in chicken tissues, using real-time PCR.

The 'gold standard' vaccine against Marek's disease in poultry is the CVI988/Rispens virus, which is not easily distinguishable, antigenically or genetically, from virulent Marek's disease herpesvirus. Accurate differential measurement of the CVI988 vaccine and virulent viruses is important to investigate mechanisms of vaccinal protection. Minimal sequence differences between CVI988 and virulent MDV strains restrict the application of molecular diagnostic methods such as real-time PCR to distinguish between these viruses. The use of bacterial-artificial-chromosome (BAC) cloned CVI988 virus, which carries the BAC vector sequences in place of the U(s)2 gene, allows its differential quantification from virulent strains using real-time PCR assays that target the BAC vector sequence and the U(S)2 gene respectively. These novel assays allowed investigation of replication of both serotype-1 vaccine virus (cloned CVI988) and challenge virus (RB-1B strain) in tissues of individual chickens in an experimental vaccination-challenge model of Marek's disease.

Comparative efficacy of BAC-derived recombinant SB-1 vaccine and the parent wild type strain in preventing replication, shedding and disease induced by virulent Marek's disease virus.

A widely used vaccine against Marek's disease (MD) in poultry is the virus SB-1, which is antigenically-related to the causative agent, Marek's disease herpesvirus. We recently cloned the SB-1 genome as an infectious bacterial artificial chromosome, BAC, (pSB-1). The protective efficacies and replication kinetics of pSB-1 and the parent strain (SB-1) were compared in an experimental model of MD induced by a virulent strain, RB-1B. Although vaccine virus replication and shedding was lower for pSB-1 than for SB-1, both vaccines reduced replication and shedding of RB-1B, and were equally effective in protecting chickens against MD. With the cloning of pSB-1, we have now generated full length genomic clones of MD vaccine virus strains belonging to each of the three serotypes. Vaccine viruses derived from each of these clones demonstrated protective efficacies at levels similar to those produced by the respective parent viruses, demonstrating their suitability to be used as vaccine candidates.

The viral envelope is a major determinant for the induction of lymphoid and myeloid tumours by avian leukosis virus subgroups A and J, respectively.

Among the six envelope subgroups of avian leukosis virus (ALV) that infect chickens, subgroups A (ALV-A) and J (ALV-J) are the most pathogenic and widespread among commercial chicken populations. While ALV-A is predominantly associated with lymphoid leukosis (LL) and less frequently with erythroblastosis (EB), ALV-J mainly induces tumours of the myeloid lineage. In order to examine the basis for the lineage specificity of tumour induction by these two ALV subgroups, we constructed two chimeric viruses by substituting the env genes into the reciprocal proviral clones. The chimeric HPRS-103(A) virus carrying the subgroup A env gene is identical to ALV-J prototype virus HPRS-103 except for the env gene, and the chimeric RCAS(J) virus carrying the subgroup J env gene is identical to the parent replication-competent ALV-A vector RCAS except for the env gene. In experimentally inoculated chickens, HPRS-103(A) virus induced LL and EB similar to ALV-A isolates such as RAV-1, while RCAS(J) virus induced myeloid leukosis (ML) and EB, similar to ALV-J, suggesting that the env gene is the major determinant for the lineage-specific oncogenicity. There were genetic differences in susceptibility to tumour induction between line 0 and line 15(I) chickens, indicating that in addition to the env gene, other viral or host factors could also serve as determinants for oncogenicity. Induction of both LL and ML by the two chimeric viruses occurred through the activation of c-myc, while the EB tumours were induced by activation of the c-erbB oncogene.

Expression of MK6a dominant-negative and C-terminal mutant transgenes in mice has distinct phenotypic consequences in the epidermis and hair follicle.

Mouse keratin 6a (MK6a) is constitutively expressed in a single cell layer of the outer root sheath (ORS) of hair follicles, but its synthesis can be induced in interfollicular epidermis including the basal cell layer in response to perturbing stimuli. A basally inducible human K6 (HK6) isoform has not been described, and it is not clear which of the known HK6 isoforms is expressed in the ORS. In this study we show that expression of a dominant-negative MK6a construct (Delta2B-P) in the interfollicular epidermis caused severe blistering and neonatal lethality, suggesting that mutations in a yet to be identified basally expressed HK6 isoform might result in a severe blistering phenotype. Surviving Delta2B-P animals showed transgene expression only in isolated epidermal cells and not in all cells of the ORS, but nevertheless developed severe alopecia. Expression of two different C-terminal mutant transgenes also caused alopecia while a third C-terminal mutant had no phenotypic conse- quences. Electron microscopy revealed that Delta2B-P expression resulted in the collapse of keratin filaments, while destruction of hair follicles in the two phenotypic C-terminal mutant lines occurred in the absence of filament abnormalities. The latter finding indicates that the innermost ORS cells are uniquely sensitive to expression of even slightly altered K6 proteins, suggesting that mutations affecting an HK6 isoform expressed in this cell layer could result in alopecia in humans as well.

Differences in both inositol 1,4,5-trisphosphate mass and inositol 1,4,5-trisphosphate receptors between normal and dystrophic skeletal muscle cell lines.

Human normal (RCMH) and Duchenne muscular dystrophy (RCDMD) cell lines, as well as newly developed normal and dystrophic murine cell lines, were used for the study of both changes in inositol 1,4,5-trisphosphate (IP3) mass and IP3 binding to receptors. Basal levels of IP3 were increased two- to threefold in dystrophic human and murine cell lines compared to normal cell lines. Potassium depolarization induced a time-dependent IP3 rise in normal human cells and cells of the myogenic mouse cell line (129CB3), which returned to their basal levels after 60 s. However, in the human dystrophic cell line (RCDMD), IP3 levels remained high up to 200 s after potassium depolarization. Expression of IP3 receptors was studied measuring specific binding of 3H-IP3 in the murine cell lines (normal 129CB3 and dystrophic mdx XLT 4-2). All the cell lines bind 3H-IP3 with relatively high affinity (Kd: between 40 and 100 nmol/L). IP3 receptors are concentrated in the nuclear fraction, and their density is significantly higher in dystrophic cells compared to normal. These findings together with high basal levels of IP3 mass suggest a possible role for this system in the deficiency of intracellular calcium regulation in Duchenne muscular dystrophy.

Formation of triads without the dihydropyridine receptor alpha subunits in cell lines from dysgenic skeletal muscle.

Muscular dysgenesis (mdg/mdg), a mutation of the skeletal muscle dihydropyridine receptor (DHPR) alpha 1 subunit, has served as a model to study the functions of the DHPR in excitation-contraction coupling and its role in triad formation. We have investigated the question of whether the lack of the DHPR in dysgenic skeletal muscle results in a failure of triad formation, using cell lines (GLT and NLT) derived from dysgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Large T antigen. Both cell lines express muscle-specific proteins and begin organization of sarcomeres as demonstrated by immunocytochemistry. Similar to primary cultures, dysgenic (GLT) myoblasts show a higher incidence of cell fusion than their normal counterparts (NLT). NLT myotubes develop spontaneous contractile activity, and fluorescent Ca2+ recordings show Ca2+ release in response to depolarization. In contrast, GLTs show neither spontaneous nor depolarization-induced Ca2+ transients, but do release Ca2+ from the sarcoplasmic reticulum (SR) in response to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha 1 subunit of the skeletal muscle DHPR, and the alpha 2 subunit is mistargeted. Nevertheless, the ryanodine receptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR alpha subunits in the T-tubules. In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced feet. These findings provide conclusive evidence that interactions between the DHPR and RyR are not involved in the formation of triad junctions or in the normal organization of the RyR in the junctional SR.