Use of nucleic acids amplification methods in Marek's disease diagnosis and pathogenesis studies.

Marek's disease (MD) is a lymphoproliferative disease of chickens with strong economic impact on poultry industry. Although successful vaccination has enabled control of the disease, outbreaks occur in commercial flocks, resulting in substantial economic losses. Together with vaccination, accurate and fast diagnosis of MD remain the most important tools for its efficient control. MD diagnosis currently relies mainly on the identification of its causative agent, Marek's disease virus type 1 (MDV-1). Nucleic acid amplification techniques have been successfully applied for identification of MDV DNA in field samples and also for studies of virus-host interactions. In this review we want to summarize recent advances in the development of standard and quantitative PCR techniques and their use in rapid MD diagnosis, including differentiation of pathogenic and vaccine MD viruses. PCR protocols have served as a useful tool for clarification of processes associated with MDV infection in chickens, such as virus spread and release, and effect of vaccine virus on progress of MD. Here, we also describe a novel multi-species qPCR methodology for identification and quantification of MDV DNA, enabling its detection in various bird species that are the most susceptible to MDV infection. Keywords: Marek's disease; MDV; diagnosis; nucleic acid detection; duplex quantitative PCR.

A chemically defined production process for highly attenuated poxviruses.

Highly attenuated poxviruses are promising vectors for protective and therapeutic vaccines. These vectors do not replicate in human cells and can therefore be safely given even to immunocompromised recipients. They can accommodate very large inserts and provide strong stimulation of the immune system against the vectored antigen. Disadvantages include that very high numbers of infectious units are required per dose for full efficacy. Because they are difficult to produce, improved cellular substrates and processes are urgently needed to facilitate programs intended to reach a large number of vaccinees. We have developed a fully scalable and very efficient chemically-defined production process for modified vaccinia Ankara (MVA), canarypox (CNPV, strain ALVAC) and fowlpox viruses (FPV) based on a continuous cell line.

Haemonchus contortus: characterization of the baculovirus expressed form of aminopeptidase H11.

Recombinant form of Haemonchus contortus aminopeptidase H11, an intestinal membrane glycoprotein considered to be in its native form the most promising vaccine candidate, was produced in insect cells, characterised and tested in pilot vaccination-challenge trial on sheep. The sequence of the cloned gene, obtained by RT PCR isolated from adult worms, showed 97% identity to the highly immunogenic H11 clone, described by Graham et al., (database accession number AJ249941.1). A 1305 bp fragment of H11 was expressed in E. coli and used to raise a specific antiserum, which recognized recombinant forms of H11 and 110 kDa protein from H. contortus extract. H11 was expressed by baculovirus recombinants in insect cells in full length and as a fusion protein with H. contortus glutathione S-transferase (GST). The baculovirus produced recombinant antigens were used without adjuvants to immunize sheep, which resulted in 30% (full length H11) and 20% (GST-H11) reduction of worm burden. These animal experiments indicated that, although the protection induced by in vitro produced protein is lower than in case of H11 isolated from worms, recombinant forms of aminopeptidase may be considered as antigens for the control of haemonchosis.

High-level expression of Marek's disease virus glycoprotein C is detrimental to virus growth in vitro.

Expression levels of Marek's disease virus (MDV) glycoprotein C (gC) are significantly reduced after serial virus passage in cell culture. Reduced gC expression coincides with enhanced MDV growth in vitro and attenuation. To analyze this phenomenon in detail, a full-length infectious MDV clone was modified by Red-based and shuttle mutagenesis in Escherichia coli. Besides a gC-negative deletion mutant harboring a kanamycin resistance gene, a markerless mutant with the U(L)44 gene deleted was constructed. On the basis of this deletion mutant, the original or a modified U(L)44 gene with a mutated start codon (AUG-->ACG) was reinserted into the authentic locus. Similarly, mutants expressing authentic gC or the start codon mutation under the control of a strong constitutive promoter were generated. In vitro studies demonstrated that gC deletion mutants induced twofold-larger plaques than the parental virus did, whereas constitutive overexpression of the glycoprotein resulted in a more than twofold reduction in plaque size. In addition, plaque sizes of the gC deletion mutant were reduced when virus was grown using supernatants from cells infected with parental virus, but supernatants obtained from cells infected with the gC deletion mutant had no measurable effect on plaque size. The results indicated that (i) expression of MDV gC, albeit at low levels in a highly passaged virus, had a significant negative impact on the cell-to-cell spread capabilities of the virus, which was alleviated in its absence and exacerbated by its overexpression, and that (ii) this activity was mediated by the secreted form of MDV gC.

Marek's disease virus research in the post-sequencing era: new tools for the study of gene functions and virus-host interactions.

Despite the fact that the causative agent of Marek's disease was described more than 30 years ago, and that subsequently many classical biological studies have been carried out on the Marek's disease virus (MDV), detailed analysis of its gene functions has been hampered by lack of suitable research tools. Information on the primary structure of MDV-1 and its serologically related viruses, MDV-2 and herpesvirus of turkeys, is now available. This review focuses on the introduction of the modern and highly efficient technology of bacterial artificial chromosome (BAC) cloning and mutagenesis for rapid manipulation of the MDV genome, with the aim of studying the functions of its genes and non-coding regions. Constructed MDV BACs carry the complete genome of MDV that can be multiplied in Escherichia coli and manipulated using the tools provided by bacterial genetics. The novel approach of MDV DNA mutagenesis using BAC technology will be explained by examples, and we will discuss gene functions in comparison with their counterparts in other herpesviruses. In addition, we have shown that MDV BAC DNA can be used as a polynucleotide vaccine to protect against Marek's disease, thus opening a new chapter in strategies for control of this disease.

A DNA vaccine containing an infectious Marek's disease virus genome can confer protection against tumorigenic Marek's disease in chickens.

A DNA vaccine containing the infectious BAC20 clone of serotype 1 Marek's disease virus (MDV) was tested for its potential to protect against Marek's disease (MD). Chickens were immunized at 1 day old with BAC20 DNA suspended either in PBS, as calcium phosphate precipitates, incorporated into chitosan nanoparticles, in Escherichia coli DH10B cells, or bound to gold particles for gene-gun delivery. Challenge infection with MDV strain EU1 was performed at 12 days old, and four out of seven birds immunized with BAC20 DNA in saline by the intramuscular route remained free of MD until day 77 after challenge infection. A delay in the development of the disease could be observed in some animals vaccinated with other BAC20 DNA formulations, but clinical MD and tumour formation were evident in all but one bird. Five out of seven animals immunized with the vaccine virus CVI988 were protected against MD, but none out of seven birds survived EU1 challenge infection after injection of negative-control plasmid DNA. In a second animal experiment, five out of 12 chickens immunized with BAC20 DNA and six out of eight birds immunized with virus reconstituted from BAC20 DNA remained free of MD after challenge infection. In contrast, none out of 12 chickens survived challenge infection after immunization with BAC20 DNA lacking the essential gE gene or with gE-negative BAC20 virus. The results suggested that an MDV BAC DNA vaccine has potential to protect chickens against MD, but that in vivo reconstitution of vaccine virus is a prerequisite for protection.

Suppression subtractive hybridisation to isolate differentially expressed genes involved in invasiveness of melanoma cell line cultured under different conditions.

We have previously analysed the invasion capacity of different melanoma cell lines in the three-dimensional dermal equivalent. The melanoma cell line M4Beu acquired invasive behaviour upon changing its cultivation conditions before the seeding on top of the collagen lattice from single cell suspension to spheroid. Based on this phenomenon SSH was used to search for the genes related to the invasive phenotype of melanoma cells. From differentially expressed clones we focused on four: fibronectin, RhoA, COXII, and H-ras-like protein. By RT-PCR the expression of these genes were tested in different populations (monolayer, spheroids on dermal equivalent) of melanoma cell line M4Beu and three additional melanoma cell lines. The expression of fibronectin was also examined by immunohistochemistry staining of co-culture spheroids-dermal equivalent.

The genome of herpesvirus of turkeys: comparative analysis with Marek's disease viruses.

The complete coding sequence of the herpesvirus of turkeys (HVT) unique long (U(L)) region along with the internal repeat regions has been determined. This allows completion of the HVT nucleotide sequence by linkage to the sequence of the unique short (U(S)) region. The genome is approximately 160 kbp and shows extensive similarity in organization to the genomes of Marek's disease virus serotypes 1 and 2 (MDV-1, MDV-2) and other alphaherpesviruses. The HVT genome contains 75 ORFs, with three ORFs present in two copies. Sixty-seven ORFs were identified readily as homologues of other alphaherpesvirus genes. Seven of the remaining eight ORFs are homologous to genes in MDV, but are absent from other herpesviruses. These include a gene with similarity to cellular lipases. The final, HVT-unique gene is a virus homologue of the cellular NR-13 gene, the product of which belongs to the Bcl family of proteins that regulate apoptosis. No other herpesvirus sequenced to date contains a homologue of this gene. Of potential significance is the absence of a complete block of genes within the HVT internal repeat that is present in MDV-1. These include the pp38 and meq genes, which have been implicated in MDV-1-induced T-cell lymphoma. By implication, other genes present in this region of MDV-1, but missing in HVT, may play important roles in the different biological properties of the viruses.