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.

Obstacles and limitations of transfer factor biological activity assay design.

Transfer factor (TF) is a heterogeneous mix of low-molecular weight molecules obtained from dialyzed leukocyte extract that is capable of transferring cell-mediated immunity. As an immunostimulatory drug TF is used to improve treatment of infectious diseases, allergies, cancer and immune deficiencies. The main benefit of TF preparations as immunotherapeutic agents is the induction of a rapid immune response and the potential of TF as an adjuvant in combination with other drugs might lead to development of novel approaches to combat various diseases in the future.

Cells transformed by murine herpesvirus 68 (MHV-68) release compounds with transforming and transformed phenotype suppressing activity resembling growth factors.

In this study, we investigated the medium of three cell lines transformed with murine herpesvirus 68 (MHV-68) in vitro and in vivo, 68/HDF, 68/NIH3T3, and S11E, for the presence of compounds resembling growth factors of some herpesviruses which have displayed transforming and transformed phenotype suppressing activity in normal and tumor cells. When any of spent medium was added to cell culture we observed the onset of transformed phenotype in baby hamster kidney cells (BHK-21) cells and transformed phenotype suppressing activity in tumor human epithelial cells (HeLa). In media tested, we have identified the presence of putative growth factor related to MHV-68 (MHGF-68).

Ovine herpesvirus 1 (OVHV-1) thymidine kinase locus sequence analysis: evidence that OVHV-1 belongs to the Macavirus genus of the Gammaherpesvirinae subfamily.

The HindIII-HincII fragment of the 5.5 kbp H11 HindIII clone of ovine herpesvirus 1 (OvHV-1) was cloned and its primary structure was determined by preparation of nested deletion subclones and their sequencing. Sequence analysis of the overlapping clones revealed that 3239 bp OvHV-1 fragment contains complete thymidine kinase (TK) gene, a partial open reading frame of ORF20 and that encoding glycoprotein H (gH).

Marek΄s disease: rapid progress in research with unclear biological implementations.

Here we would like to provide a brief overview of the modern history of Marek΄s disease (MD) research with a focus on the most recent developments in experimental work and we will try to sum up their impact on the understanding of the biological properties of Marek΄s disease type 1 (MDV-1), the only representative of the Mardivirus genus causing fatal lymphoproliferative disease in poultry. We will also compare MDV-1 with other serologically-related poultry herpesviruses, Marek΄s disease virus type 2 (MDV-2) and herpesvirus of turkeys (HVT). Although MD was first described at the beginning of the last century, proper characterization of its biological impact on poultry production and utilization of molecular biology methods for detailed characterization of causative agent MDV-1 were introduced only in recent decades.

Recombinant herpesviruses as tools for the study of herpesvirus biology.

This article is a brief summary of efforts to generate mutant herpesviruses for investigating and assigning gene functions of herpesviruses in replication and pathogenesis. While a full review of all herpesviruses is beyond the scope of this review, we focused our attention on the prototype of the herpesvirus subfamily - herpes simplex virus and murine gammaherpesvirus that serves as an excellent animal model to study human gammaherpesvirus pathogenesis. Furthermore, our present knowledge of essential, non-essential, and common genes of herpesviruses as well as of accessory genes that are currently being studied with the help of the bacterial artificial chromosome (BAC) system will also be discussed.

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.

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.

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.

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.

An enzyme-linked immunosorbent assay (ELISA) for detection of Marek's disease virus-specific antibodies and its application in an experimental vaccine trial.

An enzyme-linked immunosorbent assay (ELISA) for the detection of Marek's disease virus (MDV)-specific antibodies was developed. Chicken embryo cells (CEC) or chicken kidney cells (CKC) were infected with MDV vaccine strain CVI988/Rispens, and infected-cell lysates were prepared at day 5 post-infection by freeze-thawing. Uninfected-cell lysates served as negative controls.

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.

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.

Oncoviral bovine leukemia virus G4 and human T-cell leukemia virus type 1 p13(II) accessory proteins interact with farnesyl pyrophosphate synthetase.

G4 and p13(II) are accessory proteins encoded by the X region of bovine leukemia virus and human T-cell leukemia virus type 1 (HTLV-1), respectively. Disruption of the G4 and p13(II) open reading frames interferes with viral spread in animal model systems, indicating that the corresponding proteins play a key role in viral replication. In addition, G4 is oncogenic in primary cell cultures and is absolutely required for efficient onset of leukemogenesis in sheep.

Increased virulence of Marek's disease virus type 1 vaccine strain CV1988 after adaptation to qt35 cells.

CVI988/Rispens strain of Marek's disease virus type I (MDV-1) is widely used as efficient vaccine to control Marek's disease (MD) in chicken flocks. Similarly to other live MD vaccine viruses it is propagated in freshly prepared chicken embryo fibroblasts (CEF). In this study, MDV-1 CVI988/Rispens strain was adapted to QT35 cells.

Characterization of RKZ isolate of ovine herpesvirus 1.

Cytopathic effect (CPE) characterized mainly by foci of rounded cells was observed in cultures of primary plexus choroideus cells from healthy lamb following cryopreservation. It was possible to transmit the infectious agent to other primary cells of ovine origin by co-cultivation with infected cells. By indirect immunofluorescence microscopy it was found that high percentage of sheep (65-80% in 3 different herds from Slovakia) are infected with this infectious agent.

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.

Nucleotide sequence of the gene encoding the major capsid protein of herpesvirus of turkeys.

The gene encoding the major capsid protein (MCP) VP5 of herpesvirus of turkeys (HVT) was identified and sequenced. It has a single open reading frame of 4236 nucleotides encoding 1412 aa protein. The gene is flanked by VP23 and UL20 sequences and is localized in the unique long region (UL) within the BamHI-B fragment.

Glycoprotein gD of MDV lacks functions typical for alpha-herpesvirus gD homologues.

Glycoprotein D (gD) belongs to family of conserved structural proteins of alpha-herpesviruses. During productive infection of cells by herpes simplex virus 1 (HSV-1) gD has several important functions, is involved in virus penetration to and release from infected cells and is one of main targets of neutralizing antibodies. Similar functions are shared also by other alpha-herpesvirus gD homologues.

Sequence and characterisation of the Z gene encoding ring finger protein of the lymphocytic choriomeningitis virus MX strain.

We have cloned and characterised a cDNA encoding Z protein of recently identified MX strain of lymphocytic choriomeningitis virus (LCMV) persistently infecting human MaTu cells. Deduced amino acid sequence of LCMV MX Z protein showed 88.9% identity with that of the LCMV Armstrong (ARM) strain and 80.

Carbonic anhydrase IX, MN/CA IX: analysis of stomach complementary DNA sequence and expression in human and rat alimentary tracts.

Background & Aims: CA IX (formerly MN protein) is a carbonic anhydrase isoenzyme whose expression is associated with human tumors. However, it has also been found in normal gastric mucosa. The aim of this study was to determine differences in complementary DNAs (cDNAs), to obtain an overview of distribution in the alimentary tract, and to obtain data on expression in tumors.

Herpesvirus of turkeys homologue of HSV VP16 is structurally related to varicella zoster virus trans-inducing protein encoded by ORF 10.

Expression of the immediate-early genes of alpha-herpesviruses is stimulated by a family of trans-inducing factors represented by VP16 of HSV-1 and ORF10 gene product of VZV. We have identified and determined the nucleotide sequence of the UL48 gene encoding the herpesvirus of turkeys (HVT) homologue of HSV VP16. The gene maps to the BamHI-J fragment and appears to be expressed in a form of bicistronic transcript together with UL49.

The syn3 strain HSZP of herpes simplex virus type 1 (HSV-1) is not pathogenic for mice and shows limited neural spread.

Strain HSZP of the herpes simplex virus type 1 (HSV-1) forms large giant cells in vitro. This property was found associated with a mutation that alters the codon CGC (in the strain KOS or 17 sequence) to CAC (in the HSZP sequence), changing the amino acid 857 from arginine to histidine in the cytoplasmic domain of the glycoprotein B (gB) polypeptide chain. Giant cell formation by ANGpath was attributed to a mutation that alters the codon GCC (in KOS and strain 17 sequences) to GTC (in ANGpath sequence) changing the amino acid 854 in the same (syn3) region of the gB molecule.

Human MN/CA9 gene, a novel member of the carbonic anhydrase family: structure and exon to protein domain relationships.

We have isolated, sequenced, and characterized a human MN/CA9 gene. This gene is a novel member of the carbonic anhydrase (CA) family, which codes for widely distributed catalysts of the reversible conversion of carbon dioxide to carbonic acid. So far, MN/CA IX is the only tumor-associated CA isoenzyme.

Structure and properties of a herpesvirus of turkeys recombinant in which US1, US10 and SORF3 genes have been replaced by a lacZ expression cassette.

In the process of generating an insertional mutant of herpesvirus of turkeys (HVT) expressing lacZ at the protein kinase (PK) locus, we isolated a recombinant which contained an intact PK gene but the short unique regions US1, US10 and SORF3 had been deleted and replaced by the lacZ cassette. Moreover, the virus contained duplicate copies of gD, gI and gE in an opposite orientation flanking lacZ, US2 and PK which were contiguous. These results are of interest in relation to the flexibility of the short unique segment (Us) and of the inverted repeats flanking Us of the alpha-herpesviruses.