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. The process of TF preparation is rather simple. However, with respect to fact that TF is composed by several multifunction molecules, it is likely that during the activity measurement based only on one single parameter, other TF biological activities might be overlooked. In addition, separated TF components might display synergetic activity effect. According to recent European Pharmacopoeia there is no general protocol for immuno-stimulatory drugs (including TF) activity measurement available. Nevertheless, for the process of TF preparation, control of input material and for final pharmaceutical product batches it is inevitable to guaranty proper quality control including appropriate in vivo or in vitro test(s) for TF biological activity assay. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic problem, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. The currently used method for testing biological activity of TF is the in vitro MTT cells proliferation assay that is recognized by control authorities in Slovak Republic. Keywords: immune system; transfer factor; dialysable leukocyte extract; diseases; MTT cells proliferation assay.

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). Its bivalent properties have been blocked entirely by antisera against MHV-68 and two monoclonal antibodies against glycoprotein B (gB) of MHV-68 suggesting viral origin of MHGF-68. The results of initial efforts to separate MHGF-68 on FPLC Sephadex G15 column in the absence of salts revealed the loss of its transforming activity but transformed phenotype suppressing activity retained. On the other hand, the use of methanol-water mobile phase on RP-HPLC C18 column allowed separation of MHGF-68 to two compounds. Both separated fractions, had only the transforming activity to normal cells. Further experiments exploring the nature and the structure of hitherto unknown MHGF-68 are now in the progress to characterize its molecular and biological properties.

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). The conserved OvHV-1 TK displayed the highest similarity to homologous TK proteins encoded by members of the Macavirus genus of the Gammaherpesvirinae subfamily. These data including our previous analysis of the partial sequence of VP23 homologue might serve as further evidence that OvHV-1 should be categorized within the genus Macavirus of the Herpesviridae family.

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. This system facilitates the analysis of herpesviral genes with potential for use in gene therapy or as anti-cancer therapeutics.

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. However, many characteristics of MD infection, pathogenesis and vaccine protection mechanisms remain unclarified, though novel methods bring a challenge for better understanding of these unanswered questions.

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. Electron microscopy of cells with CPE revealed the presence of herpesvirus particles. Viral DNA was isolated from infected cells using pulse-field gel electrophoresis and further used as probe in Southern blot analysis. The probe reacted specifically only with DNA from cells infected with Ovine herpesvirus 1 (OvHV-1) but not with DNA of other ruminant herpesviruses. Some of the HindIII restriction fragments of DNA of the obtained OvHV-1 isolate denominated RKZ were cloned. Part of the H9 clone was sequenced identifying a gene that encoded a polypeptide homologous to conserved herpesvirus VP23 structural protein. From comparison of the sequence of this clone with VP23 sequences of other herpesviruses it was deduced that OvHV-1 might be classified within the Rhadinovirus genus of the Gammaherpesvirinae subfamily. The sequencing of the H9 clone of DNA of RKZ isolate enabled establishment of sensitive and highly specific polymerase chain reaction (PCR) assay for detection of OvHV-1.

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. Surprisingly, in previous studies it was found that MDV gD expression could not be detected during infection in vitro using immunological methods. In this study we have analyzed expression of MDV gD and its biological consequences. In vitro expression using rabbit reticulocyte lysate and/or overexpression in transfected cells showed that the second ATG codon is required for synthesis of mature, glycosylated gD. In addition, it was found that gD overexpression is neither toxic for transfected cells nor is involved in membrane fusion. After MDV infection of a proprietary cell line stably transfected with plasmid overexpressing MDV gD, no viral particles could be found in culture. On the other hand, cells overexpressing the MDV gD were sensitive to MDV infection in similar way as parental, non-transfected cells. From our study and results of other authors we propound the following conclusions: (i) MDV gD expression is blocked during in vitro infection at transcription level; (ii) MDV gD is lacking many important functions characteristic for other alpha-herpesvirus gD homologues; (iii) overexpression of single MDV gD does not result in production of mature infectious MDV particles.

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. METHODS: A CA9 cDNA isolated from a human stomach library was sequenced along with the cDNA derived from HeLa cells. Western blotting and immunohistochemical analyses of human and animal tissues were performed using CA IX-specific monoclonal antibody and rabbit antiserum to human CA II. RESULTS; Sequence analysis showed no differences between the stomach- and HeLa-derived cDNAs. CA IX was detected at the basolateral surface of gastric, intestinal, and gallbladder epithelia. In stomach tumor samples, expression of CA IX was lost or reduced. CONCLUSIONS: Differential distribution of CA IX in normal and tumor tissues is not associated with cDNA mutations. Evolutionary conservation in vertebrates as well as abundant expression of CA IX protein in normal human gastric mucosa, but not in derived tumors, indicate its physiological importance.

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. In contrast to the ANGpath virus, which is pathogenic (1 LD50 < 1 x 10(4) PFU) for adult DBA/2 mice after peripheral inoculation, strain HSZP was never found to be lethal for adult mice. Whereas ANGpath-infected mice which survived acute infection frequently (79%) developed latency in the regional sensory ganglion (as proved by virus reactivation during explantation), latent HSZP reactivated in ganglion culture at a considerably reduced rate (21%). Only 10-day-old DBA/2 mice were sensitive to HSZP infection. In these, HSZP spread from the site of peripheral administration mainly by hematogenous route. The neural spread of HSZP in suckling DBA/2 mice was manifested by the involvement of vegetative neurons in the wall of the small intestine and in the retroperitoneal vegetative ganglia. We conclude that HSZP, a polykaryocyte-forming strain with a mutation in the syn3 region II, shows limited neuroinvasity for mice after peripheral administration.

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. The entire genomic sequence of MN/CA9, including the 5'-flanking region, encompasses 10.9 kb. The coding sequence is divided into 11 exons, whose organization and relationships to predicted protein domains suggest that the gene arose by exon shuffling. Exon 1 encodes a signal peptide and a proteoglycan-related region. Exons 2-8 code for a CA domain with a highly conserved active site. The exon/intron pattern of the CA coding region is similar but not identical to other described animal kingdom alpha-CA genes. Exons 10 and 11 encode a transmembrane anchor and an intracytoplasmic tail, respectively. We have also determined the transcription initiation and termination sites by RNase protection assay and analyzed the 3. 5-kb region upstream of the MN/CA9 gene. Sequence of the proximate 5' end of the flanking region shows extensive homology to the long terminal repeats of HERV-K endogenous retroviruses. The putative MN/CA9 promoter immediately preceding the transcription start site does not possess a TATA box, but contains consensus sequences for the AP1, AP2, p53, and Inr transcription factors. This study will allow further investigations of the molecular events regulating expression of MN/CA IX as well as elucidation of its biological function.

Gene organization in herpesvirus of turkeys: identification of a novel open reading frame in the long unique region and a truncated homologue of pp38 in the internal repeat.

The DNA sequence of a 4.792-kb fragment comprising 3.176 kb of the long unique region (UL) and 1.605 kb of the internal repeat (IRL) flanking UL of herpesvirus of turkeys (HVT) was determined. Three potential open reading frames (ORFs) and an origin of replication have been identified. ORF 1, which maps entirely within UL, has the capacity to code for an 82K protein, 731 amino acids long, which has a counterpart in Marek's disease virus (MDV) but not in other herpesviruses. ORF 2 has the potential to encode a protein consisting of 129 amino acids with a predicted M(r) of 13.5K which appears to be unique to HVT. ORF 3 is encoded entirely within IRL and codes for an 84 amino acids long protein with a predicted M(r) of 8.5K. ORF 3 shows significant homology with the C-terminal region of the MDV-1-specific phosphoprotein pp38 and its recently identified homologue in MDV-2. Northern blot analysis of RNA extracted from HVT-infected chick embryo fibroblasts identified a 5.6-kb RNA transcribed in a leftward direction toward UL scanning ORF 1, ORF 2, and ORF 3 and a 2.8-kb also transcribed leftward toward UL which spanned only ORFs 2 and 3. In addition, a 2.3- to 2.8-kb RNA family was transcribed rightwards through the origin of replication. In vitro transcription and translation of ORF 1 and ORF 3 resulted in the synthesis of polypeptides consistent with their expected M(r), but ORF 2 failed to produce any translation product.

Marek's disease and new approaches to its control.

Marek's disease (MD) is a lymphoproliferative disorder induced by a herpesvirus. Several factors, including those virus-encoded and host-dependent, affect the course of the disease. Existing vaccination program is based on the use of attenuated strains of MD virus (MDV) serotype 1 and on strains of non-oncogenic serotype 2 (MDV2) and serotype 3 (herpesvirus of turkey-HVT) viruses. Failures resulting in disease progress have been reported and indicate need for production of new, more effective vaccines. It is likely that future development of MD vaccines will rely on recombinant molecules technology.

Identification and characterization of a cDNA clone derived from the Marek's disease tumour cell line RPL1 encoding a homologue of alpha-transinducing factor (VP16) of HSV-1.

We have identified and sequenced a 2.3 kb cDNA clone RPL(N.S) 6 derived from the Marek's disease virus (MDV)-transformed cell line RPL1, which contained open reading frames (ORFs) homologous to UL49 (VP22) and UL48 (VP16) of herpes simplex virus. Northern blot hybridization identified a 2.5 kb transcript corresponding to this cDNA clone in the total RNA from MSB1 lymphoblastoid cells, but not in RNA from the original RPL1 cells, most probably due to the very low level of its transcription. In vitro translation demonstrated that both MDV UL49 and UL48 can be expressed from a single mRNA.

Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment.

MN is a transmembrane glycoprotein that has been detected in HeLa cells and in some human carcinomas. The expression of MN protein in HeLa cells is regulated by cell density. In HeLa x fibroblast cell hybrids its expression correlates with tumorigenicity. Using a specific monoclonal antibody we have identified a cDNA clone coding for MN. Analysis of the deduced amino acid sequence revealed strong structural homology between the central region of the MN protein and carbonic anhydrases (CA). MN sequence retains the conserved zinc-binding site as well as the enzyme's active center. In accord with these findings, MN protein from HeLa cells was found to bind zinc and to have carbonic anhydrase activity. The N-terminal region of MN shares some similarity with DNA binding proteins of the helix-loop-helix (HLH) family, and the protein was found to have affinity for DNA by DNA-cellulose chromatography. The region between the CA-like domain and the putative HLH domain is rich in imperfect repeats of serine, proline, glycine and acidic residues with few hydrophobic amino acids, resembling thus an activation region of transcription factors. The fact that MN protein is detectable in several types of human carcinomas, but not in corresponding non-cancerous tissues, suggests its possible role in neoplasia. In addition, the analysis of biological consequences of MN expression of NIH3T3 cells provides the evidence in favour of MN protein involvement in control of cell proliferation and transformation.

Characterization of proteins encoded by the short unique region of herpesvirus of turkeys by in vitro expression.

Nine open reading frames mapping in the short unique (US) region of the genome of herpesvirus of turkeys (HVT) were expressed by in vitro transcription and translation. The observed M(r)s of US10, SORF3 and US2 were as predicted from the sequence but there were discrepancies between the observed and predicted M(r)s of US1, protein kinase, gI, gD and gE. These could be accounted for in most cases by post-translational and co-translational processing. Analysis of the synthesized products at different time points provided evidence for post-translational modification of HVT protein kinase. Translation in the presence of microsomal membranes resulted in co-translational processing of HVT gD, gI and gE by glycosylation and signal peptide cleavage.

Identification of novel transcripts complementary to the Marek's disease virus homologue of the ICP4 gene of herpes simplex virus.

Libraries of cDNA were generated from polyadenylated RNAs derived from Marek's disease virus (MDV)-transformed cell lines by directional cloning of oligo-(dT)-primed cDNAs in lambda gt22A. Analysis of the libraries for viral sequences showed that a number of cDNA clones originated from transcripts mapping in the BamHI A region of the MDV genome. Sequencing and fine mapping of these cDNAs suggested that the RNA transcripts expressed from this region were either in the sense or antisense direction with respect to the MDV homologue of the ICP4 gene of herpes simplex virus. The longest cDNA clone from antisense transcripts was 2756 bp long and partially overlapped the 5' end of the coding region of the ICP4 gene. The cDNA clone contained at least four introns, shown by comparison of its sequence with the sequence of the ICP4 gene. The presence of introns was confirmed by PCR analysis. All the introns have the consensus splice donor and acceptor signals at their 5' and 3' ends respectively. Northern blot analysis showed that the ICP4 gene homologue of MDV was abundantly transcribed only in lytically infected fibroblasts, whereas transcripts complementary to the ICP4 gene were the major transcripts in MDV-transformed cell lines and lymphomas. The transcripts complementary to ICP4 consist of two major RNA species approximately 15 kb and 1.32 kb long. The results suggest that there might be an inverse relationship between the abundance of ICP4 transcripts and their complementary transcripts in MDV-infected and transformed cells.

Expression of MaTu-MN protein in human tumor cultures and in clinical specimens.

MaTu is a novel agent which may be of relevance in human oncogenesis, and has 2 components. One of them, the exogenous MX (coding for protein p58X), is transmissible to human fibroblasts, to HeLa and to HeLa x fibroblast (H/F) hybrids. The other component, MN, is a cellular gene. Its product, the protein p54/58N, is inducible by infecting HeLa cells with MX or by growing them in dense cultures. This p54/58N appears to be a tumor-associated antigen: it is expressed in HeLa and in tumorigenic cells (H/F-T), but not in fibroblasts or in nontumorigenic hybrid cells (H/F-N). Proteins related to p54/58N were also found on immunoblots prepared from human carcinomas of ovary, endometrium and uterine cervix, but not from normal tissues from corresponding organs or from placenta. Using genetically engineered MN protein, we developed a radioimmunoassay for MN-specific antibodies, and for quantitative determination of MN proteins in cell extracts. In HeLa cells infected with MX we observed conspicuous ultrastructural alterations: formation of abundant filaments on the cell surface and amplification of mitochondria. Using immunogold-staining, we visualized the p54/58N on the surface microvilli and in the nucleus, particularly in nucleoli.

The 132 bp repeats are present in RNA transcripts from 1.8 kb gene family of Marek disease virus-transformed cells.

Marek's disease virus (MDV) is an oncogenic, lymphotropic herpesvirus of chickens: Loss of its tumourigenic potential is believed to be associated with amplification of the 132 bp repeats from BamHI-D and BamHI-H fragments. We prepared cDNA libraries from RPL1 and MSB1 cell line and from the latter we identified a clone which spanned the 132 bp repeats within the BamHI-H fragment. By sequencing and Northern blot analysis we confirmed the presence of the 132 bp repeats. The analysis by PCR made on the total RNA revealed two 132 bp repeats in MDV transcripts from RPL1 and two to three repeats in transcripts from MSB1 cells. These results show that sequences within the 132 bp repeats are transcribed and are not spliced out as previously reported.

Construction and properties of a turkey herpesvirus recombinant expressing the Marek's disease virus homologue of glycoprotein B of herpes simplex virus.

A herpesvirus of turkeys (HVT) recombinant containing a 3.9 kbp fragment of Marek's disease virus (MDV) DNA encoding MDV glycoprotein B (gB), stably integrated into the thymidine kinase (TK) gene of HVT, has been constructed. The replication of the recombinant in chick embryo fibroblasts (CEF) was comparable to that of wild-type HVT. The recombinant expressed authentic MDV gB and its processed forms (110K, 65K and 48K) in CEF as shown by immunoblotting using an MDV-specific anti-peptide serum. Northern blot analysis showed that MDV gB mRNA was transcribed from MDV promoter sequences flanking the MDV gB open reading frame and also from the HVT TK promoter. However, the level of replication of the recombinant in vivo appeared to be lower than wild-type HVT as shown by the titres of HVT antibodies, determined by ELISA. Pathogenicity tests showed that the recombinant was safe and did not cause microscopic or gross Marek's disease lesions or other abnormalities. The results suggest that HVT has potential as a vector for recombinant vaccines.