RESUMEN
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.
Asunto(s)
Bioensayo/normas , Inmunidad Celular , Factor de Transferencia/normas , Adyuvantes Inmunológicos , Animales , Reproducibilidad de los Resultados , EslovaquiaRESUMEN
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.
Asunto(s)
Transformación Celular Viral , Infecciones por Herpesviridae/virología , Rhadinovirus/química , Rhadinovirus/fisiología , Animales , Infecciones por Herpesviridae/patología , Ratones , Células 3T3 NIH , Rhadinovirus/genéticaRESUMEN
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.
Asunto(s)
Herpesviridae/enzimología , Timidina Quinasa/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Gammaherpesvirinae/química , Gammaherpesvirinae/clasificación , Gammaherpesvirinae/genética , Herpesviridae/química , Herpesviridae/clasificación , Herpesviridae/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Análisis de Secuencia de Proteína , Timidina Quinasa/química , Proteínas Virales/químicaRESUMEN
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.
Asunto(s)
Gammaherpesvirinae/fisiología , Simplexvirus/fisiología , Animales , Clonación Molecular/métodos , Gammaherpesvirinae/genética , Genoma Viral , Humanos , Simplexvirus/genética , Replicación ViralRESUMEN
Here we would like to provide a brief overview of the modern history of MarekÎs 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 MarekÎs 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, MarekÎs 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.
Asunto(s)
Herpesvirus Gallináceo 2/genética , Mardivirus/genética , Enfermedad de Marek/genética , Enfermedad de Marek/virología , Enfermedades de las Aves de Corral/virología , Animales , Herpesvirus Gallináceo 2/inmunología , Mardivirus/inmunología , Enfermedad de Marek/inmunología , Enfermedad de Marek/prevención & control , Vacunas contra la Enfermedad de Marek/inmunología , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/prevención & controlRESUMEN
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. Sera were used at a 1 : 100 dilution and were added in parallel to wells containing the infected and uninfected cell lysates. The optical densities at 492 nm (OD(492 nm)) were measured after detection of bound chicken antibodies with anti-chicken IgG peroxidase conjugate and colour reactions using o-phenylenediamine (OPD) as a substrate. The best results concerning the signal-to-noise ratio were obtained by using CKC cells rather than CEC for antigen preparation. The OD(492 nm) of plasma or serum samples with infected CKC was <0.02 when samples of unvaccinated and unchallenged maternal antibody-negative white leghorn chickens were tested. Sera and plasma samples of positive control birds exhibited OD(492 nm) of <0.01 when tested with uninfected CKC. The assay was used to monitor a trial that compared experimental BAC DNA vaccines and a commercial vaccine. Sustained seroconversion and antibody titers that were constantly rising until day 84 after vaccination (71 days after challenge) was observed only when chickens did not develop Marek's disease. In contrast, chickens developing the disease mounted marginal and short-lived antibody titers only. We conclude that the developed ELISA may be a valuable tool for the evaluation of the efficacy of MDV vaccination under experimental but possibly also under field conditions.
Asunto(s)
Pollos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Mardivirus/inmunología , Vacunas contra la Enfermedad de Marek/inmunología , Enfermedad de Marek/prevención & control , Animales , Anticuerpos Antivirales/análisis , Ensayo de Inmunoadsorción Enzimática/normas , Mardivirus/aislamiento & purificación , Enfermedad de Marek/diagnóstico , Valor Predictivo de las Pruebas , Vacunación/veterinariaRESUMEN
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. The adapted virus, designated QT-CVI, exhibited similar cytopathic effect (CPE) in vitro to that of parental virus propagated in CEE In contrast, QT-CVI induced MD symptoms typical for mild MDV-1 strains after injection to birds. For identification of differentially expressed transcripts that might be involved in increased virulence of QT-CVI, we performed subtractive suppression hybridization (SSH). Subtracted PCR products mapped within MDV-1 BamHI-A and -H fragments and differential gene expression was also confirmed by Northern blot analysis with probes derived from these regions. To examine possible divergence at the virus genome level, PCR analysis was carried out. The BamHI-H fragment- specific 132 bp repeats were present at variable copy number, ranging from 2 to more than 30 copies in both CVI988/ Rispens and QT-CVI DNAs. PCR assays with primers mapping at the US/lRS junction identified CVI988/ Rispens-specific insertion of 116 bp in the region upstream of the ICP4 open reading frame (ORF). PCR analysis was positive also for DNA from non-infected QT35 cells and was consistent with the observation of Yamaguchi et al. (J Virol. 74, 10176-10186, 2000) who have found that QT35 cells carry a latent MDV-1 genome. It is likely, that adaptation of CVI988/Rispens to QT35 cells resulted in reactivation of an endogenous MDV-1 or at least in induction of expression of virulence-related transcripts that have consequently led to QT-CVI pathogenicity for chickens.
Asunto(s)
Herpesvirus Gallináceo 2/genética , Enfermedad de Marek/virología , Vacunas Virales/genética , Virulencia/genética , Animales , Secuencia de Bases , Línea Celular , Pollos , Cartilla de ADN , Herpesvirus Gallináceo 2/crecimiento & desarrollo , Herpesvirus Gallináceo 2/patogenicidad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Codorniz , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Vacunas Atenuadas , Activación ViralRESUMEN
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. Comparison of amino acid homology has shown its clear position among the alpha-herpesviruses rather than beta- or gamma-herpesviruses. The VP5 is expressed from polycistronic mRNA together with the UL20 and the VP23 genes. The 7,2 kb RNA transcript is lacking any promoter elements or polyA signal in intergenomic regions between VP5 and UL20 or VP5 and VP23 genes, respectively. Multiple alignment of known major capsid protein sequences of all herpesvirus groups revealed presence of seven highly homologous clusters suggesting-that the corresponding protein domains might play an important role in folding of MCP and assembly of herpesvirus capsid.
Asunto(s)
Cápside/genética , Herpesviridae/genética , Pavos/virología , Secuencia de Aminoácidos , Animales , Northern Blotting , Southern Blotting , Proteínas de la Cápside , Línea Celular , Pollos , ADN Viral/análisis , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , ARN Viral/análisis , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Infecciones por Herpesviridae/veterinaria , Herpesviridae/clasificación , Rhadinovirus/clasificación , Enfermedades de las Ovejas/virología , Infecciones Tumorales por Virus/veterinaria , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Cápside/genética , Técnicas de Cocultivo , Técnicas de Cultivo , Efecto Citopatogénico Viral , Cartilla de ADN , Desoxirribonucleasa HindIII , Herpesviridae/genética , Herpesviridae/aislamiento & purificación , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Rhadinovirus/genética , Rhadinovirus/aislamiento & purificación , Sensibilidad y Especificidad , Homología de Secuencia de Aminoácido , Ovinos , Enfermedades de las Ovejas/sangre , Enfermedades de las Ovejas/epidemiología , Eslovaquia/epidemiologíaRESUMEN
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.
Asunto(s)
Herpesviridae/fisiología , Herpesvirus Gallináceo 2/fisiología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Línea Celular , Embrión de Pollo , Herpesviridae/genética , Herpesvirus Gallináceo 2/genética , Datos de Secuencia Molecular , Plásmidos/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Conejos , Reticulocitos/virología , Transcripción Genética , TransfecciónRESUMEN
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 80.9% identity with that of the LCMV Traub (TRA) strain. It contained conserved zinc-binding RING finger domain and C-terminal proline-rich region. Northern blot analysis of total RNA from MaTu cells revealed presence of abundant truncated forms of L RNA. Z protein-specific rabbit antibodies were produced to glutathione S-transferase (GST)-Z fusion protein expressed in E. coli and used for the detection of Z protein in MaTu cells. Western blot and immunofluorescence analyses detected relatively high levels of Z protein indicating its role in maintenance of persistent LCMV.
Asunto(s)
Genes Virales , Virus de la Coriomeningitis Linfocítica/genética , Secuencia de Aminoácidos , Northern Blotting , Línea Celular/virología , Clonación Molecular , Células HeLa , Humanos , Immunoblotting , Virus de la Coriomeningitis Linfocítica/química , Virus de la Coriomeningitis Linfocítica/aislamiento & purificación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Dedos de ZincRESUMEN
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.
Asunto(s)
Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , ADN Complementario/metabolismo , Sistema Digestivo/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Animales , Secuencia de Bases , Pollos , Femenino , Mucosa Gástrica/metabolismo , Cobayas , Células HeLa , Humanos , Inmunohistoquímica , Conejos , Ratas , Ratas Sprague-Dawley , Estómago/citología , Distribución Tisular , Células Tumorales CultivadasRESUMEN
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 deduced amino acid sequence of the protein encoded by HVT UL48 gene shows 55% identity with MDV UL48 gene product. Like the majority of related proteins in other alpha-herpesviruses, the protein encoded by HVT UL48 gene lacks the acidic C-terminal tail, known to possess the transactivation capacity of HSV VP16. Hydrophobic cluster analysis has revealed that its predicted domain composition is closely related to the transactivator protein encoded by ORF10 of VZV. However, the putative amino-terminal activation domain of the HVT homologue of HSV VP16 does not contain a typical horseshoe-like hydrophobic cluster found in other alpha-herpesvirus homologues, suggesting either that it acts as a transactivator via a different activation domain or that its transactivation potential is diminished.
Asunto(s)
Proteína Vmw65 de Virus del Herpes Simple/química , Herpesviridae/química , Herpesvirus Humano 3/química , Herpesvirus Humano 3/genética , Sistemas de Lectura Abierta/genética , Transactivadores , Pavos/virología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Genoma Viral , Datos de Secuencia Molecular , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Proteínas Virales/análisisRESUMEN
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.
Asunto(s)
Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidad , Proteínas del Envoltorio Viral/genética , Animales , Secuencia de Bases , Chlorocebus aethiops , ADN Viral , Humanos , Ratones , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Conejos , Homología de Secuencia de Ácido Nucleico , Células VeroRESUMEN
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.
Asunto(s)
Anhidrasas Carbónicas/biosíntesis , Anhidrasas Carbónicas/genética , Exones , Isoenzimas/biosíntesis , Isoenzimas/genética , Familia de Multigenes , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Southern Blotting , Clonación Molecular , Secuencia Conservada , Biblioteca Genómica , Células HeLa , Humanos , Intrones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
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. The recombinant expressed beta-galactosidase and was genetically stable in vitro and in vivo. Chickens inoculated with the virus developed antibodies to HVT antigens and to beta-galactosidase but the replication of the recombinant in vivo was impaired in comparison to parental HVT as shown by a reduction in the proportion of infected lymphocytes.
Asunto(s)
Genes Virales , Herpesviridae/genética , Operón Lac , Animales , Anticuerpos Antivirales/biosíntesis , Secuencia de Bases , Células Cultivadas , Embrión de Pollo , Pollos , Clonación Molecular , Cartilla de ADN , Regulación Viral de la Expresión Génica , Herpesviridae/fisiología , Datos de Secuencia Molecular , Mutagénesis Insercional , Plásmidos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Pavos/virología , Replicación ViralRESUMEN
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.
Asunto(s)
Enfermedad de Marek/prevención & control , Animales , Vectores Genéticos , Herpesviridae/genética , Herpesvirus Gallináceo 2/genética , Humanos , Pavos , Vacunas Sintéticas , Vacunas ViralesRESUMEN
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.
Asunto(s)
Genes Virales/genética , Sistemas de Lectura Abierta/genética , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Secuencia de Bases , Embrión de Pollo , Clonación Molecular , ADN Viral/genética , Herpesvirus Gallináceo 2/genética , Datos de Secuencia Molecular , Peso Molecular , Fosfoproteínas/genética , ARN Mensajero/análisis , ARN Viral/análisis , Origen de Réplica/genética , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Pavos/virología , Proteínas Virales/biosíntesis , Proteínas Virales/químicaRESUMEN
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.
Asunto(s)
Proteína Vmw65 de Virus del Herpes Simple/genética , Herpesvirus Gallináceo 2/genética , Proteínas Virales/genética , Proteínas Estructurales Virales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Transformada , ADN Complementario/genética , Proteína Vmw65 de Virus del Herpes Simple/química , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Viral/genética , Transcripción Genética , Células Tumorales Cultivadas , Proteínas Virales/químicaRESUMEN
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.