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Viral Proteins (viral + protein)
Selected AbstractsCharacterization of grouper nervous necrosis virus (GNNV)JOURNAL OF FISH DISEASES, Issue 1 2001S C Chi Grouper nervous necrosis virus (GNNV) was isolated from moribund grouper larvae, Epinephelus sp., using a fish cell line GF-1. The present study describes the biochemical and biophysical properties of GNNV and the expression of GNNV in diseased grouper larvae. Viral protein was detectable in most of the GNNV-infected GF-1 cells by the fluorescent antibody technique (FAT) after 12 h post-infection (p.i.), although no cytopathic effect (CPE) appeared at that time. Clear CPE developed on the third day, and complete disintegration of the monolayer occurred over the subsequent two days. The infectivity of GNNV can be blocked following treatment at 60 °C for 1 h. GNNV was sensitive to pH 3 and pH 10,12 with a 4 log10 drop in infectivity. Purified GNNV was analysed by SDS,PAGE, and then stained with periodic acid silver. The positive staining indicated that its two capsid proteins were glycoproteins. Genomic RNAs of GNNV were extracted from purified virions and analysed. The molecular weights of genomic RNAs were 1.02 × 106 and 0.50 × 106 Da. The T2 region of the coat protein gene of GNNV was amplified by polymerase chain reaction (PCR), and the multiple alignment of the T2 sequence of two GNNV isolates with four genotypes of fish nodaviruses revealed that these two isolates (GNNV9410 and GNNV9508) belong to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The tissue distribution of GNNV in naturally infected grouper larvae was investigated by in situ hybridization using a dig-labelled probe, which showed that GNNV was not only detected in the brain and retina, but also in the gill, skeletal muscle, liver, pyloric gland, intestine and blood cells in the heart. [source] The MHC class I antigen presentation pathway: strategies for viral immune evasionIMMUNOLOGY, Issue 2 2003Eric W. Hewitt Summary Presumably because of the selective pressure exerted by the immune system, many viruses have evolved proteins that interfere with antigen presentation by major histocompatibility complex (MHC) class I molecules. These viruses utilize a whole variety of ingenious strategies to inhibit the MHC class I pathway. Viral proteins have been characterized that exploit bottlenecks in the MHC class I pathway, such as peptide translocation by the transporter associated with antigen processing. Alternatively, viral proteins can cause the degradation or mislocalization of MHC class I molecules. This is often achieved by the subversion of the host cell's own protein degradation and trafficking pathways. As a consequence elucidation of how these viral proteins act to subvert host cell function will continue to give important insights not only into virus,host interactions but also the function and mechanism of cellular pathways. [source] Turnip yellow mosaic virus: transfer RNA mimicry, chloroplasts and a C-rich genomeMOLECULAR PLANT PATHOLOGY, Issue 5 2004THEO W. DREHER SUMMARY Taxonomy:,Turnip yellow mosaic virus (TYMV) is the type species of the genus Tymovirus, family Tymoviridae. TYMV is a positive strand RNA virus of the alphavirus-like supergroup. Physical properties:, Virions are non-enveloped 28-nm T = 3 icosahedrons composed of a single 20-kDa coat protein that is clustered in 20 hexameric and 12 pentameric subunits. Infectious particles and empty capsids coexist in infected tissue. The genomic RNA is 6.3 kb long, with a 5,m7GpppG cap and a 3, untranslated region ending in a tRNA-like structure to which valine can be covalently added. The genome has a distinctive skewed C-rich, G-poor composition (39% C, 17% G). Viral proteins:, Two proteins, whose open reading frames extensively overlap, are translated from the genomic RNA. p206, which contains sequences indicative of RNA capping, NTPase/helicase and polymerase activities, is the only viral protein that is necessary for genome replication in single cells. It is produced as a polyprotein and self-cleaved to yield 141- and 66-kDa proteins. p69 is required for virus movement within the plant and is also a suppressor of gene silencing. The coat protein is expressed from the single subgenomic RNA. Hosts and symptoms:, TYMV has a narrow host range almost completely restricted to the Cruciferae. Experimental host species are Brassica pekinensis (Chinese cabbage) or B. rapa (turnip), in which diffuse chlorotic local lesions and systemic yellow mosaic symptoms appear. Arabidopsis thaliana can also be used. Clumping of chloroplasts and the accumulation of vesicular invaginations of the chloroplast outer membranes are distinctive cytopathological symptoms. High yields of virus are produced in all leaf tissues, and the virus is readily transmissible by mechanical inoculation. Localized transmission by flea beetles may occur in the field. [source] Beet yellows virus: the importance of being differentMOLECULAR PLANT PATHOLOGY, Issue 2 2003Valerian V. Dolja SUMMARY Taxonomic relationship: Type member of the genus Closterovirus, family Closteroviridae. A member of the alphavirus-like supergroup of positive-strand RNA viruses. Physical properties: Virions are flexuous filaments of ,1300 nm in length and ,12 nm in diameter that are made up of a ,15.5 kb RNA and five proteins. The major capsid protein forms virion body of helical symmetry that constitutes ,95% of the virion length. The short virion tail is assembled by the minor capsid protein, Hsp70-homologue, ,64-kDa protein, and ,20-kDa protein. Viral proteins: The 5,-most ORFs 1a and 1b encode leader proteinase and RNA replicase. The remaining ORFs 2,8 are expressed by subgenomic mRNAs that encode 6-kDa membrane protein, Hsp70 homologue, ,64-kDa protein, minor and major capsid proteins, ,20-kDa protein, and ,21-kDa protein, respectively. Hosts: The principal crop plants affected by Beet yellows virus (BYV) are sugar beet (Beta vulgaris) and spinach (Spinacea oleracea). In addition, BYV was reported to infect ,120 species in 15 families. Most suitable propagation species are Nicotiana benthamiana, Tetragonia expansa, and Claytonia perfoliata. [source] Cauliflower mosaic virus: still in the newsMOLECULAR PLANT PATHOLOGY, Issue 6 2002Muriel Haas SUMMARY Taxonomic relationship:Cauliflower mosaic virus (CaMV) is the type member of the Caulimovirus genus in the Caulimoviridae family, which comprises five other genera. CaMV replicates its DNA genome by reverse transcription of a pregenomic RNA and thus belongs to the pararetrovirus supergroup, which includes the Hepadnaviridae family infecting vertebrates. Physical properties:, Virions are non-enveloped isometric particles, 53 nm in diameter (Fig. 1). They are constituted by 420 capsid protein subunits organized following T= 7 icosahedral symmetry (Cheng, R.H., Olson, N.H. and Baker, T.S. (1992) Cauliflower mosaic virus: a 420 subunit (T= 7), multilayer structure. Virology, 16, 655,668). The genome consists of a double-stranded circular DNA of approximately 8000 bp that is embedded in the inner surface of the capsid. Figure 1. Electron micrograph of CaMV virions. Courtesy of J. Menissier de Murcia, Ecole Supérieure de Biotechnologie de Strasbourg. Viral proteins: The CaMV genome encodes six proteins, a cell-to-cell movement protein (P1), two aphid transmission factors (P2 and P3), the precursor of the capsid proteins (P4), a polyprotein precursor of proteinase, reverse transcriptase and ribonuclease H (P5) and an inclusion body protein/translation transactivator (P6). Hosts: The host range of CaMV is limited to plants of the Cruciferae family, i.e. Brassicae species and Arabidopsis thaliana, but some viral strains can also infect solanaceous plants. In nature, CaMV is transmitted by aphids in a non-circulative manner. [source] Regulation of cell death during infection by the severe acute respiratory syndrome coronavirus and other coronavirusesCELLULAR MICROBIOLOGY, Issue 11 2007Yee-Joo Tan Summary Both apoptosis and necrosis have been observed in cells infected by various coronaviruses, suggesting that the regulation of cell death is important for viral replication and/or pathogenesis. Expeditious research on the severe acute respiratory syndrome (SARS) coronavirus, one of the latest discovered coronaviruses that infect humans, has provided valuable insights into the molecular aspects of cell-death regulation during infection. Apoptosis was observed in vitro, while both apoptosis and necrosis were observed in tissues obtained from SARS patients. Viral proteins that can regulate apoptosis have been identified, and many of these also have the abilities to interfere with cellular functions. Occurrence of cell death in host cells during infection by other coronaviruses, such as the mouse hepatitis virus and transmissible porcine gastroenteritis virus, has also being extensively studied. The diverse cellular responses to infection revealed the complex manner by which coronaviruses affect cellular homeostasis and modulate cell death. As a result of the complex interplay between virus and host, infection of different cell types by the same virus does not necessarily activate the same cell-death pathway. Continuing research will lead to a better understanding of the regulation of cell death during viral infection and the identification of novel antiviral targets. [source] Synergistic activation of interferon-, gene transcription by the viral FLICE inhibitory protein of Kaposi's sarcoma-associated herpesvirus and type,I IFN activatorsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2007Nathalie Cloutier Abstract Expression of Kaposi's sarcoma-associated herpesvirus v-FLIP leads to the spindle-shape morphology of endothelial cells and is essential for the survival of primary effusion lymphoma cells. Activation of the NF-,B transcription factor by v-FLIP is responsible for these effects. Considering that the interferon-, (ifn- ,) gene is regulated partly through NF-,B, we sought to determine whether v-FLIP would activate the expression of the ifn -, gene. Our results indicate that when v-FLIP is expressed by itself it has no effect on ifn -, gene activation but when it is combined with known IFN-, inducers, a synergistic activation of the ifn -, gene occurs. This effect is strictly dependent on NF-,B and is mediated through the positive regulatory domain,II of the IFN-, promoter. Furthermore, we report that protection from Fas-induced cell-death by v-FLIP is observed whether or not the type,I IFN signaling pathway is activated. Our work therefore contributes to increase our knowledge on v-FLIP, highlighting the complex immunomodulatory properties of this anti-apoptotic viral protein. [source] Caspase-dependent induction of apoptosis in barramundi, Lates calcarifer (Bloch), muscle cells by grouper iridovirusJOURNAL OF FISH DISEASES, Issue 12 2009P P Chiou Abstract We recently reported that grouper iridovirus (GIV) can induce apoptosis in barramundi, Lates calcarifer, muscle (BM) and swim bladder (BSB) cell lines. In this paper, we further characterize the molecular mechanism underlying apoptotic death in BM cells triggered by GIV. DNA-laddering and apoptotic cells were observed in BM cells infected with UV-irradiated or untreated GIV but was absent in cells infected with heat-inactivated GIV, indicating the involvement of viral protein in the apoptosis event. In GIV-infected BM cells, the conversion of procaspase-3 to caspase-3 was evident and the level of caspase-8 and -9 increased as early as 30 min post-infection. When treated with a pancaspase inhibitor, the GIV-induced apoptosis event was abolished. These observations indicate that GIV-induced apoptosis is caspase-dependent, and that both the external and internal routes in the caspase-dependent pathway are likely involved in the apoptosis process. [source] Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt-head seabream, Sparus aurata L.JOURNAL OF FISH DISEASES, Issue 2 2009I Cano Abstract Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques have been used for the detection of lymphocystis disease virus (LCDV) in formalin-fixed, paraffin-embedded tissues from gilt-head seabream, Sparus aurata L. Diseased and recovered fish from the same population were analysed. IHC was performed with a polyclonal antibody against a 60-kDa viral protein. A specific digoxigenin-labelled probe, obtained by PCR amplification of a 270-bp fragment of the gene coding the LCDV major capsid protein, was used for ISH. LCDV was detected in skin dermis and gill lamellae, as well as in several internal organs such as the intestine, liver, spleen and kidney using both techniques. Fibroblasts, hepatocytes and macrophages seem to be target cells for virus replication. The presence of lymphocystis cells in the dermis of the skin and caudal fin, and necrotic changes in the epithelium of proximal renal tubules were the only histological alterations observed in fish showing signs of the disease. [source] Detection of human bocavirus in respiratory, fecal, and blood samples by real-time PCR,JOURNAL OF MEDICAL VIROLOGY, Issue 3 2009Sarah J. Tozer Abstract Human bocavirus (HBoV) has been detected worldwide in respiratory samples. Two real-time PCR assays, targeting the non-structural protein (NP-1) and viral protein (VP-1) genes, were designed and validated to detect HBoV in patients with respiratory disease, gastroenteritis, or systemic illness. Sensitivity of the NP-1 and VP-1 assays were equal to the conventional PCR assay previously described by Allander et al. [2005: Proc Natl Acad Sci USA 102: 12891,12896] being 100%, and giving specificity of 94% and 93%, respectively. There was no cross-reaction identified with unrelated respiratory agents, or to human DNA. The limits of detection were 10 copies of genomic DNA equivalents per reaction for both assays. The assays were used to screen three different sample populations, combined nose, and throat swabs (n,=,96) from children with acute respiratory disease, fecal samples (n,=,375) from adults, and children with gastroenteritis and whole blood (n,=,229) collected from 31 immunocompromised children taken over an 18-month period. In total 17 (18%) respiratory samples and 18 (4.8%) fecal samples were identified as having HBoV present. Of the pediatric whole blood specimens investigated, HBoV was detected in six (2.6%) samples from four patients. In summary, two real-time PCR assays targeting different genes were designed and validated for use as screening methods for the detection of HBoV. HBoV was found in three different specimen types: parent-collected combined nose,throat swabs, fecal samples collected from symptomatic individuals and whole blood from immunocompromised children. J. Med. Virol. 81:488,493, 2009. © 2009 Wiley-Liss, Inc. [source] Presence and expression of JCV early gene large T Antigen in the brains of immunocompromised and immunocompetent individualsJOURNAL OF MEDICAL VIROLOGY, Issue 12 2008Serena Delbue Abstract JC virus (JCV) is a polyomavirus that asymptomatically infects up to 80% of the worldwide human population and establishes latency in the kidney. In the case of host immunodeficiency, it can cause progressive multifocal leukoencephalopathy (PML), which is a fatal demyelinating disease of the central nervous system. In an attempt to understand better PML pathogenesis and JCV infection, the presence of the JCV genome and expression of the early viral protein in the brain of deceased individuals, with and without HIV infection, was investigated. Sixty autopsy samples of brain tissues were collected from 15 HIV-positive PML patients, 15 HIV-positive patients with other neurological diseases, 15 HIV-positive patients without neurological disorders, and 15 HIV-negative individuals who died from diseases unrelated to the central nervous system. By means of specific Real Time Polymerase Chain Reaction, the JCV genome was detected in 14 of 15 PML brains, three of 15 HIV-positive brains (with and without neurological diseases), and 1 of 15 HIV-negative brains. JCV genotyping was also performed. Expression of the early JCV protein T Antigen was verified by a specific immunohistochemistry assay, and it was found in the brain tissues from 12 PML cases and one case with other neurological disease. The data obtained demonstrate that infection of the brain with JCV can also be observed in the brains of HIV-negative individuals, without neurological disorders. However, viral protein expression was limited to PML brains and to one brain from a patient with other neurological disease, suggesting that JCV can also be present in the brains of patients without PML. J. Med. Virol. 80:2147,2152, 2008. © 2008 Wiley-Liss, Inc. [source] Double-stranded RNA-activated protein kinase inhibits hepatitis C virus replication but may be not essential in interferon treatmentLIVER INTERNATIONAL, Issue 2 2010Jin-Hai Chang Abstract Background: Double-stranded RNA-activated protein kinase (PKR), an interferon (IFN)-stimulated gene, is activated by binding with double-stranded RNA, a putative replicative intermediate of the hepatitis C virus (HCV). Activated PKR phosphorylates the , subunit of eukaryotic initiation factor-2 to inhibit the translation of viral protein. Aims/methods: We established stable PKR knockdown Huh7 cells using RNA interference and investigated the effect of PKR against HCV replication using a subgenomic replicon that expressed luciferase reporter protein and the JFH1 full-length HCV genome. Results: In stable PKR knockdown cells that harboured a subgenomic replicon, luciferase activity was approximately three times higher than that of control cells, indicating that the subgenomic replicon replicated with a higher efficiency in stable PKR knockdown cells than that in control cells. Furthermore, stable PKR knockdown cells secreted significantly more HCV particles than did control cells after transfection with the full-length HCV genome. The replication of the subgenomic replicon was suppressed by the addition of IFN-, in both cells. Although the extent of suppression was significantly lower in stable PKR knockdown than control cells using a low concentration (2.5,5 U/ml) of IFN-,, even 10 U/ml IFN-, suppressed the replication of subgenomic replicon by >98% in both cells. Conclusions: Double-stranded RNA-activated protein kinase plays an important role in suppressing HCV replication in an innate state, but may not be essential in IFN therapy. [source] Selective human enterovirus and rhinovirus inhibitors: An overview of capsid-binding and protease-inhibiting moleculesMEDICINAL RESEARCH REVIEWS, Issue 4 2004Shin-Ru Shih Abstract The absence of effective vaccines for most viral infections highlights an urgent necessity for the design and development of effective antiviral drugs. Due to the advancement in virology since the late 1980s, several key events in the viral life cycle have been well delineated and a number of molecular targets have been validated, culminating in the emergence of many new antiviral drugs in recent years. Inhibitors against enteroviruses and rhinoviruses, responsible for about half of the human common colds, are currently under active investigation. Agents targeted at either viral protein 1 (VP1), a relatively conserved capsid structure mediating viral adsorption/uncoating process, or 3C protease, which is highly conserved among different serotypes and essential for viral replication, are of great potential to become antipicornavirus drugs. © 2004 Wiley Periodicals, Inc. Med Res Rev, 24, No. 4, 449,474, 2004 [source] Protection of red sea bream Pagrus major against red sea bream iridovirus infection by vaccination with a recombinant viral proteinMICROBIOLOGY AND IMMUNOLOGY, Issue 3 2010Hajime Shimmoto ABSTRACT Megalocytivirus infections cause serious mass mortality in marine fish in East and Southeast Asian countries. In this study the immunogenicity of crude subunit vaccines against infection by the Megalocytivirus RSIV was investigated. Three capsid proteins, 18R, 351R and a major capsid protein, were selected for use as crude subunit vaccines. High homology among Megalocytivirus types was found in the initial sequence examined, the 351R region. Red sea bream (Pagrus major) juveniles were vaccinated by intraperitoneal injection of recombinant formalin-killed Escherichia coli cells expressing these three capsid proteins. After challenge infection with RSIV, fish vaccinated with the 351R-recombinant bacteria showed significantly greater survival than those vaccinated with control bacteria. The 351R protein was co-expressed with GAPDH from the bacterium Edwardsiella tarda in E. coli; this also protected against viral challenge. A remarkable accumulation of RSIV was observed in the blood of vaccinated fish, with less accumulation in the gills and spleen tissues. Thus, the 351R-GAPDH fusion protein is a potential vaccine against Megalocytivirus infection in red sea bream. [source] Insights into the function of the WhiB-like protein of mycobacteriophage TM4 , a transcriptional inhibitor of WhiB2MOLECULAR MICROBIOLOGY, Issue 3 2010Jan Rybniker Summary WhiB-like proteins of actinomycetes are known to co-ordinate iron-sulfur (Fe-S) clusters and are believed to have regulatory functions in many essential bacterial processes. The systematic determination of the genome sequences of mycobacteriophages has revealed the presence of several whiB -like genes in these viruses. Here we focussed on the WhiB-like protein of mycobacteriophage TM4, WhiBTM4. We provide evidence that this viral protein is capable of co-ordinating a Fe-S cluster. The UV-visible absorption spectra obtained from freshly purified and reconstituted WhiBTM4 were consistent with the presence of an oxygen sensitive [2Fe-2S] cluster. Expression of WhiBTM4 in the mycobacterial host led to hindered septation resembling a WhiB2 knockout phenotype whereas basal expression of WhiBTM4 led to superinfection exclusion. The quantification of mRNA-levels during phage infection showed that whiBTM4 is a highly transcribed early phage gene and a dominant negative regulator of WhiB2. Strikingly, both apo-WhiB2 of Mycobacterium tuberculosis and apo-WhiBTM4 were capable of binding to the conserved promoter region upstream of the whiB2 gene indicating that WhiB2 regulates its own synthesis which is inhibited in the presence of WhiBTM4. Thus, we provide substantial evidence supporting the hypothesis of viral and bacterial WhiB proteins being important Fe-S containing transcriptional regulators with DNA-binding capability. [source] Turnip yellow mosaic virus: transfer RNA mimicry, chloroplasts and a C-rich genomeMOLECULAR PLANT PATHOLOGY, Issue 5 2004THEO W. DREHER SUMMARY Taxonomy:,Turnip yellow mosaic virus (TYMV) is the type species of the genus Tymovirus, family Tymoviridae. TYMV is a positive strand RNA virus of the alphavirus-like supergroup. Physical properties:, Virions are non-enveloped 28-nm T = 3 icosahedrons composed of a single 20-kDa coat protein that is clustered in 20 hexameric and 12 pentameric subunits. Infectious particles and empty capsids coexist in infected tissue. The genomic RNA is 6.3 kb long, with a 5,m7GpppG cap and a 3, untranslated region ending in a tRNA-like structure to which valine can be covalently added. The genome has a distinctive skewed C-rich, G-poor composition (39% C, 17% G). Viral proteins:, Two proteins, whose open reading frames extensively overlap, are translated from the genomic RNA. p206, which contains sequences indicative of RNA capping, NTPase/helicase and polymerase activities, is the only viral protein that is necessary for genome replication in single cells. It is produced as a polyprotein and self-cleaved to yield 141- and 66-kDa proteins. p69 is required for virus movement within the plant and is also a suppressor of gene silencing. The coat protein is expressed from the single subgenomic RNA. Hosts and symptoms:, TYMV has a narrow host range almost completely restricted to the Cruciferae. Experimental host species are Brassica pekinensis (Chinese cabbage) or B. rapa (turnip), in which diffuse chlorotic local lesions and systemic yellow mosaic symptoms appear. Arabidopsis thaliana can also be used. Clumping of chloroplasts and the accumulation of vesicular invaginations of the chloroplast outer membranes are distinctive cytopathological symptoms. High yields of virus are produced in all leaf tissues, and the virus is readily transmissible by mechanical inoculation. Localized transmission by flea beetles may occur in the field. [source] HLA Type-Independent Method to Monitor Polyoma BK Virus-Specific CD4+ and CD8+ T-Cell ImmunityAMERICAN JOURNAL OF TRANSPLANTATION, Issue 3 2006M.H. Hammer (Re)activation of quiescent viral diseases is a major problem in immunosuppressed transplant patients. Polyoma BK virus-associated nephropathy (PVAN) caused by active polyoma BK virus (BKV) infection became a main reason for graft loss in kidney transplantation. After diagnosis, most transplant centers react by reducing immunosuppression (IS) to allow the immune system to control the infection. However, the impact of reduced IS on BKV immunity is not well researched. Here we present an HLA type-independent method to monitor BKV-specific T-cell immunity. Applying our method, viral protein 1-specific CD4+ and CD8+ T-cell responses were detected in patients with serum BKV-DNA levels >250 000 copies/mL. In addition, specific T-cell responses were also found in allograft-infiltrating cells. The method can be used to assess the impact of decreased immunosuppresson on BKV immunity and to clarify the role of specific T cells in the pathogenesis of PVAN. We strongly recommend its implementation in future clinical studies. [source] A simple method for purifying the White Spot Syndrome Virus using ultrafiltrationAQUACULTURE RESEARCH, Issue 6 2009Martina Hilda Gracia-Valenzuela Abstract A very simple and efficient method was developed for isolating intact White Spot Syndrome Virus (WSSV) particles from infected Litopenaeus vannamei tissue. No density gradient centrifugation, ultracentrifugation or protease inhibitors were required for the purification of intact WSSV virions using microfilters (100 kDa cut-off) combined with several steps of conventional centrifugation procedures. A mortality assay was run using healthy shrimp to prove that the virions obtained were infective. The concentrated viral preparations were further studied using polyacrylamide gel electrophoresis (PAGE). At least five distinct protein bands were detected when intact purified WSSV virions were found by sodium dodecyl sulphate-PAGE, followed by Coomassie Brilliant R-250 staining. The estimated molecular weights of these proteins were 23, 24, 29, 32 and 42-kDa, which could correspond to viral protein. Using this method, the virus does not lose its ability to infect healthy shrimp. [source] Quantifying Viral Propagation in Vitro: Toward a Method for Characterization of Complex PhenotypesBIOTECHNOLOGY PROGRESS, Issue 6 2001Karen A. Duca For a eukaryotic virus to successfully infect and propagate in cultured cells several events must occur: the virion must identify and bind to its cellular receptor, become internalized, uncoat, synthesize viral proteins, replicate its genome, assemble progeny virions, and exit the host cell. While these events are taking place, intrinsic host defenses activate in order to defeat the virus, e.g., activation of the interferon system, induction of apoptosis, and attempted elicitation of immune responses via chemokine and cytokine production. As a first step in developing an imaging methodology to facilitate direct observation of such complex host/virus dynamics, we have designed an immunofluorescence-based system that extends the traditional plaque assay, permitting simultaneous quantification of the rate of viral spread, as indicated by the presence of a labeled viral protein, and cell death in vitro, as indicated by cell loss. We propose that our propagation and cell death profiles serve as phenotypic read-outs, complementing genetic analysis of viral strains. As our virus/host system we used vesicular stomatitis virus (VSV) propagating in hamster kidney epithelial (BHK-21) and murine astrocytoma (DBT) cell lines. Viral propagation and death profiles were strikingly different in these two cell lines, displaying both very different initial titer and cell age effects. The rate of viral spread and cell death tracked reliably in both cell lines. In BHK-21 cells, the rate of viral propagation, as well as maximal spread, was relatively insensitive to initial titer and was roughly linear over several days. In contrast, viral plaque expansion in DBT cells was contained early in the infections with high titers, while low titer infections spread in a manner similar to the BHK-21 cells. The effect of cell age on infection spread was negligible in BHK-21 cells but not in DBTs. Neither of these effects was clearly observed by plaque assay. [source] A new look at viruses in type 1 diabetesDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 1 2003Hee-Sook Jun Abstract Type 1 diabetes (T1D) results from the destruction of pancreatic beta cells. Genetic factors are believed to be a major component for the development of T1D, but the concordance rate for the development of diabetes in identical twins is only about 40%, suggesting that nongenetic factors play an important role in the expression of the disease. Viruses are one environmental factor that is implicated in the pathogenesis of T1D. To date, 14 different viruses have been reported to be associated with the development of T1D in humans and animal models. Viruses may be involved in the pathogenesis of T1D in at least two distinct ways: by inducing beta cell-specific autoimmunity, with or without infection of the beta cells, [e.g. Kilham rat virus (KRV)] and by cytolytic infection and destruction of the beta cells (e.g. encephalomyocarditis virus in mice). With respect to virus-mediated autoimmunity, retrovirus, reovirus, KRV, bovine viral diarrhoea-mucosal disease virus, mumps virus, rubella virus, cytomegalovirus and Epstein-Barr virus (EBV) are discussed. With respect to the destruction of beta cells by cytolytic infection, encephalomyocarditis virus, mengovirus and Coxsackie B viruses are discussed. In addition, a review of transgenic animal models for virus-induced autoimmune diabetes is included, particularly with regard to lymphocytic choriomeningitis virus, influenza viral proteins and the Epstein-Barr viral receptor. Finally, the prevention of autoimmune diabetes by infection of viruses such as lymphocytic choriomeningitis virus is discussed. Copyright © 2002 John Wiley & Sons, Ltd. [source] Effect of detergent on electromigration of proteins: CE of very low density lipoprotein receptor modules and viral proteinsELECTROPHORESIS, Issue 20 2007Leopold Kremser Dr. Abstract The different electrophoretic behavior of the members of two groups of proteins with respect to the absence or presence of detergent additives in the BGE was explored. Recombinant soluble concatemers of repeat 3 of the very low density lipoprotein (VLDL)-receptor fused at their N -terminus to maltose-binding protein (MBP) exhibited different electrophoretic mobilities in borate buffer (pH,8.3) in the absence and in the presence of dodecyl-PEG ether (D-PEG). This enabled the separation of the receptor fragments from MBP after enzymatic cleavage. In the presence of SDS, the mobilities of all proteins approached the same values with increase in detergent concentrations. In contrast, viral capsid proteins of a human rhinovirus (HRV) exhibited different migration in the presence of the additive. For the receptor proteins, extreme apparent high plate numbers were observed when the SDS concentration in the sample and the separation buffer differed. This effect might be erroneously interpreted as a high efficiency. However, it is due to the conductivity boundaries caused by the sample and leads to a total loss of separation. [source] Construction of a cDNA library of Bemisia tabaci for use in the ,yeast two-hybrid screen' methodEPPO BULLETIN, Issue 1 2002S. Ohnesorge The molecular mechanisms involved in the circulative, non-propagative transmission pathway of TYLCV through its vector the whitefly Bemisia tabaci have hardly been studied. Points requiring investigation include the specific adhesion of virus coat protein to insect structures, the proteins involved in membrane passage in the insect and the possibility of replication of the virus in the vector. To isolate the insect proteins which are involved in transmission by interaction with viral proteins, we propose to use the ,yeast two-hybrid screen' genetic method. For this method, it is indispensable to have a ,cDNA library' of the organism concerned, cloned in plasmids, and our first step has been to develop this. A new method was developed for isolating whitefly mRNA. From this mRNA, cDNA was synthesized, ligated in the plasmid pGADT7 (Clontech) and transformed in bacteria to amplify the plasmid DNA. The number of independent clones and average insert size of the plasmids were determined. [source] Differential effects of US2, US6 and US11 human cytomegalovirus proteins on HLA class,Ia and HLA-E expression: impact on target susceptibility to NK cell subsetsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2003Manuel Llano Abstract We compared in an inducible expression system the individual effect of US2, US6 and US11 human cytomegalovirus (HCMV) proteins on HLA-E and HLA class,Ia surface expression, assessing in parallel their influence on target susceptibility to NK cell clones. To this end, the RPMI,8866 B,lymphoma cell line (HLA-A2, HLA-A3, HLA-B7, HLA-Cw7, HLA-ER, HLA-EG) was stably cotransfected with the ecdysone receptor, together with the US sequences under the control of an ecdysone-inducible promoter. Biosynthesis of viral proteins was turned on by incubating transfectants with Ponasterone,A. US6 down-regulated expression of all class,I molecules, hampering target resistance to NK cell clones controlled by the CD94/NKG2A, KIR2DL2 and/or CD85j (ILT2 or LIR-1) inhibitory receptors. By contrast, US11 reduced the surface levels of class,Ia molecules but preserved HLA-E; this rendered US11+ cells sensitive to NK clones under the control of KIR2DL2 and/or CD85j, while their resistance to CD94/NKG2A+KIR2DL2, effector cells was maintained. US2 preserved as well HLA-E expression but selectively targeted class,Ia molecules; in fact, HLA-A and HLA-C allotypes were down-modulated whereas HLA-B7 remained unaltered. US2+ targets became sensitive to KIR2DL2+ cells but remained resistant to CD94/NKG2A+CD85j+ NK clones. The differential effects of US proteins on HLA class,Ia and HLA-E likely reflect the evolutionary adaptation of HCMV to counteract NK-mediated surveillance. [source] Possible involvement of epidermodysplasia verruciformis human papillomaviruses in the immunopathogenesis of psoriasis: a proposed hypothesisEXPERIMENTAL DERMATOLOGY, Issue 6 2003Slawomir Majewski Abstract:, We have shown previously in psoriasis a very high prevalence of epidermodysplasia verruciformis-associated human papillomavirus 5 (EVHPV5) DNA and antibodies to human papillomavirus 5 (HPV5) virus-like particle (VLP)L1, and we suggested that this benign hyperproliferative disorder could be a reservoir for EVHPVs. Here we provide new data confirming the expression of EVHPVs in psoriasis and present our hypothesis on their possible involvement in the immunopathogenesis of the disorder. The new important finding was detection by a radioimmunoprecipitation assay of a very high prevalence of antibodies to E6/E7 HPV5 oncoproteins, known to enhance keratinocyte proliferation. More recently, EV genes were identified, EVER1 and EVER2, whose mutations are responsible for epidermodysplasia verruciformis. Epidermodysplasia verruciformis-associated human papillomaviruses are harmless to the general population as a result of genetic restriction, which in psoriasis appears to be partly alleviated, and this may allow the viral gene expression. We hypothesize that induction of keratinocyte proliferation in psoriasis by various stimuli initiates the EVHPV life cycle with expression of early (E6/E7) and late (L1) viral proteins. The early proteins may, in turn, enhance the keratinocyte proliferation, and the late proteins could serve as a target for specific B- and T-cell-mediated responses. Immune responses against the viral antigens in the epidermis may result in chemoattraction of leukocytes and Munro abscess formation, as well as in production of proinflammatory cytokines, leading to self perpetuation of the psoriatic process. The novel immunomodulatory therapies could also inhibit immune responses against EVHPV proteins, leading to decreased cytokine production, keratinocyte proliferation and EVHPV expression. Thus the beneficial effect of these therapies is not discordant with the proposed hypothesis of possible involvement of EVHPVs in the immunopathogenesis of psoriasis. [source] Replication of Theiler's virus requires NF-,B-activation: Higher viral replication and spreading in astrocytes from susceptible miceGLIA, Issue 9 2008Min Hyung Kang Abstract To investigate viral replication and cell,cell spreading in astrocytes, recombinant Theiler's murine encephalomyelitis virus (TMEV) expressing green fluorescent protein (GFP) during the replication was generated. GFP and TMEV proteins were processed correctly in infected cells and production of viral proteins could be tracked by fluorescent microscopy. Viral replication of both wild-type TMEV and GFP-TMEV was dependent on the activation of NF-,B and partially MAP kinase, based on chemical inhibition studies. Viral replication was significantly reduced in primary astrocytes from NF-,B1 (p105)-deficient mice compared with that from wild-type control mice, whereas cytokine production was enhanced. These results suggest an association of canonical NF-,B subunits in viral replication, but not cytokine production. Viral replication was also suppressed in both IKK, and IKK,-deficient mouse embryonic fibroblasts (MEFs), compared with that in wild-type MEF. However, the inhibition was significantly greater in IKK,-deficient MEF, suggesting that IKK, plays a stronger role in supporting viral replication. Interestingly, viral replication and spreading in primary astrocytes from susceptible SJL/J mice were several-fold higher than those in astrocytes from resistant C57BL/6 mice, suggesting that higher viral replication levels in astrocytes may also contribute to the viral persistence in the central nervous system (CNS) of susceptible SJL/J mice. A relatively higher level of activated NF-,B was found in the nuclei of virus-infected SJL astrocytes compared with C57BL/6 astrocytes suggest that the NF-,B activation level affects on viral replication. © 2008 Wiley-Liss, Inc. [source] Favorable effect of adefovir on the number and functionality of myeloid dendritic cells of patients with chronic HBV,HEPATOLOGY, Issue 4 2006Renate G. van der Molen In patients with chronic hepatitis B virus (HBV), 2 predominant precursor dendritic cell (DC) subtypes, the myeloid dendritic cell (mDC) and the plasmacytoid dendritic cell (pDC), were recently found to be functionally impaired. HBV DNA was found to be present in the DC subtypes, but no viral replication could be detected. The question remains whether simply the presence of the virus and viral proteins causes this dysfunction of DCs. To address this issue, the effect of viral load reduction resulting from treatment with the nucleotide analogue adefovir dipivoxil on the number and functionality of circulating DCs was studied during 6 months of treatment. Treatment resulted in a mean 5 log10 decrease in the viral load and normalization of alanine aminotransferase within 3 months. The number of mDCs, but not of pDCs, increased significantly over 6 months of treatment to a level comparable to that of uninfected healthy controls. The allostimulatory capacity of isolated and in vitro matured mDCs increased significantly after 3 months of treatment. Accordingly, mDCs exhibited an increased capacity to produce tumor necrosis factor alpha and interleukin-12 after 3-6 months of treatment. There was no change in interferon alpha production by pDCs during treatment. In conclusion, adefovir treatment results in an improvement in the number and functionality of mDCs, but not of pDCs. Our findings provide clues for the reasons why current antiviral therapy does not lead to consistently sustained viral eradication. (HEPATOLOGY 2006;44:907,914.) [source] The MHC class I antigen presentation pathway: strategies for viral immune evasionIMMUNOLOGY, Issue 2 2003Eric W. Hewitt Summary Presumably because of the selective pressure exerted by the immune system, many viruses have evolved proteins that interfere with antigen presentation by major histocompatibility complex (MHC) class I molecules. These viruses utilize a whole variety of ingenious strategies to inhibit the MHC class I pathway. Viral proteins have been characterized that exploit bottlenecks in the MHC class I pathway, such as peptide translocation by the transporter associated with antigen processing. Alternatively, viral proteins can cause the degradation or mislocalization of MHC class I molecules. This is often achieved by the subversion of the host cell's own protein degradation and trafficking pathways. As a consequence elucidation of how these viral proteins act to subvert host cell function will continue to give important insights not only into virus,host interactions but also the function and mechanism of cellular pathways. [source] Regulation of growth signalling and cell cycle by Kaposi's sarcoma-associated herpesvirus genesINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 6 2004Shamindra Direkze Summary Kaposi's sarcoma-associated herpesvirus (KSHV) is the primary aetiological agent of at least three malignancies associated with HIV infection and immunosuppression: Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. KSHV encodes proteins that deregulate key checkpoints in the signalling pathways governing cell proliferation, which may ultimately contribute to the virus' oncogenic potential. To alter cellular signalling associated with proliferation, these viral proteins function like growth factor ligands/receptors, signal transduction proteins, transcription factors and cell cycle regulators. This review focuses on the mechanisms by which some KSHV-encoded proteins activate signalling pathways and cell proliferation and their role in the pathogenesis of KSHV-driven mechanisms. [source] Viral appropriation of apoptotic and NF-,B signaling pathwaysJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2004Andrew G. Bowie Abstract Viruses utilize a variety of strategies to evade the host immune response and replicate in the cells they infect. The comparatively large genomes of the Orthopoxviruses and gammaherpesviruses encode several immunomodulatory proteins that are homologous to component of the innate immune system of host cells, which are reviewed here. However, the viral mechanisms used to survive host responses are quite distinct between these two virus families. Poxviruses undergo continuous lytic replication in the host cytoplasm while expressing many genes that inhibit innate immune responses. In contrast, herpesviruses persist in a latent state during much of their lifecycle while expressing only a limited number of relatively non-immunogenic viral proteins, thereby avoiding the adaptive immune response. Poxviruses suppress, whereas latent gammaherpesviruses activate, signaling by NF-,B, yet both viruses target similar host signaling pathways to suppress the apoptotic response. Here, modulation of apoptotic and NF-,B signal transduction pathways are examined as examples of common pathways appropriated in contrasting ways by herpesviruses and poxviruses. © 2004 Wiley-Liss, Inc. [source] Persistent parvovirus B19 infection detected by specific CD4+ T-cell responses in a patient with hepatitis and polyarthritisJOURNAL OF INTERNAL MEDICINE, Issue 3 2009G. Pongratz Abstract. We, here, report the case of a parvovirus B19 infection in an immunocompetent male patient presenting with acute hepatitis and polyarthritis. To follow the course of infection, we used a previously established enzyme-linked immunosorbent spot assay (ELISPOT) technique to detect CD4+ T cells specific for viral proteins. Even though symptoms of arthritis and hepatitis resolved in the immunocompetent individual within a few weeks, viral DNA in serum and CD4+ T cells specific for the viral protein VP1 unique region were still detectable more than 6 month after the onset of symptoms, thus pointing to a persistent state of infection. On the basis of this observation, we hypothesize that the intensity of liver involvement correlates with the likelihood of developing persistent parvovirus B19 infection. The described ELISPOT technique to detect virus-specific CD4+ T cells provides an excellent tool to analyse the state of parvovirus B19 infection for future studies to test this hypothesis. [source] | |||||||||||||||||||||||||||||||