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At least 19 recordsLinked to original sources
PubMed2004

Inactivation of West Nile virus, vaccinia virus and viral surrogates for relevant and emergent viral pathogens in plasma-derived products.

BACKGROUND AND OBJECTIVES: Human plasma is the source of a wide variety of therapeutic proteins, yet it is also a potential source of viral contamination. Recent outbreaks of emergent viral pathogens, such as West Nile virus, and the use of live vaccinia virus as a vaccine have prompted a reassessment of the viral safety of plasma-derived products. The purpose of this study was to evaluate the efficacy of current viral inactivation methods for West Nile and vaccinia viruses and to reassess the use of model viruses to predict inactivation of similar viral pathogens. MATERIALS AND METHODS: Virus-spiked product intermediates were processed using a downscaled representation of various manufacturing procedures. Virus infectivity was measured before and after processing to determine virus inactivation. RESULTS: The results demonstrated effective inactivation of West Nile virus, vaccinia virus and a model virus, bovine viral diarrhoea virus, during pasteurization, solvent/detergent treatment and caprylate treatment. Caprylate provided rapid and effective inactivation of West Nile virus, vaccinia virus, duck hepatitis B virus and Sindbis virus. Inactivation of West Nile virus was similar to that of bovine viral diarrhoea virus. CONCLUSIONS: This study demonstrates that procedures used to inactivate enveloped viruses in manufacturing processes can achieve inactivation of West Nile virus and vaccinia virus. In addition, the data support the use of model viruses to predict the inactivation of similar emergent viral pathogens.

Animals↗
PubMed1975

Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid.

Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha.

Adenoviridae↗
PubMed1968

Sensitivity of herpes simplex virus, vaccinia virus, and adenoviruses to deoxyribonucleic acid inhibitors and thiosemicarbazones in a plaque suppression test.

Herpes simplex and vaccinia viruses and adenovirus types 1, 2, 5, and 7 were tested by plaque suppression methods for sensitivity to halogenated deoxyuridines (5-iodo-, 5-bromo-, 5-chloro-, and 5-fluoro-), cytosine arabinoside, isatin-beta-thiosemicarbazone, and N-methylisatin-beta-thiosemicarbazone. After incubation for 12 days in HeLa cell cultures, vaccinia virus plaques were still readily suppressed by deoxyribonucleic acid (DNA) inhibitors and thiosemicarbazones. Herpes simplex virus plaques were likewise suppressed by at least three DNA inhibitors. Adenovirus plaques were not suppressed by DNA inhibitors or thiosemicarbazones. 5-Fluoro-2'-deoxyuridine could not be shown to have any antiviral activity, but it did produce a substantial lethal action on the cells.

Adenoviridae↗
PubMed1998

Inactivation of human immunodeficiency virus type 1, hepatitis A virus, respiratory syncytial virus, vaccinia virus, herpes simplex virus type 1, and poliovirus type 2 by hydrogen peroxide gas plasma sterilization.

BACKGROUND: Studies were conducted to determine the capability of a hydrogen peroxide gas plasma sterilization process to inactivate several types of viruses. Six test agents were used: HIV type 1, human hepatitis A virus, respiratory syncytial virus, vaccinia, herpes simplex virus type 1, and poliovirus type 2. METHODS: The test viruses were suspended in cell culture medium and dried on the bottom of sterile glass petri dishes. The inoculated dishes were processed in the hydrogen peroxide gas plasma system for half the normal sterilization cycle time. Four inoculated carriers for each virus were used in two separate half cycles. Infectivity of the test viruses and cytotoxicity to the indicator cell lines were assayed. RESULTS: The hydrogen peroxide gas plasma sterilization process produced inactivation of the six viral test agents under these experimental conditions. The reduction in viral titers ranged from 2.5 log10 to 5.5 log10, a 99.68% to 99.999% decrease. CONCLUSIONS: These results clearly demonstrate the virucidal effectiveness of the hydrogen peroxide gas plasma sterilization process against both lipid and nonlipid viruses.

Cells, Cultured↗
PubMed1996

Viral status and antibody response in cattle inoculated with recombinant bovine leukemia virus-vaccinia virus vaccines after challenge exposure with bovine leukemia virus-infected lymphocytes.

OBJECTIVE: To determine the bovine leukemia virus (BLV) antibody response and infectivity status in BLV-vaccinated cattle after challenge exposure with BLV-infected lymphocytes. DESIGN: Steers were inoculated with vaccinia virus constructs expressing the gag, pol, and env genes of the BLV or the env gene only of the BLV genome, then challenge exposed with BLV-infected lymphocytes. The steers' BLV antibody and infectivity status was monitored. ANIMALS: Fifteen 8- to 9-month-old Holstein steers previously determined to be BLV antibody and BLV negative. PROCEDURE: 1 month after second after inoculation, steers were challenge exposed with 10(6) BLV-infected lymphocytes from a highly infective BLV-positive cow. Serum and blood lymphocytes were obtained regularly for 6 months. The agar gel immunodiffusion assay, ELISA, and serum neutralization assay were used to detect BLV antibody in serum of steers. The sheep infectivity and syncytium-forming assays were used to determine the viral status of the steers. RESULTS: Differences were seen in antibody responses between the BLV-vaccinated and non-BLV-vaccinated control groups. All cattle were susceptible to infection when challenge exposed with BLV-infected lymphocytes. CONCLUSION: Despite the enhanced immune response in the BLV-vaccinated cattle after challenge exposure, none of the BLV-vaccinated cattle was protected from BLV infection. CLINICAL RELEVANCE: Vaccination is not an effective way to protect cattle from BLV infection.

Animals↗
PubMed1998

PCR-based method for the introduction of mutations in genes cloned and expressed in vaccinia virus.

Vaccinia virus expression systems allow efficient expression of genes and facilitate functional studies of expressed proteins in cultured mammalian cells. We designed and tested a rapid method to introduce defined mutations in genes inserted and expressed in vaccinia virus. PCR mutagenesis is used to construct a recombination cassette that contains: (i) the mutated exogenous gene, (ii) recombination flanks to direct insertion into the virus genome and (iii) a selectable gene to allow easy isolation of recombinant viruses. To generate recombinant viruses, the recombination cassette is transfected into vaccinia virus-infected cells. The procedure does not require cloning, and the mutated gene versions are inserted directly into the vaccinia virus genome downstream of a vaccinia virus strong promoter. The method was tested by introducing a point mutation into Aequorea victoria green fluorescent protein (GFP), known to alter the fluorescence absorption and emission spectra of the protein. This system should facilitate and speed the isolation of virus recombinants expressing mutated versions of any given gene and can be adapted to random mutagenesis procedures, exon shuffling or other PCR-based mutagenesis protocols.

Cell Line↗
PubMed1992

RNA polymerase-associated transcription specificity factor encoded by vaccinia virus.

Vaccinia virus encodes a multisubunit DNA-dependent RNA polymerase (EC 2.7.7.6) that is packaged in the infectious virus particle. This polymerase was found to contain a submolar polypeptide of approximately 85 kDa in addition to the core subunits, which consist of two larger and several smaller polypeptides. The polymerase containing the 85-kDa polypeptide was separated from the core polymerase by column chromatography. Although the core polymerase actively transcribed heterologous single-stranded DNA, only the form with the associated 85-kDa polypeptide could act in conjunction with an early stage-specific factor to transcribe double-stranded DNA containing a vaccinia virus early promoter. Peptide sequencing established that the RNA polymerase-associated 85-kDa protein was derived from the vaccinia virus H4L open reading frame, which encodes a 94-kDa polypeptide that we named RAP94. RAP94 is not closely related to prokaryotic sigma 70 or eukaryotic RAP30 RNA polymerase-binding proteins, although there are short regions of sequence similarity. The specific association of RAP94 with viral RNA polymerase was corroborated with antibody raised to a recombinant fusion protein. Unlike the previously defined subunits of vaccinia virus RNA polymerase, RAP94 is synthesized exclusively late in infection, and synthesis could be prevented by a DNA replication inhibitor. The role of RAP94 in mediating specific transcription was demonstrated by using an extract from cells in which the H4L open reading frame had been transiently expressed.

Amino Acid Sequence↗
PubMed1995

Species specificity of ectromelia virus and vaccinia virus interferon-gamma binding proteins.

Interferon-gamma functions within the immune system as a potent anti-viral and immunoregulatory cytokine. In order to successfully replicate within a host cell, poxviruses have evolved a number of strategies to counteract the pleiotropic effects of interferon-gamma. In particular, the leporipoxvirus myxoma virus was shown to express an extracellular soluble interferon-gamma receptor homolog, denoted M-T7, which is capable of inhibiting the anti-viral activities of rabbit interferon-gamma (C. Upton, K. Mossman, and G. McFadden, 1992, Science 258, 1369-1372). Here, we demonstrate that expression of soluble interferon-gamma receptor homologs appears to be characteristic of all poxviruses tested, including Shope fibroma virus, vaccinia virus (strains WR and IHDW), ectromelia virus, cowpox virus, and rabbitpox virus. We have cloned, sequenced, and characterized the interferon-gamma binding protein in supernatants from ectromelia virus-infected cells, and demonstrate the capability of this soluble protein to bind human, murine, and rabbit interferon-gamma with similar affinity. We also investigate the properties of the vaccinia virus interferon-gamma binding protein and demonstrate that this protein binds human and rabbit interferon-gamma with similar affinity and binds murine interferon-gamma with a significantly lower relative affinity. The implications of these studies with respect to viral pathogenesis and the evolutionary relationship between a virus and its host are discussed.

Amino Acid Sequence↗
PubMed1980

Phosphorylation of myelin basic protein by vaccinia virus.

Vaccinia virus phosphorylates myelin basic protein in the myelin membrane in vitro. In the presence of vaccinia virus cores maximally 1.5 mol and in the presence of intact virus 0.7 mol phosphate residues were incorporated into 1 mol of myelin basic protein in the myelin membrane. The peptides of myelin basic protein which were phosphorylated by the vaccinia virus kinase were clearly all different from the peptides which were phosphorylated by the endogenous myelin protein kinase. The viral modification of the encephalitogenic protein and its significance to immunological events is discussed.

Myelin Basic Protein↗
PubMed1999

Immune modulation by proteins secreted from cells infected by vaccinia virus.

Vaccinia virus comprises the live vaccine that was used for vaccination against smallpox. Following the eradication of smallpox, vaccinia virus was developed as an expression vector that is now used widely in biological research and vaccine development. In recent years vaccinia virus and other poxviruses have been found to express a collection of proteins that block parts of the host response to infection. Some of these proteins are secreted from the infected cell where they bind and neutralise host cytokines, chemokines and interferons (IFN). In this paper three such proteins that bind interleukin (IL)-1 beta, type I IFNs and CC chemokines are described. The study of these immunomodulatory molecules is enhancing our understanding of virus pathogenesis, yielding fundamental information about the immune system, and providing new molecules that have potential application for the treatment of immunological disorders or infectious diseases.

Animals↗
PubMed2004

An oligonucleotide microarray for the detection of vaccinia virus.

Vaccinia virus is a member of the orthopoxvirus group, to which also belongs variola virus, one of the most hazardous pathogens known to man. To establish a model system to detect orthopoxviruses, a vaccinia oligonucleotide microarray is designed, produced and tested. Vaccinia virus is used to test the prepared microarrays. The virus DNA samples in different propagation phases are extracted and hybridised with the oligonucleotide microarray. The results showed that the oligonucleotide microarray can detect vaccinia virus with high specificity and sensitivity.

Base Sequence↗
PubMed1991

Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus.

Vaccinia virus has been used as a vector to express foreign genes for the production of functional and posttranslationally modified proteins. A procedure is described here that allows the rapid native purification of vaccinia-expressed proteins fused to an amino-terminal tag of six histidines. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2+.nitrilotriacetic acid (Ni2+.NTA)-agarose and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. In the case of the human serum response factor (SRF), a transcription factor involved in the regulation of the c-fos protooncogene, the vaccinia-expressed histidine-tagged SRF (SRF-6His) could be purified solely by this step to greater than 95% purity. SRF-6His was shown to resemble authentic SRF by functional criteria: it was transported to the nucleus, bound specifically the c-fos serum response element, interacted with the p62TCF protein to form a ternary complex, and stimulated in vitro transcription from the serum response element. Thus, the combination of vaccinia virus expression and affinity purification by Ni2+.NTA chromatography promises to be useful for the production of proteins in a functional and posttranslationally modified form.

Amino Acid Sequence↗
PubMed1996

Active specific immunotherapy of pulmonary metastasis with vaccinia melanoma oncolysate prepared from granulocyte/macrophage-colony-stimulating-factor-gene-encoded vaccinia virus.

Vaccinia melanoma oncolysate (VMO) prepared with recombinant vaccinia virus encoding the gene of murine granulocyte/macrophage-colony-stimulating factor (GM-CSF) was tested for its therapeutic effect on melanoma pulmonary metastasis. The murine pulmonary metastasis model was established by injecting 2 x 10(5) B16F10 melanoma cells into the tail vein of a C57BL/6 mouse. Intraperitoneal injection of VMO was performed in tumor-bearing mice 3 and 10 days after B16F10 cell inoculation. The results showed that treatment with VMO prepared with GM-CSF-gene-encoded vaccinia virus (GM-CSFVMO) significantly decreased the number of murine pulmonary metastases and prolonged the survival of the tumor-bearing mice. Lymphocytes isolated from fresh blood and spleen of GM-CSFVMO-treated mice showed higher cytolytic activity against B16F10 melanoma cells when compared with lymphocytes from the mice of other treatment groups. Natural killer activity remained unchanged in the GM-CSFVMO-treated group. Cytotoxic activities of peritoneal macrophages were found to be greatly elevated in mice treated with GM-CSFVMO. Further study illustrated that the increased tumor necrosis factor and nitric oxide release from macrophages may contribute to their cytotoxic effects. These results suggest that the tumor oncolysate vaccine prepared with GM-CSF-gene-encoded vaccinia virus has a potent therapeutic effect on tumor metastasis through the efficient induction of antitumor immunity of the host, mainly through the cytotoxic effects of cytotoxic T lymphocytes and macrophages.

Animals↗
PubMed2006

Differences and similarities in viral life cycle progression and host cell physiology after infection of human dendritic cells with modified vaccinia virus Ankara and vaccinia virus.

Modified vaccinia virus Ankara (MVA) is an attenuated strain of vaccinia virus (VV) that has attracted significant attention as a candidate viral vector vaccine for immunization against infectious diseases and treatment of malignancies. Although MVA is unable to replicate in most nonavian cells, vaccination with MVA elicits immune responses that approximate those seen after the administration of replication-competent strains of VV. However, the mechanisms by which these viruses elicit immune responses and the determinants of their relative immunogenicity are incompletely understood. Studying the interactions of VV and MVA with cells of the human immune system may elucidate these mechanisms, as well as provide a rational basis for the further enhancement of the immunogenicity of recombinant MVA vectors. Toward this end, we investigated the consequences of MVA or VV infection of human dendritic cells (DCs), key professional antigen-presenting cells essential for the generation of immune responses. We determined that a block to the formation of intracellular viral replication centers results in abortive infection of DCs with both VV and MVA. MVA inhibited cellular protein synthesis more rapidly than VV and displayed a distinct pattern of viral protein expression in infected DCs. MVA also induced apoptosis in DCs more rapidly than VV, and DC apoptosis after MVA infection was associated with an accelerated decline in the levels of intracellular Bcl-2 and Bcl-X(L). These findings suggest that antigen presentation pathways may contribute differentially to the immunogenicity of VV and MVA and that targeted modifications of virus-induced DC apoptosis may further increase the immunogenicity of MVA-vectored vaccines.

Apoptosis↗
PubMed2005

Evasion of innate immunity by vaccinia virus.

Vaccinia virus, a member of the Poxviridae, expresses many proteins involved in immune evasion. In this review, we present a brief characterisation of the virus and its effects on host cells and discuss representative secreted and intracellular proteins expressed by vaccinia virus that are involved in modulation of innate immunity. These proteins target different aspects of the innate response by binding cytokines and interferons, inhibiting cytokine synthesis, opposing apoptosis or interfering with different signalling pathways, including those triggered by interferons and toll-like receptors.

Apoptosis↗
PubMed2003

Evidence against an essential role of COPII-mediated cargo transport to the endoplasmic reticulum-Golgi intermediate compartment in the formation of the primary membrane of vaccinia virus.

Vaccinia virus assembles two distinct lipoprotein membranes. The primary membrane contains nonglycosylated proteins, appears as crescents in the cytoplasm, and delimits immature and mature intracellular virions. The secondary or wrapping membrane contains glycoproteins, is derived from virus-modified trans-Golgi or endosomal cisternae, forms a loose coat around some intracellular mature virions, and becomes the envelope of extracellular virions. Although the mode of formation of the wrapping membrane is partially understood, we know less about the primary membrane. Recent reports posit that the primary membrane originates from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). According to this model, viral primary membrane proteins are cotranslationally inserted into the ER and accumulate in the ERGIC. To test the ERGIC model, we employed Sar1(H79G), a dominant negative form of the Sar1 protein, which is an essential component of coatomer protein II (COPII)-mediated cargo transport from the ER to the ERGIC and other post-ER compartments. Overexpression of Sar1(H79G) by transfection or by a novel recombinant vaccinia virus with an inducible Sar1(H79G) gene resulted in retention of ERGIC 53 in the ER but did not interfere with localization of viral primary membrane proteins in factory regions or with formation of viral crescent membranes and infectious intracellular mature virions. Wrapping of intracellular mature virions and formation of extracellular virions did not occur, however, because some proteins that are essential for the secondary membrane were retained in the ER as a consequence of Sar1(H79G) overexpression. Our data argue against an essential role of COPII-mediated cargo transport and the ERGIC in the formation of the viral primary membrane. Instead, viral membranes may be derived directly from the ER or by a novel mechanism.

Animals↗