- Volume 84, Issue 7, 2003
Volume 84, Issue 7, 2003
- Review
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Mechanisms of CD4+ T lymphocyte cell death in human immunodeficiency virus infection and AIDS
More LessAIDS, caused by the retroviruses human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2), has reached pandemic proportions. Therefore, it is critical to understand how HIV causes AIDS so that appropriate therapies can be formulated. Primarily, HIV infects and kills CD4+ T lymphocytes, which function as regulators and amplifiers of the immune response. In the absence of effective anti-retroviral therapy, the hallmark decrease in CD4+ T lymphocytes during AIDS results in a weakened immune system, impairing the body's ability to fight infections or certain cancers such that death eventually ensues. The major mechanism for CD4+ T cell depletion is programmed cell death (apoptosis), which can be induced by HIV through multiple pathways. Death of HIV-infected cells can result from the propensity of infected lymphocytes to form short-lived syncytia or from an increased susceptibility of the cells to death. However, the apoptotic cells appear to be primarily uninfected bystander cells and are eradicated by two different mechanisms: either a Fas-mediated mechanism during activation-induced cell death (AICD), or as a result of HIV proteins (Tat, gp120, Nef, Vpu) released from infected cells stimulating apoptosis in uninfected bystander cells. There is also evidence that as AIDS progresses cytokine dysregulation occurs, and the overproduction of type-2 cytokines (IL-4, IL-10) increases susceptibility to AICD whereas type-1 cytokines (IL-12, IFN-γ) may be protective. Clearly there are multiple causes of CD4+ T lymphocyte apoptosis in AIDS and therapies that block or decrease that death could have significant clinical benefit.
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- Animal
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- RNA viruses
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Construction and in vivo infection of a new simian/human immunodeficiency virus chimera containing the reverse transcriptase gene and the 3′ half of the genomic region of human immunodeficiency virus type 1
More LessA new simian/human immunodeficiency virus (SHIV) chimera with the reverse transcriptase (RT)-encoding region of pol, in addition to the 3′ region encoding vpr, vpu, tat, rev, env and nef of HIV-1, on an SIVmac (SIV from a macaque monkey) background was constructed. This new SHIV chimera, named SHIVrt/3rn, could replicate in monkey peripheral blood mononuclear cells (PBMCs) as well as in the human and monkey CD4+ T-cell lines M8166 and HSC-F. Since SHIVrt/3rn contains the RT gene of HIV-1, replication of the virus in M8166 cells was inhibited by an HIV-1-specific non-nucleoside RT inhibitor, MKC-442, with a sensitivity similar to that of HIV-1. To investigate the replication competence of SHIVrt/3rn in vivo, two rhesus monkeys were inoculated intravenously with the virus. At 2 to 4 weeks post-inoculation (p.i.), plasma viral RNA loads of both monkeys showed a peak value of more than 104 copies ml−1. Infectious virus was isolated from the PBMCs of one monkey at 2 and 3 weeks p.i. and from the other at 4 weeks p.i. Moreover, proviral DNA was detected constantly throughout the observation period, starting from 3 weeks p.i. An antibody response, detected first at 3 weeks p.i., was maintained at high titres. These results indicate that SHIVrt/3rn can infect and replicate in vivo. SHIVrt/3rn, having part of HIV-1 pol in addition to the 3′ part of the HIV-1 genome is genetically more close to HIV-1 than any of the other monkey-infecting SHIVs reported previously.
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Infection of macaques with simian immunodeficiency virus induces a species-specific antibody response to major histocompatibility complex class I and class II molecules
More LessEnvelopes of retroviruses, including human immunodeficiency virus and simian immunodeficiency virus (SIV), contain host cell proteins that potentially represent novel targets for vaccine development. We show here that sera from rhesus macaques recognized simian major histocompatibility complex (MHC) molecules in response to infection with SIV. Antibodies from these animals did not cross-react with human MHC antigens on mitogen-activated peripheral blood mononuclear cells. The development of antibodies to MHC class I α-chain did not correlate with anti-SIV envelope antibody responses, suggesting that these antibodies did not arise through molecular mimicry. In contrast to the species-specific response in infected animals, sera from animals vaccinated with inactivated human cell-grown SIV reacted to both human and rhesus MHC class I and class II molecules.
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Gene expression analysis of murine cells producing amphotropic mouse leukaemia virus at a cultivation temperature of 32 and 37 °C
More LessCultivation of retrovirus packaging cells at 32 °C represents a common procedure to achieve high titres in mouse retrovirus production. Gene expression profiling of mouse NIH 3T3 cells producing amphotropic mouse leukaemia virus 4070A revealed that 10 % of the 1176 cellular genes investigated were regulated by temperature shift (37/32 °C), while 5 % were affected by retrovirus infection. Strikingly, retrovirus production at 32 °C activated the cholesterol biosynthesis/transport pathway and caused an increase in plasma membrane cholesterol levels. Furthermore, these conditions resulted in transcriptional activation of smoothened (smo), patched (ptc) and gli-1; Smo, Ptc and Gli-1, as well as cholesterol, are components of the Sonic hedgehog (Shh) signalling pathway, which directs pattern formation, diversification and tumourigenesis in mammalian cells. These findings suggest a link between cultivation at 32 °C, production of MLV-A and the Shh signalling pathway.
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Requirement for cyclophilin A for the replication of vesicular stomatitis virus New Jersey serotype
More LessSeveral host proteins have been shown to play key roles in the life-cycle of vesicular stomatitis virus (VSV). We have identified an additional host protein, cyclophilin A (CypA), a chaperone protein possessing peptidyl cis-trans prolyl-isomerase activity, as one of the cellular factors required for VSV replication. Inhibition of the enzymatic activity of cellular CypA by cyclosporin A (CsA) or SDZ-211-811 resulted in a drastic inhibition of gene expression by VSV New Jersey (VSV-NJ) serotype, while these drugs had a significantly reduced effect on the genome expression of VSV Indiana (VSV-IND) serotype. Overexpression of a catalytically inactive mutant of CypA resulted in the reduction of VSV-NJ replication, suggesting a requirement for functional CypA for VSV-NJ infection. It was also shown that CypA interacted with the nucleocapsid (N) protein of VSV-NJ and VSV-IND in infected cells and was incorporated into the released virions of both serotypes. VSV-NJ utilized CypA for post-entry intracellular primary transcription, since inhibition of CypA with CsA reduced primary transcription of VSV-NJ by 85–90 %, whereas reduction for VSV-IND was only 10 %. Thus, it seems that cellular CypA binds to the N protein of both serotypes of VSV. However, it performs an obligatory function on the N protein activity of VSV-NJ, while its requirement is significantly less critical for VSV-IND N protein function. The different requirements for CypA by two serologically different viruses belonging to the same family has highlighted the utilization of specific host factors during their evolutionary lineages.
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Antibody-mediated growth of infectious salmon anaemia virus in macrophage-like fish cell lines
More LessInfectious salmon anaemia virus (ISAV), a pathogen in marine aquaculture, belongs to the genus Isavirus, family Orthomyxoviridae. There is limited information on how ISAV interacts with host defences. To study ISAV–antibody interactions, virus neutralization (VN) assays were performed in the cell lines CHSE-214, SHK-1 and TO using three strains of ISAV and rabbit or fish anti-ISAV sera. Homologous VN titres of >1 : 1280 in CHSE-214 cells corresponded to titres of only 1 : 80 in the macrophage-like fish cell lines SHK-1 and TO, despite using 1000 and 2000 times less virus, respectively. However, rabbit antiserum to infectious pancreatic necrosis virus (IPNV) had a VN titre of 1 : 10 260 against IPNV in both CHSE-214 and TO cells. Poor ISAV neutralization in TO cells was attributed to Fc receptors mediating virus infectivity, because (1) neutralization by rabbit antiserum to ISAV was increased 48-fold in the presence of staphylococcal Protein A and (2) when using FITC-labelled virus and spectrofluorometry, a significant increase (P=0·018) in the intensity of fluorescence of intracellular virus was observed in assays of virus–antiserum mixtures in the absence of Protein A as compared to those in the presence of Protein A. Neutralization of ISAV with fish antisera was observed only in CHSE-214 cells, as Protein A could not restore neutralization in TO cells. These findings demonstrate for the first time antibody-mediated infection of macrophage-like fish cell lines by a fish virus, ISAV, and, as ISAV in Atlantic salmon targets leukocytic and endothelial cells, this may have implications for ISA pathogenesis and vaccination.
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The membrane proteins of flaviviruses form ion-permeable pores in the target membrane after fusion: identification of the pores and analysis of their possible role in virus infection
More LessRecently, we presented evidence that the E1 fusion protein of the alphavirus Semliki Forest virus forms ion-permeable pores in the target membrane after fusion. We proposed that the homologous fusion proteins of flaviviruses and hepatitis C virus form similar pores. To test this hypothesis for the E fusion protein of flaviviruses, the release of [3H]choline from liposomes by the flavivirus West Nile (WN) virus was determined. [3H]Choline was released at mildly acid pH. The pH threshold depended on the lipid composition. Release from certain liposomes was activated even at neutral pH. To identify the generation of individual pores, single cells were investigated with the patch-clamp technique. The formation of individual pores during low pH-induced WN virus entry at the plasma membrane occurred within seconds. These experiments were performed in parallel with Semliki Forest virus. The results indicated that, similar to alphavirus infection, infection with flaviviruses via endosomes leads to the formation of ion-permeable pores in the endosome after fusion, which allows the flow of protons from the endosome into the cytoplasm during virus entry. However, in vitro translation experiments of viral cores showed that, in contrast to alphaviruses, which probably need this proton flow for core disassembly, the genome RNA of WN virus present in the viral core is directly accessible for translation. For entry of flaviviruses, therefore, a second pathway for productive infection may exist, in which fusion of the viral membrane is activated at neutral pH by contact with a plasma membrane of appropriate lipid composition.
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Antibody-dependent enhancement of Murray Valley encephalitis virus virulence in mice
Enhancement of flavivirus infection in vitro in the presence of subneutralizing concentrations of homologous or heterologous antiserum has been well described. However, the importance of this phenomenon in the enhancement of flavivirus infection in vivo has not been established. In order to study antibody-mediated enhancement of flavivirus infection in vivo, we investigated the effect of passive immunization of mice with Japanese encephalitis virus (JE) antiserum on the outcome of infection with Murray Valley encephalitis virus (MVE). We show that prior treatment of mice with subneutralizing concentrations of heterologous JE antiserum resulted in an increase in viraemia titres and in mortality following challenge with wild-type MVE. Our findings support the hypothesis that subneutralizing concentrations of antibody may enhance flavivirus infection and virulence in vivo. These findings are of potential importance for the design of JE vaccination programs in geographic areas in which MVE co-circulates. Should subneutralizing concentrations of antibody remain in the population following JE vaccination, it is possible that enhanced disease may be observed during MVE epidemics.
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Common host genes are activated in mouse brain by Japanese encephalitis and rabies viruses
More LessThis study identified nine genes whose expression is upregulated in the central nervous system (CNS) of mice during Japanese encephalitis virus (JEV) infection. These include: cathepsin S, oligoadenylate synthetase (OAS), GARG49/IRG2, lymphocyte antigen-6A (Ly-6A), macrophage activation gene-2 (Mpa2), early growth response gene1 (Egr1), pyrimidine 5′-nucleotidase (P5N), apolipoprotein D (ApoD) and STAT1. Activation of all nine genes during JEV infection was confirmed by Northern blot analysis. JEV replication was inhibited in the majority of mice immunized with Biken JEV vaccine, and these mice also exhibited reduced expression of JEV-inducible CNS genes. Thus, there is a good correlation between virus load and upregulation of host CNS genes. It was also demonstrated that all the CNS genes activated by JEV are also upregulated during rabies virus infection. In addition, GARG49, STAT1, cathepsin S and ApoD are known to be upregulated in the CNS by Sindbis virus, an alphavirus, and this supports the proposal that common host cell pathways are activated in the CNS by different neurotropic viruses.
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Interference in Japanese encephalitis virus infection of Vero cells by a cationic amphiphilic drug, chlorpromazine
More LessEntry of Japanese encephalitis virus (JEV) into cells was analysed by using the vertebrate cell line Vero. Vero cells were treated with chlorpromazine, nystatin or cytochalasin D, which inhibit clathrin- and caveola-dependent endocytosis, and macropinocytosis of the cells, respectively. Productive JEV infection was inhibited by pretreatment with chlorpromazine; the number of JEV antigen-positive cells was less than one-fifth of that in untreated cultures, but was not significantly decreased by pretreatment with nystatin or cytochalasin. Viral antigens were detected in the membrane fractions, but not in the endosome fractions from chlorpromazine-treated JEV-inoculated cells. When the cells were treated with chlorpromazine, clathrin heavy chain antigen and JEV antigen were not detected in cytoplasm by indirect immunofluorescence staining. These results indicate that JEV is taken up by cells through the clathrin-dependent endocytic pathway, and this process leads to infection.
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Hepatitis C virus dynamics and pathology: the role of CTL and antibody responses
More LessThis paper investigates the role of CTL and antibody responses in hepatitis C virus (HCV) dynamics and pathology. Mathematical models suggest that a strong CTL response is required for resolution of HCV infection and that a weak CTL response can result in persistent infection. According to the model, establishment of persistent infection is accompanied mainly by an ongoing antibody response, while CTLs are not maintained at high levels. In the model, this outcome correlates with absence of pathology. Persistent infection in the face of an ongoing antibody response can result in evolution of antigenic escape. According to the model, evolution towards escape from antibodies can shift the balance of immune responses so that the weak CTL levels become increasingly more dominant relative to antibodies. This shift results in onset of liver pathology as the virus evolves towards increased levels of antigenic escape. Therefore, the relative balance of the immune response can be a decisive factor that determines whether patients are asymptomatic or whether pathology is observed. Virus evolution can shift this balance towards pathology over time. Theoretical results are discussed in the context of published data.
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Characterization of the expression of the hepatitis C virus F protein
Hepatitis C virus (HCV) is an important human pathogen that affects 170 million people worldwide. The HCV genome is approximately 9·6 kb in length and encodes a polyprotein that is proteolytically cleaved to generate at least 10 mature viral protein products. Recently, a new protein, named F, has been described to be expressed through a ribosomal frameshift within the capsid-encoding sequence, a mechanism unique among members of the family Flaviviridae. Here, expression of the F protein was investigated in an in vitro transcription/translation assay. Its expression in mammalian cells was confirmed using specific recombinant vaccinia viruses; under these conditions, protein expression is dependent on the HCV IRES. The F protein was tagged with firefly luciferase or the Myc epitope to facilitate its identification. Ribosomal frameshifting was dependent on the presence of mutations in the capsid-encoding sequence. No frameshifting was detected in the absence of any mutation. Furthermore, analysis of the F protein in time-course experiments revealed that the protein is very unstable and that its production can be stabilized by the proteasome inhibitor MG132. Finally, indirect immunofluorescence studies have localized the F protein in the cytoplasm, with notable perinuclear detection.
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Analysis of hepatitis C virus/classical swine fever virus chimeric 5′NTRs: sequences within the hepatitis C virus IRES are required for viral RNA replication
More LessHepatitis C virus (HCV) is classified in the genus Hepacivirus of the family Flaviviridae, whose members have a single-stranded RNA genome of positive polarity, which encodes a single polyprotein. Within this family, HCV is closely related to viruses of the genus Pestivirus, which includes classical swine fever virus (CSFV). Translation of the hepaci- and pestiviral polyprotein is initiated by internal entry of ribosomes, promoted by the 5′NTR. The secondary and tertiary RNA structures of the HCV and pestivirus 5′NTRs are well conserved, despite the fact that their sequences differ significantly from one another. By analogy with other positive-stranded RNA viruses, the 5′NTR of HCV is likely to contain cis-acting determinants for replication as well as the determinants for translation. Studies on both signals could be complicated, as these signals might overlap. In this study, this problem was addressed by constructing chimeric HCV/CSFV 5′NTRs. A two-step analysis of these 5′NTRs was performed: (a) in a translation assay, which provided the possibility to study translation independently of the possible effects on replication; and (b) in a replication assay, in which were studied only the chimeric 5′NTRs for which IRES-dependent translation was demonstrated. An overlap was observed between HCV RNA elements involved in these processes. Exchange of domain II had a minor effect on the translation efficiency of the chimeric 5′NTRs, while replication of subgenomic replicons with these chimeric 5′NTRs was abolished. Exchange of domain III subdomains severely decreased translation activity, while replication was maintained.
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Differential effects of bovine viral diarrhoea virus on monocytes and dendritic cells
More LessVarious pathogens have been shown to infect antigen-presenting cells and affect their capacity to interact with and stimulate T-cell responses. We have used an antigenically identical pair of non-cytopathic (ncp) and cytopathic (cp) bovine viral diarrhoea virus (BVDV) isolates to determine how the two biotypes affect monocyte and dendritic cell (DC) function. We have shown that monocytes and DCs are both susceptible to infection with ncp BVDV and cp BVDV in vitro. In addition, monocytes infected with ncp BVDV were compromised in their ability to stimulate allogeneic and memory CD4+ T cell responses, but DCs were not affected. This was not due to down-regulation of a number of recognized co-stimulatory molecules including CD80, CD86 and CD40. Striking differences in the response of the two cell types to infection with cytopathic virus were seen. Dendritic cells were not susceptible to the cytopathic effect caused by cp BVDV, whereas monocytes were killed. Analysis of interferon (IFN)-α/β production showed similar levels in monocytes and DCs exposed to cp BVDV, but none was detected in cells exposed to ncp BVDV. We conclude that the prevention of cell death in DCs is not associated with enhanced production of IFN-α/β, as proposed for influenza virus, but is by a distinct mechanism.
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Characterization of formaldehyde-inactivated poliovirus preparations made from live-attenuated strains
More LessFormaldehyde-inactivated virus samples from type 1 poliovirus live-attenuated strains were prepared in the laboratory. The effect of treatment with formaldehyde on virus infectivity and immunogenicity in mice was investigated and the results compared with those from Mahoney wild-type poliovirus strain, the common type 1 component in commercial inactivated polio vaccines (IPV). Differences in the potency and specificity between these experimental vaccines were identified in both normal mice and transgenic mice expressing the human poliovirus receptor. The possible advantages/disadvantages of using live-attenuated strains for IPV production are discussed in the context of the global polio eradication initiative. A novel transgenic mouse model to study in vivo the immune protection induced by IPV preparations is described.
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Flock house virus replicates and expresses green fluorescent protein in mosquitoes
More LessFlock house virus (FHV) is a non-enveloped, positive-sense RNA virus of insect origin that belongs to the family Nodaviridae. FHV has been shown to overcome the kingdom barrier and to replicate in plants, insects, yeast and mammalian cells. Although of insect origin, FHV has not previously been shown to replicate in mosquitoes. We have tested FHV replication in vitro in C6/36 cells (derived from neonatal Aedes albopictus) and in vivo in four different genera of mosquitoes, Aedes, Culex, Anopheles and Armigeres. FHV replicated to high titres in C6/36 cells that had been subcloned to support maximum growth of FHV. When adult mosquitoes were orally fed or injected with the virus, FHV antigen was detected in various tissues and infectious virus was recovered. Vectors developed from an infectious cDNA clone of a defective-interfering RNA, derived from FHV genomic RNA2, expressed green fluorescent protein in Drosophila cells and adult mosquitoes. This demonstrates the potential of FHV-based vectors for expression of foreign genes in mosquitoes and possibly other insects.
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The cypovirus Diadromus pulchellus RV-2 is sporadically associated with the endoparasitoid wasp D. pulchellus and modulates the defence mechanisms of pupae of the parasitized leek-moth, Acrolepiopsis assectella
More LessDiadromus pulchellus is a solitary endoparasitoid wasp that parasitizes the pupae of the leek-moth, Acrolepiosis assectella (Lepidoptera). Hitherto, every individual D. pulchellus from France that has been investigated was infected by an orthoreovirus, DpRV-1, and an ascovirus, DpAV-4. Recently, a new strain of D. pulchellus, established from a French field population, was found to be able to develop on leek-moth pupae, but lacked both DpRV-1 and DpAV-4. However, all these wasps were infected with a new cypovirus, DpRV-2. This cypovirus is transmitted to the A. assectella pupae at each wasp oviposition and is replicated mainly in the gut cells of the parasitized pupae. DpRV-2, like the ascovirus DpAV-4, is able to inhibit the defence reaction of A. assectella pupae and so contributes to the parasitic success of D. pulchellus wasps.
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Early interactions of marine birnavirus infection in several fish cell lines
More LessMarine birnavirus (MABV), a member of the genus Aquabirnavirus, family Birnaviridae, is an unenveloped icosahedral virus with two genomes of double-stranded RNA. The mechanisms of MABV adsorption and penetration are still undetermined. This work examined MABV infection in susceptible and resistant fish cell lines. MABV adsorbed not only onto the cell surfaces of susceptible (CHSE-214 and RSBK-2) cells but also onto resistant (FHM and EPC) cells. Furthermore, the virus entered the cytoplasm through the endocytotic pathway in CHSE-214, RSBK-2 and FHM cells but did not penetrate EPC cells. Thus, restriction of the MABV replication cycle is different between resistant FHM and EPC cells. The virus was found to bind to an around 250 kDa protein on CHSE-214, RSBK-2, FHM and EPC cells. Thus, this 250 kDa protein may be a major MABV receptor that exists in the plasma membranes of all four cell lines examined. This result suggests further that another receptor for virus penetration may exist in CHSE-214, RSBK-2 and FHM cells but not in EPC cells.
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- DNA viruses
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Functional dissection of the baculovirus late expression factor-8 gene: sequence requirements for late gene promoter activation
More LessThe late expression factor-8 gene (lef-8) of Autographa californica M nucleopolyhedrovirus encodes the largest subunit of the virally encoded DNA-directed RNA polymerase specific for the transcription of late and very late viral genes. The sequence of lef-8 predicts a C-terminal motif of 13 amino acids that is conserved in other polymerases. Detailed mutagenesis throughout lef-8 was performed, including this C-terminal motif, to define sequences required for late promoter activation. It was found that the conserved C-terminal motif was critical for late gene expression. In addition, regions throughout the entire lef-8-encoding sequence were important for optimal function, suggesting complex protein–protein and protein–DNA interrelationships in the late gene-specific viral transcriptosome.
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Comparative analysis of the genomes of Rachiplusia ou and Autographa californica multiple nucleopolyhedroviruses
More LessThe Rachiplusia ou multiple nucleopolyhedrovirus (RoMNPV) is a variant of Autographa californica MNPV (AcMNPV) but is significantly more virulent against several major agricultural pests. The genome sequence of the R1 strain of RoMNPV was determined and compared to that of AcMNPV strain C6. The RoMNPV genome is approximately 131·5 kbp with a G+C content of 39·1 %. The homologous repeat regions (hrs) described for AcMNPV-C6 are present in RoMNPV-R1 but the hrs of RoMNPV have fewer palindromic repeats. The RoMNPV-R1 nucleotide sequence is almost completely collinear with the sequence of AcMNPV-C6 and contains homologues of 150 of the 155 ORFs described for AcMNPV-C6. Deletions, insertions and substitutions have resulted in the loss of homologues for AcMNPV ORFs ac2 (bro), ac3 (ctl), ac97, ac121 and ac140 from the RoMNPV genome. The average amino acid sequence identity between RoMNPV and AcMNPV ORFs is 96·1 % and there are differences in promoter motif composition for 23 of these ORFs. Maximum-likelihood analysis of selection pressures on AcMNPV and RoMNPV ORFs indicate that ORFs ro18/ac20-ac21 (arif-1) and ro135/ac143 (odv-e18) have undergone positive selection.
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Inactivated parapoxvirus ovis (Orf virus) has antiviral activity against hepatitis B virus and herpes simplex virus
It is known that some viruses are able to induce vigorous immune reactions. This study shows that inactivated parapoxvirus ovis (Orf virus), strain D1701 (PPVO), induces an autoregulatory cytokine response that involves the upregulation of IL-12, IL-18, IFN-γ and other T helper 1-type cytokines and their subsequent downregulation, which is accompanied by induction of IL-4. An increase in IL-10 expression was also found in the livers of PPVO-treated mice. PPVO protects mice from lethal herpes simplex virus type 1 infection and guinea pigs from recurrent genital herpes disease. With dosages as low as 500 000 virus particles, PPVO is more potent than the current standard 3TC therapy in hepatitis B virus transgenic mice. No signs of inflammation or any other side effects were observed. PPVO induces IL-12, TNF-α and, together with a suboptimal concentration of Concanavalin A, IFN-γ in human peripheral blood leukocytes as well. The principle of an autoregulatory cytokine induction by an inactivated virus might have advantages over existing immune therapies and it is concluded that inactivated PPVO should be investigated further for its potential use in antiviral therapy.
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Neutralizing antibody to gB2 human cytomegalovirus does not prevent reactivation in patients with human immunodeficiency virus infection
More LessThe incidence of human cytomegalovirus (CMV) genotype gB2 (UL55) is high in patients with human immunodeficiency virus (HIV) infection in the San Francisco Bay area of California. Virus neutralizing antibody (NAb) to human CMV strain Ad169, a gB2 laboratory strain, was measured prospectively in HIV-infected patients, with CD4 T-lymphocyte counts <200, who were at risk for CMV-associated disease. Patients were grouped according to CMV DNA copy number, as quantified by PCR, and presence or absence of CMV-induced retinitis. Mean NAb titres were similar in all patient groups and unrelated to either virus load or outcome of CMV infection. Both gB2 and mixtures of gB2 with other gB genotypes were represented in isolates from blood and/or urine, even in the presence of high titres of antibody to the gB2 genotype challenge virus.
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Differential effects of glycoprotein B epitope-specific antibodies on human cytomegalovirus-induced cell–cell fusion
More LessAttachment of, and cell–cell fusion induced by, human cytomegalovirus were studied in the presence of neutralizing monospecific antibodies against antigenic domains 1 (AD-1) or 2 (AD-2) of glycoprotein B (gB, gpUL55). Efficient inhibition of the virion-mediated fusion event was consistently observed for the human AD-2-specific antibody as determined by a reporter gene activation assay based on permissive astrocytoma cells. In contrast, antibodies directed against the major neutralizing gB epitope AD-1 reduced fusion only by 20–60 %. Virus attachment via heparan sulfate was unaffected by the antibodies under the conditions used. Virus receptor binding as examined by heparin treatment of adsorbed virus was significantly reduced only if the virus had been coated with the AD-2-specific antibody. Neutralization of virus infectivity by the AD-2-specific antibody thus seems most likely to result from interference with a receptor-binding event during initial virus–host cell interaction.
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No role for Epstein–Barr virus in Dutch hepatocellular carcinoma: a study at the DNA, RNA and protein levels
Epstein–Barr virus (EBV) has been suggested to play a role in hepatocellular carcinoma (HCC). However, reports on detailed EBV transcript analyses in HCCs are limited. It was shown recently that expression of the transforming BARF1 (BamHI A rightward open reading frame 1) gene of EBV is restricted to latently EBV-infected epithelial malignancies, i.e. nasopharyngeal carcinoma and gastric carcinoma. The aim of this study was to test the presence of EBV in Dutch HCCs. A semiquantitative DNA PCR-enzyme immunoassay (PCR-EIA) for the BamHI W fragment of EBV was used to assess the presence of EBV in frozen and paraffin-embedded tissues of 16 HCCs. In addition, several RNA detection techniques, i.e. nucleic acid sequence-based amplification (NASBA), RT-PCR, RNA in situ hybridization (RISH) and immunohistochemistry (IHC), were applied. Five of 16 HCCs and two of four hepatitis C virus hepatitis samples were weakly positive for EBV DNA by PCR-EIA. Using sensitive RNA transcription techniques, no transcripts were found for BARF1, EBNA-1 and BARTs (BamHI A rightward transcripts) in any of the liver tissues tested. In addition, RISH for EBER1/2 and BARTs and IHC for EBNA-1, LMP-1 and ZEBRA, performed on the paraffin-embedded tissue of the PCR-EIA-positive cases and on adjacent non-neoplastic liver tissues, were negative. The absence of epithelial-specific BARF1 transcripts and other EBV transcripts and proteins in the EBV DNA PCR-positive cases argues strongly against a role for EBV in HCC.
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Localization of the Epstein–Barr virus protein LMP 1 to exosomes
More LessThe Epstein–Barr virus latent membrane protein (LMP 1) functions as a constitutively active signalling molecule and associates in lipid rafts clustered with other signalling molecules. Using immunofluorescent confocal microscopy, LMP 1 was shown to have an heterogeneous distribution among individual cells which was not related to the cell cycle stage. LMP 1 was shown to localize to intracellular compartments in cells other than the plasma membrane. Co-labelling of cells with both an LMP 1 antibody and an antibody to the Golgi protein GS15 revealed that the intracellular LMP 1 partly co-localized with the Golgi apparatus. Further confirmation of intracellular LMP 1 localization was obtained by immunoelectron microscopy with rabbit polyclonal LMP 1 antibodies and cryosectioning. As well as being present in intracellular foci, LMP 1 co-localized in part with MHC-II and was present on exosomes derived from a lymphoblastoid cell line. Preparations of LMP 1 containing exosomes were shown to inhibit the proliferation of peripheral blood mononuclear cells, suggesting that LMP 1 could be involved in immune regulation. This may be of particular relevance in EBV-associated tumours such as nasopharyngeal carcinoma and Hodgkin's disease, as LMP 1-containing exosomes may be taken up by infiltrating T-lymphocytes, where LMP 1 could exert an anti-proliferative effect, allowing the tumour cells to evade the immune system.
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Prevalence and type spectrum of human papillomaviruses in healthy skin samples collected in three continents
In order to investigate whether previous findings of ubiquitous skin papillomavirus infection in Caucasians apply to populations from other parts of the world, skin swab samples from Bangladesh, Japan, Ethiopia and Zambia were analysed in parallel with Swedish samples. The prevalence of HPV DNA in the material from Bangladesh was 68 %, Japan 54 %, Ethiopia 52 %, Zambia 42 % and Sweden 70 %. A great multiplicity of genotypes was demonstrated by the finding of 88 HPV types or putative types in 142 HPV DNA-positive samples in total. Double or multiple genotypes were frequently found in the same sample. The most prevalent HPV type was HPV-5, with an overall prevalence of 6·5 %. This was also the only type that was found in samples from all of the countries in the study. The results presented show that commensal skin HPV infections have a worldwide distribution with a very broad spectrum of genotypes.
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A role of the TATA box and the general co-activator hTAFII130/135 in promoter-specific trans-activation by simian virus 40 small t antigen
The small t antigen (st-ag) of simian virus 40 can exert pleiotropic effects on biological processes such as DNA replication, cell cycle progression and gene expression. One possible mode of achieving these effects is through stimulation of NFκB-responsive genes encoding growth factors, cytokines, transcription factors and cell cycle regulatory proteins. Indeed, a previous study has shown that st-ag enhanced NFκB-mediated transcription. This study demonstrates that promoters possessing a consensus TATA box (i.e. TATAAAAG) in the context of either NFκB- or Sp1-binding sites are trans-activated by st-ag. Overexpressing the general transcription factor hTAFII130/135, but not hTAFII28 or hTAFII80, stimulated the activity of promoters in a consensus TATA box-dependent mode. Converting the consensus TATA motif into a non-consensus TATA box strongly impaired activation by st-ag and hTAFII130/135. Conversely, mutating a non-consensus TATA motif into the consensus TATA box rendered the mutated promoter inducible by st-ag and hTAFII130/135. Mutation of the TATA box had no effect on TNFα- or RelA/p65-mediated induction of NFκB-responsive promoters, indicating a specific st-ag effect on hTAFII130/135. St-ag stimulated the intrinsic transcriptional activity of hTAFII130/135. Substitutions in the conserved HPDKGG motif in the N-terminal region or a mutation that impaired the interaction with protein phosphatase 2A abrogated the ability of st-ag to activate hTAFII130/135-mediated transcription. These results indicate that trans-activation of promoters by st-ag may depend on a consensus TATA motif and suggest that such promoters recruit the general transcription factor hTAFII130/135.
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Simian virus 40 VP1 capsid protein forms polymorphic assemblies in vitro
The simian virus 40 (SV40) capsid is composed of 72 pentamers of VP1, the major protein of SV40. These pentamers are arranged in a T=7d icosahedral surface lattice, which is maintained by three types of appropriately arranged, non-equivalent interactions between the pentamers. However, it remains unclear how these interactions are achieved. In this study, the in vitro assembly of recombinant VP1 was analysed. Electron microscopy observations revealed that these recombinant VP1 proteins assembled into structurally polymorphic particles depending on environmental conditions. VP1 pentamers assembled efficiently into virus-like particles (VLPs) when high concentrations of ammonium sulfate were present. However, in the presence of 1 M NaCl and 2 mM CaCl2 at neutral pH, VP1 pentamers formed not only VLPs but also produced tiny T=1 icosahedral particles and tubular structures. The exclusion of CaCl2 resulted in the exclusive formation of tiny particles. In contrast, in the presence of 150 mM NaCl at pH 5, the VP1 pentamers produced only extraordinarily long tubular structures. VP1 is thus quite unique in that it can assemble into such diverse structures. These observations provide clues that will help elucidate the mechanisms underlying SV40 capsid formation.
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Antisense RNAs transcribed from the upstream region of the precore/core promoter of hepatitis B virus
The bidirectional activity of the precore/core promoter of hepatitis B virus (HBV) has been demonstrated in cultured cell lines. However, HBV antisense transcripts (asRNAs) have not been demonstrated in vivo. In the present study using liver tissue from patients with chronic hepatitis, an anchored 5′RACE mapping the 5′ ends at position 1680/1681, 1655 or 1609/1602 was carried out. In limited cases, RLM-3′RACE detected asRNAs to terminate at four or five consecutive dT residues in the 0·7 kb downstream region. PCR of oligo(dT)-primed cDNA did not amplify a typical polyadenylated asRNA. RT-PCR using various primers did not detect any spliced forms. Competitive RT-PCR estimated the copy numbers of the asRNAs to be 0·05–0·4 % of total sense RNAs. All sequenced asRNAs had ORF6 but, in one patient, the asRNA initiating at position 1680/1681 had additional initiation and termination codons in front of ORF6. Therefore, asRNAs are transcribed by RNA polymerase III at a low level, encompass a dispensable ORF6 gene and might be retained in the nucleus. The endogenous asRNAs complementary to the common ends of all sense RNAs suggest antisense-mediated self-regulation of hepadnavirus.
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Hepatitis B virus: predominance of genotype D in primitive tribes of the Andaman and Nicobar islands, India (1989–1999)
To understand the possible origin of hepatitis B virus (HBV), three of the four hyperendemic, primitive accessible tribes of the Andaman and Nicobar islands, India, were investigated. The Nicobarese tribe was investigated in 1989 and 1999. The S gene from 65 HBV isolates was amplified by PCR and sequenced. Genotyping and serotyping were carried out on the basis of phylogenetic and amino acid analyses of S gene. All 20 Nicobarese-89 isolates, nine Onges-99 isolates and the single Andamanese-99 HBV isolate were classified as genotype D. Of the Nicobarese-99 isolates, 32 (91·4 %) and three (8·6 %) were genotypes D and A, respectively. Per cent nucleotide identity between the S sequences representing different tribes varied from 98·06 to 98·59 % and varied from mainland isolates by 1·6–2·0 %. Although southeast Asian origin is postulated for the Nicobarese tribe, the presence of different genotypes suggests introduction of HBV after migration to these islands, probably from mainland India, 200 years back, when these islands became inhabited as a part of penal settlement during the British regimen.
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Interaction of replicase components between Cucumber mosaic virus and Peanut stunt virus
More LessCucumber mosaic virus (CMV) and Peanut stunt virus (PSV) each have genomes consisting of three single-stranded RNA molecules: RNA 1, 2 and 3. RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are necessary for replication of the viral genome. Although RNA 3 is exchangeable between CMV and PSV, exchange of RNA 1 and 2 between the two viruses has been unsuccessful. In this study, reassortants containing PSV RNA 1 and CMV RNA 2 together with RNA 3 of CMV or PSV were shown to be able to replicate their genomic RNA, but not to transcribe subgenomic RNA 4 in tobacco protoplasts. Conversely, the reassortant consisting of CMV RNA 1 and PSV RNA 2 together with RNA 3 of CMV or PSV could not replicate. Subsequently, a yeast two-hybrid system was used to analyse the in vivo interaction between the 1a and 2a proteins. The C-terminal half of PSV-1a protein interacted with the N-terminal region of 2a protein of both PSV and CMV, but the C-terminal half of CMV-1a and the N-terminal region of PSV-2a did not interact. These results suggest that RNA replication in the interspecific reassortant between CMV and PSV requires compatibility between the C-terminal half of the 1a protein and the N-terminal region of the 2a protein, and this compatibility is insufficient for transcription of subgenomic RNA 4.
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Improved conformation-dependent immunoassay: suitability for human prion detection with enhanced sensitivity
More LessThe presence of pathogenic prion protein (PrPSc) in lymphoid tissues of variant Creutzfeldt–Jakob disease (vCJD) patients raises questions as to whether prions may be present in bodily fluids as well. Currently, transgenic mice are highly sensitive in vivo tools for the study of prions in tissues or fluids containing high levels of normal prion protein (PrPC). We report here an in vitro assay with virtually equivalent sensitivity incorporating a capture antibody into a sandwich conformation-dependent immunoassay (CDI), resulting in 30- to 100-fold increased sensitivity compared with the original, direct CDI. Furthermore, spiking plasma with vCJD prions in different preparations demonstrated that sandwich CDI detects prions with different biophysical properties at high sensitivity, even without proteinase K pretreatment of samples. Thus, sandwich CDI represents a powerful tool to study prions in bodily fluids of CJD/vCJD patients, with a turnaround time of less than 24 h.
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Prion diseases: infectious and lethal doses following oral challenge
More LessA brain homogenate prepared from a terminally ill hamster infected with scrapie strain 263K was serially diluted and administered orally to groups of hamsters. The undiluted brain homogenate led to clinical scrapie in all animals inoculated. The attack rate decreased with dilutions of the homogenate, and subclinical infections were identified among the healthy survivors at 520 days post-infection by Western blotting. The number of animals succumbing to disease and the combined number of Western blot-positive survivors plus diseased hamsters were used to calculate the LD50 and ID50 of the inoculum. The model system represents an approximation to the transmission of TSEs such as new variant Creutzfeldt–Jakob disease (vCJD) via dietary exposure to the infectious agent and suggests that, due to the rather small difference between the calculated LD50 and ID50, the number of clinical cases will not be vastly exceeded by the number of subclinical carriers of the disease.
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Volumes and issues
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Volume 105 (2024)
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Volume 104 (2023)
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Volume 8 (1970)
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Volume 7 (1970)
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Volume 6 (1970)
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Volume 5 (1969)
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Volume 4 (1969)
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Volume 3 (1968)
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Volume 2 (1968)
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Volume 1 (1967)