- Volume 77, Issue 10, 1996
Volume 77, Issue 10, 1996
- Review Article
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- Articles
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- Animal
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- RNA viruses
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Bimodal down-regulation of CD4 in cells expressing human immunodeficiency virus type 1 Vpu and Env
More LessWe analysed clones of HeLa cells stably expressing the human immunodeficiency virus (HIV-1) envelope gene (env) and the HIV-1 receptor, CD4. Surprisingly, individual clones were found to consist of two distinct populations of cells differing by about 10-fold in the level of surface CD4. When high and low CD4-expressing cells were separated by FACS, each subpopulation gave rise to a mixture of high and low CD4-expressing cells after several days in culture. High and low CD4-expressing subpopulations did not differ with respect to the amount of intracellular Env, but there was an inverse correlation between CD4 and another HIV-1 protein encoded by the same segment of the HIV genome, Vpu. High surface CD4 cells had high levels of intracellular CD4, largely in the perinuclear region, and low levels of Vpu with a diffuse staining pattern. Conversely, low surface CD4 cells had low levels of intracellular CD4 with a diffuse staining pattern, and high levels of Vpu, largely in the perinuclear region. Vectors containing mutant versions of either Env or Vpu failed to down-regulate surface CD4. The phenomenon of bimodal expression of a surface protein in cells derived from single clones provides a simple model of differentiation in vitro. We show how a hypothetical interaction between CD4 and a multimer of Vpu, the multimerization of which is cooperative, would lead to bimodal expression of CD4. This model may be generalized and could explain other cellular ‘switches’.
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Loss of antibody reactivity directed against the V3 domain of certain human immunodeficiency virus type 1 variants during disease progression
We have previously shown that in AIDS patients a predominant species of infectious virus can be found which is not neutralized by homologous serum. The presence of the infectious virus was associated with the lack of type-specific antibody directed against the V3 domains of these virions. In contrast to this lack of V3-specific antibody, the other V3 domains of non-infectious virions were well recognized by antibody. To determine whether the lack of a V3-specific antibody response is due to a progressive loss of antibody during human immunodeficiency virus type 1 (HIV-1) infection, we monitored the anti-V3 antibody response in 90 patients over time. Anti-V3 antibodies were monitored by a V3-specific ELISA using 21 different V3 domains as a fusion with glutathione S-transferase (GST-V3) based upon sequences from 11 HIV-1 patient isolates and 10 sequences from an HIV-1 B subtype consensus-like GST-V3 expression library. This strictly heterologous screening showed a loss of V3-specific antibodies in 20 out of the 90 patients tested. To study the in vivo relevance of these findings we analysed V3 antibody loss in two patients. This strictly autologous antibody screening was performed based upon V3 sequences of the patients’ cell-free virions. In both patients the loss of a V3-specific antibody could be detected in parallel to a decline of CD4+ T cells. Moreover, the escape of a distinct V3 variant was shown to correlate closely with the loss of the V3-specific antibody.
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Human immunodeficiency virus type 1 RNA populations in faeces with higher homology to intestinal populations than to blood populations
More LessTo determine whether human immunodeficiency virus type 1 (HIV-1) in faeces is representative of the HIV-1 population in intestinal tissue, we studied HIV-1 V3 variation in faeces, intestinal biopsies and serum from two individuals. Phylogenic analysis of HIV-1 V3-coding RNA in faeces from one individual showed three distinct genotypes. Viruses belonging to all three genotypes were also present in sigmoidal tissue and in serum. Jejunal tissue contained two of these three genotypes. Analysis of the V3-coding RNA in faeces of the other individual showed five distinct genotypes. One of these genotypes was present in all specimens from this individual. Besides this shared genotype, jejunal tissue and serum contained sequences belonging to one other genotype. In addition, one of the other three V3 variants was detected in sigmoidal tissue. For both persons the shared HIV-1 RNA genotypes in faeces and serum displayed a distinctly different frequency distribution. In one individual, the genotype which was detected in a majority of the clones in faeces (59%) and as a minority in serum (11%), was the most abundant genotype in jejunal and sigmoidal tissue (61% and 80%, respectively). For the other individual the genotype that was present in faeces in a significant number of clones (43%) was detected in serum as a minority (8%), whereas this genotype composed 47% of the clones isolated from jejunal tissue. Taken together these data suggest that faeces contain HIV-1 sequences that are derived from local HIV-1 replication in intestinal tissue.
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Replication and cytopathogenicity of human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus agm3 chimeric viruses in human and monkey cells: the 5′ half of the HIV-1 genome is responsible for virus cytopathogenicity
Two chimeric viruses were constructed between human immunodeficiency virus type 1 (HIV-1) and an apathogenic simian immunodeficiency virus (SIVagm3mc) from African green monkeys. One of the chimeras, HE-A391, expressed the HIV-1-derived env, vpu, tat and rev genes and the SIVagm3mc-derived LTR and the gag, pol and vif genes. The other chimera, SE-H13, contained the SIVagm3mc-derived env, tat and rev genes and the HIV-1-derived LTR and the gag, pol, vif and nef genes. Both constructs yielded infectious viruses and their phenotypes (growth-competence and cell-killing capacity) were examined in various CD4+ cells including human and monkey PBMCs. The results indicated that the replicative properties of the chimeras were mainly dependent on the 5′-genomic half of the parental viruses, and the determinant for viral cytopathogenicity was located within the 5′ half of the HIV-1 genome.
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T cell apoptosis in human immunodeficiency virus type 2- and simian immunodeficiency virus-infected macaques
Recent evidence suggests that T cell apoptosis could be involved in the pathogenesis of HIV infection. In addition, lymphocyte apoptosis has been described in SIV-infected macaques that developed simian AIDS. To investigate further the role of apoptosis in AIDS pathogenesis, we studied lymphocytes of HIV-2-infected cynomolgus macaques that did not develop simian AIDS. We compared apoptosis of lymphocytes from animals infected with non-pathogenic HIV-2 to that in macaques infected with pathogenic SIV. Unfractionated peripheral blood mononuclear cells of SIV- and HIV-2-infected macaques showed evidence of apoptosis by electron microscopy, flow cytometry (terminal dUTP nick end labelling) and visualization of DNA fragmentation. Between 30–50% apoptotic cells could be detected in SIV-infected animals, compared to approximately 30% in HIV-2-infected and 5–12% in uninfected monkeys. However, separation of PBMC into T cell subpopulations revealed striking differences in apoptosis between SIV- and HIV-2-infected macaques. In SIV-infected monkeys both CD4 and CD8 cells underwent apoptosis to a large extent. In contrast, in the HIV-2-infected macaques apoptosis was restricted to the CD8 cell compartment. The lack of apoptosis in CD4 cells of healthy HIV-2-infected macaques implies an important role for CD4 cell apoptosis in AIDS pathogenesis.
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Rabies virus binding to the nicotinic acetylcholine receptor α subunit demonstrated by virus overlay protein binding assay
More LessA virus overlay protein binding assay was used to study binding of 125I-labelled rabies virus to the acetylcholine receptor (AChR) from Torpedo californica electric organ membranes. After gel electrophoresis of electric organ membranes and transfer of proteins to nitrocellulose, 125I-labelled α-bungarotoxin, a curaremimetic neurotoxin, bound to a 40 kDa band and 125I-labelled rabies virus bound to 51 kDa and 40 kDa bands. Binding of rabies virus to the 40 kDa band was inhibited by unlabelled α-bungarotoxin. In blots of affinity-purified AChR, labelled virus bound to the 40 kDa α subunit and was competed by α-bungarotoxin. Based on binding of rabies virus to the α subunit and the ability of α-bungarotoxin to compete for binding, rabies virus appears to bind to the neurotoxin-binding site of the nicotinic AChR α subunit.
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Mother to fry, successful transfer of immunity against infectious haematopoietic necrosis virus infection in rainbow trout
More LessWe have tested whether immunity can be transferred from a mother fish to its fry. Rainbow trout mother fish were inoculated against infectious haematopoietic necrosis virus (IHNV) by intraperitoneal injection of a fragment of the IHNV glycoprotein spanning amino acids 31 to 310. This protein fragment was obtained by isolating the specific cDNA from Japanese IHNV strain HV7601 and expressing it in Escherichia coli. Fry from immunized and control fish were exposed to IHNV at various intervals after hatching, and their mortality monitored. Survival of the fry of immunized fish was significantly greater when exposure to virus occurred 7 days after hatching, and some immunity appeared to persist until at least 25 days after hatching.
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Th1 and Th2 cytokine induction in pulmonary T cells during infection with respiratory syncytial virus
More LessHelper T (Th) cells can be classified functionally into two main types. Broadly, Th1 cells play a major role in eliminating viral pathogens, while Th2 cells mediate anti-parasite immunity and allergic responses. These functions are thought to depend on characteristic and distinct patterns of cytokine production. Infection with human respiratory syncytial virus, an important common cold virus, causes transient lymphocytic bronchiolitis in mice. Activated T cells are partly responsible for this disease, but also eliminate the virus. To show whether polarized cytokine production occurs in individual cells during viral bronchiolitis, we sampled murine bronchoalveolar lavage and mediastinal lymph node cells before and after infection. RT-PCR of cellular mRNA and flow cytometric analysis of intracellular cytokine production showed a rapid IFN-γ response at both sites, which persisted for more than 3 weeks in the lung. Most IFN-γ-producing cells were CD8+. Some early CD4+ IFN-γ-producing cells also made IL-10. Only low levels of IL-2, IL-4 and IL-5 mRNA or protein expression were detected at any time at either site. No cytokines were detected in B cell populations at either site. These novel techniques show the true complexity of cytokine production patterns on a cell-by-cell basis, allowing T cells to be reclassified according to function.
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Interaction between nucleocapsid protein (NP) and phosphoprotein (P) of human parainfluenza virus type 2: one of the two NP binding sites on P is essential for granule formation
The paramyxovirus phospho-(P) and nucleocapsid (NP) proteins are involved in transcription and replication of the viral genome. To study the interaction between NP and P proteins, we established HeLa cell lines that constitutively expressed the NP and/or P proteins of human parainfluenza virus type 2 (hPIV-2). Co-immunoprecipitation assays revealed that the NP and P proteins can form complexes in HeLa cells expressing both proteins (HeLa-NP + P cells) and in mixed cell lysates of HeLa-NP and HeLa-P cells. Deletion mutant analysis of the P protein was performed to identify the regions of P protein that interact with NP protein. The results indicate that two independent NP-binding sites exist on P protein: one is located in the N-terminal part of the protein, aa 1–47, and the other in the C-terminal part, aa 357–395. In addition, cells co-expressing NP and P proteins with N-terminal deletions showed immunofluorescence staining patterns (granular pattern) similar to those found in hPIV-2-infected cells. However, cells co-expressing NP and P proteins with C-terminal deletions showed a different immunofluorescence staining pattern (diffuse pattern), indicating that the C-terminal region is required for granule formation.
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Rescue of Sendai virus cDNA templates with cDNA clones expressing parainfluenza virus type 3 N, P and L proteins
More LessSeveral years ago, we reported that a Sendai virus (SeV) defective genome (DIH4UV) could be rescued in vivo with human parainfluenza virus type 1 (hPIV1) and bovine PIV3 but not by measles virus or vesicular stomatitis virus. It was concluded that the cis-acting RNA sequences were conserved within the SeV/PIV1/PIV3 group but that interactions between the polymerase complex (P-L) and the template protein N were unique for each virus. We have re-examined these conclusions using proteins expressed from cloned N, P and L genes for SeV and PIV3. The results demonstrate the specificity of the protein-protein interactions between polymerase and template, and confirm the prediction of the earlier work that PIV3 N, P and L proteins are capable of assembling and replicating SeV minigenomes also expressed from a cDNA clone.
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Mucosal and systemic immune responses to measles virus haemagglutinin in mice immunized with a recombinant vaccinia virus
More LessThe immune response to a vaccinia virus recombinant expressing the measles virus haemagglutinin (VV-HA) was compared after parenteral or mucosal immunizations in mice. Parenteral immunizations with 106 p.f.u. VV-HA induced HA-specific antibody-producing cells (IgG > IgA) and HA-specific class I-restricted cytotoxic T lymphocytes (CTL) in the spleen. In contrast, intranasal administrations of 106 p.f.u. of VV-HA induced HA-specific spot-forming cells in the spleen (IgG > IgA) and the lungs (IgA > IgG), and HA-specific CTL in the spleen. Co-immunization by the nasal route with VV-HA and cholera toxin enhanced HA-specific immune responses. Oral immunizations with 108 p.f.u. of VV-HA generated low numbers of HA-specific IgA-producing cells in the lamina propria of the gut, and a weak HA-specific CTL activity in the spleen and mesenteric lymph nodes. Oral co-immunization with VV-HA and cholera toxin greatly enhanced the level of HA-specific spot-forming cells in the lamina propria (IgA ≫ IgG). Interestingly, intrajejunal immunizations with 108 p.f.u. VV-HA alone induced high levels of anti-HA IgG-producing cells in the spleen and anti-HA IgA-secreting cells in the lamina propria of the gut. These data show that (i) VV-HA by the nasal route is immunogenic and generates a measles-specific mucosal immune response in the lung, which represents the primary site of replication of measles virus and that (ii) VV-HA can also induce measles-specific immunity in the intestine provided that it is protected from degradation in the gastrointestinal tract, or that cholera toxin is used as an adjuvant.
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Protection against measles virus encephalitis by monoclonal antibodies binding to a cystine loop domain of the H protein mimicked by peptides which are not recognized by maternal antibodies
After immunization with measles virus (MV) several monoclonal antibodies (MAbs) were obtained, which reacted with peptides corresponding to the amino acids 361–410 of the haemagglutinin protein (MV-H). Three of these MAbs (BH6, BH21 and BH216) inhibited haemagglutination, neutralized MV in vitro and protected animals from a lethal challenge of rodent-adapted neurotropic MV. These MAbs reacted with the 15-mer peptides H381 and H386 defining their overlapping region 386–395 as a sequential neutralizing and protective epitope, which can be imitated by a short peptide. H381 and H386 share two Cys residues (C386KGKIQALC394ENPEWA) and for optimal MAb binding of peptide (or MV) disulphide bonds were required in addition to a linear C-terminal extension. Other MAbs bound to peptides C- (BH147, BH195) and N-terminally (BH168, BH171) adjacent to the loop but did not neutralize or protect. When sera from measles patients or from women of child-bearing age were tested with the peptides corresponding to this haemagglutinating and neutralizing epitope (HNE), none of the sera recognized the 15-mer peptides of this region, while some reactivity was found to 30-mers homologous to different wild-type mutants. Its lack of recognition by maternal antibodies and its high degree of conservation would make the HNE loop an attractive candidate to include into a subunit vaccine, which could be administered during early childhood, independent of immune status.
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Antigenic and genetic variation of the HN protein of mumps virus strains
More LessThe relationships between different strains of mumps virus were established by determination of the sequence of the HN gene. They closely resemble those established from other portions of the genome, suggesting that the viruses are not recombinants over the areas examined. The relationships were consistent with those established by reaction with monoclonal antibodies raised against the Urabe strain, which has a similar antigenic structure to previously studied laboratory strains, and largely consistent with the specificity of the serological response of naturally infected or vaccinated human subjects.
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Studies on the molecular basis for loss of the ability of recent influenza A (H1N1) virus strains to agglutinate chicken erythrocytes
More LessRecent strains of influenza A but not B viruses have lost the ability to agglutinate chicken red blood cells (CRBC). The H1N1 viruses isolated in Japan during the 1991/92 season could be divided into two groups. Group 1 viruses (A/Aichi/4/92 and A/Aichi/7/92) agglutinated goose red blood cells (GRBC) and CRBC, while group 2 viruses (A/Aichi/24/92 and A/Aichi/26/92) did not agglutinate CRBC. There were no amino acid differences between them in the haemagglutinin (HA) polypeptide. Reassortment experiments between a group 1 virus (A/Aichi/4/92) or a group 2 virus (A/Aichi/24/92) and the A/WSN/33 influenza A (H1N1) virus strain suggested that the HA gene products of the viruses of both groups had lost the capacity to agglutinate CRBC. The HA proteins expressed on Cos cells by transfecting the cDNAs of the virus HA gene of A/Aichi/4/92 and A/Aichi/24/92 agglutinated GRBC but not CRBC. These experiments indicated that the HA proteins of H1N1 viruses of both groups isolated in 1992 had lost the ability to agglutinate CRBC even though the group 1 virions showed haemagglutinating capacity with CRBC. By using the cDNAs of the HA gene of seven natural isolates obtained from 1977 to 1992, it was found that the expressed HA proteins of influenza A (H1N1) viruses isolated since 1988 had lost the ability to agglutinate CRBC. Experiments with chimeric and point-mutated HA cDNAs of A/Aichi/24/92 showed that an amino acid change at residue 225, which occurred after 1986, and a cluster of amino acid changes at residues 193, 196 and 197, which occurred before 1986, were responsible for loss of the ability to agglutinate CRBC. Egg-adapted virus derived from A/Aichi/24/92 had one amino acid change at residue 225 compared to the parental virus.
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Virus entry into a polarized epithelial cell line (MDCK): similarities and dissimilarities between influenza C virus and bovine coronavirus
More LessWe have analysed the uptake of influenza C virus and bovine coronavirus (BCV) by a polarized epithelial cell line, Madin-Darby canine kidney (MDCK) cells. Both viruses use N-acetyl-9-O-acetyl-neuraminic acid as a receptor determinant for attachment to cells. Virus binding assays with immobilized proteins indicated that a glycoprotein of 40 kDa is the major surface protein containing the receptor determinant for the two viruses. MDCK cells grown on filters for permeable support were found to have differential sensitivity to infection by these viruses. Both viruses were able to initiate infection via the apical domain of the plasma membrane, but only influenza C virus also accomplished infection via the basolateral plasma membrane. The resistance of MDCK cells to BCV infection from the basal filter chamber was overcome when the cell polarity was abolished by maintaining the cells in calcium-free medium. This finding indicates that the resistance to basolateral infection by BCV is a property of the cell line and not due to a technical problem related to the use of filters. Our results indicate that two viruses which use the same receptor for attachment to cells may differ in their ability to enter polarized cells. The possible involvement of an accessory molecule in the entry of BCV is discussed.
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Characterization of functional domains in the human coronavirus HCV 229E receptor
More LessHuman aminopeptidase N (hAPN or CD13) and porcine aminopeptidase N (pAPN) are functional receptors for human coronavirus (HCV) 229E and porcine transmissible gastroenteritis virus (TGEV), respectively. However, hAPN cannot function as a receptor for TGEV and pAPN cannot function as a receptor for HCV 229E. In this study, we constructed a series of chimeric hAPN/pAPN genes and expressed the corresponding proteins in transfected cells. Subsequently, we identified the chimeric proteins that can function as a receptor for HCV 229E. The results show that replacement of a small region of pAPN sequence (pAPN amino acids 255–348) with the corresponding hAPN sequence (hAPN amino acids 260–353) converts pAPN into a functional receptor for HCV 229E. The region of hAPN that we have defined in this way does not correspond to the region of pAPN that has been identified as essential for the TGEV-receptor interaction. We conclude that although both viruses use a homologous receptor protein, different regions of the protein are required to mediate susceptibility to infection with HCV 229E and TGEV.
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Nucleotide sequence analysis of a major antigenic domain of the E1 glycoprotein of 22 rubella virus isolates
More LessWe have determined the nucleotide sequence of the region of the rubella virus genome which encodes amino acids 195–296 of the E1 glycoprotein (E1-195–296) from a panel of 22 rubella viruses obtained from Europe, USA and Asia between 1963–1995. E1-195–296 contains neutralizing and haemagglutinating determinants, and may represent a major antigenic domain. The nucleotide sequence divergence of the 22 rubella viruses compared to the Therien strain sequence ranged from 0.65–7.14%. The greatest sequence divergence occurred in two rubella viruses of Indian origin, and was more than twofold greater than that previously reported for rubella virus. The majority of nucleotide changes occurring in the 22 viruses did not effect the deduced amino acid sequence of E1-195–296. Two rubella viruses isolated from cases of reinfection in pregnancy did not exhibit nucleotide sequence variation resulting in changes in the deduced amino acid sequence of E1-195–296, suggesting that antigenic change within this region of E1 is not associated with rubella reinfection. A rubella virus isolated from a synovial fluid sample exhibited a nucleotide substitution in a putative neutralization domain contained within E1-195–296. Phylogenetic analysis was performed to study the relationship between E1-195–296 coding sequences of the 22 viruses in this report and corresponding sequences of other rubella viruses in the databank.
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Human recombinant antibodies specific for hepatitis C virus core and envelope E2 peptides from an immune phage display library
More LessHepatitis C virus (HCV) is the aetiological agent responsible for most cases of non-A non-B hepatitis. Hepatitis C is a disease of clinical importance because of its high infection rate in blood donors and its persistence as chronic infections which may lead to cirrhosis and hepatocellular carcinoma in the long term. The variability of the HCV genome has posed difficulties in serological detection and vaccine design. The recent advance in phage technology offers a means of cloning human anti-HCV antibodies of a defined specificity that may have potential therapeutic use. We now report the generation of a phage display library using the VH genes of a HCV-infected patient and the VL genes of two non-immune individuals. From this library we were able to obtain specific IgG single-chain Fvs (scFvs) that recognize viral core and envelope proteins by selection on synthetic peptides derived from the core sequence PKARRPEGRTWAQPG and the envelope E2 sequence RPIDDFDQGWGPITY. The specificity of the scFvs was demonstrated by their specific reactions with homologous peptides in ELISA and the specific blocking of scFv binding by homologous peptides, in a dose-dependent manner, in inhibition ELISA. The binding of the anticore 4c2 to homologous peptide was blocked by HCV-positive human sera in an antibody-concentration-dependent manner, suggesting that the scFv recognizes a similar if not identical epitope to those of one or more of the polyclonal antibodies present in the sera.
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A single nucleotide change in the E protein gene of dengue virus 2 Mexican strain affects neurovirulence in mice
More LessEleven clones of dengue virus 2 Mexican strain were selected by the size of their lytic plaques. Nucleotide sequences of the clones producing large plaques (D2ML2, D2ML3 and D2ML4) revealed 11 mutations, 10 of which were silent. The substitution at nucleotide 1168 (G → C) generates one amino acid difference at residue 390 (Asp → His) of the envelope protein (E). These clones showed high virulence in suckling mice when inoculated intracerebrally (⩾ 70% mortality). However, the clones which showed small lytic plaques (D2MS1, D2MS2 and D2MS4) displayed a substitution from Asp → Asn at the same positio nand had attenuated virulence. Based on these data, we suggest that substitution of Asp → His at residue 390 perhaps affects a functionally important structural element that could be a determinant of dengue neurovirulence. This substitution falls in domain III of the E protein, which plays an important role in viral binding; therefore, we propose that the substitution affects virulence and cellular tropism.
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Dengue 1 virus binding to human hepatoma HepG2 and simian Vero cell surfaces differs
More LessWe analysed the binding and infectivity of dengue virus serotype 1 (DEN-1) for the human hepatoma cell line HepG2 in comparison with the simian kidney cell line Vero. The higher susceptibility of Vero cells to DEN-1 correlated with greater binding affinity of DEN-1 to these cells. In contrast, the capacity of virus attachment was higher for HepG2 than for Vero cells. Profiles of DEN-1 binding at different pH were markedly different between the two cell types. A type-specific neutralizing monoclonal antibody reduced initial virus binding to both cell types similarly but complex- and group-specific neutralizing antibodies affected virus adhesion differently. Altogether, these results suggest the involvement of different receptors or receptors presented in a different environment on the cell surface in the two cell lines. The sensitivity to proteolytic enzymes and to ionic detergent of the binding sites on the two cell types was tested and results indicated that they may be multimeric proteins or protein complexes.
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Distribution and genetic heterogeneity of Puumala virus in Sweden
Small mammals trapped in Sweden were analysed for specific antibody responses against three hantavirus serotypes and for the presence of viral antigen. To determine the genetic identity of viral RNA in lungs of seropositive bank voles (Clethrionomys glareolus), polymerase chain reactions and subsequent partial sequencing of both the M and S segments were employed. The sequences obtained were all identified as Puumala (PUU) virus, with a high degree of heterogeneity between the different geographical localities. Alignment of nucleotide and deduced amino acid sequences, together with phylogenetic analysis, showed that PUU viruses circulating in central Sweden were distinct from those in the northern region. The localization of the two distinct PUU virus genotypes was shown to correlate with the postglacial recolonization of Sweden by bank voles.
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- DNA viruses
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In vivo complementation studies of a glycoprotein H-deleted herpes simplex virus-based vector
More LessThe utilization of herpes simplex virus (HSV) as a vector for gene delivery to the nervous system or as a live vaccine delivery system is dependent on the construction and characterization of disabled virus mutants which are unable to cause disease. Under certain circumstances, however, replication-defective vectors may carry a potential risk if they can be efficiently complemented by a co-infecting wild-type virus. Stocks of defective vectors should, therefore, be free from replication-competent virus, and helper cell lines should be incapable of generating replication-competent virus by recombination between the vector and the complementary gene. We describe a glycoprotein H-negative (gH−) virus/helper cell line combination which generates helper-free defective virus stocks containing replication-competent virus at a frequency no higher than 1 in 109 p.f.u. This virus/helper cell system provides a suitable background for the construction of safe replication-defective gene delivery vectors. In vivo studies demonstrate that gH− virus is unable to initiate disease in mice and establishes latency at low efficiency compared to wild-type HSV. To determine whether gH− virus can be complemented by wild-type virus in vivo, mice were infected with a variety of mixtures of these viruses. Complementation was observed in a minority of animals infected with more than 106 p.f.u. of both wild-type and defective virus but the most common observation was that the presence of defective virus suppressed entry of wild-type virus into the nervous system.
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Characterization of herpes simplex virus type 1 recombinants with mutations in the cytoplasmic tail of glycoprotein H
More LessHerpes simplex virus (HSV) type 1 glycoprotein H is essential for fusion of virus envelopes with cellular membranes and for the fusion of an infected cell membrane with an uninfected neighbour. Previous studies have pointed to a requirement for certain amino acid residues of the cytoplasmic tail of gH in these processes. Results from transient transfection experiments suggested that the serine-valine-proline (SVP) motif in the cytoplasmic tail may be important for gH-mediated fusion. HSV recombinants expressing gH molecules with mutations in the cytoplasmic tail were constructed and analysed in terms of their abilities to fuse cellular membranes and to function in virus entry. Viruses containing a deletion of the SVP motif, or in which the valine residue of this triplet was replaced by alanine, entered cells less efficiently than wild-type virus and were unable to induce syncytium formation on Vero cells.
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A murine RNA polymerase I promoter inserted into the herpes simplex virus type 1 genome is functional during lytic, but not latent, infection
More LessThe development of herpes simplex virus as a vector for neuronal gene delivery is dependent upon the identification and characterization of promoter elements capable of driving long-term expressio nduring latency. The majority of RNA polymerase II (pol II) promoters studied are active during acute infection but silenced during latency. In order to investigate the potential of a murine RNA polymerase I (pol I) promoter to drive reporter gene expressio nduring lytic and latent infection, we describe the construction and characterization of two recombinant viruses; SC16 LAT neo and SC16 US5 neo. These viruses contain a pol I-encephalomyocarditis virus internal ribosome entry site (EMCV IRES)-neomycin phosphotransferase gene (neo r ) cassette inserted into the non-essential major latency associated transcript (LAT) and US5 regions respectively. Pol I promoter activity could be detected in the rodent BHK cell line, but not the primate derived Vero cell line — consistent with the known species specificity of such promoters. This activity was specific to a virus containing an active pol I promoter. However, in situ hybridization analyses of latently infected cervical dorsal root ganglia failed to detect pol I mediated transcription of the reporter gene indicating that the murine pol I promoter is silenced following the establishment of latency. Insertion of the pol I-EMCV IRES-neo R cassette into the major LAT locus resulted in the production of a hybrid LAT transcript during latency which was translocated to the cytoplasm of latently infected neurones.
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Reactivation of herpes simplex virus type 1 in the mouse trigeminal ganglion: an in vivo study of virus antigen and immune cell infiltration
More LessThe corneas of latently infected mice were UV irradiated to induce reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG). On days 1 to 4 after irradiation, TG were removed, serially sectioned and double stained to identify immune cells and virus antigens. Virus antigen was detected in small numbers (most commonly one) of neurons per ganglion as early as day 1, confirming the rapidity of reactivation and the neuron as the likely site of this event. The immune response was also rapid and effective since virus antigen was identified in immune cells at day 1 and by day 4 all samples were negative. The predominant infiltrating cells on days 1 and 2, when virus antigen was present and being cleared, were T cells, both CD4+ and CD8+. Later, large numbers of B cells appeared, suggesting that local antibody production may also be involved in controlling the reactivated infection. The observations suggest that a significant proportion of reactivation events do not result in disease of the eye or shedding of virus in the tear film. However, they also suggest that as little as one reactivating neuron in the ganglion may be sufficient to lead to such disease and/or shedding.
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Expression of human cytomegalovirus ppUL83 (pp65) in a stable cell line and its association with metaphase chromosomes
More LessAstrocytoma cells were transfected with an expression vector for the structural phosphoprotein ppUL83 (pp65) of human cytomegalovirus (HCMV). Once made, pp65 showed the same characteristics as naturally produced protein and was stable for several days in astrocytoma cells, as it is during HCMV replication in fibroblasts. In permanently transfected cells, pp65 was homogeneously dispersed in the cell nucleus and showed a preferential methanol-stable association with condensed chromatin throughout cell division. Furthermore, pp65 could be detected bound to metaphase-arrested chromosomes in pp65-expressing astrocytoma cells, and in fibroblasts and astrocytoma cells during productive virus infection.
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Evidence for the role of cell protein phosphorylation in human cytomegalovirus/host cell fusion
More LessThe mechanism by which human cytomegalovirus (HCMV) enters cells is unknown. We sought evidence that protein phosphorylation plays a role in HCMV infection in two ways: (1) by determining whether the degree of phosphorylation of a constitutively phosphorylated 92.5 kDa putative cell membrane receptor for HCMV gH is changed following exposure to HCMV or monoclonal anti-idiotype antibodies (MAb2) that antigenically mimic HCMV gH, and (2) by studying the effects of protein kinase inhibition on receptor phosphorylation and HCMV adsorption or fusion. Phosphorylation of the 92.5 kDa cell membrane protein was specifically increased within 10 min of incubation with HCMV or MAb2 that had been crosslinked by goat anti-mouse antibodies. In addition, fusion of viral envelope with the cell membrane was inhibited by certain protein kinase inhibitors which also inhibited receptor phosphorylation, while the adsorption of [3H]HCMV to human embryonic lung cells was not affected. Tyrosine kinase inhibitors inhibited virus/cell fusion to a greater extent than protein kinase C (PKC) inhibitors, and an inhibitor which primarily affects cAMP and cGMP kinases had little effect. In addition, fusion was stimulated by preincubating cells with agents that stimulated receptor phosphorylation including a phorbol ester, tyrosine phosphatase inhibitor and serine/threonine phosphatase inhibitor. These data indicate that increased phosphorylation of a 92.5 kDa putative cell membrane protein receptor for gH is an early event in response to HCMV, and that cell protein phosphorylation by tyrosine kinase(s) and PKC may facilitate HCMV/cell membrane fusion.
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Effect of host genotype in determining the relative roles of natural killer cells and T cells in mediating protection against murine cytomegalovirus infection
More LessThe influence of host genotype on the relative importance of T cell subsets and natural killer (NK) cells in controlling murine cytomegalovirus (MCMV) replication has been investigated. Genetically susceptible BALB/c and A/J, moderately resistant C57BL/10, and resistant CBA/CaH mouse strains were treated with monoclonal antibodies (MAb) to the CD4 and CD8 markers and the extent of MCMV replication in major target tissues was determined. Both mouse strain-specific and tissue-specific effects were observed. CBA/CaH and C57BL/10 mice were found not to require CD4+ or CD8+ T cells for control of MCMV replication in the spleen or liver. In contrast, in A/J mice, as well as BALB/c mice, the CD8+ T cell population was primarily responsible for the clearance of virus from these tissues. However, in all strains of mice, CD4+ T cells were required for delayed type hypersensitivity and antibody responses, and for virus clearance in the salivary glands. The dependence of mice with the BALB genetic background on CD8+ T cells for limitation of acute MCMV infection was found to be negated in the BALB.B6-Cmv1r congenic strain, in which an effective NK cell response has been generated through the introduction of the resistant Cmv1r allele from C57BL/6 mice. Depletion of NK cells in the BALB.B6-Cmv1r strain using anti-NK1.1 MAb restored the role of CD8+ T cells in mediating viral clearance. These analyses demonstrate that some, but not all, strains of mice use CD8+ T cells to controlMCMV replication and that even when CD8+ T cell-dependence exists, this can be circumvented by an appropriate NK cell response.
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Effect of natural sequence variation at the H-2Ld-restricted CD8+ T cell epitope of the murine cytomegalovirus ie1-encoded pp89 on T cell recognition
More LessThe amino acid sequence YPHFMPTNL of pp89, the ie1-encoded product of murine cytomegalovirus (MCMV; Smith strain), constitutes an immunodominant T cell epitope recognized in association with H-2Ld. Nucleotide sequencing of MCMV isolates derived from wild mice identified variation between amino acids 147–192 of pp89 in 19 of 27 isolates, including the region encompassing the CTL epitope (amino acid residues 168–176). Four groups of isolates with naturally occurring variant sequences for the CTL epitope were defined: (1) YPHFMPPNL; (2) YPHFMPPSL; (3) YPHFIPPSL; and (4) YLDFMPPNL. The remaining isolates, and the laboratory strains K181 and Vancouver, showed complete identity with the Smith strain. Polyclonal pp89 (Smith strain)-specific CTL only weakly recognized target cells infected with MCMV from most variant groups. No lysis of cells infected with isolate N1 from group 4 was detected. Analyses of cross-reactive recognition of YPHFMPTNL peptide-coated targets by CTL primed with variant MCMV isolates showed that the group 2 and 3 isolates, G4 and K6, respectively, but not the group 4 isolate N1, elicited CTL that exhibited a cross-reactive response. Furthermore, while the group 2 and 3 isolates G4 and K6 were able to prime CTL responses that displayed reactivity to homologous pp89 variant nonapeptides, the group 4 isolate N1 failed to do so. Finally, while immunization of mice with the nonapeptide YPHFMPTNL conferred significant protection against the laboratory strain KI81, no evidence of protection was observed for the group 2 and 4 variants G4 and N1, respectively. These observations raise the possibility that clinical isolates of HCMV may also differ in sequence from potential vaccine strains at immunodominant epitopes for CD8+ T cells thus reducing the efficacy of vaccination.
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Granulocyte-macrophage colony stimulating factor promotes prolonged survival and the support of virulent infection by African swine fever virus of macrophages generated from procine bone marrow and blood
More LessLong-surviving cultures of non-adherent cells of the monocyte-macrophage lineage were established from the bone marrow and blood of weanling pigs by culturing cells from these tissues in the presence of recombinant procine granulocyte-macrophage colony stimulating factor (GM-CSF). The cells increased in number, principally during the first 4 weeks of culture, bound monoclonal antibodies recognizing porcine macrophage antigens and avidly phagocytosed latex particles. The GM-CSF generated mononuclear phagocytes were highly infectable with a virulent Malawi isolate of African swine fever virus (ASFV) and able to generate levels of virus progeny similar to those produced by freshly isolated pig macrophages. The cultured cells retained their susceptibility to ASFV infection for as long as the cultures survived i.e. for up to 3 months.
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- Insect
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Host haemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene
More LessPolydnaviruses produced by the hymenopteran endoparasitoid Cotesia rubecula are deposited together with the egg into the lepidopteran host Pieris rapae and are apparently involved in the suppression of the host’s defence system. Around 6 h post-parasitization host haemocytes change their surface properties, actin cytoskeleton structure and adhesion properties. Here we show that a single polydnavirus gene is expressed inside the caterpillar haemocytes in a transient fashion between 4 to 8 h post-parasitization.
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- Plant
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The molecular structure of Iresine viroid, a new viroid species from Iresine herbstii (‘beefsteak plant’)
More LessA viroid was isolated from Iresine herbstii plants using the bidirectional PAGE method for analysis of small circular RNA molecules. The viroid was transmitted to viroid-free Iresine herbstii plants of the cultivar ‘Aureoreticulata’ by mechanical inoculation. Infected plants did not develop any symptoms. The new viroid species has been named Iresine viroid (IrVd). It is a member of the potato spindle tuber viroid group and consists of 370 nucleotides, 227 G+C, 143 A+U (GC content, 61.4%). The most stable rod-like secondary structure of this viroid has 86 G:C, 34 A:U and 12 G:U base pairs with a minimum free energy of -153.4 kcal/mol (-641.2 kJ/mol). The sequence and the position of the terminal conserved region (TCR) within the rod-like secondary structure of IrVd differ from the other known TCRs. The structure of the left terminal domain of IrVd may reflect the result of an intramolecular RNA recombination event.
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The nucleotide sequence of RNA1 and RNA2 of olive latent virus 2 and its relationships in the family Bromoviridae
More LessThe complete nucleotide sequence of RNA1 and RNA2 of olive latent virus 2 (OLV-2), a virus with quasi-spherical to bacilliform particles and a non-polyadenylated tripartite ssRNA genome, was determined. RNA1 consists of 3126 nucleotides and contains a single open reading frame (ORF) coding for a polypeptide with a molecular mass of 102689 Da (p1a). RNA2 is also a monocistronic molecule, 2734 nt in length, coding for a polypeptide with a molecular mass of 90631 Da (p2a). The translation products of RNA1 and RNA2 possess the motifs proper to helicase, methyltransferase (RNA1) and RNA polymerase (RNA2), suggesting that both are involved in the replication of the viral RNA. The similarities found between OLV-2 and members of the Bromoviridae in some properties and in the sequences of all genomic products (including p1a and p2a) are strongly indicative that it belongs in this family. OLV-2, however, did not show a direct relationship with any of the current genera in the family. Rather, it revealed homologies in diverging directions with one or other of the Bromoviridae genus, thus qualifying as the possible representative of a new taxon in this family.
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The nucleotide sequence and genomic organization of grapevine virus B
More LessGrapevine virus B (GVB) is a tentative member of the genus Trichovirus. The 5′-terminal region of the RNA genome of GVB comprises 5437 nucleotides and has been sequenced by the dideoxynucleotide chain termination method. Evidence was obtained that the RNA is capped. Two putative open reading frames (ORFs) were identified. ORF 1 coded for a 194.7 kDa polypeptide with conserved motifs of replication-related proteins of positive-strand RNA viruses (i.e. methyltransferase, helicase and RNA-dependent RNA polymerase, in that order from the N to the C terminus). ORF 2 encoded a 20 kDa polypeptide that did not show any significant sequence homology with protein sequences from the databases. The biological function of this polypeptide was not determined. Although the 20 kDa product was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and an antiserum produced, it could not be identified in GVB-infected plant tissue extracts. The GVB genome had the same size as that of apple chlorotic leaf spot virus (ACLSV), the type species of the genus Trichovirus, but differed substantially in the number (five compared to three), size and order of genes. Differences existed also in the extent of sequence homology between polymerases, which did not cluster together in tentative phylogenetic trees. The results of this study show that definitive and tentative trichovirus species differ molecularly to an extent that may warrant a taxonomic revision of the genus.
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- Other Agents
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Infectivity in extraneural tissues following intraocular scrapie infection
More LessIntraocular (i.o.) infection of mice with scrapie produces strain-specific targeting of replication and subsequent pathology within the visual system projection areas in the CNS, but also initiates an extraneural infection. Following i.o. infection with ME7 scrapie, infectivity was detected 24 h later in the Harderian gland, the superficial cervical lymph nodes (SCLNs) and the spleen, but not until 20 days in Peyer’s patches and inguinal lymph nodes (ILNs). Persistent low levels of infectivity were found in the Harderian gland (which lies within the orbit), but the presence of PrP could not be confirmed by immunolabelling or Western blotting. SCLNs contained maximal amounts of infectivity by 20 days post-infection and remained at this level throughout the incubation period. ILNs reached a similar plateau at 60 days, as did Peyer’s patches at 80 days and spleen at 100 days. Further investigation of the role of the spleen in pathogenesis showed that in contrast to ME7 scrapie, mice infected with 79A scrapie had high levels of infectivity in the spleen by 20 days post-infection, irrespective of the route of infection. In addition, the disease developed more rapidly following direct intrasplenic infection with ME7 scrapie than with intraperitoneal infection. Splenectomy at 7 days either before or after i.o. infection had no effect on the incubation period. These results indicate that the rate of replication of infectivity is both tissue and scrapie-strain dependent, and that extraneural spread of infection can occur via the lymphatic system.
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PrP genotype contributes to determining survival times of sheep with natural scrapie
More LessSeveral allelic variants of the sheep PrP gene are associated with scrapie susceptibility. However, it is not known whether, and to what extent, the PrP genotype contributes to determining survival times of scrapie sheep. We therefore determined the PrP genotype and life spans of over 50 Flemish and Swifter sheep within a single scrapie-affected flock. Eighty-three per cent of the scrapie sheep were homozygous for the PrPVQ allele (polymorphic amino acids at codons 136 and 171 are indicated) and these sheep died from scrapie at a mean age of 25 months. In sheep heterozygous for PrPVQ, development of scrapie was delayed or did not occur. Sheep with at least one PrPAR allele, including PrPVQ/PrPAR sheep, did not develop scrapie. No scrapie sheep were found without a PrPVQ allele. We conclude that the PrP genotype contributes to determining survival times of sheep with natural scrapie. Additionally, we describe two novel sheep PrP allelic variants.
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