- Volume 78, Issue 6, 1997
Volume 78, Issue 6, 1997
- Articles
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Genetic variation among strains of wild-type yellow fever virus from Senegal.
We have examined and compared at the molecular level three strains of wild-type yellow fever (YF) virus isolated from Senegal in 1927, 1953 and 1965, termed French viscerotropic virus, Rendu and Dak1279 respectively. Over the structural protein genes, Rendu differed from the other two strains by 8% at the nucleotide level. Rendu also differed antigenically, possessing a ‘vaccine’- specific envelope (E) protein epitope (i.e. an epitope previously shown to be found on 17D and French neurotropic vaccine viruses only and not wild-type strains of YF virus). Consequently, we propose that at least two distinct genotypes of wild-type YF virus have been present in Senegal. Since Rendu virus was isolated from a fatal case of YF, it would indicate that the vaccine-specific epitope on the E protein is not associated with attenuation of the viscerotropism of wild-type YF virus.
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Yellow fever virus envelope protein has two discrete type-specific neutralizing epitopes.
More LessTwo monoclonal antibody neutralization resistant (MAbR) variants of the yellow fever (YF) 17D-204 vaccine virus strain were selected using YF type- specific MAb B39. These B39R variants were compared with the variant virus selected by Lobigs et al. (Virology 161,474–478, 1987) using a second YF- type specific MAb (2E10) which mapped to amino acid position 71/72 in the envelope (E) protein. Neutralization assays with a panel of MAbs suggested that these two YF-type-specific epitopes are located in two discrete regions of the folded E protein. Each of the B39R variants had a single nucleotide mutation which encoded an amino acid substitution at either position E-155 or E-158. Thus, YF type-specific epitopes map to both domain I (B39) and II (2E10) of the YF virus E protein. The B39 defined epitope represents the first flavivirus neutralizing epitope localized to this region of domain I of the E protein.
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Phylogenetic analysis of pestiviruses from domestic and wild ruminants.
More LessInfections with pestiviruses occur in cattle, sheep, pigs and also in numerous other ungulate species. In the present study, pestiviruses from goat, buffalo, deer and giraffe were analysed at the molecular level; unusual strains from cattle and pigs were also included. A phylogenetic analysis of the respective pestiviruses was undertaken on the basis of a fragment from the 5′ noncoding region as well as the gene encoding autoprotease Npro. Statistical analyses of the respective phylogenetic trees based on the 5′ NCR revealed low confidence levels for most of the branches, while the structure of the tree based on the Npro gene was supported by high bootstrap values. Accordingly, the isolates from goat, buffalo and deer can be grouped together with bovine viral diarrhoea virus (pestivirus type 1); within this genotype three subgroups and one disparate virus have been identified. One isolate from pig and one from cattle belong to the group of ‘true’ border disease virus (pestivirus type 3), which can be further subdivided into two major subgroups. Interestingly, the giraffe isolate does not belong to one of the four established pestivirus genotypes. The phylogenetic analysis strongly suggests that genotype 1 pestiviruses occur world-wide in many ruminant species. Furthermore, phylogenetic trees based on the Npro gene nucleotide sequences show that the respective sequences do not segregate into discrete lineages based on host-species origin.
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Solubilized and cleaved VP7, the outer glycoprotein of rotavirus, induces permeabilization of cell membrane vesicles.
More LessIt has been previously shown that rotavirus triplelayered particles induce permeabilization of liposomes and membrane vesicles. These effects were mediated by one or both of the solubilized outer-capsid proteins, VP4 and VP7. Permeabilization was dependent on trypsin treatment of the viral particles, suggesting that VP4 was involved. To analyse the respective roles of the outer-capsid proteins in this permeabilization process, we have used membrane vesicles loaded with carboxyfluorescein and virus-like particles derived from insect cells co-expressing various sets of capsid proteins. Virus-like particles containing VP2, VP6 and VP7 (VLP2/6/7) are as efficient in permeabilizing vesicles as triple-layered particles. As with double-layered particles, virus-like particles made of VP2 and VP6 had no effect on vesicle permeabilization. Permeabilization of membrane vesicles required trypsinization of the VP7 solubilized from VLP2/6/7. These results show that solubilized and trypsinized VP7 is able to induce membrane permeabilization, independently of the presence of VP4.
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A novel human rotavirus serotype with dual G5-G11 specificity.
More LessRotavirus serotype G5 isolates were recently recovered from children with diarrhoea in Brazil. Like most human strains, they exhibited long electro- pherotypes and subgroup II and Wa-like VP4 specificity. We report the successful propagation and the molecular and antigenic characterization of one of these isolates (IAL-28). Cross-neutralization of IAL-28 and a single gene reassortant, UK × IAL- 28, which contains the gene encoding the IAL-28 VP7 in the UK genomic background, with prototype G1 to G14 rotaviruses demonstrated that IAL-28 has antigenic determinants specific for both G5 and G11 serotypes. Sequence analysis of the gene encoding VP7 suggested that one or two amino acid substitutions at positions 96 and 100 on IAL-28 VP7 were possibly responsible for the additional G11 specificity. G5 rotaviruses are found in horses and predominate in piglets, whereas G11 has been identified exclusively as a swine pathogen.
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Cloning, sequence analysis and expression of the major outer capsid protein gene of an aquareovirus.
More LessThe nucleotide and deduced amino acid sequences of genome segment 10 of aquareovirus strain SBR, encoding the major outer capsid protein (VP7), have been determined. Genome segment 10 of SBR virus is 986 nucleotides long and encodes a polypeptide of 298 amino acids with a predicted molecular mass of 32430 Da. There are 26 non- translated nucleotides at the 5′ end and 66 non- translated nucleotides at the 3′ end. Using a recombinant baculovirus system, the VP7 protein of SBR virus was expressed to a high level. The baculovirus-produced VP7 protein was similar both in its size and antigenic properties to the authentic aquareovirus VP7 protein. Antiserum from a rabbit immunized with the baculovirus-produced VP7 protein failed to neutralize the homologous aquareovirus strain. As determined by Western blotting, this antiserum reacted with aquareovirus strains belonging to the same genogroup as SBR virus, but did not react with aquareovirus strains belonging to the other genogroups.
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Identification of a new genogroup of aquareovirus by RNA-RNA hybridization.
More LessThe relative mobilities of the 11 dsRNA genomic segments of 22 aquareovirus isolates from fish and shellfish obtained from different geographical areas of the world were compared by PAGE. Using reciprocal RNA-RNA dot blot hybridization, a new sixth genetic group of aquareovirus (genogroup F) was identified. Genogroup A was represented by eight and genogroup B by 12 isolates. The remaining two isolates represented the new sixth genogroup (genogroup F). The genetic relationship of these aquareoviruses with mammalian rotavirus group A (SA11) was also examined by reciprocal RNA-RNA blot hybridization but none was found under any of the stringency conditions used.
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Degenerate and specific PCR assays for the detection of bovine leukaemia virus and primate T cell leukaemia/lymphoma virus pol DNA and RNA: phylogenetic comparisons of amplified sequences from cattle and primates from around the world
Degenerate and specific PCR assays were developed for bovine leukaemia virus (BLV) and/or primate T cell leukaemia/lymphoma viruses (PTLV). The degenerate assays detected all major variants of the BLV/PTLV genus at a sensitivity of 10-100 copies of input DNA; the specific systems detected 1–10 copies of input target. Sensitivity was 100% in specific DNA-PCR assays done on peripheral blood from seropositive BLV-infected cattle and HTLV-I- or HTLV-II-infected humans, and 62% in RNA/DNA- PCR assays on sera from BLV seropositive cattle. The pol fragments from 21 different BLV strains, isolated from cattle in North and Central America, were cloned and sequenced, and compared to other published BLV and PTLV pol sequences. BLV and PTLV sequences differed by 42%. Sequence divergence was up to 6% among the BLV strains, and up to 36% among the PTLV strains (with PTLV-I and PTLV-II differing among themselves by 15% and 8%, respectively). Some cows were infected with several BLV strains. Among retroviruses, BLV and PTLV sequences formed a distinct clade. The data support the interpretation that BLV and PTLV evolved from a common ancestor many millennia ago, and some considerable time before the PTLV- I and PTLV-II strains diverged from each other. The dissemination of the BLV strains studied probably resulted from the export of European cattle throughout the world over the last 500 years. The relatively similar mutation rates of BLV and PTLV, after their various points of divergence, suggest that there could be a much wider genetic range of BLV than has currently been defined.
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Identification and characterization of bovine herpesvirus-1 glycoproteins E and I
More LessTo identify the products of the bovine herpesvirus- 1 (BHV-1) gE and gI genes, we constructed baculo- virus recombinants containing the putative gE or gI genes. These recombinant viruses synthesized BHV-1 gE and gI with apparent molecular masses of 84 and 41 kDa, respectively. Polyclonal antibodies against these recombinant gE or gI proteins were produced and by using these antibodies, we showed the presence of gE and gI with apparent molecular masses of 94 and 45 kDa, respectively, in purified BHV-1 virions. We also demonstrated that like their herpes simplex virus-1 and pseudorabies virus counterparts BHV-1 gE and gI form a complex. A gI BHV-1 mutant failed to express gI but gE was found in the virions. On the other hand, neither gE nor gI was found in the virions of a gE BHV-1 mutant. In th gE BHV-1 mutant, gI was produced but released into the medium without being integrated in the virions.
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Herpesvirus saimiri-immortalized human T-cells support long-term, high titred replication of human immunodeficiency virus types 1 and 2
More LessHerpesvirus saimiri strain C488 transforms human CD4 T-lymphocytes to continuous interleukin- 2-dependent growth. Unlike human T-cell lines derived from tumours or those transformed by human T-lymphotropic virus 1, herpesvirus saimiri- immortalized T-cells (HVS T-cells) retain many functions of primary activated T-lymphocytes. We have characterized the course of human immunodeficiency virus types 1 and 2 (HIV-1/-2) infection in three HVS T-cell lines. Our results confirm that HVS T-cells are highly permissive to both HIV-1/-2 prototype viruses and to poorly replicating HIV-2 strains of restricted cell tropism. However, the infection was persistently productive for up to 5 months. The down-regulation of surface CD4 molecules was delayed and virus yields significantly exceeded those obtained in T-cell lines.
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Identification of a cis-acting element within the herpesvirus saimiri ORF 6 promoter that is responsive to the HVS.R transactivator
More LessWe have previously demonstrated that two distinct transcripts are produced from ORF 50, the major transcriptional activating gene of herpesvirus saimiri. The products of these transcripts trans- activate the delayed-early ORF 6 promoter, though to different degrees. Deletion analysis demonstrated that the ORF 50 responsive elements are contained in a 132 bp fragment situated 127–259 bp from the transcription initiation site within the ORF 6 promoter. This fragment conferred ORF 50-responsiveness on an enhancerless simian virus 40. Gel retardation analysis further mapped the responsive elements to a 38 bp fragment.
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Direct demonstration of persistent Epstein-Barr virus gene expression in peripheral blood of infected common marmosets and analysis of virus-infected tissues in vivo
Epstein-Barr virus (EBV) infection in animal model systems has been studied previously in marmosets and tamarins using serology and PCR of saliva. Here we directly demonstrated long-term persistence of EBV in the peripheral blood of marmosets by assaying EBER RNA expression. A new reverse transcription-PCR assay, able to distinguish a naturally occurring strain polymorphism in EBER 2 that may be useful as a strain marker for monitoring persistence and interactions between multiple strains in the same animal or person, has been developed. In situ hybridization and immunohisto-chemistry have also been used to search for EBV-infected cells in the animals. The carrier state in the common marmoset is similar to that of humans in that it is asymptomatic, long-lived and displays a very low level of circulating virus-infected cells. It differs from the human in lacking the characteristic antibody response to EBNA 1.
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Genetic content and preliminary transcriptional analysis of a representative region of murine gammaherpesvirus 68
Murine gammaherpesvirus 68 (MHV-68) is a relatively recently discovered pathogen of wild rodents and provides a unique opportunity to explore in detail the interactions of a gammaherpesvirus with its natural host. It may also provide a much needed small animal model for human gammaherpes-viruses. As a step in the detailed analysis of virus gene structure and expression we have sequenced over 20 kb of the MHV-68 genome and mapped gene transcripts by Northern blot hybridization. The region we chose to analyse contains several conserved gene blocks as well as some less well conserved genes and allowed us to estimate the relationship of this virus to other herpesvirus family members. Of particular interest is the fact that none of the characteristic Epstein-Barr virus (EBV) genes is present at this genomic locus although MHV-68 does have one gene encoding a membrane glycoprotein, gp150, which shows similarities to the major membrane glycoprotein of EBV. Our results further confirm that MHV-68 is a gammaherpesvirus marginally more closely related to a cluster of gammaherpesviruses including herpesvirus saimiri than to EBV. Northern analysis shows that the temporal regulation of expression is broadly similar to that of other herpesviruses in this region of the genome. We also show that like other gammaherpesviruses, MHV-68 splices its homologue of the EBV transcriptional activator gene BMRF1.
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Self-assembly of the JC virus major capsid protein, VP1, expressed in insect cells
The major capsid protein of human polyomavirus JC virus, VP1, has been cloned into a baculovirus genome and expressed in insect cells. The VP1 protein was expressed in the cytoplasm and transported into the nucleus. It was then purified by a sucrose cushion and CsCl density gradient centrifugation to near homogeneity. Electron microscopy showed that isolated recombinant VP1 protein self-assembled into a capsid-like structure similar to the natural empty capsid. Both chelator (EDTA) and reducing agent (DTT) are required to disrupt the capsid structure into the pentameric capsomeres, as demonstrated by haemagglutination assay and electron microscopy. These results suggest that JC virus VP1 can be transported into the nucleus and self-assembled to form capsid-like particles without the involvement of the viral minor capsid proteins, VP2 and VP3. In addition, metal ions and disulphide bonds appear to be important in maintaining the integrity of the viral capsid structure.
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Interaction of human papillomavirus type 16 and adeno-associated virus type 2 co-infecting human cervical epithelium
Recently, we hypothesized that the tumour-suppressive, human helper-virus-dependent, adeno- associated parvoviruses (AAV) may interfere with transforming functions of human papillomaviruses (HPV) in the development of cervical carcinoma. Here, we demonstrate that in cervical epithelium containing papillomavirus DNA, AAV DNA can be detected in a replication-competent form and that AAV proteins are expressed. In cultured cells con-taining integrated AAV-2 DNA, transfection of HPV- 16 DNA induced rescue of infectious AAV-2, revealing helper functions of HPV-16. Similarly, cotransfection of HPV-16 and AAV-2 DNAs into human epithelial cell lines led to replication of AAV-2, and, in keratinocytes, to a cytopathic effect. These data suggest an interaction of the two viruses, possibly influencing the development of HPV-related lesions.
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Assembly of adeno-associated virus type 2 capsids in vitro
More LessCapsid proteins VP1, VP2 and VP3 of adeno- associated virus type 2 (AAV-2) were separately expressed by recombinant baculoviruses, purified under denaturing conditions and renatured in the presence of 0·5 M arginine, followed by dialysis against buffers of physiological ionic strength. At a protein concentration of 0·05 mg/ml, the three capsid proteins predominantly formed monomers and, to a lesser extent, oligomers, as determined by sedimentation analysis. Oligomerization increased at higher protein concentrations. The capsid protein oligomers consisted of globular, non-capsid-like structures, as detected by electron microscopy. Addition of a HeLa cell extract significantly stimulated oligomerization of the capsid proteins, probably due to interactions with HeLa cell proteins. Characterization of structures sedimenting around 60S by immunoprecipitation and electron microscopy showed that, in addition to other aggregates, empty capsid-like structures were formed in vitro. The identity of these structures as empty AAV capsidswasverified by immunoelectron microscopy. Analysis of capsid formation in HeLa cells by transfection of VP expression constructs allowing separate expression of VP1, VP2 and VP3 showed that they were able to form capsids, although with a reduced efficiency as compared to VP proteins expressed from the wt cap gene. This finding suggests that the mutations introduced to allow separate capsid protein expression reduced the efficiency of capsid assembly in vivo and might also explain the reduced recovery of empty capsids employing the in vitro assembly procedure.
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Production of hepatitis B virus covalently closed circular DNA in transfected cells is independent of surface antigen synthesis
More LessCovalently closed circular DNA (cccDNA) is the first hepatitis B virus (HBV) DNA replicative intermediate formed from the genomic DNA and serves as the template for synthesis of viral mRNA and pregenomic RNA. It also appears to be produced by intracellular recycling of relaxed circular DNA intermediates. Here, we report that none of the forms of HBV surface antigen affect this intracellular recycling of HBV DNA. Elimination of the initiation codons for the large and middle surface proteins did not affect the detection of surface antigen in culture supernatants. In contrast, detection of surface antigen was eliminated by the removal of, or the introduction of two stop codons downstream of, the initiation codon of the small (major) surface antigen. None of the mutations affected the production, in transfected HepG2 cells, of HBV DNA replicative intermediates, including cccDNA.
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Hepatitis B virus X gene 1751 to 1764 mutations: implications for HBeAg status and disease
More LessA translational stop in the hepatitis B virus (HBV) precore codon 28 and specific changes in the core promoter region of the X gene have been suggested to influence the level of circulating HBeAg in patients. We analysed the core promoter region and precore sequences from 59 HBV strains (including 14 from the databank) of different genotypes and from patients with different HBeAg/anti-HBe patterns. The initiator and TATA elements for transcription of precore and pregenomic RNA were highly conserved. The majority of X gene deletions in the core promoter region would lead to translational frame-shifts and stops, truncating the C- terminal end of the X protein. We found significant associations between specific changes in core promoter positions 1762 to 1764, or in precore codon 28, and absence of circulating HBeAg. For the core promoter mutations alone, this association was related to the apparent degree of liver damage (as estimated by alanine aminotransferase levels) at the time of sampling. Mutations at nucleotides 1762 and/or 1764 were often accompanied by point mutations at positions 1751 to 1755. Since mutations at nucleotide positions 1762 and 1764 have recently been shown by in vitro studies to suppress HBeAg production with a concomitant enhancement of virus production, disappearance of the HBeAg- positive phenotype associated with 1762 to 1764 mutations may thus have at least as much significance for the course of infection as HBeAg absence associated with precore codon 28 stop mutations. These observations are considered against a secondary structural model for the 3′ end of HBV pregenomic RNA which also predicts enhancement of virus replication after mutation at positions 1762 and 1764.
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Reduced antigen production by hepatitis B virus harbouring nucleotide deletions in the overlapping X gene and precore-core promoter
More LessHepatitis B virus (HBV) genomes with deletions in the precore-core (preC-C) promoter have been detected in HBV infections without serological markers. To address whether the mutations are responsible for the reduced production of virus antigens, either an 8 bp (8d, position 1763 to 1770) or a 20 bp (20d, 1753 to 1772) deletion was created in a wild-type (wt) HBV clone. Both mutations cause premature termination of the overlapping X ORF. When introduced into HepG2 cells, both mutants produced reduced amounts of HBsAg, HBcAg and HBeAg, but released the same or more virion- associated DNA compared with the wt. A co-transfection of the 20d mutant with a small amount of intact X gene resulted in a 3-fold increase of HBcAg production compared to transfection with either the 20d or wt alone. When the promoter region was cloned into CAT plasmids, the 8d preC promoter showed weak activity and its initiation site was shifted 6 to 10 bp downstream. The preC promoter activity of 20d was not detectable by CAT ELISA and 5′ RACE. The levels of C transcripts of both mutants were higher than that of the wt, and their start sites were not altered. Therefore, the deletions cause the reduction of HBsAg, HBcAg and HBeAg although the mutant viruses can still replicate in cultured cells. The reduction of HBeAg is due to both the reduced preC promoter activity and the defect in HBx. The reduction of HBcAg is due to the disrupted X gene, despite augmented C promoter activity.
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The hepatitis B virus MHBst167 protein is a pleiotropic transactivator mediating its effect via ubiquitous cellular transcription factors
C-terminally truncated surface proteins of hepatitis B virus (HBV) are frequently translated from genomically integrated viral sequences. They may be relevant for hepatocarcinogenesis by stimulating gene expression. First, we examined the transactivating potential of middle hepatitis B surface protein truncated at amino acid (aa) position 167 (MHBst167) on the HBV regulatory element. In transient cotransfection assays using Chang liver or HepG2 cell lines and chloramphenicol acetyl- transferase (CAT) reporter constructs only the HBV enhancer I, but no other HBV regulatory elements like the X promoter, the S1 or S2 promoter or the enhancer II/core promoter could be stimulated by MHBst167. Since there is no evidence for a direct interaction of MHBst167 with DNA, we subsequently analysed whether cellular transcription factors were involved in mediating transactivation. This was tested both with isolated transcription-factor-binding sites and in the natural context of viral and cellular promoter elements. Deletion analysis and electrophoretic mobility shift assays revealed that Sp1,AP1 and NF-kB can mediate transactivation by MHBst167. No involvement of CREB, NF1 or the liver- specific factor C/EBP was found. These data indicate that MHBst167 is a pleiotropic, non-liver-specific transactivator which exerts its effect via ubiquitous cellular transcription factors that are also involved in the regulation of expression of cellular genes relevant for proliferation and inflammation.
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Volumes and issues
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