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Volume 77,
Issue 10,
1996
Volume 77, Issue 10, 1996
- Animal
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- RNA viruses
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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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