- Volume 74, Issue 8, 1993
Volume 74, Issue 8, 1993
- Animal
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Inducible expression of a foreign gene inserted into the human cytomegalovirus genome
More LessWe previously described the insertion of a foreign gene into a non-essential region of human cytomegalovirus (HCMV) by homologous recombination. Here we report insertion of the Escherichia coli lacZ gene downstream of the mouse metallothionein promoter into the HindIII-O region of HCMV by replacement-type recombination. Expression of the lacZ gene in the recombinant was independent of viral growth, but dependent on induction by heavy metals. Of several metals tested for β-galactosidase induction and also for their toxicity to HEL cells, Zn was found to be the most suitable for use as an inducer. In HEL cells infected with the recombinant in the presence of 50 μm-Zn, β-galactosidase activity was maximal 3 days after infection, and reached levels 27 times higher than the value obtained in the absence of Zn.
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Lipopolysaccharide inhibits the production of lymphocytic choriomeningitis virus in a human monocytic cell line
More LessThe human monocytic cell line THP-1 was used as a model to study the mechanism of infection in the monocyte/macrophage, a natural target of lymphocytic choriomeningitis virus (LCMV) infection in vivo. Both the virulent strain, LCMV. WE, and the avirulent strain, LCMV. ARM, infected THP-1 cells, but did not stimulate THP-1 cells to secrete interleukin 1 (IL-1) or tumour necrosis factor (TNF-α). When lipopolysaccharide (LPS) was added to THP-1 cells together with LCMV, an 80 to 90% reduction in the number of infected cells (measured by immunofluorescence) and a 90% reduction in viral plaques was observed 5 to 6 days postinfection. Neither interferon α (IFN-α) nor IFN-β were detected in supernatants from THP-1 cells after the addition of LCMV, LPS, or LPS plus LCMV. In contrast, the same levels of IL-1 and TNF-α were observed in the presence of LPS and LCMV, or LPS alone. However, antibodies to IL-1, TNF-α, interleukin 6 and IFN-α did not block the antiviral effect of LPS. In kinetic studies, LPS added 1 day after adding LCMV to THP-1 cells was still effective in reducing the number of infected cells. Our findings suggest that LPS alters cellular metabolism, possibly through the induction of IFN-α, and that IFN-α in the absence of LPS suppresses virus production.
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Molecular and antigenic characterization of HV114, a hantavirus isolated from a patient with haemorrhagic fever with renal syndrome in China
More LessThe relationship of a Hantaan-like virus (HV114), isolated from a patient with haemorrhagic fever with renal syndrome in Hubei Province, People’s Republic of China, to other pathogenic hantaviruses was evaluated by cross-neutralization studies and nucleotide sequence analysis of the M genome segment. Plaque reduction neutralization assays indicated that HV114 is closely related to prototype Hantaan (HTN) virus, strain 76-118, which was originally isolated from an Apodemus field mouse in Korea. Comparison of the M genome segments of HTN 76-118 and HV114 revealed sequence identity of 84·7% and 95·4% for nucleotides and deduced amino acids, respectively. These data demonstrate that HV114 and 76-118 are two closely related but different isolates of HTN virus, establishing the scientific basis for testing and future use in China of a recombinant vaccine expressing the genome of HTN virus strain 76-118.
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Construction of an antigenic map of the haemagglutinin–esterase protein of influenza C virus
More LessFour different antigenic sites (A-1, A-2, B-1, B-2) have been identified previously on the haemagglutinin–esterase (HE) glycoprotein of influenza C/Ann Arbor/1/50 virus with seven monoclonal antibodies (MAbs). In this study we produced 30 additional anti-HE MAbs, nine of which demonstrated at least one of the following activities: haemagglutination inhibition, receptor-destroying enzyme inhibition, haemolysis inhibition, and neutralization (group A). The remaining had none of these activities (group B). These antibodies, and those previously isolated, were used to construct a more complete antigenic map of the HE molecule. Operational and topological analyses showed that a minimum of nine non-overlapping or partially overlapping antigenic sites were present on the HE protein, five recognized by group A MAbs (A-1 to A-5) and four by group B (B-1 to B-4). Among these antigenic sites, site A-5 was unique in that MAbs to this site inhibited the receptor-destroying activity without influencing the receptor-binding activity, which supports the idea that the sites responsible for these two functions are separate. It was also found that several group B MAbs were cross-reactive with host cell antigens.
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Location on the evolutionary trees of the non-structural protein (NS) and neuraminidase (NA) genes of late human influenza A (H2N2) viruses: parental viruses of the NS and NA genes of Hong Kong influenza A (H3N2) viruses
More LessThe nucleotide sequences of the non-structural protein (NS) and neuraminidase (NA) genes of human influenza A (H2N2) viruses isolated in 1967 and 1968 in Europe, Asia and North and South America were located on evolutionary trees in order to identify the parental virus of Hong Kong influenza A (H3N2) viruses, which appeared in the human population in 1968. From the evolutionary trees, the H2N2 viruses isolated during the 1967 to 1968 period were divided into two groups. Group I includes the A/Tokyo/3/67, A/Hachioji/1/67, A/Perg/1/68, A/Cordoba/522/67, A/Texas/2/68 and A/Berkeley/1/68 viruses, whereas group II includes the A/Georgia/1/67, A/England/10/67 and A/Poland/6/67 viruses. The NS and NA genes of Hong Kong H3N2 viruses isolated in 1968 were genetically closer to those of group II viruses and closest to those of A/Poland/6/67.
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Production of the M2 protein of influenza A virus in insect cells is enhanced in the presence of amantadine
More LessRecombinant baculoviruses that express the M2 protein from the genes of either the amantadine-sensitive, influenza A/Ann Arbor/6/60 virus or a laboratory-derived, amantadine-resistant mutant of this virus were constructed. Addition of amantadine or rimantadine at 2 µg/ml to cultures of Sf9 cells infected with the recombinant baculoviruses increased the yield of the M2 protein from the amantadine-sensitive virus approximately 10-fold, but did not increase the yield of the M2 protein from the amantadine-resistant virus. Flow cytometry demonstrated that the increased production of M2 in the presence of amantadine resulted in increased cell surface expression of the M2 protein. Pulse-chase experiments indicated that whereas the rate of synthesis of the M2 protein increased in the presence of amantadine, the M2 protein was stable in both the presence and absence of amantadine. Addition of amantadine to Sf9 cells as late as 72 h after infection with the recombinant virus increased the production of M2 protein. These data suggest that the M2 protein exerts some biological activity in Sf9 cells.
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Characterization of the gene encoding the A-type inclusion protein of camelpox virus and sequence comparison with other orthopoxviruses
More LessA gene was identified in camelpox virus strain CP-1 that is similar to the 160K gene of cowpox virus strain Brighton (BR) that encodes the A-type inclusion body protein (ATIP). The CP-1 gene was mapped, sequenced, and the presence of the ATIP-specific mRNA was demonstrated. The open reading frame [2178 nucleotides (nt)] was found at a similar position in the CP genome as the one reported for the cowpox virus 160K ATI gene. DNA sequence comparison revealed a deletion of two adjacent adenine residues relative to cowpox virus BR, generating a reading frame shift accompanied by the formation of a translational stop codon. An identical deletion has been described for vaccinia virus strain Western Reserve. The DNA sequence of the corresponding region of monkeypox virus strain Copenhagen revealed a deletion leading to a putative stop codon 75 nt upstream of the same stop codons in the camelpox and vaccinia virus genes. These findings are consistent with the expression of truncated ATIPs, of 94K in vaccinia and camelpox viruses and of 92K in monkeypox virus. In addition, a deletion of 789 bp could be localized downstream of the ATI open reading frame in camelpox virus isolates of different origin. This causes the transcription of a shortened ATI-specific mRNA (3·7 kb) relative to vaccinia and cowpox viruses (both 4·5 kb). The similarity observed in ATIP-encoding and flanking sequences might suggest that vaccinia and camelpox viruses are descended from a common ancestor.
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Antiviral effect of short hyperthermic treatment at specific stages of vesicular stomatitis virus replication cycle
More LessStarting from the observation that the antiviral activity of cyclopentenone prostaglandins is associated with the synthesis of a 70K heat shock protein (HSP70), we have analysed the effect of short hyperthermic treatment (HT) on HSP70 induction and virus production in monkey epithelial cells during the replication of vesicular stomatitis virus (VSV). The heat shock response, as determined by HSP70 synthesis, appeared to be unaltered in VSV-infected cells in the first 4 h following virus infection, after which time it started to decline. No induction of HSP70 synthesis was observed when HT was applied 8 h after VSV infection. A 20 min HT at 45 °C was effective in suppressing VSV multiplication by more than 90% when applied at specific stages of the virus replication cycle. Synthesis of virus proteins was not affected by HT, indicating that the target for the treatment is a post-translational event. The HT-induced block of virus replication appeared to be associated with inhibition of VSV G protein maturation and HSP70 induction.
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Sindbis virus pathogenesis: phenotypic reversion of an attenuated strain to virulence by second-site intragenic suppressor mutations
More LessMonoclonal antibodies (MAbs) specific for the E2c neutralizing antigenic site on the Sindbis virus E2 glycoprotein define a pathogenesis domain that affects neonatal mouse virulence. Sequence analysis of E2c MAb escape mutants showed that the domain included E2 amino acids 62, 96 and 159. The pathogenesis domain is also influenced by changes at E2 position 114. Mutation of E2 residues Asn 62 to Asp or Lys 159 to Glu results in suppression of the attenuated phenotype conferred by a mutation from Ser to Arg at E2 position 114. Possible mechanisms of phenotypic suppression within the E2c pathogenesis domain were investigated by using site-directed mutagenesis to determine the effects of specific combinations of positively charged, negatively charged and uncharged amino acid substitutions at E2 positions 62, 114 and 159. Phenotypic reversion to virulence by second-site suppressor mutations at E2 amino acids 62 or 159 was not dependent on ionic interaction with the residue at E2 114. Rather, suppression appeared to be the result of independent virulence effects mediated by specific residues.
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Subgenomic RNAs of Lelystad virus contain a conserved leader-body junction sequence
More LessDuring the replication of Lelystad virus in alveolar lung macrophages, a 3′-coterminal nested set of six subgenomic RNAs (RNA2 to RNA7) is formed. These contain a common leader sequence derived from the 5′ non-coding region of the genomic RNA. In this study, the sequence of the junction sites, i.e. the sites where the leader sequence joins to the body of the subgenomic RNA, was determined for all six subgenomic RNAs. For each subgenomic RNA, six to nine cDNA clones were isolated by means of reverse transcription and PCR. The nucleotide sequence at the junction site was identical for all eight cDNA clones derived from subgenomic RNA4. However, heterogeneity was observed in the nucleotide sequence surrounding the junction sites of the cDNA clones derived from subgenomic RNAs 2, 3, 5, 6 and 7. This heterogeneity suggests that the fusion of the leader to the body of the subgenomic RNA may be imprecise. The junction sites of the six subgenomic RNAs had a conserved sequence motif of six nucleotides (UCAACC or a highly similar sequence). The distance between the junction site and the translation initiation codon of the downstream open reading frame varied from nine to 83 nucleotides.
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St Louis encephalitis virus establishes a productive, cytopathic and persistent infection of Sf9 cells
More LessThe Sf9 cell line, commonly used for gene expression by recombinant baculovirus, has been productively infected by St Louis encephalitis (SLE) virus, a flavivirus. SLE viral infection produced a c.p.e. in the Sf9 cells characterized by giant cells and the presence of 10-fold fewer cells in the infected cultures after the first week of infection compared with uninoculated control cultures. Infected Sf9 cells expressed SLE viral antigens, and intracellular virus particles were observed by electron microscopy. Titres of cell-associated SLE virus rose slightly over an 8 week period, whereas titres of cell-free virus remained stable, suggesting that SLE virus establishes a productive and persistent infection of Sf9 cells. The SLE virus produced by the Sf9 cells could be neutralized by SLE virus-immune mouse ascitic fluid, and no evidence of escape mutants was detected. Sf9 cells persistently infected with SLE virus could be superinfected with a recombinant baculovirus and expressed recombinant antigen. The successful infection of Sf9 cells by SLE virus represents the first report of production of c.p.e. by SLE virus in insect cells under routine cell culture conditions and of the infection of Sf9 cells by a human pathogen.
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Nucleotide sequence comparison of the VP8* gene of rotaviruses possessing the AU-1 gene 4 allele
Of the five currently recognized alleles of the human rotavirus VP4 gene, the AU-1 allele has captured attention because of its possible non-human origin. The 5′ 750 nucleotide region of the VP4 gene, encoding the VP8* fragment [amino acids (aa) 1 to 241] and the connecting peptide (aa 242 to 247), from 13 human and two feline rotavirus strains possessing the AU-1 allele was highly conserved both at the nucleotide sequence (93·8 to 99·7% identity) and amino acid level (95·5 to 100% identity) irrespective of the year and the place of isolation or of the host species from which these viruses were isolated. This is consistent with the hypothesis that the AU-1 allele of the VP4 gene has been maintained in both human and feline rotavirus gene pools.
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- Plant
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A strong-stop DNA in rice plants infected with rice tungro bacilliform virus
Yiming Bao and Roger HullA virus-specific small nucleic acid (strong-stop DNA) was identified in rice plants infected with rice tungro bacilliform virus, but not in the virus particles. This nucleic acid was shown to consist of about 595 deoxyribonucleotides with about 70 ribonucleotides covalently linked at the 5ʹ end. Hybridization with sequence-specific oligonucleotides showed that the ribonucleotides were from the plant cytoplasmic tRNAMet i sequence. PCR analysis detected hairpin structures at the 3ʹ end of the DNA.
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Sequence and structure of defective interfering RNAs associated with cucumber necrosis virus infections
More LessThe presence of symptom-attenuating defective interfering (DI) RNAs in a laboratory culture of cucumber necrosis tombusvirus (CNV) was confirmed. Sequencing of cDNA clones of these DI RNAs revealed that CNV DI RNAs retained sequences from the CNV 5′-untranslated and 3′-terminal regions as well as a portion of the coding region for the 92K protein. Similar sequence arrangements were also observed in symptom-attenuating DI RNAs generated de novo from synthetic wild-type CNV transcripts. A comparison of the sequences and biological activities of these CNV DI RNAs is presented. In co-infections of synthetic wild-type CNV and CNV DI RNAs, prominent RNA species of a higher M r than the DI RNA used in the co-infection were found. The possible nature of these RNA species is discussed.
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The involvement of cowpea mosaic virus M RNA-encoded proteins in tubule formation
More LessOn the surface of cowpea protoplasts inoculated with cowpea mosaic virus (CPMV), tubular structures containing virus particles have been found. Such tubular structures are thought to be involved in cell-to-cell movement of CPMV in cowpea plants. To study the involvement of the 58K/48K and capsid proteins of CPMV in the formation of the tubular structures, mutations were introduced into M cDNA clones from which infectious transcripts could be derived. No tubules were found on protoplasts inoculated with a mutant that fails to produce the 48K protein nor with a mutant that has a deletion in the 48K coding region, suggesting that the 48K protein is essential for this process. However, a possible role of the 58K protein in tubule formation could not be excluded. A mutant that fails to produce the capsid proteins did produce tubules and therefore the capsid proteins are not involved in the formation of the tubular structures. Electron microscopic analysis revealed that the tubules produced by this mutant are, apart from the absence of virus particles, morphologically identical to the tubules formed by the wild-type virus.
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