- Volume 69, Issue 6, 1988
Volume 69, Issue 6, 1988
- Bacterial
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Genome Homology and Host Range of Some SPβ-related Bacteriophages of Bacillus Subtilis and Bacillus amyloliquefaciens
More LessSummaryTemperate Bacillus subtilis bacteriophages SBβ, ø3T and SPR and B. amyloliquefaciens phage H2 were compared with respect to DNA-DNA homology by Southern blot analysis to each other and to members of the genus Bacillus. The results show that H2 is a distantly related member of the group III B. subtilis phages. Detectable homology to group III phages could be found in the DNA of several other Bacillus species, including B. natto and B. amyloliquefaciens, demonstrating the widespread occurrence of this group of phages. The host ranges for the phages SPβ, SPR and H2 were determined by adsorption efficiency and by the ability of erythromycin resistance- and chloramphenicol resistance-transducing phages to convert susceptible host strains. Of the three phages examined, only H2 was capable of infecting B. amyloliquefaciens. Based on these results we propose that group III phages should be divided into three subgroups: SPβ, ø3T, Rho11, IG1, IG3 and Z (subgroup 1), SPR (subgroup 2) and H2 (subgroup 3).
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- Animal
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Early Events of Importance in Determining Host Cell Permissiveness to Mouse Hepatitis Virus Infection
More LessSummaryThree categories of cell lines are described which differ with respect to their permissiveness to mouse hepatitis virus (MHV), strain A59. Fully permissive L-2 cells gave rise to 100- to 1000-fold higher numbers of infectious centres than did semipermissive LM, LM-K or C-1300 cells, whereas non-permissive Vero or C-6 cells were refractory to MHV infection. On an infected cell basis, semi-permissive cells (LM, LM-K or C-1300) were as efficient in replicating viral RNA, protein and progeny virions as fully permissive L-2 cells. This result suggested that LM, LM-K and C-1300 cells were deficient in their ability to permit full expression (as compared to L-2 cells) of an early event in MHV infection. Assays of radiolabelled MHV binding to cells of all three categories (L-2, LM, LM-K and C-6) and of infectious MHV binding to L-2 and LM-K cells showed no correlation between virion binding and degree of permissiveness to MHV infection. Internalization of MHV virions into L-2 and LM-K cells, as assayed by proteinase K-resistant infectious centres, showed that, in both cases, maximum virion uptake was complete by approximately 40 min post-inoculation. Direct assays of infectious virion uptake showed similar numbers of internalized viruses (only a threefold difference between L-2 and LM-K cells, as compared to a 500-fold difference in infectious centres). Attempts to enhance MHV uptake into LM-K cells relative to L-2 cells, with DEAE-dextran or the cytoskeleton-disrupting drugs colchicine and cytochalasin B, were unsuccessful, further suggesting that the ability of LM-K cells to internalize the virus was not lacking. The results suggest that MHV infection of at least some semi-permissive cells, such as the LM-K line, is limited by a process which chronologically correlates with virion uncoating. Since LM-K cells have been shown previously to be resistant to membrane fusion in MHV infection, it is postulated that they may also restrict uncoating of MHV by limiting the degree of normal endosomal membrane fusion with the viral envelope.
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Interaction of Polylysine with the Cellular Receptor for Herpes Simplex Virus Type 1
More LessSummaryWe earlier reported that neomycin blocked reversibly the binding of herpes simplex virus type 1 (HSV-1) to the receptor of BHK cells, while the binding of HSV-2 to the receptor was unaffected. We could not determine whether the effect was on the virus particle, the receptor, or both. We have now tested several other cationic substances, and report that polylysine (and polyarginine) block the binding of HSV-1 to the receptor by interfering with the cellular receptor function; higher molecular weight polylysines were more potent than those of lower molecular weight. Polylysine and neomycin showed additive effects. In vitro, polylysine showed the same strong binding to the plasma membrane phosphoinositides as did neomycin. Together these data suggest that the drugs may have a common target in the cell membrane. The HSV-1 and HSV-2 virus particles were unaffected by the drugs, as was the cellular HSV-2 receptor.
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Excretion of Non-infectious Virus Particles Lacking Glycoprotein H by a Temperature-sensitive Mutant of Herpes Simplex Virus Type 1: Evidence that gH Is Essential for Virion Infectivity
More LessSummaryA temperature-sensitive mutant of herpes simplex virus type 1, tsQ26, was shown to contain an amino acid substitution in glycoprotein H (gH). The mutant entered cells efficiently at the non-permissive temperature and replicated to give nearly normal yields of intracellular infectivity. The intracellular virions contained, predominantly, an immature form of gH and no gH was found on the surface of infected cells. Excreted virions were devoid of gH and were not infectious. Virions excreted at the permissive temperature were infectious and contained gH and no loss of gH resulted from incubation of these virions at the non-permissive temperature. The temperature-sensitive phenotype apparently results from the loss of gH from virions during their transport to the cell surface, and since loss of gH is accompanied by loss of infectivity we conclude that gH is an essential component of the infectious virion.
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Limited Efficacy of Inhibitors of Herpes Simplex Virus DNA Synthesis in Murine Models of Recrudescent Disease
More LessSummaryThe herpesvirus DNA polymerase inhibitor foscarnet, applied topically, and the anti-herpesvirus guanosine analogue buciclovir, given orally, decreased virus replication and disease development in primary skin infections of mice caused by herpes simplex virus type 1 (HSV-1). If the same tissues were infected via sensory nerves, following zosteriform spread of the virus the same treatments showed strongly decreased efficacy, or were inefficacious, when started before development of clinical signs in the infected tissues. These results were obtained in murine models of zosteriform spread of HSV-1 to the ear (following inoculation of the ventral side of the neck) or to the lower flank (following inoculation of the upper flank). In these models the immune system played a dominant role in virus clearance. The topically applied foscarnet could not prevent disease development in these models of recrudescent disease even when applied before the virus was detected in the skin, but a decrease in virus titre was obtained. Orally administered buciclovir lost efficacy when administered at the time of virus entry into the skin, i.e. 1 or 2 days before development of clinical signs. In the flank model, measuring lesion development, orally administered acyclovir also had a strongly decreased efficacy, when compared with its effect during infections in which lesion development did not involve translocation of virus through nerves. In the presence of developing immunity the inhibitors could not accelerate the clearance of virus from infected tissues. Furthermore, all treatments (topical foscarnet and oral buciclovir or acyclovir) were without effect on disease development when treatment was initiated on appearance of the first clinical signs of disease. As disease development following zosteriform spread of HSV resembles that in recurrent herpes in humans, and as the limited efficacy of the inhibitors observed resembles the poor results obtained with inhibitors of herpesvirus DNA synthesis in clinical studies on the treatment of symptomatic recurrent herpes, we suggest the use of animal models of zosteriform spread for pre-clinical evaluation of new antiherpes drugs.
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Inhibition of Transcription of Herpes Simplex Virus Immediate Early Genes in Interferon-treated Human Cells
More LessSummaryThe effect of interferon (IFN) treatment on the early stages of herpes simplex virus type 1 (HSV-1) replication in three types of human cells was investigated. Interferon pretreatment was shown to reduce the steady state levels of both total and polysome-bound HSV-1 immediate early α mRNAs. Using the nuclear run-off transcription assay, we showed that IFN selectively inhibited transcription of the HSV-1 genes, with no effect on transcription of total cellular RNA or that of the β-tubulin RNA. Thus, IFN appears to inhibit the initiation of HSV-1 α gene transcription rather than affect the stability of the respective mRNAs. IFN did not prevent the HSV-1-induced early shut-off of host cellular protein synthesis caused by a structural protein of the infecting virus. This observation indicated that the IFN-mediated inhibition of HSV-1 replication is at a stage beyond viral penetration into the cytoplasm. These results suggested that IFN blocked HSV-1 replication primarily at a very early stage, during the onset of α mRNA transcription.
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Induction of Gene Expression under Human Cytomegalovirus Immediate Early Enhancer-Promoter Control by Inhibition of Protein Synthesis Is Cell Cycle-dependent
More LessSummaryIn this paper we describe stably transfected rat cell lines which harbour either the human cytomegalovirus (HCMV) immediate early (IE) gene encoding the 72K IE nuclear antigen (IEA) or the bacterial chloramphenicol acetyltransferase (CAT) gene both under transcriptional control of the HCMV IE enhancer-promoter (-484 to -19 relative to the IE cap site, +1). In these cell lines IE gene or CAT gene expression is repressed but can be induced by heat-shock, by sodium arsenite and by inhibitors of protein synthesis such as cycloheximide (CH). In addition, we present evidence suggesting that CH-mediated activation is cell cycle-dependent. Thus CH-mediated induction of the 72K IEA as well as CAT gene expression was impaired and accumulation of mRNAs did not occur when cellular DNA synthesis was inhibited. Activation of IE genes by CH occurred almost exclusively in those cells which were in S-phase. In contrast, activation of gene expression by sodium arsenite occurred independently of cellular DNA synthesis and was not restricted to cells in S-phase. The data are consistent with, but not proof of, the hypothesis that the activation of IE transcription, brought about by inhibition of protein synthesis, resulted from a disturbed chromatin conformation due to DNA synthesis continuing in the absence of a supply of chromatin-organizing proteins. The possible relevance of these observations with regard to HCMV latency and reactivation is discussed.
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Prokaryotic Expression of Immunogenic Polypeptides of the Large Phosphoprotein (pp150) of Human Cytomegalovirus
SummaryThe large phosphorylated matrix protein pp150 of human cytomegalovirus (HCMV) is the polypeptide most frequently reactive in immunoblotting analyses with human antisera when compared with other viral proteins. Several defined regions of pp150 were expressed as β-galactosidase fusion proteins and these were tested for their immunoreactivity with human sera and their immunogenicity. One antigenic region could be expressed in large amounts and was found to carry immunodominant epitopes, as shown by immunoblotting and ELISA. A rabbit antiserum raised against recombinant pp150 antigens produced in bacteria proved to be useful for immunofluorescence and immunohistochemistry studies of HCMV-infected cells and tissues. The results suggest that this anti-pp150 serum will help to elucidate the process of virus assembly and antigen detection in infected cells.
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Characterization of a Human Cytomegalovirus Glycoprotein Complex (gcI)
More LessSummaryThree distinct families of glycoprotein complexes present in the envelopes of human cytomegalovirus and designated gcI, gcII and gcIII have been described recently. The synthesis of the gcI family was analysed using either inhibitors of glycoprotein processing and transport or endoglycosidase treatments of purified glycoproteins. The initial step in gcI synthesis involved the glycosylation of a 95K protein (p95) to form a high-mannose, simple N-linked glycoprotein of M r 158K (gp158), which was detected only in the presence of the glycoprotein processing inhibitor castanospermine. This intermediate was rapidly trimmed in the virus-infected cell to form a more stable simple N-linked precursor glycoprotein of M r 138K (gp138). Treatment of either gp158 or gp138 with endoglycosidase H produced p95. Both molecules, gp158 and gp138, were found in disulphide-linked complexes which are presumably infected cell precursors to gcI since they were not found in virions. The processing of these complexes involved complete cleavage of gp138 and conversion of some but not all of its oligosaccharide to complex N-linked chains. Both processing events were inhibited by the ionophore monensin. Mature gcI contained the gp138 cleavage product, gp55, in a disulphide-linked complex with a heterogeneous glycoprotein designated gp93-130. The latter glycoprotein could be separated into two electrophoretic forms, gp93 and gp130. The deglycosylated form of gp55 had a discrete banding pattern with an apparent M r of 46K (p46). In contrast, the deglycosylated forms of gp93 and gp130 had diffuse banding patterns with apparent M r values of 46K to 56K (p46–56) and 60K to 70K (p60–70) respectively. Peptide profiles comparing gp93 with gp130 indicated that they have highly similar polypeptide backbones. Since the deglycosylated forms of gp55 and gp130, 46K and 60K to 70K, respectively, together exceed the 95K precursor/deglycosylated intermediate in M r, we propose that the above glycoproteins are derived by an alternative proteolytic cleavage of the precursor. The heterogeneous electrophoretic properties of the deglycosylated forms of gp93 and gp130 may be due to additional post-translational modifications other than glycosylation.
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Characterization of the Serological Response in Man to the Latent Membrane Protein and the Six Nuclear Antigens Encoded by Epstein-Barr Virus
More LessSummaryA total of 116 sera from healthy individuals and from patients with Burkitt’s lymphoma (BL), nasopharyngeal carcinoma (NPC) or rheumatoid arthritis (RA) were studied with respect to antibody responses to each of the seven known transformation-associated Epstein-Barr virus (EBV)-encoded antigens [latent membrane protein (LMP) and six nuclear proteins (EBNAs 1 to 6)]. The antibodies were detected using modified standard immunoblotting techniques. Antibodies to LMP were detected for the first time in sera from 6/27 (22%) healthy, EBV-immune individuals (seropositive for the viral capsid antigens). An increased incidence of anti-LMP antibodies was found in EBV-immune sera from patients with BL (17/24 positive; 71%), NPC (21/33; 64%), and RA (16/21; 76%). Antibodies to EBNA 1 were detected in all EBV-immune sera at a standard 1:20 dilution. Antibodies to the other EBNAs were detected in only a proportion of these sera (20 to 95%) at the same dilution. Only minor disease-associated differences in the incidence of these antibodies were observed, the most consistent being that RA sera had a higher incidence of antibodies to EBNAs 2 to 6 compared with healthy controls. Testing of the sera at a 1:100 dilution suggested that there were some disease-related differences in the titres of anti-EBNA antibodies. At this serum dilution, a reduced incidence of antibodies to EBNA 2 was seen in NPC (6/31) compared with RA (18/19) and healthy EBV-seropositives (16/26); antibodies to EBNA 3 were detected at an increased incidence in BL (8/15) and NPC (16/31) compared with control sera (7/26); antibodies to EBNA 4 were detected at increased incidence in BL (5/15) and RA (6/19) compared with control sera (1/26); and antibodies to EBNA 6 were detected at increased incidence in NPC (19/31) and RA (7/19) compared with control sera (3/26).
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The Response of Infants with Bronchiolitis to the Proteins of Respiratory Syncytial Virus
More LessSummaryAcute phase sera were collected from 28 infants hospitalized with bronchiolitis due to respiratory syncytial (RS) virus and convalescent sera were collected from 24 of them. The sera were assayed for neutralizing antibodies by plaque inhibition, for antibodies to the viral proteins by Western blot against partially purified RS virus, and for their ability to inhibit attachment and fusion. Among the 28 acute phase sera, 27 had antibody to the attachment glycoprotein (G), 16 had antibody to the fusion glycoprotein (F), but none had antibody to the matrix protein (VPM). Both the geometric mean anti-G titre, and the geometric mean anti-F titre correlated with the 50% neutralizing dose (ND50) titre in the acute phase serum. Among the 24 convalescent sera, only four exhibited an increase in neutralizing antibody titre. The response to G appeared to be related to the acute phase ND50 titre. Of 17 infants with acute phase titres of less than 100 ND50/ml, 10 responded to G while there was no response to this protein in seven infants with acute phase titres greater than 100 ND50/ml. While only one infant responded to F, 18 responded to the phosphorylated nucleocapsid protein, VP32, and none responded to VPM. The ability of the acute phase sera to inhibit virus attachment to HeLa cells and to inhibit fusion correlated with the anti-G titre and the anti-F titre, respectively. However, there was no correlation between the inhibition of fusion and the anti-F titre in the convalescent sera, almost all of which inhibited fusion. These results suggest that the infected infants were responding to RS virus, but that their response to the viral proteins was either masked or slowed by residual maternal antibody. The inability to detect VPM in the acute and convalescent phase sera, as well as in 20 paired maternal and cord sera at a 1:50 dilution suggested that VPM, although it is one of the most prevalent viral proteins in both the virion and the infected cell, may be poorly antigenic in humans.
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Reduction of Yellow Fever Virus Mouse Neurovirulence by Immunization with a Bacterially Synthesized Non-structural Protein (NS1) Fragment
More LessSummaryPart of a yellow fever virus-specified non-structural protein (NS1) was expressed in Escherichia coli as a fusion protein with β-galactosidase. Immunization of mice with this partially purified NS1-β-galactosidase fusion protein induced yellow fever virus-specific antibodies and provided some protection against intracerebral challenge with the virus.
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Association between the pH-dependent Conformational Change of West Nile Flavivirus E Protein and Virus-mediated Membrane Fusion
More LessSummaryThe major envelope protein (E) of West Nile virus mediates fusion between the membranes of the viral envelope and the target cell at optimum pH values of just below neutrality. The fusion is critical for the entry mechanism, allowing virus to escape from the acidic endosomal compartment. To define the role of the viral E protein in the fusion reaction, the conformational change in E and concomitant change of viral infectivity were studied quantitatively, using protease digestion of the E protein and assay of viral infectivity. The results showed that the conformational change occurred in a pH-dependent manner with an upper threshold of pH 7.0 and maximum conversion occurring at pH 6.4 and below. The conversion was rapid and reached a half-maximal value within 15 s after acidification. The exposure of free or cell-bound virions to acid pH resulted in the loss of infectivity in an almost identical pH-dependent manner. Based on these findings, it is suggested that there are two distinct viral modes of entry into macrophages, i.e. infectious endocytosis and non-infectious viral fusion with plasma membranes, with the pH of the extracellular medium determining which of these predominates. The implications of these observations for the role of the E protein in membrane fusion and the probable localization of fusion epitopes are discussed.
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Protein Coding Assignment for the Genome of Epizootic Haemorrhagic Disease Virus
More LessSummaryViral genomic RNA was purified from BHK-21 cells infected with epizootic haemorrhagic disease virus and the 10 dsRNA genome segments were isolated by polyacrylamide gel electrophoresis. These genome segments were translated in vitro using the rabbit reticulocyte lysate system and the synthesized proteins were detected by immune precipitation and gel electrophoresis. This allowed the assignment of protein coding to the genome segments and the identification of two additional virus-specified proteins not readily detectable in lysates of virus-infected cells.
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Analysis of the L1 Gene Product of Human Papillomavirus Type 16 by Expression in a Vaccinia Virus Recombinant
More LessSummaryThe L1 open reading frame of human papillomavirus type 16 (HPV16) has been expressed in vaccinia virus under the control of both the 7.5K early and late promoter, and the 4b major late promoter. Antibodies to a β-galactosidase fusion protein containing a C-terminal portion of the HPV16 L1 gene product were used to compare the levels of L1 expression in the two recombinants, and showed that greater levels of expression were obtained when the gene was placed under the control of the 4b late promoter. Immunofluorescence studies revealed a nuclear location of the L1 gene product when expressed in vaccinia virus. Antibodies to the β-galactosidase fusion protein detected a major polypeptide species of 57K and a minor species of 64K in Western blots of recombinant-infected cell lysates. The 64K species was not detected when cells were infected in the presence of tunicamycin, indicating that the primary translation product of the HPV16 L1 open reading frame is modified by N-linked glycosylation when expressed in vaccinia virus. Whereas antibodies to HPV16 L1 fusion proteins and to a peptide containing amino acids from the C terminus of HPV16 L1 reacted well in Western blots with the HPV16 L1 target expressed in vaccinia virus, no reactivity was observed with antibodies to bovine papillomavirus type 1 particles or to a HPV6b fusion protein.
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Comparison of a Conserved Region in Fowlpox Virus and Vaccinia Virus Genomes and the Translocation of the Fowlpox Virus Thymidine Kinase Gene
More LessSummaryThe DNA sequence of a clustered set of genes which are conserved in orthopoxviruses has been determined for the avipoxvirus, fowlpox virus. The arrangement of the genes in fowlpox virus is nearly identical to that in vaccinia virus, and genes which are overlapping in vaccinia virus overlap in fowlpox virus. One major difference exists however, as the thymidine kinase (TK) gene is absent in fowlpox virus from the position it occupies within this cluster of genes in vaccinia virus. Instead, in fowlpox virus there is a 32 bp non-coding region present between the genes that flank the TK gene in vaccinia virus. The fowlpox virus TK gene has been cloned and sequenced. The sequences immediately flanking the TK gene show no homology to any previously reported poxvirus gene. These results are discussed in terms of genome stability in poxviruses and the use of the TK gene as a non-essential region for the introduction of foreign genes into poxviruses.
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Physical Maps and Comparative DNA Hybridization of Mamestra Brassicae and Panolis Flammea Nuclear Polyhedrosis Virus Genomes
More LessSummaryThe genome DNAs from the Panolis flammea (Pf) multiple nucleocapsid nuclear polyhedrosis virus (MNPV) and the Mamestra brassicae (Mb) MNPV were analysed with the restriction endonucleases BamHI, BglII, KpnI, HindIII, SmaI and XhoI. The profiles produced by each enzyme for the two virus genomes were quite dissimilar with very few comigrating fragments. The size of PfMNPV DNA was calculated to be 145 kilobase pairs (kbp) and that of MbMNPV 150 kbp. Physical maps of the two genomes were constructed utilizing the above enzymes. The two maps were oriented in relation to their putative polyhedrin genes. Alignment of the two restriction maps for PfMNPV and MbMNPV was achieved by performing cross blot hybridization between XhoI digests of the two virus genomes. This showed that despite differences in the physical maps the two genomes shared overall similarity in gene organization. The two viruses were also compared using dot blot hybridization analysis to quantify homology with Autographa californica (Ac) MNPV. These data showed that both PfMNPV and MbMNPV were distantly related to AcMNPV but exhibited a high degree of homology to each other (nearly 100% in 20% formamide, 70% in 50% formamide).
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Isolation, Complementation and Partial Characterization of Temperature-sensitive Mutants of the Baculovirus Hyphantria Cunea Nuclear Polyhedrosis Virus
More LessSummaryTwelve temperature-sensitive mutants were isolated from Hyphantria cunea nuclear polyhedrosis virus grown in Spodoptera frugiperda cells and were sorted into four groups by their properties in plaque assays at the non-permissive temperature (32 °C). The phenotypes of the four groups were as follows: (i) failure to make polyhedra, (ii) few polyhedra formed, (iii) reduced plaquing efficiency, (iv) small plaque size. Ten mutants had reduced plaque size and polyhedra formation at 32 °C. One mutant formed plaques without polyhedra, had a reduced infectious virus titre at 32 °C and also showed a defect in late gene function. Two mutants formed small plaques with few polyhedra and were temperature-sensitive with respect to production of extracellular non-occluded virions at 32 °C. Other phenotypes were also distinguished. The formation of polyhedra by all the mutants was 2 to 4 h faster at 32 °C than at 25 °C. After temperature shift-up from 25 °C to 32 °C at 12 h post-infection polyhedron formation was still 2 to 4 h faster. Complementation analyses based on polyhedron formation in double infections at 32 °C distinguished four complementation groups.
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The Genome of the Multicapsid Baculovirus of Orgyia Pseudotsugata: Restriction Map and Analysis of Two Sets of GC-rich Repeated Sequences
More LessSummaryFive cosmids containing inserts that comprise the complete genome of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata were mapped with four restriction enzymes (BglII, ClaI, SstI, XhoI). From these cosmid maps, composite maps of the complete genome were constructed for each restriction enzyme. A region containing repeats of the sequence GGC downstream of the polyhedrin gene was used to probe the genome. It cross-hybridized with a region which, upon sequence analysis, was found to be a highly repetitive GC-rich region of nearly 500 nucleotides. The two GC-rich regions appeared to be evolutionarily unrelated.
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The Complete Nucleotide Sequence of the Genome of a Hepatitis B Virus Isolated from a Naturally Infected Chimpanzee
More LessSummaryThe complete nucleotide sequence of a strain of hepatitis B virus, originally isolated from a naturally infected chimpanzee, has been determined. Interesting features of the sequence include the presence of an in-phase stop codon in the ‘pre-core’ region of the core antigen open reading frame. The sequence shows approximately 10% nucleotide divergence from all of the other hepatitis B virus sequences previously published and the possibility that this divergence is the result of passage through chimpanzees is discussed.
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Partial Nucleotide Sequence and Deduced Amino Acid Sequence of the Structural Proteins of Dengue Virus Type 2, New Guinea C and PUO-218 Strains
More LessSummaryThe nucleotide sequence and the deduced amino acid sequence for the genes encoding the structural proteins of two strains of dengue virus type 2 (DEN-2) were determined from cDNA clones. The genes for C, prM(M) and E proteins were sequenced for the prototype DEN-2 virus, the New Guinea C strain. Also sequenced were the prM(M) and E genes of PUO-218. This strain of DEN-2 was isolated during 1980 in Bangkok and had received a limited number of laboratory passages. Comparisons of the newly determined sequences with those published for the Jamaica 1409 and Puerto Rico PR-159 (S1 vaccine candidate) strains revealed a close relationship between New Guinea C virus and both the Jamaica and PUO-218 viruses (greater than 96% similarity in nucleotides of the E gene), whereas S1 virus was the most divergent.
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Influence of Interferons α11 and γ and of Tumour Necrosis Factor on Persistent Infection with Bovine Viral Diarrhoea Virus in Vitro
More LessSummaryNon-cytopathic strains of bovine viral diarrhoea virus (BVDV) readily establish persistent infections in cells of bovine origin. The involvement of endogenous interferon (IFN) on the maintenance of the infection level, as well as the effect of exogenous IFN and tumour necrosis factor alpha (TNF-α), was studied. Although exogenous IFN suppressed the spread and replication of virus, it did not cure the infection, even when continuously present over many cell passages. TNF-α alone had no antiviral effect in this system. However, both TNF-α and IFN enhanced the cytopathic effect of cytopathic BVDV, and induced a BVDV-like cytopathic effect in cells infected with non-cytopathic BVDV. These data are discussed with regard to possible mechanisms of pathogenesis during fatal BVDV disease in cattle.
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Effect of Herpes Simplex Virus Type 2 Infection on Mitochondrial Gene Expression
More LessSummaryA cDNA clone derived from the mitochondrial cytochrome oxidase I gene has been used to show that the level of mitochondrially encoded RNA species declines during herpes simplex virus type 2 infection in a manner similar to that for RNA species derived from nuclear genes. In contrast to the situation for nuclear genes, however, no change in the transcription rate of the mitochondrial genome during infection was detected, indicating that post-transcriptional processes alone are responsible for the decline in the levels of mitochondrial RNA species during infection. Two stages in this post-transcriptional degradation have been defined, only one of which is dependent upon viral protein synthesis in the infected cell.
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Protection of Mice from Lethal Infection with Aujeszky’s Disease Virus by Immunization with Purified gVI
More LessSummaryVirus envelope glycoprotein gVI (gp50) of Aujeszky’s disease virus (ADV) was purified from a Nonidet P40-solubilized lysate of ADV-infected Vero cells by immunoaffinity chromatography using a monoclonal antibody against gVI. Mice immunized by the purified gVI produced virus-neutralizing antibody and were successfully protected against subsequent challenge with ADV.
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One Defective Interfering Particle per Cell Prevents Influenza Virus-mediated Cytopathology: An Efficient Assay System
More LessSummaryThe titre of defective interfering (DI) influenza virus measured by an assay based on the inhibition of cytopathology caused by A/WSN (H1N1) influenza virus in MDCK cells was 320000-fold greater than titres measured by inhibition of infectious centre formation. Interference was less in other types of cell. By electron microscopy, we have shown that the ratio between physical particles and DI units in preparations of the DI virus was approximately unity, which suggested that one or few DI particles is/are required to confer resistance of a MDCK cell to viral cytopathology. This human DI virus interfered heterotypically with an avian H7N1 influenza virus.
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Electron Microscopic Evidence for Bridges between Bovine Respiratory Syncytial Virus Particles
More LessSummaryElectron microscopic examination of ultrathin sections of a continuous cell line of ovine kidney (OK) origin, infected by bovine respiratory syncytial virus (BRSV), revealed the presence of well defined bridges between virus particles. This is the first report of this novel structure. Observation of ultrathin sections of human RSV Long strain also grown on OK cells did not show inter-particle bridges and therefore suggested that this structure could be specific to BRSV. The biological significance of these bridges is not clear at this time; a possibility is that the bridges are formed by the fusion protein of BRSV which is known to cause cell fusion. Besides the structural implications, the importance is in relation to purification strategies for this virus, which must now take into account that most of the viral particles occur in large aggregates.
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Ultrastructure of Human Immunodeficiency Virus Type 2
More LessSummaryThe ultrastructure of human immunodeficiency virus type 2 (HIV-2) was determined by negative stain and thin section electron microscopy (EM). Some virus particles had surface projections about 10 nm in length which were evenly spaced. Nonidet P40-treated particles which were penetrated by stain revealed a distinctive off-centre cone-shaped core and, in addition, free-lying cores were also seen in detergent-treated preparations. The surface of the cores was composed of a layer of small subunits. The structure of HIV-2 determined by thin section EM was the same as that deduced by negative stain EM.
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Transformation of Rat 3Y1 Cells by a Deletion DNA of Human Papillomavirus Type 16 Molecularly Cloned from Genomic DNA of a Cervical Carcinoma
More LessSummaryHuman papillomavirus type 16 (HPV 16) DNA integrated within the cell DNA in cervical carcinomas is frequently defective, but commonly retains its putative transcriptional control region and E6 and E7 open reading frames (ORFs) intact. One clone of such a partially deleted HPV 16 DNA was tested and found to be transforming for rat 3Y1 cells. The result indicates that HPV 16 DNA containing E6 and E7 ORFs with the homologous promoter is sufficient for inducing focal transformation of immortalized rat fibroblasts.
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Is Vertical Transmission Sufficient to Maintain Junin Virus in Nature?
More LessSummaryThe quantitative contribution of vertical transmission to the prevalence rate of Junin virus infection in subsequent generations of its natural reservoir, Calomys musculinus, was analysed. Data on mortality and reproduction of C. musculinus infected at birth with a wild strain of Junin virus were used to estimate the infection-dependent relative survival rate (β = 0.4849) and relative fertility of the infected host (α = 0.2088). Prevalence rates of infection, obtained by mathematical simulation in optimal conditions of vertical transfer, dropped steadily to zero in a few generations. Vertical transmission was found to be insufficient to overcome the effect of highly depressed survival and fertility of the infected host and maintain a stabilized prevalence of Junin virus infection in successive generations; this suggested that viral maintenance is mainly dependent upon horizontal transmission.
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The Use of Monoclonal Antibodies to Detect Wheat Soil-borne Mosaic Virus
More LessSummaryStable hybridoma cell lines secreting monoclonal antibodies (MAbs) to wheat soilborne mosaic virus (WSBMV) were produced by fusing spleen cells of immunized BALB/c mice and mouse myeloma cell line P3-X63-Ag8.653. Hybridoma clones produced antibodies of the IgG2a, IgG2b and IgG3 subclasses. Epitope analysis suggested that all the antibodies tested reacted with the same or closely adjacent antigenic sites. The MAbs reacted with particles of four isolates of WSBMV, but not with particles of 13 other viruses. The serological reactivities of the MAbs were compared with those of rabbit polyclonal antibodies for the detection of WSBMV in leaf tissue by ELISA. ELISA using both polyclonal antibodies and MAbs was superior to assays using either source of antibody alone. The MAbs of the IgG2b subclass worked well in a Protein A sandwich ELISA, and a dot immunobinding assay using only MAbs was also satisfactory for the detection of WSBMV.
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The Use of Cloned Sequences for the Identification of Coconut Foliar Decay Disease-associated DNA
More LessSummarySingle-stranded circular DNA associated with foliar decay disease of coconut palm in Vanuatu (FDD-DNA) has been purified and three fragments have been cloned in plasmid pUC19. Clones labelled with 32P by nick translation were used as specific probes for FDD-DNA in dot blot and Southern transfer hybridization assays. These assays were more sensitive than the polyacrylamide gel electrophoresis assay developed previously for diagnosis of FDD. Hybridization tests showed that FDD-DNA had no detectable sequence similarity to the geminivirus infecting Digitaria sanguinalis which grows alongside FDD-infected coconut palms; that Hibiscus tiliaceus, which is the host plant of the vector of FDD, Myndus taffini, is not infected with FDD-DNA; and that the symptomless coconut variety, Vanuatu Tall, is susceptible to infection with FDD-DNA. Native FDD-DNA migrates as two distinct bands in polyacrylamide gels, and both hybridize to the probe. Previous estimates and data presented here show that FDD-DNA is approximately half the size of the D. sanguinalis geminivirus DNA (approx. 2350 nucleotides) and support the view that FDD is caused by a virus not typical of any plant virus taxonomic group.
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Plasmid DNA Containing a Copy of RNA3 Can Substitute for RNA3 in Alfalfa Mosaic Virus RNA Inocula
More LessSummaryThe infectivity of a DNA copy of RNA3 from strain S of alfalfa mosaic virus inserted in a pT7-1 plasmid vector (clone 3pT7-1) was tested as circular DNA by co-inoculation with RNAs 1, 2 and 4 from strain B (mix I0) on young tobacco leaves. Virus multiplication was monitored by serological detection of the coat protein. Progeny RNA3 derived from circular plasmid DNA 3pT7-1 was of the same length as naturally occurring RNA3-S. Analysis by primer extension demonstrated that it had the 5′-terminal sequence of RNA3-S and not that of RNA3-B. Mix I0 also became infectious when mixed with M13 single-stranded DNA carrying RNA3-S inserted in the (-) strand orientation, probably because traces of RNA3-B present in mix I0 were protected from degradation by base-pairing and were therefore active.
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Characterization of a Subgenomic DNA Isolated from Triticum Aestivum Plants Infected with Wheat Dwarf Virus
More LessSummaryA subgenomic DNA has been isolated from wheat tissue infected with a Swedish isolate of wheat dwarf virus and cloned. Restriction endonuclease analysis and nucleotide sequence determination indicated that the subgenomic DNA (1472 nucleotides) was derived from the genomic DNA (2749 nucleotides) by two separate deletions. The subgenomic DNA had lost open reading frames (ORFs) encoding the virus coat protein and a putative protein of M r 10146, but retained an ORF and an open reading region encoding putative proteins of M r 30156 and 17292, respectively, and two structural features thought to be important for virus DNA replication i.e. a potentially stable stem-loop structure containing the conserved TAATATTAC sequence and a putative primer initiation site.
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Priming of Complementary DNA Synthesis in Vitro by Small DNA Molecules Tightly Bound to Virion DNA of Wheat Dwarf Virus
More LessSummaryDNA isolated from purified preparations of wheat dwarf virus (WDV) has been shown to contain tightly bound small DNA molecules which can act as primers for the synthesis of full-length complementary DNA in vitro. The small DNA molecules are bound in the terminating intergenic region of the WDV genome between the end of an open reading frame encoding a putative protein of M r 17292 and a conserved ‘A-T’ box containing putative transcriptional polyadenylation signals. Evidence that the small DNA molecules contain ribonucleotides at their 5′ termini is presented and their possible role in the priming of virus DNA synthesis in vivo is discussed.
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Identification of the Coat Protein Gene of Tomato Golden Mosaic Virus
SummaryAn open reading frame, designated AR1, on DNA component A of tomato golden mosaic virus has been identified as the virus coat protein gene on the basis of the molecular weight and amino acid composition of the virus capsid polypeptide, the mass spectra and N-terminal sequences of peptides produced by cyanogen bromide cleavage of the capsid polypeptide and by the binding of antibodies, induced by immunization of a rabbit with a β-galactosidase-AR1 fusion protein, to the capsid polypeptide in a Western blot.
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Transcriptional Mapping of the Coat Protein Gene of Tomato Golden Mosaic Virus
More LessSummaryPolyadenylated RNA was isolated from Nicotiana benthamiana plants infected with tomato golden mosaic virus (TGMV). Northern hybridization with a strand-specific probe of cloned TGMV DNA A revealed a 0.9 kb transcript with the same orientation as virion DNA. The positions of the 5′ and 3′ termini of the transcript, which were mapped by nuclease protection and primer extension techniques, indicated that it corresponded to the virus coat protein mRNA and enabled the likely functional promoter and polyadenylation signals to be identified.
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The Fate of the Transport Protein of Tobacco Mosaic Virus in Systemic and Hypersensitive Tobacco Hosts
SummaryThe transport protein of tobacco mosaic virus (TMV) (M r 30000, 30K non-structural protein) was detected on Western blots, using an antiserum to a synthetic C-terminal nonapeptide. Accumulation of this protein in subcellular fractions of inoculated leaves was measured during TMV infection of Nicotiana tabacum cv. Samsun and cv. Samsun NN. In cv. Samsun, a systemic host, the 30K protein appeared transiently in a crude membrane fraction but accumulated more stably in cell walls. In cv. Samsun NN, which is a hypersensitive host giving only localized infection, the early accumulation (up to 40 h; before any necrosis was visible) was the same as in cv. Samsun. However, as soon as necrosis was visible, the amount of 30K detected in the cell wall fraction decreased sharply and coat protein synthesis stopped. This drop in the amount of 30K protein is most easily interpreted as a side-effect of the hypersensitive reaction and may explain why TMV infection becomes localized in leaves of cv. Samsun NN.
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