- Volume 73, Issue 11, 1992
Volume 73, Issue 11, 1992
- Articles
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- Animal
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Nucleotide sequence and transcriptional analysis of the polyhedrin gene of Spodoptera exigua nuclear polyhedrosis virus
More LessThe nucleotide sequence of a 1.1 kbp fragment of the multiple nucleocapsid nuclear polyhedrosis virus (MNPV) of Spodoptera exigua (Se) containing the polyhedrin gene was determined. An open reading frame (ORF) of 738 nucleotides (nt) was detected. This ORF encoded a protein of 246 amino acids with a predicted M r of 29K. The nucleotide and amino acid sequences were compared with the sequences of eight other NPV polyhedrins. The SeMNPV polyhedrin protein was most closely related to S. frugiperda MNPV polyhedrin with differences in only five amino acids, and most distantly related to the Lymantria dispar MNPV polyhedrin. The size of the mRNA was approximately 1000 nt, as determined by Northern blot analysis. Using primer extension assays and S1 nuclease mapping the transcriptional start and stop sites of the polyhedrin mRNA were located. The 5′ regulatory sequence appeared to be 44 nt in length with the mRNA start site predominantly at the first A of the TAAG consensus start sequence. Two degenerate poly(A) signals were found immediately downstream of the translational stop signal. The transcriptional stop was located approximately 230 nt downstream from the translational stop signal, in an AT-rich sequence that appears to be common to all baculovirus poly-hedrin genes. The SeMNPV polyhedrin mRNA does not appear to be polyadenylated.
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Identification of viral structural polypeptides of Thogoto virus (a tick-borne orthomyxo-like virus) and functions associated with the glycoprotein
More LessThogoto (THO) virus is a tick-borne virus which shares morphological and genetic features with members of the Orthomyxoviridae family although the viral glycoprotein appears to be related to gp64 of baculoviruses. Characterization of THO virus was undertaken to clarify its taxonomic position. Purified virus preparations contained at least six virus-encoded polypeptides with apparent M r values ranging from 29K to 92K. A 75K polypeptide was identified as an envelope-associated glycoprotein by Triton X-100 and salt dissociation studies, and by proteolytic degradation of the exposed proteins of the virion. By the same criteria, the nucleoprotein and the matrix protein were identified as the 52K and 29K polypeptides, respectively. Immunofluorescence studies using monoclonal antibodies (MAbs) located the glycoprotein on the external cell membrane and the nucleoprotein in the nucleus of infected cells indicating that virus replication involved a nuclear phase. In addition, the virus displayed haemagglutination and haemolytic activities with an optimum at pH 6. These activities are functions of the viral glycoprotein since they were inhibited by antiglycoprotein MAbs. The data reported here support the notion that THO virus is a member of the Orthomyxoviridae family but that it should be classified in a group distinct from the other influenza viruses.
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Fusion of influenza virus particles with liposomes: requirement for cholesterol and virus receptors to allow fusion with and lysis of neutral but not of negatively charged liposomes
More LessInfluenza virus particles are able to fuse with liposomes composed of negatively charged or neutral phospholipids, as shown by using fluorochrome-labelled virions and fluorescence dequenching methods. Fusion with liposomes composed of only phosphatidylcholine (PC) was dependent on the presence of cholesterol (Chol), whereas fusion with liposomes containing negatively charged phospholipids, such as phosphatidylserine (PS), or of PC and phosphatidylethanolamine (PE) occurred in the absence of Chol. Fusion of influenza virions with PC:Chol liposomes was observed at pH 5.0, but not at pH 7.4, whereas a low degree of fusion with negatively charged liposomes or those containing PE was observed at pH 7.4. In addition, non-fusogenic influenza virions or HA0 influenza virions fused with PS- or PE-containing liposomes, especially at pH 5.0. Influenza virus particles were also able to induce the release of the fluorochrome calcein from negatively charged calcein-loaded liposomes at pH 5.0, as well as at pH 7.4, but failed to do so with PC:Chol liposomes. Lysis of PC:Chol by influenza virions was dependent on the presence of virus receptors, namely gangliosides (sialoglycolipids), and was observed only at pH 5.0. The results show that fusion of influenza virions with negatively charged or PE-containing liposomes does not reflect the biological activity of the virus needed for penetration and infection of living cells. On the other hand, fusion with PC:Chol liposomes is probably due to the activity of the viral fusion protein, the haemagglutinin glycoprotein.
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Partial dissociation of subgroup C phenotype and in vivo behaviour in feline leukaemia viruses with chimeric envelope genes
Feline leukaemia viruses (FeLVs) are classified into subgroups A, B and C by their use of different host cell receptors on feline cells, a phenotype which is determined by the viral envelope. FeLV-A is the ubiquitous, highly infectious form of FeLV, and FeLV-C isolates are rare variants which are invariably isolated along with FeLV-A. The FeLV-C isolates share the capacity to induce acute non-regenerative anaemia and the prototype, FeLV-C/Sarma, has strongly age-restricted infectivity for cats. The FeLV-C/Sarma env sequence is closely related to that of common, weakly pathogenic FeLV-A isolates. We now show by construction of chimeric viruses that the receptor specificity of FeLV-A/Glasgow-1 virus can be converted to that of FeLV-C by exchange of a single env variable domain, Vr1, which differs by a three codon deletion and nine adjacent substitutions. Attempts to dissect this region further by directed mutagenesis resulted in disabled proviruses. Sequence analysis of independent natural FeLV-C isolates showed that they have unique Vr1 sequences which are distinct from the conserved FeLV-A pattern. The chimeric viruses which acquired the host range and subgroup properties of FeLV-C retained certain FeLV-A-like properties in that they were non-cytopathogenic in 3201B feline T cells and readily induced viraemia in weanling animals. They also induced a profound anaemia in neonates which had a more prolonged course than that induced by FeLV-C/Sarma and which was macrocytic rather than non-regenerative in nature. Although receptor specificity and a major determinant of pathogenicity segregate with Vr1, it appears that sequences elsewhere in the genome influence infectivity and pathogenicity independently of the subgroup phenotype.
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Analysis of a 9.6 kb sequence from the 3′ end of canine coronavirus genomic RNA
More LessWe have analysed the organization of the 3′ end of the genomic RNA of canine coronavirus (CCV), a virus which has a close antigenic relationship to transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV) and feline infectious peritonitis virus (FIPV). Genomic RNA isolated from CCV strain Insavc-1-infected A72 cells was used to generate a cDNA library. Overlapping clones, spanning approximately 9.6 kb [from the 3′ end of the polymerase gene, 1b, to the poly(A) tail] were identified. Sequencing and subsequent analyses revealed 10 open reading frames (ORFs). Three of these code for the major coronavirus structural polypeptides S, M and N; a fourth codes for a small membrane protein, SM, a putative homologue of the IBV structural polypeptide 3c, and five code for polypeptides, designated 1b, 3a, 4, 7a and 7b, homologous to putative non-structural polypeptides encoded in the TGEV or FIPV genomes. An extra ORF which had not hitherto been identified in this antigenic group of coronaviruses was designated 3x. Pairwise alignment of these ORFs with their counterparts in TGEV, PRCV and FIPV revealed high levels of identity and highlighted the close relationship between the members of this group of viruses.
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Evidence of genomic variations between infectious pancreatic necrosis virus strains determined by restriction fragment profiles
More LessInfectious pancreatic necrosis virus (IPNV) is the aetiological agent of an important disease in hatchery-reared salmonid fish in North America, Europe and Japan. It belongs to the family Birnaviridae and shows a high degree of antigenic heterogeneity. However, genomic variations between the 10 identified serotypes have not yet been studied. In order to correlate genomic heterogeneity with the different serotypes, oligonucleotides were synthesized according to the published sequence of the Jasper strain (serotype A9). They were used as primers for the amplification of a 359 bp cDNA fragment of the viral genome using the polymerase chain reaction. Fragments amplified from 37 strains were digested with five different restriction enzymes. Restriction fragment profiles obtained on agarose gels showed heterogeneity not only between strains of different serotypes, but also among those belonging to serotype A1. A cluster analysis of the restriction patterns showed that IPNV strains can be divided into three major groups, corresponding approximately to serotypes A1, A2 and A3, and 10 subgroups which do not correlate with the serotyping of the strains.
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Bovine polyomavirus, a cell-transforming virus with tumorigenic potential
More LessThe early region of bovine polyomavirus (BPyV) was tested for its cell transformation potential employing an assay of dense focus formation. Dense foci of morphologically transformed cells were observed upon transfection of primary rodent cells with a plasmid construct encoding the complete early region of BPyV under the transcriptional control of the long terminal repeat of Rous sarcoma virus. No transformation of primary rodent cells was observed upon transfection of these cells with a plasmid encoding the complete early region of BPyV under the control of its own transcriptional regulatory sequences. In BPyV-transformed cells, the viral sequences had become integrated into the cellular genome, and expression of large T antigen could be detected in a high percentage of cells. The transformed cells were demonstrated to be capable of anchorage-independent growth and to be oncogenic in immunocompromised newborn rats. Therefore BPyV should be considered as a potentially tumorigenic polyomavirus. Since many commercial batches of calf serum have been shown to be contaminated with BPyV, our observations may have implications for the use of calf serum in cell culture.
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Analysis of splice sites in the early region of bovine polyomavirus: evidence for a unique pattern of large T mRNA splicing
More LessThe genetic organization of the early region of bovine polyomavirus (BPyV) was studied by analysis of the splice sites used in early mRNA maturation, using reverse transcription-polymerase chain reaction and DNA sequencing techniques. When compared to other polyomaviruses, the BPyV early region appears to have an uncommon organization. In the major early mRNA molecule two small intron sequences of 71 and 77 nucleotides, separated from one another by an 80 nucleotide exon sequence, were identified. Through splicing out both introns, a mRNA molecule is generated that contains an open reading frame with the capacity to encode 619 amino acids. Comparisons with the simian virus 40 large T antigen suggested that this mRNA molecule encodes the BPyV large T antigen. Remarkably, no mRNA product encoding a protein with a size comparable to that of the small t antigens of other polyomaviruses was detected. Another transcript was observed from which only the 77 nucleotide intron sequence had been removed, thereby creating a mRNA molecule with the capacity to encode only 45 amino acids. Whether this mRNA product represents a mature transcript which is translated in BPyV-infected cells or is an intermediate in the formation of the large T mRNA molecule is not known. Analysis of BPyV-specific early mRNA products isolated from BPyV-transformed murine cells revealed only the amplification product representing the putative large T antigen transcript.
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Nucleotide sequence of 21.8 kbp of variola major virus strain Harvey and comparison with vaccinia virus
More LessA 21.8 kbp region of the genome of variola major virus (strain Harvey), a virus that caused haemorrhagic-type smallpox, has been sequenced and shown to possess 96% nucleotide identity to the corresponding region of vaccinia virus, the smallpox vaccine. Overall the gene arrangement in the two viruses is highly similar and individual open reading frames (ORFs) display a high degree of amino acid identity, for instance 26 of the 32 variola virus ORFs have ≥90% identity with their vaccinia virus counterparts. A remarkable difference is the disruption of seven vaccinia virus ORFs into small fragments in variola virus. These include the variola virus homologue of vaccinia virus SalF2R, which encodes a protein related to C-type animal lectins, and SalF7L, which encodes an active 3β-hydroxysteroid dehydrogenase enzyme that contributes to vaccinia virus virulence. Upstream of the variola virus haemagglutinin gene there is a deletion of 1910 bp so that the equivalent of vaccinia virus gene SalF17R is truncated, and SalF16R, which shows amino acid similarity to the tumour necrosis factor receptor, is absent. The region sequenced includes the genes for thymidylate kinase and DNA ligase both of which are active in vaccinia virus and are highly conserved in variola virus. Other conserved ORFs with interesting homologies are those encoding profilin, superoxide dismutase and part of guanylate kinase. Two vaccinia virus genes encoding glycoproteins of the outer envelope of extracellular enveloped virus are also conserved in variola virus and this homology is likely to have contributed to the immunological protection which vaccinia virus evoked against smallpox. Lastly, there are multiple instances in which short oligonucleotide direct repeats flank a region absent from either variola or vaccinia virus.
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Nucleotide sequence analysis of a unique near-terminal region of the tumorigenic poxvirus, Shope fibroma virus
More LessShope fibroma virus (SFV), a tumorigenic poxvirus, has a DNA genome of approximately 160 kb. Previous DNA sequence analysis of SFV has been mainly limited to the terminal inverted repetitions (about 12 kb at each end of the genome) and immediately adjacent regions. We have sequenced a 4 kb fragment located approximately 20 kb from the right-terminal hairpin. Within this region three complete and two partial open reading frames (ORFs) have been identified. Each of the putative polypeptides has sequence similarity to one or more previously identified poxvirus or cellular proteins, with homology to protein kinases, erythrocyte ankyrin and a vaccinia virus virulence-related protein (ORF N1L). The potential significance of these gene products with regard to the phenotype of SFV is discussed.
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Assembly of conformation-dependent neutralizing domains on glycoprotein B of human cytomegalovirus
More LessWe analysed the antigenic properties of human cytomegalovirus (CMV) glycoprotein B (gB) by constructing a set of deletion derivatives lacking different portions of the carboxy terminus and reacting them with a panel of monoclonal antibodies with neutralizing activity. We found that two novel antigenic domains that bind neutralizing antibodies were assembled on truncated forms of gB, one in the aminoterminal half and one that spans the midregion of the molecule. Assembly of the conformation-dependent epitopes occurred independently of residues in the carboxy-terminal half of the molecule and did not depend on proteolytic cleavage of the molecule between amino acids 460 and 461. Ten antibodies recognized a derivative with 447 amino-terminal residues; their failure to recognize a derivative 411 residues long suggested that the amino acids required for assembly of these epitopes either were incorrectly folded, or had been totally or partially deleted in this derivative. Epitopes for three antibodies with complement-independent neutralizing activity were assembled when amino acids from the midregion of gB between residues 447 and 476 were present. Two other antigenic domains were formed by the addition of residues 476 to 618 and 619 to 645 from the carboxy-terminal half of gB. Our results underscore the importance of conformation in the antigenic structure and functional properties of both the amino- and carboxy-terminal portions of gB.
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The lower matrix protein pp65 is the principal viral antigen present in peripheral blood leukocytes during an active cytomegalovirus infection
During an active infection with human cytomegalovirus (HCMV), viral antigen is consistently present in peripheral blood leukocytes. Two monoclonal antibodies (MAbs), CMV-C10 and CMV-C11, are commonly used in the HCMV antigenaemia assay to detect these cells in the peripheral blood of patients suspected of having an active HCMV infection. We demonstrate that the viral antigen detected by these MAbs is the viral structural protein pp65 and not an immediate early antigen as previously reported. Furthermore, significantly fewer leukocytes were found to be positive with MAbs specific for immediate early antigens.
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Glycoprotein 300 is encoded by gene 28 of equine herpesvirus type 1: a new family of herpesvirus membrane proteins?
A portion of equine herpesvirus type 1 (EHV-1) gene 28, which is homologous to herpes simplex virus type 1 gene UL32, was expressed using a prokaryotic system to yield a fusion protein which reacted on Western blots with P19, a monoclonal antibody (MAb) that reacts with EHV-1 glycoprotein 300 (gp300), confirming that this gene encodes gp300. Hydrophobicity analysis showed that gp300 is a glycoprotein with multiple hydrophobic domains that might interact with, or span, the membrane several times. As such, it may represent the first member of a new family of herpesvirus glycoproteins to be identified as a virus structural component. Gp300 was also shown to be modified by palmitic acid residues, and a second MAb (1G12) directed against gp300 inhibited fusion between EHV-1-infected cells.
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Characterization and transcript mapping of a bovine herpesvirus type 1 gene encoding a polypeptide homologous to the herpes simplex virus type 1 major tegument proteins VP13/14
More LessUsing in vitro translation of hybrid-selected mRNA, we have previously shown that bovine herpesvirus type 1 HindIII fragment M encodes an abundant 94K polypeptide. Using immunoprecipitation and sequencing analyses, it has now been shown that the polypeptide is related to the major tegument protein VP8 and is homologous to the herpes simplex virus type 1 major tegument proteins VP13/14. The sequence of the VP8 gene (field isolate 34) is reported and compared to published data. Several differences between the sequences were detected, resulting particularly from base insertions/deletions generating three major frameshifts affecting an area of 87 amino acid residues of the encoded protein. In addition, sequence comparison revealed 29 single base alterations, excluding frameshift regions, producing 17 amino acid substitutions. Overall, 14.1% of the deduced amino acid sequences were divergent. We have also established that the last 152 nucleotides of the previously reported sequence correspond to the sequence of the minus not the sense strand. Finally, we report that the 4.4 kb transcript of the VP8 gene is initiated 39 nucleotides upstream from the translation start codon.
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Cyclic AMP-mediated inhibition of vesicular stomatitis virus and herpes simplex virus replication in mouse macrophage-like cells
More LessIn this study, we have analysed the effects of cAMP inducers on the multiplication of vesicular stomatis virus (VSV) and herpes simplex virus type 1 (HSV-1) in mouse macrophage-like cells. The addition of dibutyryl cAMP (dB-cAMP) or cholera toxin to resting peritoneal macrophages aged in vitro or P388D1 cells resulted in a 10- to 100-fold reduction of VSV yield compared to control cultures. In contrast, no cAMP-dependent inhibition was found in VSV-infected L929 cells. In macrophage-like cells, the dB-cAMP-induced antiviral state was not inhibited by antibodies to interferon (IFN)-α/β and did not correlate with any increase in the intracellular levels of 2–5 oligo(A) synthetase. Dibutyryl cAMP did not inhibit virus yields in mouse macrophages infected with encephalomyocarditis virus. In P388D1 cells, the addition of dB-cAMP resulted in an approximately 10-fold inhibition of HSV-1 replication with respect to control cultures, as evaluated both by TCID50 and plaque assays on Vero cells. Dibutyryl cAMP did not affect VSV binding or entry into mouse macrophages and the cAMP-mediated anti-VSV state was significantly reduced by inhibitors of protein kinase C (i.e. staurosporine and H7). These data suggest that macrophages may acquire resistance to infection by VSV and HSV-1 after treatment with cAMP inducers. This cAMP-mediated antiviral activity does not depend on the modulation of the endogenous IFN system, suggesting that macrophages exhibit multiple resistance mechanisms (i.e. IFN-dependent and IFN-independent) to maintain their intrinsic antiviral activity.
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Antiviral properties of a dominant negative mutant of the herpes simplex virus type 1 regulatory protein ICP0
More LessDominant negative or trans-dominant mutants of viral proteins represent a new and exciting potential approach to antiviral therapy. Unfortunately, the extreme specificity of a given dominant negative mutant limits its general utility in treating a broad spectrum of viral diseases, since it can typically interfere with the activity of only a single viral polypeptide encoded by a single virus. However, it seems likely that dominant negative mutants of promiscuous viral trans-activator proteins, which by definition would repress rather than activate gene expression, should be able to inhibit infectious virus production for a number of different viruses. One such dominant negative mutant, derived from the herpes simplex virus type 1 (HSV-1) regulatory protein ICP0, was found previously to behave as a powerful repressor of gene expression from an assortment of HSV-1 and non-HSV-1 promoters in transient expression assays. In the present study, this ICP0 mutant was found to be capable of inhibiting the replication of both HSV-1 and a completely unrelated virus, human immunodeficiency virus, in cell culture. The properties of this dominant negative mutant indicate that it may have potential as a means of treating diseases caused by a number of DNA and RNA viruses. Moreover, a truncated form of ICP0 which can hypothetically be created by alternative splicing was found to possess similar inhibitory capabilities, suggesting that a virus-encoded version of this dominant negative mutant may play a role in down-regulating HSV-1 gene expression during infection in vivo.
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Nucleotide sequence analysis of genes encoding glycoproteins D and J in simian herpes B virus
More LessThe gene encoding glycoprotein D (gD) of simian herpes B virus (SHBV) was identified by hybridization with the gD gene of herpes simplex virus type 1 (HSV-1). The gene probe bound to a 2.6 kbp SalI-EcoRI fragment of SHBV DNA, which was cloned into a plasmid vector. The nucleotide sequence of the SHBV DNA fragment was determined. Two complete and one partial open reading frames (ORFs) were found. The nucleotide sequences of the two complete ORFs are 57% and 69% identical to HSV-1 genes US5 (encoding gJ) and US6 (encoding gD), respectively. The partial ORF showed 64% similarity with HSV-1 US7 (encoding gI). The SHBV gD gene revealed many features which are also found in the gD homologues of other herpesviruses. The positions of cysteine residues and receptor-binding sites for the predicted protein are shown to be highly conserved.
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Human sera from varicella-zoster virus (VZV) infections cross-react with human T cell leukaemia virus type 1 (HTLV-1): common epitopes in VZV gene 22 protein and HTLV-1 p19 gag protein
Twenty-nine of 100 sera from patients recently infected with varicella-zoster virus (VZV) were found to cross-react with human T cell leukaemia virus type 1 (HTLV-1) antigen in the particle agglutination (PA) assay using HTLV-1 antigen-coated gelatin particles. Anti-VZV IgM antibodies were shown to be responsible for this cross-reactivity. Western blot analysis revealed that PA-positive anti-VZV sera reacted with the HTLV-1 gag p19 protein in HTLV-1-infected cells and recombinant p19 protein produced in Escherichia coli. By using a truncated p19, the cross-reactive region was located to the C-terminal 17 amino acids of p19. One oligopeptide derived from the C terminus, PQIP-PPYVEPT (amino acids 115 to 125), was capable of inhibiting PA, suggesting that this peptide carries the cross-reactive epitope. A homologous sequence was found in the VZV gene 22 protein by database analysis, and the oligopeptide TNIPPPLALLR (amino acids 1330 to 1340) had the ability to inhibit PA. These findings suggest that some IgM antibodies against the VZV gene 22 protein produced in the early phase of VZV infection are cross-reactive with the HTLV-1 gag p19 protein because they recognize an antigenic determinant containing an IPPP tetrapeptide.
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Open reading frames 1 and 2 of adenovirus region E4 are conserved between human serotypes 2 and 5
More LessThe E4 region of human adenovirus type 2 is predicted to encode seven proteins as judged from its nucleotide sequence and the pattern of differential splicing of its transcript. Two of the open reading frames (ORFs), ORF1 and ORF2, had been identified as being disrupted in the recently published sequence of the related serotype 5 virus. These ORFs were resequenced and found to be intact in the wt300 strain of adenovirus type 5.
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Volumes and issues
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Volume 105 (2024)
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