- Volume 72, Issue 3, 1991
Volume 72, Issue 3, 1991
- The Fourteenth Fleming Lecture
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Varicella-zoster virus
More LessOver the past 15 years or so, we have witnessed a dramatic increase in the generation of nucleic acid sequence data. The impact of this process has been felt particularly in animal virology, so that we now have complete genome sequences for representatives of most of the virus families. It is apparent that complete sequence determination of any virus genome is now a practical short or medium term goal. This is illustrated most clearly by the herpesviruses, whose large genomes have provided a challenge suited to serious genome sequencers. Thus, four complete herpesvirus sequences have been published to date. Those for Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) were derived at the MRC Laboratory of Molecular Biology in Cambridge, U.K. (Baer et al., 1984; Chee et al., 1990) and those for varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) were obtained at the MRC Virology Unit in Glasgow, U.K. (Davison & Scott, 1986; McGeoch et al., 1988).
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Two vaccinia virus proteins structurally related to the interleukin-1 receptor and the immunoglobulin superfamily
More LessThe structures of two vaccinia virus genes (B15R and B18R) from near the right inverted terminal repeat are described. These genes encode proteins of 36.5K and 40.7K, respectively, which have an N-terminal hydrophobic sequence, possible sites for attachment of N-linked carbohydrate and a short string of hydrophobic residues near the C terminus. These properties are consistent with the mature proteins being either virion, cell surface or secretory glycoproteins. Protein sequence comparisons established that the two gene products are related to each other (20% identity) and to the immunoglobulin (Ig) superfamily. Intriguingly, the nearest homologues of these proteins in the SWISS-PROT (version 14) database are the human and murine interleukin-1 receptors, although both proteins are related to a wide range of Ig superfamily members, including the interleukin-6 receptor. The product of one of these genes is known to be expressed on the cell surface early during infection and immunity directed against it confer resistance to virus infection without directly neutralizing virus infectivity. We propose a novel method for virus immune evasion in which the product of one or both of these proteins may bind interleukin-1 and/or interleukin-6 and prevent these cytokines reaching their natural receptors. In consequence the inflammatory response would be diminished and virus replication enhanced.
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Relative enhancer activity and transforming potential of authentic human papillomavirus type 6 genomes from benign and malignant lesions
More LessHuman papillomavirus type 6 (HPV-6) is predominantly associated with benign genital warts whereas HPV-16 and HPV-18 are detected predominantly in carcinomas of the lower genital tract; HPV-6 is found rarely in such carcinomas. Experiments were designed to discriminate between two hypotheses concerning the role of HPV-6 in the genesis of a genital tract carcinoma: (i) the HPV-6 in the carcinoma (HPV6-T70) differs genetically from HPV-6 in a benign lesion (HPV6-W50), giving HPV6-T70 properties similar to those of HPV-16/18; (ii) HPV6-T70 and HPV6-W50 have similar biological activity, suggesting that the role of HPV-6 in oncogenesis is different from that of HPV-16/18. Restriction enzyme digestion and DNA sequence determination established that HPV6-T70 differs from HPV6-W50 in the upstream regulatory region (URR) but not in the proteins encoded by open reading frames (ORFs) E5, E6 or E7, ORFs implicated in oncogenesis. To determine whether the difference in the URR sequence could alter the level of expression of viral genes, the URRs were cloned into the enhancer-less plasmid pSVEcat. Analysis of chloramphenicol acetyltransferase activity after transfection into HeLa and Vero cells showed that the URRs had comparable enhancer activity. Cotransfection of baby rat kidney (BRK) cells with HPV6-T70 and an activated ras gene indicated that, in contrast to HPV-16 and HPV-18, this HPV-6 genome could not cooperate with ras to transform BRK cells. The data suggest that HPV6-T70 and HPV6-W50 have similar enhancer activity and transforming potential.
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The influence of antisense RNA on transcriptional mapping of the 5′ terminus of a baculovirus RNA
More LessS1 nuclease protection and primer extension analyses were used to determine the 5′ end of the Autographa californica nuclear polyhedrosis virus 603 open reading frame (ORF) transcript upstream of and on the opposite strand to the polyhedrin gene. These analyses suggested that the 5′ end of the 603 ORF was located near the initiation site for polyhedrin gene transcription. Primer extension products of reverse transcription of 603 RNA were dependent on the presence of the TAAG sequence, an essential polyhedrin promoter element. The results could be interpreted as indicating bidirectional transcription from the TAAG element. However, the data could also be due to an artefact of antisense transcripts and RNA duplex formation in this region. Since the bidirectional transcriptional model is not consistent with other mapping data and Northern blot analysis, we conclude that the presence of antisense RNA can result in mapping artefacts and that caution must be taken in interpreting data where overlapping sense and antisense RNAs are present.
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Studies on the genetic control of resistance of black hooded rats to Borna disease
More LessIn contrast to Lewis (LEW) and Wistar rats, black hooded (BH) rats inoculated with Borna disease (BD) virus developed neither encephalitis nor clinical disease despite persistent replication of the virus in the central nervous system. In comparison to LEW rats, production of virus-specific antibodies was significantly delayed in BD-resistant BH rats, even though identical titres were finally reached. The different susceptibility in LEW and BH rats was studied further by investigating responses of F1 hybrid animals. Although these rats developed encephalitis, they did not become sick. The differences in host responses for BD virus were found to be genetically determined but were independent of class I or class II major histocompatibility complex gene products or to genes responsible for lymphocyte differentiation.
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Rotavirus infection alters Na+ and K+ homeostasis in MA-104 cells
Infection of MA-104 cells with the OSU strain of rotavirus induced an increase in Na+ and a decrease in K+ intracellular concentrations, starting at 4 h post-infection. These changes were not related to an inhibition of the Na+/K+ pump since ouabain-sensitive 86Rb uptake was augmented in rotavirus-infected cells compared to control cells, whereas the [3H]ouabain binding and Na+/K+ ATPase activity in the cell homogenate were unaffected. Furosemide-sensitive 86Rb uptake (Na+/K+/2Cl− cotransport) was not modified by the infection. Passive 86Rb efflux and 22Na influx were augmented in infected cells suggesting an increase in the plasma membrane permeability. The increase in intracellular Na+ concentration might be responsible for the observed stimulation of the Na+/K+ pump. This effect was dependent upon the synthesis of viral proteins because it was abolished by addition of cycloheximide up to 4 h post-infection. Prevention of the increase in intracellular Na+ by the use of low Na+-containing media did not modify the pattern of protein synthesis. This suggests that changes in intracellular Na+ and K+ concentrations were not related to shutoff of cellular protein synthesis. Alterations of ion contents in the rotavirus-infected enterocytes might impair intestinal absorptive capacity before the appearance of histopathological lesions.
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Antigenic relatedness between arenaviruses defined at the epitope level by monoclonal antibodies
More LessMonoclonal antibodies (MAbs) were produced against two African arenaviruses, Lassa virus and Mopeia virus. Competitive binding analysis of MAbs identified four antigenic sites on the nucleoprotein (NP), two on glycoprotein 1 (GP1) and six on glycoprotein 2 (GP2) of the Josiah strain of Lassa virus. 64 virus isolates from western, central and southern Africa were all consistently distinguishable by MAbs to certain epitopic sites on GP1, GP2 and NP viral proteins. Furthermore, MAbs to Lassa virus GP1 and NP uniformly distinguished viruses from the West African countries of Sierra Leone, Liberia and Guinea from those of Nigeria. GP2-directed MAbs to two African arenaviruses reacted broadly with South American arenaviruses demonstrating that an epitopic site on GP2 may be the most highly conserved antigen in the arenavirus group.
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Identification of linear epitopes on Semliki Forest virus E2 membrane protein and their effectiveness as a synthetic peptide vaccine
Semliki Forest virus (SFV) infection of mice was used as a model to study the applicability of synthetic peptides containing only linear epitopes as viral vaccines. The identification of linear epitopes with vaccine potential on the E2 membrane protein of SFV was based on the binding of SFV-specific antibodies to a set of overlapping synthetic hexapeptides (Pepscan) representing the whole E2 amino acid sequence. The 14 available E2-specific monoclonal antibodies which were protective in vivo proved to be unsuitable for the identification of linear epitopes because they recognized only conformational epitopes, as indicated by their lack of reactivity with unfolded, reduced E2 protein on immunoblots. Three epitopes were detected with polyclonal anti-SFV serum at amino acid positions 135 to 141, 177 to 185 and 240 to 246 of the E2 protein. Synthetic peptides containing these epitopes were coupled to a carrier protein and tested as a vaccine. Mice immunized with the peptide containing amino acids 240 to 255 of protein E2 were protected against a challenge with virulent SFV but protection of mice immunized with the peptides containing amino acids 126 to 141 or 178 to 186 was only marginally better than that of controls. The prechallenge sera of most peptide-immunized mice reacted with SFV-infected cells but none of these sera neutralized the virus in vitro. However, protection of mice correlated well with SFV-specific antibody titre, suggesting antibody-mediated protection.
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Evidence for dissimilar properties of comoviral and picornaviral RNA polymerases
More LessThe poliovirus RNA polymerase has been synthesized in Spodoptera frugiperda cells by using the baculovirus expression system. Crude sonicates of these cells exhibited an RNA-elongating activity of a synthetic oligo(U) primer with poly(A) or cowpea mosaic virus (CPMV) RNA as a template. A similar polymerase activity was found in extracts of insect cells in which foot-and-mouth disease virus (FMDV) proteins, including the putative polymerase, were produced. The analogous CPMV 87K protein and several of its precursors, synthesized in S. frugiperda cells, did not show any detectable polymerase activity in the same assay under a variety of conditions. The results indicate that, in contrast to the picornaviral polymerases, the CPMV polymerase is unable to function in an oligo(U)-primed polymerase assay.
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Persistent infection of Vero cells by the flavivirus Murray Valley encephalitis virus
More LessMurray Valley encephalitis (MVE) virus strain OR2 was serially passaged on Vero cells to establish a persistent infection which was maintained for over 300 days. Supernatants from infected cells protected Vero cells from c.p.e. and caused up to a 95% reduction of wild-type virus yield. These protective and interfering effects suggest that defective interfering (DI) particles are responsible for the establishment and maintenance of the MVE virus persistent infection. The persistently infected cell supernatant preparations shared several features with DI particle preparations from other viral systems, such as their amplification to detectable levels after two to four passages of virus. However, results from this study suggest that DI particles of MVE virus differ from other studied systems in that they are able to affect only moderately the yield of infectious wild-type virus. The genetic drift of the parental virus during the course of a long term persistent infection in vitro appears to be minimal.
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Sequence comparison of the 5′ end of mRNA 3 from transmissible gastroenteritis virus and porcine respiratory coronavirus
More LessAnalysis of porcine transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) mRNA species indicated a deletion in mRNA 3 of PRCV. Polymerase chain reaction (PCR) was used to clone the 5′ end of mRNA 3 from PRCV for comparison with the equivalent region in TGEV. Small deletions were observed within and around the PRCV sequence equivalent to the putative open reading frame (ORF) ORF-3a identified in TGEV. The potential RNA polymerase-leader complex binding site (leader RNA binding site), ACTAAAC, found upstream of ORF-3a in TGEV, was absent from the PRCV genome but a potential site was found in the PRCV genome upstream of a gene equivalent to TGEV ORF-3b. PCR analysis, using primers corresponding to sequences within the ORF-3b gene and the leader RNA sequence, confirmed that the leader RNA binding site was upstream of a gene equivalent to TGEV ORF-3b on PRCV mRNA 3 but upstream of ORF-3a on TGEV mRNA 3. The presence of the new leader RNA binding site would be responsible for generating the smaller mRNA 3 species found in PRCV-infected cells.
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Epidemiological and experimental studies on a new incident of transmissible mink encephalopathy
More LessEpidemiological investigation of a new incident of transmissible mink encephalopathy (TME) in Stetsonville, Wisconsin, U.S.A. in 1985 revealed that the mink rancher had never fed sheep products to his mink but did feed them large amounts of products from fallen or sick dairy cattle. To investigate the possibility that this occurrence of TME may have resulted from exposure to infected cattle, two Holstein bull calves were injected intracerebrally with mink brain from the Stetsonville ranch. Each bull developed a fatal spongiform encepha-lopathy 18 and 19 months after inoculation, respectively, and both bovine brains passaged back into mink were highly pathogenic by either intracerebral or oral inoculation. These results suggest the presence of a previously unrecognized scrapie-like infection in cattle in the United States.
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The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis
More LessMouse lines which are congenic for Sinc, the major gene controlling scrapie incubation period, have been produced by selective breeding from the inbred C57BL(Sinc s7) and VM(Sinc p7) strains; the s7 allele of Sinc has been introduced into a VM background by 18 serial backcrosses, at each generation selecting on the basis of the incubation period with the ME7 scrapie strain. The characteristics of the disease produced by seven scrapie strains have been compared in Sinc s7 and Sinc p7 congenic mice and in the F1 cross between them. As previously found in non-congenic mice, each scrapie strain has a characteristic, precisely reproducible incubation period pattern in the three Sinc genotypes. The Sinc gene controls the incubation period for all scrapie strains tested but the direction of allelic action and the apparent dominance pattern differs between scrapie strains. Comparison with non-congenic mice shows that other genes also have a minor effect on incubation period. The distribution of vacuolar degeneration in the brain depends mainly on the scrapie strain but is also influenced by Sinc and other unspecified mouse genes. Restriction fragment length polymorphism analysis has already shown that the close linkage between Sinc and the gene encoding PrP has been maintained in the Sinc congenic lines, strengthening the possibility that PrP is the Sinc gene product. The present study confirms that scrapie strains carry information which is independent of the host but nevertheless suggests that host PrP protein interacts with this information to regulate the progression of the disease.
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Further characterization of the gapped DNA intermediates of human spumavirus: evidence for a dual initiation of plus-strand DNA synthesis
We recently reported the presence of linear duplex DNA intermediates with a gap in the middle of the molecules in the replicative cycle of human (HSRV) and simian (SFV1) spumaviruses. The polypurine tract (PPT), at the 5′ boundary of the 3′ long terminal repeat, was found to be duplicated in the gap region. By molecular analysis of HSRV proviral DNA with region- and strand-specific probes, we have now determined that the gap is located on plus-strand DNA and that it is 120 bases long with the 3′ end mapping at the duplicated PPT site. Kinetic analysis of proviral DNA provided evidence that the gap did not result from processing of a complete, full-length DNA molecule. These data strongly suggest that plus-strand DNA synthesis is initiated at both PPT sites.
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Analysis of Fv-1 restriction in two murine embryonal carcinoma cell lines and a series of differentiated derivatives
More LessWe have used antibiotic-resistant retrovirus vectors rescued by Fv-1-sensitive murine leukaemia viruses (MuLV) to examine the Fv-1 phenotype of two undifferentiated embryonal carcinoma (EC) cell lines derived from teratocarcinomas of mouse strain 129. In addition, a set of EC cell-derived differentiated cell lines was analysed. Restriction of both B-tropic and endogenous N-tropic virus is characteristic of the Nr-type restriction reported in mouse strain 129. However, results indicate that Fv-1 restriction is not expressed in the PCC4.aza1R EC cell line. In contrast, the F9 EC cell line showed a strong restriction of the B-tropic pseudotyped vector but failed to restrict endogenous N-tropic pseudotypes. The Fv-1 gene thus seems to be differentially expressed in two EC cell lines derived from the same mouse strain. Furthermore, the selective restriction of B-tropic but not endogenous N-tropic MuLV in F9 cells suggests that these activities function independently of each other. Analysis of PCC4.aza1R-derived differentiated cell lines revealed that three fibroblast cell lines derived by retinoic acid-induced differentiation were also phenotypically silent for Fv-1. However, a pre-adipocyte line established following simultaneous exposure to retinoic acid and 5-azacytidine showed strong restriction of both B-tropic and endogenous N-tropic MuLV. Although additional data suggest that there is no correlation between the differentiated pre-adipocyte phenotype and Fv-1 expression, our results nonetheless show that Nr restriction can be observed in some derivatives of PCC4.aza1R cells, presumably by activating expression of the Fv-1 gene.
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Characterization of two monoclonal antibodies against feline immunodeficiency virus gag gene products and their application in an assay to evaluate neutralizing antibody activity
More LessMonoclonal antibodies (MAbs) 3B7 and 1C11 were produced against the gag gene products of feline immunodeficiency virus (FIV). These MAbs reacted strongly with FIV p24 in Western blots (immunoblots) and recognized p50 with a lower intensity. They specifically bound antigens in the cytoplasm of FIV-infected cells as determined by indirect immuno-fluorescence and immunocytochemistry. Although neither MAb inhibited viral replication in vitro, they were useful in a simple assay for the detection and quantification of infectious virus and neutralizing antibody activity. The assay utilizes Crandell feline kidney cells and requires 4 days for completion. Neutralizing antibodies in cats were detected 3 to 4 weeks after experimental infection with FIV. Antibody titres progressively increased during the first year of infection reaching high titres which were maintained 2.5 years post-infection. The MAbs produced should be valuable reagents for the monitoring of viral replication in cells or tissues from FIV-infected cats and for other in vitro applications.
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Analysis of mutations in the thymidine kinase genes of drug-resistant varicella-zoster virus populations using the polymerase chain reaction
More LessWe have applied the polymerase chain reaction (PCR) technique to analyse mutations in the thymidine kinase (TK) gene of varicella-zoster virus (VZV) associated with resistance to the 5-bromovinyl (BVaraU) and 5-propynyl (PYaraU) analogues of arabinofuranosyl deoxyuridine. The results from this study allow three clear conclusions to be drawn. Firstly, the technique clearly shows that populations of VZV derived from plaque purification were truly clonal only when the plaques were initiated from cell-free virus (representing a tiny fraction of infectious virus) and plaques initiated by infected cells contained a mixture of variants. Secondly, despite the background mutations caused by errors of the Taq DNA polymerase, mutations relevant to drug resistance can easily be distinguished. The BVaraU-resistant mutant, 7-1, contained an aspartic acid to asparagine mutation at residue 18 and a single base deletion (position 65298 of the VZV DNA sequence), resulting in a frameshift and premature termination of the polypeptide chain, was found in the BVaraU-resistant mutant YSR. PYaraU-resistant virus populations contained viruses with one or more of three independent mutations, i.e. single base substitutions resulting in mutations from leucine to proline at residue 92, histidine to arginine at residue 97 and a deletion of 20bp (residues 65135 to 65154). Finally, the technique has uncovered novel sites in the virus TK associated with drug resistance. We conclude that in vitro amplification using the PCR combined with cloning and sequencing is a relatively rapid method for identifying mutations in small virus populations even when they are not homogeneous.
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Herpes simplex virus type 1 deletion variants 1714 and 1716 pinpoint neurovirulence-related sequences in Glasgow strain 17+ between immediate early gene 1 and the ‘a’ sequence
More LessDideoxynucleotide sequence analysis of a spontaneously isolated deletion variant (1714) of Glasgow strain 17+ of herpes simplex virus type 1 (HSV-1) demonstrates that the deletion is 759 bp in length and is located within each copy of the BamHI s fragment (0 to 0.02 and 0.81 to 0.83 map units) of the long repeat region of the genome. The deletion removes one complete copy of the 18 bp DR1 element of the ‘a’ sequence and terminates 1105 bp upstream of the 5′ end of immediate early (IE) gene 1. The variant grows to high titre, is not temperature-sensitive and is not host cell type-restricted in vitro. In vivo studies demonstrate that 1714 is totally avirulent for BALB/c mice following intracerebral inoculation, with an LD50 of 7 × 106 p.f.u./mouse compared to < 10 p.f.u./mouse for the parental wild-type strain 17+. In vivo growth kinetics show that the non-neurovirulent phenotype is due to an inability to replicate in mouse brain. Because 1714 was in a genomic background in which the four XbaI sites had been removed and because the phenotype was thymidine kinase-negative, the 759 bp deletion was introduced into an otherwise totally wild-type background. The resulting variant (1716) is nonneurovirulent for mice, with an LD50 of 7 × 106 p.f.u./mouse. The deletion does not prevent the virus from establishing a latent infection or reactivating from it in vitro. The results demonstrate that sequences between IE-1 and the ‘a’ sequence produce neurovirulence in Glasgow strain 17+ and, in conjunction with the nonneurovirulence of the HSV-2 HG52 variant JH2604, identify a common function conserved in HSV-1 and -2.
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Investigation of herpes simplex virus type 1 (HSV-1) gene expression and DNA synthesis during the establishment of latent infection by an HSV-1 mutant, in1814, that does not replicate in mouse trigeminal ganglia
In previous studies, the herpes simplex virus type 1 (HSV-1) mutant, in1814, which lacks the trans-inducing function of Vmw65, did not replicate in the trigeminal ganglia of mice following corneal inoculation but did establish a reactivatable latent infection in the ganglia 12 to 24 h after ocular infection. Since in1814 did not replicate in vivo, the molecular events during the establishment phase of latent HSV-1 infection could be characterized without the complications of concurrent productive viral infection. In comparison to parental HSV-1 strain 17+, the expression of viral immediate early (IE), early and late genes and the levels of viral DNA in the trigeminal ganglia of mice following in1814 infection were greatly reduced. However, accumulation of latency-associated transcripts, a prominent feature of latent HSV-1 infection, occurred in a wild-type fashion. Furthermore, low levels of viral gene expression and an increase in the level of viral DNA in the in1814-infected ganglia were not detected until 1 to 2 days after the establishment of HSV-1 latency. Thus, IE gene expression and replication of viral DNA in the trigeminal ganglia are not prerequisites for the establishment of HSV-1 latency. These results suggest that the pathways leading to productive and latent infections in neurons may diverge at an early stage of the host-HSV-1 interaction and that the level of viral IE gene expression has a key role in determining the outcome of infection.
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Construction and characterization of herpes simplex type 1 viruses without introns in immediate early gene 1
More LessHerpes simplex virus type 1 (HSV-1) encodes at least 70 distinct genes in a DNA genome sequence of about 150 kb. In contrast to most cellular genes and those of several other DNA viruses, the overwhelming majority of HSV-1 transcripts are not spliced. One exception is immediate early (IE) gene 1, which contains two introns in the Vmw110 coding region. This study investigated the possibility that IE-1 intron sequences have a role during HSV-1 infection. IE-1 genes lacking the first, second or both introns were constructed by site-directed deletion mutagenesis and recombined into the viral genome. Viruses lacking the IE-1 introns were essentially indistinguishable from the parent virus in terms of growth, particle to p.f.u. ratio or viral polypeptide expression in a variety of cell types. The lack of introns did not affect the time-course or efficiency of expression of Vmw110 either during normal infection or in cycloheximide reversal experiments. In contrast, in transfection assays, the loss of both intron sequences resulted in the elimination of the ability of a plasmid-encoded IE-1 to activate gene expression. These results imply that in certain situations the introns in IE-gene 1 may contribute to the efficient expression of Vmw110 but such an effect is not readily apparent using a recombinant virus in the tissue culture systems tested. In the course of this work a mutant of Vmw110 was fortuitously isolated which had lost the majority of an extremely acidic section of the polypeptide; this mutation appeared to have little effect on Vmw110 function.
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Identification and characterization of a novel non-infectious herpes simplex virus-related particle
More LessDuring gradient purification of herpes simplex virus type 1 (HSV-1) two bands of particles were observed: a sharp lower band and a more diffuse upper band. The lower band contained almost exclusively HSV-1 virions (H particles) whereas the upper band consisted of membrane-enclosed particles (L particles). These L particles resembled the virions in appearance, but lacked the viral nucleocapsid and were not infectious. Many polypeptides of the viral envelope and the tegument were common to both types of particles. The H particles had polypeptide profiles typical of HSV virions. The L particles contained at least three phosphoproteins (175K, 92K and 55K) and a further two phosphorylated polypeptides not normally observed in virion profiles which comigrated with the 134K and 60K glycoproteins. This clearly indicates that the novel L particles were not merely virions which had formed without the inclusion of a nucleocapsid or virions which had subsequently lost their nucleocapsid during preparative handling. Thus these novel L particles are genuine products of the infectious processes occurring when HSV-1 replicates.
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The G protein of human respiratory syncytial virus: significance of carbohydrate side-chains and the C-terminal end to its antigenicity
More LessThe reactivities of eighteen monoclonal antibodies with different glycosylated forms of the human respiratory syncytial (RS) virus G protein were tested in Western blots. Only five antibodies recognized the unglycosylated precursor. The majority of antibodies, however, reacted with the O-glycosylated form of the G protein, emphasizing the importance of this type of modification for the antigenicity of the mature molecule. Human antisera, which recognized the RS virus G protein in Western blots, failed to inhibit the binding of anti-G antibodies to the virus but inhibited the binding of anti-F antibodies in the same type of assay. The human antibodies, however, did not recognize the G protein of some neutralization-resistant mutants selected with one anti-G monoclonal antibody. These mutants contain drastic amino acid sequence changes in the C-terminal end of the G molecule. The results are discussed in terms of the G protein antigenic structure.
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Sequence of the major nucleocapsid protein gene of pneumonia virus of mice: sequence comparisons suggest structural homology between nucleocapsid proteins of pneumoviruses, paramyxoviruses, rhabdoviruses and filoviruses
More LessThe complete nucleotide sequence of gene 3 of pneumonia virus of mice has been determined, and the 5′ end of the mRNA mapped using a modification of the polymerase chain reaction technique. The gene contains a single open reading frame, beginning with a 5′-proximal AUG initiation codon, encoding a polypeptide with a predicted M r of 43141. Expression of the gene 3 protein in Escherichia coli and in vitro showed that it reacted with virus-specific antiserum and comigrated with the major nucleocapsid (N) polypeptide. The predicted amino acid sequence has extensive identity with that of the N protein of human respiratory syncytial virus. Comparisons with the amino acid sequences of N proteins of other paramyxo-viruses, vesicular stomatitis virus and Ebola virus suggest that these proteins may have retained much of the same structure. These regions of conserved structure would most likely have the common functions of RNA binding and protein/protein interactions in the virus nucleocapsid.
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B95a, a marmoset lymphoblastoid cell line, as a sensitive host for rinderpest virus
More LessWe reported earlier that B95a, an Epstein-Barr virus-transformed marmoset B lymphoblastoid cell line, is more susceptible to infection with measles virus than other cells. The cell line also was found to be susceptible to infection with the lapinized Nakamura III (L) strain of rinderpest virus and various strains derived from it. The B95a cell line was therefore the only host cell system available for the propagation and quantification of the L strain. In contrast to the adaptation of the L strain to Vero cells which results in a diminution of virulence in rabbits, the propagation of the virus in B95a cells preserved the virulence and some other properties in rabbits. Furthermore, when Vero cell-adapted variants of the L strain with diminished virulence were serially passaged in B95a cells, virulence in rabbits was gradually regained.
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Evolutionary pathways of N2 neuraminidases of swine and human influenza A viruses: origin of the neuraminidase genes of two reassortants (H1N2) isolated from pigs
The complete nucleotide sequences of the neuraminidase (NA) genes of two reassortant (H1N2) and two H3N2 influenza A viruses isolated from pigs were determined and phylogenetic relationships between these and previously reported N2 NA genes were investigated. On the basis of pairwise nucleotide sequence identity, the NA genes of two reassortants, A/sw/Kanagawa/2/78 and A/sw/Ehime/1/80, were most closely related to those of human influenza A virus strains isolated in 1972 and the earliest available swine H3N2 influenza A viruses, respectively. Phylogenetic trees showed that the NA genes can be segregated into three groups, including lineages for (i) swine strains, (ii) the earliest human strain and (iii) recent human strains. The evolutionary tree for the 11 nucleotide and amino acid sequences suggested that the NAs of A/sw/HK/4/76 and A/sw/Kanagawa/2/78 belong to the lineage for recent human viruses. In contrast, the NA genes of the A/sw/HK/3/76 and H1N2 reassortant A/sw/Ehime/1/80 viruses were found to be of a swine lineage. The swine virus NA genes were further characterized by the cocirculation of two distinct lineages. Although the rates of synonymous (silent) substitutions for the swine and human viruses were nearly identical (0.00946 to 0.00884 per site per year), the rate of non-synonymous (amino acid changing) substitutions for swine virus NA genes was about 60% of that for the human virus.
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Homotypic and heterotypic protection against influenza virus infection in mice by recombinant vaccinia virus expressing the haemagglutinin or nucleoprotein gene of influenza virus
Recombinant vaccinia virus expressing the influenza virus haemagglutinin (HA) or nucleoprotein (NP) genes from A/SW/Hong Kong/1/74 (H1N1) under the control of a hybrid promoter containing the P7.5 early promoter element and promoter of the gene encoding the major protein of cowpox virus A type inclusion body was constructed to investigate protective immunity against homologous and heterologous viruses in mice. These recombinant vaccinia viruses produced authentic influenza virus HA and NP in infected cells. The recombinant vaccinia virus-influenza virus HA conferred efficient subtype-specific protection although mice challenged with heterologous influenza viruses underwent initial infection. By contrast, immunization with the recombinant vaccinia-influenza virus NP limited virus multiplication in the lungs against challenge infection with all H1N1 and H3N2 influenza viruses examined, although less efficiently. These results will prompt the re-examination of the possibility of using the recombinant vaccinia virus-influenza virus NP as a cross-protective vaccine
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Nature of the endogenous pyrogen (EP) induced by influenza viruses: lack of correlation between EP levels and content of the known pyrogenic cytokines, interleukin 1, interleukin 6 and tumour necrosis factor
More LessFever in influenza results from the release of endogenous pyrogen (EP) following virus-phagocyte interaction and its level correlates with the differing virulence of virus strains. However, the different levels of fever produced in ferrets by intracardial inoculation of EP obtained from the interaction of different virus strains with ferret or human phagocytes did not correlate with the levels of interleukin 1 (IL-1), IL-6 or tumour necrosis factor in the same samples as assayed by conventional in vitro methods. Hence, the EP produced by influenza virus appears to be different to these cytokines.
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Prevalence of antibody to influenza C virus among pigs in Hyogo Prefecture, Japan
More LessThe prevalence of influenza C virus among pigs in Hyogo Prefecture, Japan, was investigated by serological techniques. Out of 240 sera tested, 45 (19%) showed haemagglutination inhibition (HI) to influenza C virus. Pig sera with high HI titres also scored high in neutralization tests and ELISAs. When fractionated by sucrose density gradient ultracentrifugation, the HI/ELISA reactivities corresponded to antibodies of the IgM and IgG classes. Radioimmunoprecipitation tests revealed that some, but not all, of the pig sera with high HI activities precipitated HEF glycoprotein of influenza C virus. These results suggested that the HI activities of pig sera in Hyogo Prefecture were due to the presence of antibody to influenza C virus. Sera with IgM class antibody to influenza C virus were found throughout the year. However, the question of whether or not pigs serve as a natural reservoir for human influenza C virus still remains to be solved.
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Antigenic and genomic identity between simian herpesvirus aotus type 2 and bovine herpesvirus type 4
Herpesvirus aotus type 2 (HVA-2) was isolated from a culture of kidney cells from a healthy owl monkey (Aotus trivirgatus). Bovine herpesvirus type 4 (BHV-4) is frequently isolated from diseased and even healthy cattle and occasionally from sheep, wild ruminants and cats. The two viruses are related antigenically, as was revealed by an indirect fluorescent antibody test using polyclonal antisera from experimentally infected rabbits or monoclonal antibodies raised against six BHV-4 proteins, three of which were glycosylated. The genome structures of the two viruses consist of a unique central sequence flanked at both ends by G+C-rich tandem repeats. Restriction maps (produced using EcoRI, BamHI and HindIII) of these two viruses were nearly identical but the unique sequence of the HVA-2 genome possessed two additional BamHI sites. Four genomic regions of variable size were detected, two located in the unique part, one in the repetitive part and one in the left junction between the unique and the repeated part of the genome; these slight variations were similar to those observed between various BHV-4 isolates. These results suggest that HVA-2 and BHV-4 belong to the same virus species; HVA-2 could be either a BHV-4 contaminant of owl monkey kidney cell cultures or an isolate from an owl monkey accidentally infected with BHV-4.
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Nucleotide sequence of a Guinea-Bissau-derived human immunodeficiency virus type 2 proviral clone (HIV-2CAM2)
M. Tristem, F. Hill and A. KarpasWe report the complete nucleotide sequence of a human immunodeficiency virus type 2 (HIV-2) isolate from Guinea-Bissau (HIV-2CAM2). The genomic organization of HIV-2CAM2 is identical to that of other HIV-2 isolates but contains a stop codon in the pol gene. The deduced amino acid sequences of the viral proteins show variation of 20% in the gag, pol and vpx regions, and 25 to 45% in the tat, env and nef regions when compared to other isolates of HIV-2. This is greater than the variation observed between isolates of HIV-1.
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Antigenic and genomic comparison between non-cytopathic and cytopathic bovine viral diarrhoea viruses isolated from cattle that had spontaneous mucosal disease
More LessAntigenic and genomic relationships between five pairs of non-cytopathic and cytopathic bovine viral diarrhoea (BVD) viruses isolated from cattle with mucosal disease were examined. Antigenic similarity was evaluated by studying the binding characteristics of 10 monoclonal antibodies (MAbs) directed against the gp53 BVD virus glycoprotein. The MAb binding match between members of the same virus pair ranged from 10/10 to 7/10. The genomic relationship was evaluated by studying the hybridization characteristics of two cDNA probes and six complementary 20 base oligomer probes with virus DNA, selected on the basis of conservation between published BVD virus sequences; cDNA probes were hybridized at two stringencies (60 °C and 45 °C). The match of hybridization results between members of the same virus pair ranged from 4/4 to 1/4 with the cDNA probes and from 6/6 to 3/6 with the oligomer probes. The results indicate that members of the same virus pair can differ at both the antigenic and genomic levels.
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Human papillomavirus type 6 and 11 E4 gene products in condyloma acuminata
The human papillomavirus type 6 (HPV-6) E4 gene was expressed in Escherichia coli as a fusion protein with E. coli β-galactosidase (E4-β-Gal), and rabbit antibody against the E4-β-Gal was prepared. By Western blotting with this antibody, we detected E4 gene products in six out of 18 condyloma acuminata specimens. In four specimens (C-1, C-13, C-14 and C-19), the E4 protein was found as a 10K/11K doublet, but in other specimens (C-8 and C-23), only the 11K protein was detected. By Southern blot analysis, it was found that C-13 harboured HPV-6 DNA but that C-1 and C-8 harboured HPV-11 DNA, indicating that the E4 proteins of HPV-6 and -11 have cross-reactive antigenicity. After incubation at 37 °C of the C-23 tissue specimen, the 10K protein was clearly detected. These results suggest that the 10K protein may be derived from the 11K protein by a modification such as proteolytic cleavage before and/or after specimens were taken.
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Nucleotide sequence analysis of three different hepatitis delta viruses isolated from a woodchuck and humans
More LessWe have investigated the extent of hepatitis delta virus (HDV) genetic variability after serial passages in chimpanzees and woodchucks and between different human isolates. A complete HDV genome, isolated from a woodchuck liver, was cloned after five serial transmissions. The 1679 nucleotide long genome revealed only point mutations and a nucleotide divergence of 0.65% and 0.89% with previously published sequences of two epidemiologically related HDVs. We have obtained partial nucleotide sequences of two unrelated human HDV cDNAs by using the polymerase chain reaction. When compared to the woodchuck HDV strain and other previously reported isolates, a perfectly conserved region of 90 nucleotides was shown in the region encompassing the delta antigen antigenomic self-cleavage site. In woodchuck and human HDV strains, the two forms of delta protein (195 and 214 amino acids) were potentially expressed. Our study indicates that only a limited genetic variability is generated by several passages in animals despite significant modification of pathogenicity during these transmissions.
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In vitro propagation of parvovirus B19 in primary foetal liver culture
More LessThe culture of parvovirus B19 in foetal liver tissue has been described recently. We have established the technique in our laboratory and studied parameters affecting the yield of B19 virus. Replication of the virus was detected by radioimmunoassay for B19 antigen and dot blot hybridization assay of B19 DNA, and the virus was localized by immunofluorescence and thin section electron microscopy. B19 DNA and antigen production became detectable at day 2 and reached a maximum at day 5. Virus particles were seen mainly in cell nuclei, but some cytoplasmic membranes were lined with virus particles. The amount of virus produced depended on the age of the foetus and the cell culture and the concentration of erythropoietin and interleukin 3 in the culture medium.
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Myristoylation of foot-and-mouth disease virus capsid protein precursors is independent of other viral proteins and occurs in both mammalian and insect cells
More LessThe myristoylation of the foot-and-mouth disease virus (FMDV) capsid precursor P1-2A and its amino-terminal cleavage product 1AB, expressed from subgenomic cDNA, has been analysed. The modification reaction is independent of other FMDV proteins and occurs in both mammalian and insect cells. Blocking of the myristoylation site does not prevent efficient processing of the FMDV capsid precursor. A cDNA cassette in which the leader protease sequence is substituted by an ATG codon produces myristoylated 1AB, indicating correct removal of the novel N-terminal methionine residue.
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Synthesis of Newcastle disease virus (NDV)-like envelopes in insect cells infected with a recombinant baculovirus expressing the haemagglutinin-neuraminidase of NDV
More LessElectron microscopical examination of negatively stained extracellular fluids (ECF) from Spodoptera frugiperda cell cultures infected with a recombinant baculovirus expressing the Newcastle disease virus (NDV) haemagglutinin-neuraminidase (HN) revealed NDV-like envelopes which resembled the envelopes of authentic NDV. Immunogold staining with anti-NDV HN monoclonal antibodies demonstrated HN antigen in spikes on the NDV-like envelopes. The ECF from the recombinant-infected cultures also contained baculovirus particles which resembled standard baculovirus particles except that some showed polar protrusions of the envelope. It was concluded that the HN of NDV, in the absence of the matrix protein, might be able to initiate and control the production of viral envelopes which are morphologically identical to those of authentic NDV.
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A temporal study of the expression of the capsid, cytoplasmic inclusion and nuclear inclusion proteins of tobacco etch potyvirus in infected plants
More LessYoung leaves of tobacco, systemically infected by tobacco etch potyvirus (TEV), were examined for the presence and distribution of four virus encoded proteins [capsid, cytoplasmic inclusion (CI) and two nuclear inclusion (NI) proteins] at various time periods after inoculation of expanded leaves of the plants. The analyses were carried out by ELISA and by immunogold electron microscopy of thin sections of the leaves. All four proteins were detected simultaneously in the systemic leaves for the first time on the fifth day after inoculation of the expanded leaves. All four proteins increased in concentration until the seventh day and then showed no further increase with the exception of the capsid protein which continued to accumulate. The CI protein was first detected in association with the plasmalemma/cell wall and was subsequently found mostly in the form of pinwheels in the cytoplasm. The two NI proteins were found at all times after infection within the nucleus, although small concentrations were detected in the cytoplasm. These experiments suggest that both the NIa and NIb proteins are transported into the nucleus immediately after synthesis. At the earliest time periods after infection, high concentrations of these proteins (NIa and NIb) were found in their noninclusion form in the nucleolus. At 14 days after infection, both proteins were found only as inclusions in the nucleus. The capsid protein was found at all stages of infection only in the cytoplasm.
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Cylindrical inclusion bodies of wheat streak mosaic virus and three other potyviruses only self-assemble in mixed infections
More LessPotyviruses produce cylindrical inclusions (CIs) in the cytoplasm of infected cells. Immunogold labelling and electron microscopy of embedded and sectioned wheat and maize cells doubly infected by different potyviruses revealed no mixing of inclusion proteins in CIs. The viruses were wheat streak mosaic virus (WSMV), agropyron mosaic virus and hordeum mosaic virus, in wheat, and WSMV and maize dwarf mosaic virus in maize. The three viruses in wheat were indistinguishable morphologically and in ultrastructural features but can be separated by serology and host range. The absence of phenotypic mixing in CIs showed that in the presence of CI proteins of other potyviruses, assembly was either highly virus-specific, or that no opportunity existed for CI proteins to assemble into hybrid CIs in mixed infections.
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cDNA cloning and nucleotide sequence of the wheat streak mosaic virus capsid protein gene
The 3′-terminal region of wheat streak mosaic virus (WSMV) genomic RNA was cloned and a cDNA sequence of 1809 nucleotides upstream of the poly(A) tract was determined. The sequence contains a single open reading frame of 1662 nucleotides and a 3′ untranslated region of 147 nucleotides. Translation products from WSMV RNA and WSMV cDNA transcripts were immunoprecipitated by WSMV capsid protein antiserum, indicating that the 3′-terminal region of WSMV RNA encodes the capsid protein. Five potential N-terminal capsid protein protease cleavage sites were identified, which would yield proteins ranging from 31.7K to 46.8K. Alignment of the deduced amino acid sequence of the WSMV capsid protein with those of other potyviruses showed significant, but limited, identity as compared to the alignment of two or more aphid-transmitted potyviruses. Although WSMV has characteristics distinct from poty-viruses, because of its particle morphology, translation strategy apparently based on polyprotein processing, the ability to form cytoplasmic cylindrical inclusions and the degree of capsid protein homology with aphidtransmitted potyviruses, it should be considered a member of the potyvirus group.
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De novo generation of cymbidium ringspot virus defective interfering RNA
More LessNicotiana clevelandii plants were inoculated with cymbidium ringspot tombusvirus RNA synthesized in vitro, after which further passages were made by sap inoculation. During the third passage, low M r RNA species appeared which had the characteristics of deletion mutants of genomic RNA. Sequence analysis of several of these defective interfering RNAs suggested a possible evolution of smaller from larger molecules. Computer-generated secondary structures of sequences surrounding recombination sites were extensive and stable and these sites occurred in interior or hairpin loops, thus providing a possible explanation for discontinuous RNA transcription and the formation of deletions in genomic RNA.
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