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Volume 67,
Issue 9,
1986
Volume 67, Issue 9, 1986
- Review Article
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- Animal
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The Complete DNA Sequence of Varicella-Zoster Virus
More LessSummaryThe entire DNA sequence of varicella-zoster virus (VZV) was determined using the M13-dideoxynucleotide technology. The genome is variable in size, but the sequence which was obtained comprises 124884 bp. Analysis of the sequence indicated that the genome contains 70 genes distributed about equally between the two DNA strands. The genes are organized compactly, but regions of overlap between protein-coding regions are not extensive. Many of the genes are arranged in 3′-coterminal families, and at least one is spliced. The discerned organization of VZV genes and that deduced for herpes simplex virus type 1 (HSV-1) from published transcript mapping data indicate that these two members of the Alphaherpesvirinae are very similar in gene layout. Comparisons of the predicted amino acid sequences of VZV proteins with those available for HSV-1 proteins generally suggest evolution from an ancestral genome, and allow the functions of several VZV genes to be deduced, although limited regions where the genomes differ in functional organization were also identified.
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Analysis of Varicella-Zoster Virus DNAs of Clinical Isolates by Endonuclease HpaI
More LessSummaryThe DNAs of 20 strains of varicella-zoster virus (VZV) isolated from epidemiologically unrelated individuals, and of 15 strains isolated from vesicles of vaccinees with varicella or zoster after vaccination, were compared by restriction enzyme cleavage using HpaI. Differences were found in the sizes of the HpaI-F, -G and -K fragments of the wild strains. The gel migration patterns of the HpaI-F and -G fragments, but not of the HpaI-K fragment, were polymorphic in the different strains isolated from the vaccinees. The effects of serial passages in vitro and in humans on the genome stability of VZV were investigated by HpaI analysis. The DNA profiles of the HpaI-K fragments from six isolates recovered from room-mates infected in a single outbreak were identical, but the mobilities of their HpaI-F and -G fragments varied. The DNA profiles of the Oka vaccine virus after 10 and 85 passages in human embryo cells differed only in the HpaI-F fragment. The profiles of these fragments in DNA derived from two isolates obtained at different times from a vaccinee with varicella followed by zoster were compared with those of the Oka (parental) and Oka (vaccine) strains, and identical results were obtained for the two viruses. In addition, the same DNA profiles of HpaI fragments were obtained from three sequential isolates from one person and also from two isolates from another with varicella and zoster. Thus, it was concluded that: (i) three variable fragments (HpaI-K, -F and -G) were not changed in the DNAs of isolates derived from the same patient; (ii) HpaI-K was stable both on passage in vitro and after human transmission in the case of the same outbreak, but was different among all wild-type strains isolated in epidemiologically unrelated outbreaks; (iii) HpaI-F was very unstable both on passage in vitro and in human infections by either vaccine or wild-type strains; (iv) HpaI-G was not influenced by passage in vitro but varied among wild-type strains. Using physical maps of VZV DNA established by others, three variable regions on the viral genome were identified. One was located near the 0.16 coordinate, which is covered by HpaI-K (variable region I, VRI). Another was represented by HpaI-F (VRII), the most unstable fragment, and mapped at about the 0.35 coordinate. The third was VRIII near the right terminus, covered by HpaI-G.
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The Effect of Triterpenoid Compounds on Uninfected and Herpes Simplex Virus-infected Cells in Culture. II. DNA and Protein Synthesis, Polypeptide Processing and Transport
More LessSummaryThe effect of the triterpenoid drugs carbenoxolone sodium (CBX) and cicloxolone sodium (CCX) on DNA and protein synthesis in uninfected and herpes simplex virus (HSV)-infected BHK-21 or Flow 2002 cells has been studied. No consistent effect of 500 µm-CBX or 300 µm-CCX on HSV DNA synthesis was identified. With 300 µm-CCX, some inhibition of cellular DNA synthesis was observed, but this was relatively slight. The restriction enzyme digest profiles of HSV DNA from drug-treated cells appeared normal. The synthesis of several HSV-specified polypeptides was much reduced by treatment with CBX or CCX and the transport of certain proteins between the nuclear and cytoplasmic compartments of infected cells was also affected. CBX or CCX treatment strongly inhibited post-translational glycosylation and sulphation of both host- and HSV-specified proteins, but the phosphorylation of only a few proteins appeared affected. The drugs induced quantitative changes in the synthesis of some BHK cell polypeptides, but these were not considered important. CCX treatment of Flow 2002 cells, however, induced the synthesis of several new polypeptides, some of which had the same apparent molecular weights as identified Flow 2002 cell stress proteins. When treated with concentrations greater than 50 µm-CCX, the plasma membranes of both uninfected and HSV-infected cells became increasingly leaky. The SDS-PAGE polypeptide profile of purified virus particles made in BHK cells treated with 300 µm-CCX differed markedly from that synthesized under drug-free conditions. The greatly reduced amount of infectious virus made in cells treated with 300 µm-CCX was more thermolabile at 42 °C than virus produced in the absence of the drug. Our results indicate that the antiviral activity of the triterpenoid drugs is non-specific and operates by interfering with or changing the normal function of cell membranes so that cells, although retaining their viability, can no longer assemble the virus components efficiently into infectious particles. As a consequence, the population of virus particles made is of inferior quality. While we have no evidence that there is also a specific anti-HSV effect, this possibility cannot yet be ruled out.
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Zosteriform Spread of Herpes Simplex Virus Type 2 Genital Infection in the Guinea-pig
More LessSummaryA model of herpes simplex virus type 2 (HSV-2) infection was developed in the guinea-pig that permits investigation of the role of neural spread of virus in the pathogenesis of genital skin disease. After HSV-2 inoculation of an abraded area lateral to the external genital skin, virus was first detected in the ipsilateral (to the inoculation site) peripheral nerves or genital skin on day 2 or 3 post-inoculation followed by detection in the ipsilateral dorsal root ganglia and spinal cord on day 3 or 4. Virus also spread to the contralateral dorsal root ganglia, contralateral peripheral nerves and contralateral genital skin on day 4 or 5 although lesions were only rarely detected on the contralateral side. Genital skin lesions first developed on day 5 and were followed by the development of lesions along the medial aspect of the ipsilateral hindlimb. Virus was first detected in the vaginal vault on day 4 or 5. The development of lesions appeared to be unrelated to vaginal virus replication but was associated with the recovery of virus from peripheral nerves. Recurrent genital skin lesions were seen more commonly on the inoculated side than the contralateral side. Since the development of skin lesions appeared to result from virus emerging from nerve endings, an event similar to the terminal event that is believed to be involved in the development of recurrent lesions, we now have a model which may be useful in further exploring the pathogenesis of herpetic disease.
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Genetically Determined Difference in the Antiviral Action of α/β Interferon in Cells from Mice Resistant or Susceptible to Herpes Simplex Virus Type 2
More LessSummaryResistance of mice to infection with herpes simplex virus type 2 (HSV-2) is genetically determined. Embryonic cells from susceptible BALB/c and resistant C57BL/6 mice were equally sensitive to infection with HSV-2 as judged by plaque area, plaquing efficiency, endpoint titration and virus yield. Cells from C57BL/6 mice showed a higher sensitivity than cells from BALB/c mice to the protective action of two preparations of α/β interferon against challenge with HSV-2. This was evident both from c.p.e. inhibition and yield reduction experiments. The difference in sensitivity was dependent on virus dose and was greatest (up to 50-fold) with low virus doses. An analysis of the genetics of the α/β interferon sensitivity in cells from embryos of parental mice and embryos derived from reciprocal matings between HSV-2-resistant and -susceptible mice suggested that interferon sensitivity is inherited as a co-dominant autosomal trait. The induction of the interferon-induced enzyme 2′-5′-oligoadenylate synthetase was also different in cells from the two mouse strains, since significant levels were only detected in cells from C57BL/6 mice. It is suggested that differential interferon sensitivity of cells from HSV-2-resistant and -susceptible mice might be a factor of importance for the course of the infection.
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An Antiviral State Induced in HeLa Cells by Microinjected Poly(rI).Poly(rC)
More LessSummaryThe direct introduction with micropipettes of poly(rI).poly(rC) into the cytoplasm of several human cell lines inhibited the multiplication of vesicular stomatitis virus. This antiviral activity was at least partly due to interferon (IFN) production and secretion from the injected cells since it was species-specific, partly neutralized by IFN antibodies and was transmissible to non-adjacent cells. This suggests a mechanism of IFN induction involving the internalization of poly(rI).poly(rC) and its interaction with an intracellular target.
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Autologous Monoclonal Antibodies Recognize Tumour-associated Antigens in X-irradiated C57BL/6 Mice
SummaryX-irradiation of C57BL/6 mice induces thymic lymphosarcomas which sometimes contain retroviruses which upon injection into normal mice mimic the effect of the irradiation. We examined whether specific antigenicities, viral or cellular, were expressed by tumour cells that could be recognized by antibodies from the irradiated animals. We developed monoclonal antibodies (MAbs) using splenocytes of the diseased animal. The reactivity of such MAbs towards thymoma cell lines established in vitro was investigated by means of an ELISA. At least 10 antibody specificities were detected on the 13 tumours investigated, allowing separation of the MAbs into three classes: (i) those recognizing the autologous tumour, heterologous tumours as well as normal thymic tissue, (ii) those specific for the autologous tumour, and (iii) those specific for one tumour, but not ones of autologous origin. The last two classes corresponded to specific tumour-associated antigens. Our panel of MAbs defined each tumour by the particular pattern of antigens harboured. It is striking that most of the antigens were present in the normal thymus and that only two tumours had additional antigenicities. Additionally, quantitative variations were observed in the levels of expression of these antigens.
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Immunoprevention of Friend Leukaemia Virus-induced Erythroleukaemia by Vaccination with Aggregated gp70
More LessSummaryVaccines prepared from Friend leukaemia virus envelope and core polypeptides were compared for their efficiency in preventing erythroleukaemia in mice. High doses (100 µg) of gp85, the micellar complex of the envelope polypeptides gp70 and p15E, completely protected STU mice. The same dose of purified gp70 still protected about 80% of the animals, while p15E did not affect the cumulative mortality. The internal viral polypeptide p30 was ineffective. Serological examination indicated that immunity against death from leukaemia was mediated by specific antibodies. These leukaemia-preventing antibodies were predominantly induced by immunization with the gp70 env gene product, since p15E showed only minor protection. Glycoprotein gp70, however, was more effective when given as the gp85 micellar complex. An even more potent vaccine was obtained when gp70 was coupled to keyhole limpet haemocyanin (KLH) by glutaraldehyde. Ten µg gp70 coupled to KLH was enough to save more than 90% of Friend leukaemia virus-infected mice from erythroleukaemia. KLH may also be a suitable experimental carrier for subunits of gp70 or synthetic oligopeptides for viral vaccines.
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The Expression of Human Papillomavirus Type 18 E6 Protein in Bacteria and the Production of Anti-E6 Antibodies
More LessSummaryHuman papillomavirus type 18 (HPV-18) has recently been closely linked with human malignant cervical lesions. The early region of the genome of the related bovine papillomavirus (BPV) has been shown to be important for the production of the transformed phenotype. BPV E6 has been shown to be a transforming protein. We report the primary structure of the HPV-18 E6 open reading frame and its predicted amino acid sequence. Both E6 protein and E6-β-galactosidase fusion protein were synthesized in bacteria. Antisera were raised against the E6-β-galactosidase fusion protein and against an E6 N-terminal peptide which was 14 amino acids long. We show that these antisera reacted on Western blots against E6 synthesized in bacteria. The HPV E6 antigen and antibodies described here will be useful in understanding HPV expression and its association with human malignancies and may also be diagnostically useful.
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Nucleotide Sequence Analysis of the Haemagglutinin-Neuraminidase Gene of Newcastle Disease Virus
More LessSummaryThe nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV) has been determined. The HN gene is 2031 nucleotides long, approximately 13.5% of the viral genome. The nucleotide sequence contains a single long open reading frame which would encode a protein of 577 amino acids, with a mol. wt. of 63149. This is in good agreement with estimates of the molecular weight of the unglycosylated HN protein. Analysis of the amino acid sequence reveals six potential glycosylation sites and shows the major hydrophobic region to be close to the N terminus. This provides evidence for the N-terminal attachment of HN to the viral membrane. The hydrophilic nature of the extreme N-terminal amino acids suggests the absence of a cleaved signal sequence. Analysis of the long non-coding region at the 3′ end of the mRNA encoded by the HN gene of NDV suggests a possible explanation for the origin of HN0 in extremely avirulent strains of NDV. There are regions of high homology between the deduced amino acid sequence of the NDV HN glycoprotein and the HN glycoproteins of two other paramyxoviruses, Sendai virus and simian virus 5 (SV5). An alignment of the HN amino acid sequences of these viruses shows 32% of amino acid residues are conserved between NDV and SV5, and 23% between NDV and Sendai virus. In contrast, only very limited homology is found between NDV HN and the influenza virus glycoproteins.
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Immunological Relationships between Mumps Virus and Parainfluenza viruses Studied with Monoclonal Antibodies
More LessSummaryThe immunological relationships between mumps virus and parainfluenza viruses were investigated with 74, 78 and 80 previously developed monoclonal antibodies directed against five major structural proteins of mumps virus, Sendai virus (a murine parainfluenza type 1 virus) and parainfluenza type 3 virus. These monoclonal antibodies were reacted with the three viruses, with parainfluenza type 2 virus and with Newcastle disease virus (NDV) in ELISA and immunofluorescence (IF) tests. In addition, immunoprecipitation tests with [35S]methionine-labelled extracellular virions were carried out with cross-reacting monoclonal antibodies. None of all 232 monoclonal antibodies against the three viruses cross-reacted with either parainfluenza type 2 virus or NDV in ELISA and IF tests. In the collection of 74 mumps virus monoclonal antibodies, three directed against the nucleocapsid (NP) protein, polymerase protein, and fusion protein cross-reacted with Sendai virus. Two Sendai virus monoclonal antibodies directed against two different epitopes of the haemagglutinin-neuraminidase (HN) protein cross-reacted with parainfluenza type 3 virus. Six other Sendai virus monoclonal antibodies directed against four different epitopes of the HN protein and one directed against the NP protein cross-reacted with mumps virus. Eight out of 80 monoclonal antibodies directed against parainfluenza type 3 virus cross-reacted with Sendai virus. One was directed against the HN protein, four were directed against a minimum of two epitopes of the matrix protein and three were directed against three different epitopes of the NP protein. The different cross-reactions found show that Sendai virus is antigenically related to both mumps virus and parainfluenza type 3 virus. In contrast, no antigenic relationship could be demonstrated between mumps virus and parainfluenza type 3 virus.
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The Uncoating and Infectivity of the Flavivirus West Nile on Interaction with Cells: Effects of pH and Ammonium Chloride
More LessSummaryInfectivity of the West Nile virus (WNV; Flaviviridae) was inactivated on exposure for brief periods (90 s) to pH 6.6 and below. This inactivation was not due to decreased interaction between cells and acid-treated virus. The RNA of [3H]uridine-labelled virus particles prebound to the cell surface before acidic pH treatment underwent rapid uncoating within 1 min at 37 °C at the same pH values that inactivated virus particles. The uncoating of [3H]uridine-labelled virus particles was also studied over longer time periods after synchronized internalization by P388D1 cells. At pH 7.6 uncoating occurred rapidly after a reproducible time lag of 1 min on warming to 37 °C and was essentially complete by 15 to 30 min after the start of internalization, leaving uncoated RNA in an infectious form. In contrast, at pH 6.2 viral uncoating occurred rapidly without any time lag and the uncoated RNA appeared to be far less infectious than that uncoated at pH 7.6. Ammonium chloride could almost totally inhibit both the infectivity and uncoating of virus particles on synchronized internalization into P388D1 cells, with a pH optimum of 8.0. These results suggest that the uncoating of virus particles is dependent on an acidic pH, although the location of uncoating (prelysosomal endosome or plasma membrane) decides whether the uncoated RNA will be infectious or not. Essentially the same results were obtained when infections were carried out in the presence of enhancing antibody.
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Molecular Determinants of Alphavirus Neurovirulence: Nucleotide and Deduced Protein Sequence Changes during Attenuation of Venezuelan Equine Encephalitis Virus
More LessSummaryThe nucleotide and deduced amino acid sequences of the structural proteins of the TC-83 vaccine strain of Venezuelan equine encephalitis (VEE) virus have been determined from a cDNA clone containing the 26S mRNA coding region. A cDNA clone encoding the equivalent region of the virulent parent VEE virus [Trinidad donkey strain (TRD)] has been sequenced previously. Comparison of the sequences of the TC-83 and TRD cDNA clones revealed 13 nucleotide differences. Neither the organization of the structural proteins (5′-capsid-E3-E2-6K-E1-3′) nor the length (3762 nucleotides) of the open reading frame coding for the viral polyprotein precursor was altered during attenuation. Of the 13 nucleotide differences between the cDNA clones of TC-83 and TRD, nine occurred in the dominant population of the respective genomic RNAs from plaque-purified viruses. Six of the nine mutations were clustered in the E2 surface glycoprotein gene. All five of the nucleotide changes which produced non-conservative amino acid substitutions in the encoded proteins were located in the E2 gene. Two mutations occurred in the E1 glycoprotein gene; one was silent and the other did not alter the chemical character of the E1 protein. One nucleotide difference was found in the non-coding region immediately preceding the 5′-end of the 26S mRNA. The E2 and non-coding region mutations are candidates for the molecular determinants of VEE virus neurovirulence.
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Molecular Analysis of Campoletis sonorensis Virus DNA in the Lepidopteran Host Heliothis virescens
More LessSummaryNucleic acid hybridization techniques were used to analyse the fate of Campoletis sonorensis virus (CsV) DNAs in naturally parasitized and virus-injected Heliothis virescens larvae. Viral DNA persisted in injected H. virescens larvae from 0 to 10 days post-injection but no increase in the amount of viral DNA could be detected. Similarly, no increase in the amount of viral DNA was detected in naturally parasitized H. virescens larvae before the development of the C. sonorensis pupae. However, a dramatic increase of viral DNA was detected in pharate and newly emerged (0 to 48 h) adult C. sonorensis wasps. The results from these in vivo molecular analyses suggest that CsV replication does not occur in naturally parasitized or virus-injected H. virescens and that viral replication may be restricted to tissues of C. sonorensis wasps.
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Characterization of Clones for the Sixth (L) Gene and a Transcriptional Map for Morbilliviruses
More LessSummarycDNA clones of the largest RNA transcript of the canine distemper and measles morbilliviruses were characterized. This presumably codes for the L protein of these viruses. mRNA 4 was identified as coding for the haemagglutinin protein of measles virus. From an analysis of readthrough transcripts representing tandem copies of two or three genes we established a transcriptional map and the gene order on the negative strand genome of the morbilliviruses to be 3′-N-P+C-M-F-H-L-5′. The data exclude the presence of small intervening genes between the six major genes of morbilliviruses and indicate the gene order to be similar to that of Sendai virus and different from that of simian virus 5.
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Measles Virus Persistent Infection: Modification of the Virus Nucleocapsid Protein
More LessSummaryThe synthesis of intracellular measles virus proteins in persistently infected human cell cultures was studied. The virus-induced proteins were analysed after radioimmuno-precipitation by one- and two-dimensional polyacrylamide gel electrophoresis. The measles virus-induced nucleoprotein (NP) synthesized in persistently infected cells had a reduced binding capacity with measles virus antibodies (human convalescent serum) compared to the NP protein induced by the virus used to initiate the infection. In contrast, monospecific rabbit serum prepared against the original virus NP, or monoclonal anti-NP antibodies, precipitated NP proteins from acutely and persistently infected cells with equal efficiency. When the NP in acutely or persistently infected cells was labelled with either 14C- or 3H-amino acids and subjected to two-dimensional gel analysis, significant charge differences were observed between the virus proteins. When measles virus-infected cells were examined for virus protein synthesis at 40 °C, although no change was found in acutely infected cells, NP was not detected in the persistent infection.
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Transcriptional Map of the Measles Virus Genome
SummaryWe have constructed a recombinant DNA library of the measles virus genome and identified gene-specific clones containing sequences coding for portions of each of the six viral structural proteins, as well as clones coding for intercistronic sequences. By Northern blotting, we could order the clones according to the pattern of individual gene-specific and readthrough mRNAs. Clones corresponding to the N, P/C and M genes were identified by correlation of the mRNAs with their in vitro translation products; clones corresponding to the H, F and L genes were identified by indirect evidence. The results indicated that the gene order in measles virus is that of a typical paramyxovirus (3′-N,P/C,M,F,H,L-5′), but that the M and F transcripts each contain 1.5 times the coding capacity needed for synthesis of these proteins in vivo.
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Detection and Characterization of Infectious DNA Intermediates of a Primate Foamy Virus
More LessSummaryDNA from human T-lymphoid (Molt-4) and hamster kidney (BHK-21) cells infected with the T-lymphotropic simian foamy virus LK-3 was shown to be infectious, when assayed by transfection of BHK-21 cells. The proviral genome was further characterized by blot hybridization to a specific cDNA probe, which had been prepared by reverse transcription in vitro using viral RNA and RNA-dependent DNA polymerase present in cytoplasmic extracts of infected BHK-21 cells. This probe hybridized to a DNA species of 14 kbp in extracts from LK-3-infected diploid human fibroblasts, Molt-4 and BHK-21 cells, whereas no hybridization occurred with DNA from the respective uninfected controls. No integrated proviral DNA could be demonstrated, and the 14 kbp DNA was shown not to represent circular DNA. The patterns of restriction endonuclease and S1 nuclease fragments indicated a unique configuration of linear double-stranded DNA containing a single-stranded section separating two subunits one of which may be sufficient to transmit LK-3 by transfection with DNA.
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Suppression of Friend Virus-induced Leukaemia in Mice by Tuftsin
More LessSummaryA significant decrease in mortality was observed when 25 µg of the tetrapeptide tuftsin was given to DBA/2J mice 5 days before infection with Friend leukaemia virus (FLV). The same effect was observed when tuftsin was given 5 days before and twice a week for 3 weeks after FLV infection. No effect was observed when the same amount of tuftsin was given 1 day before infection. A 5 µg dose of tuftsin given 5 days before and twice-weekly for 3 weeks had no effect on leukaemia induced by FLV infection. These findings showed that time and dosage were critical to the protective effect of tuftsin against virus-induced leukaemia.
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