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Volume 70,
Issue 8,
1989
Volume 70, Issue 8, 1989
- Bacterial
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Isolation and Morphological Characterization of E1 Tor Cholera Phages
More LessSUMMARYFive bacteriophages have been isolated for phage typing of Vibrio cholerae biotype E1 Tor. The morphology of these phages has been studied by electron microscopy.
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- Animal
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Role of Adenovirus Type 2 Early Region 1B 19K Protein Stability in Expression of the cyt and deg Phenotypes
More LessSUMMARYThe cell line KB18 constitutively expresses adenovirus type 2 (Ad2) early region 1B (E1B) genes in the absence of E1A expression and thus is useful for understanding the function and properties of E1B gene products. We report here that KB18 cells complement the cyt and deg phenotypes of Ad2 cytocidal (cyt) mutants and an Ad12 cyt mutant. Thus the E1B 19K polypeptide in KB18 is functional. Expression of E1B mRNA and synthesis of the 19K polypeptide were studied in wild-type Ad2- and Ad2cyt15-infected KB cells at 15 h post-infection, and in KB18 cells. Although E1B mRNA synthesized in KB18 cells at 5 to 7% of the level in Ad2-infected KB cells, the amount of E1B 19K polypeptide formed was similar. In contrast, the amount of 19K protein in Ad2cyt15-infected KB cells was about one-tenth of that in Ad2-infected cells, although expression of E1B mRNA was comparable to that during Ad2 infection. The half-lives of the 19K polypeptide in wild-type-infected KB cells, in Ad2cyt15-infected KB cells and in KB18 were approximately 90 min, 25 min and 22 h, respectively. The cyt phenotype was expressed at 30 to 35 h post-infection, thus showing that the instability of the 19K polypeptide in Ad2cyt15-infected cells is not due to alteration of cell morphology or cell destruction.
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Bovine Leukaemia Virus Packaging Cell Line for Retrovirus-mediated Gene Transfer
More LessSUMMARYRetroviral packaging cell lines were constructed by using the gag–pol gene of spleen necrosis virus, the gag–pol gene of Moloney murine leukaemia virus and the env gene of bovine leukaemia virus. The plasmids containing the gag–pol genes and the plasmid containing the env gene were cotransfected into NIH/3T3 and D17 cells. The cells containing the helper virus constructs were tested for their ability to package replication-defective murine leukaemia and avian reticuloendotheliosis retrovirus vectors. The titre of vector virus produced by each of the retroviral packaging cell lines was about 102 colony-forming units per ml of medium. Tests for events that might result in intact replication-competent retroviruses showed no evidence for the generation of such viruses. The vector viruses were able to infect dog and rat cells. Bovine cells were infected only after their cocultivation with the retroviral packaging cell lines producing murine leukaemia virus vectors, perhaps as a result of a low concentration of receptors.
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A Transcriptional Map of Visna Virus: Definition of the Second Intron Structure Suggests a rev-like Gene Product
More LessSUMMARYVisna virus is the prototype lentivirus, with a genome structure similar to that of the human immunodeficiency viruses HIV-1 and HIV-2. We have analysed in vitro the transcription pattern of this virus in lytic infections of choroid plexus cells. Northern blot analysis shows the presence of spliced subgenomic mRNA species of 4·9, 4·3, 4·0, 1·7 and 1·4 kb. Use of appropriate subgenomic probes shows that the first three of these species encode envelope protein (but also potentially the small open reading frames Q and S). The 1·7 kb RNA could contain S. In order to elucidate the translational coding potential of the smallest RNA, and to characterize further all the transcripts, S1 mapping was performed across those parts of the genome which were close to exon/intron boundaries. This allowed the definition of acceptor splice sites following both introns 1 and 2 as well as donor sites preceding intron 2. The data suggest that the 1·4 kb RNA encodes a protein derived from the F reading frame that may form part of a precursor protein and also contains sequences with some degree of homology to the rev (trs/art) protein of HIV, as well as a typical protease cleavage site between these sequences and the F protein.
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The Characterization of Equine Encephalosis Virus and the Development of Genomic Probes
More LessSUMMARYEquine encephalosis virus (EEV) is an orbivirus associated with a peracute illness of horses in southern Africa. The virus has now been partially purified for the first time and characterized on a molecular level. The virion is composed of 10 dsRNA segments and a protein capsid consisting of at least seven structural proteins that vary in M r from 36 000 to 120 000. Partial clones of six of the dsRNA segments of EEV serotype Cascara were obtained and analysed for possible use as serotype-specific or group-specific probes in the detection of EEV dsRNA. Cloned fragments of genome segments 3, 8 and 10 were found to show high conservation of these segments, hybridizing to dsRNA from the six EEV serotypes under conditions that indicated more than 90% sequence homology. The genome segment 2-specific probe did not hybridize with dsRNA from any of the other EEV serotypes, suggesting that this segment encodes the serotype-specific antigen of EEV. Cross-hybridization of probes from genome segments 3 and 5 with dsRNA from bluetongue virus (BTV), epizootic haemorrhagic disease virus (EHDV) and African horse sickness virus (AHSV) indicated that EEV is more closely related to BTV and EHDV than to AHSV. Both probes can be used to distinguish between EEV and AHSV dsRNA.
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The Genomic Identity of Different Strains of Mouse Scrapie Is Expressed in Hamsters and Preserved on Reisolation in Mice
More LessSummary263K is the most widely used strain of agent in scrapie research because it produces very short incubation periods in golden hamsters and exceptionally high infectivity titres in clinically affected brain. 263K is also remarkable in having a very low pathogenicity for mice. Evidence is presented that 263K originated as a mutant that was strongly selected on passage in hamsters. Seven new passage lines have been established in hamsters using well characterized strains of mouse scrapie representing the ‘drowsy goat’ and SSBP/1 families of scrapie strains, and one natural scrapie source. Considerable differences between scrapie strains were found in hamsters using incubation period criteria alone. There was evidence that the parent strain of 263K might be 79V or a strain like it in the ‘drowsy goat’ family. Four of the hamster passage lines were established from scrapie strains that had been cloned in mice. Reisolates in mice were compared with original strains. By the criteria used, two of the reisolates were the same as the original strains. Two others were mutants with incubation periods longer than those of their parental strains but the mutants were different from one another. It is concluded that passage between mice and hamsters can select mutants that would otherwise be lost but there is also clear evidence that the genotypic identity of some scrapie strains is preserved on passage between different host species. These findings are important in the search for the putative nucleic acid genome of the scrapie agent.
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Examination of Eight Cases of Multiple Sclerosis and 56 Neurological and Non-neurological Controls for Genomic Sequences of Measles Virus, Canine Distemper Virus, Simian Virus 5 and Rubella Virus
More LessSUMMARYIn situ hybridization studies have been carried out on brain samples from eight cases of multiple sclerosis (MS) and 56 non-neurological and neurological controls, using single-stranded 35S-labelled RNA probes prepared against genomic RNA sequences of measles virus, canine distemper virus, rubella virus and simian virus 5. Foci of hybridization were found using probes against the measles virus nucleocapsid protein (N), phosphoprotein and fusion protein gene sequences in two of the MS cases, and also in one control, a case of disseminated cytomegalovirus infection with spinal cord necrosis. This result was confirmed using biotinylated probes prepared against the measles virus N genomic sequence. No hybridization was found in any of the MS or control cases using any of the other viral genome-specific probes.
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Molecular Characterization of a Neutralizing Domain of the Japanese Encephalitis Virus Structural Glycoprotein
SUMMARYExpression of antigenic fragments of the Japanese encephalitis virus envelope protein (E) in Escherichia coli has been used to define the boundaries of an antigenic domain that contains the binding sites for 10 anti-E monoclonal antibodies (MAbs). All of these antibodies neutralized the virus in vitro and some of them passively protected mice from a fatal virus challenge. We have shown previously that nine of these antibodies react with the antigenic determinants encoded by a 405 bp fragment of viral cDNA. To determine the amino acid sequences of specific determinants, truncated polypeptides were expressed as fusion proteins in E. coli following progressive Bal 31 exonuclease digestion of the 5′ and 3′ ends of the cDNA fragment. Examination of the immunoreactivity of these polypeptides revealed that the region from methionine 303 to tryptophan 396 was the shortest sequence capable of reacting with any of the 10 MAbs or with a polyclonal, antiviral hyperimmune mouse ascitic fluid. Biochemical tests showed that an intramolecular disulphide cross-linkage between cysteine 304 and cysteine 335 of the E protein sequence was required for presentation of the binding site(s) for these MAbs. Although this 95 amino acid antigenic domain appeared to be capable of forming several conformational neutralizing epitopes, it was not an effective immunogen for inducing neutralizing or protective antibodies in mice.
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Replication Kinetics and Cytopathic Effect of Hepatitis A Virus
More LessSUMMARYThe replication kinetics and c.p.e. of hepatitis A virus (HAV) strain HM-175 were shown to depend upon the passage level of the cell line, and the passage level and method of selection of the virus population. Maximum virus production under singlestep growth curve conditions occurred as early as 24 to 28 h or as late as 10 days post-infection. Although rapid replication of an isolate of HM-715 (pHM-175) occurred initially in BS-C-1 cells, its most pronounced c.p.e. was induced in FRhK-4 cells. The replication kinetics of pHM-175 in BS-C-1 cells were similar to those in FRhK-4 cells, although a higher yield of virus was obtained in the latter. The HAV that generated c.p.e. in FRhK-4 cells was obtained by two different selection processes: virus passage, or cloning of large focus-forming variants from the radioimmunofocus assay. The c.p.e. and yield of infectious pHM-175 in FRhK-4 cells could be reduced by 3 mm-guanidine. Another HAV isolate, strain MD-1, isolated directly from contaminated ground water in cell culture demonstrated c.p.e. in FRhK-4 cells after passage as persistently infected A-549 cells.
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Complete Cloning and Molecular Organization of a Rabies-related Virus, Mokola Virus
More LessSUMMARYMokola virus is a rabies-related virus responsible for both animal and human encephalitis cases in Africa. We report here the construction of a genomic library containing overlapping cDNA clones encompassing the entire genome. Five overlapping clones were sufficient to cover the genome (about 12 kb in size). Mokola virus was shown to share the same genomic organization as the rabies virus genome and also identical transcription signals. cDNA probes characterized six different transcripts by Northern blotting experiments; five of them corresponded in size and location to rabies virus mRNAs, and one of particular interest corresponded to a bicistronic RNA which included the genes for the phosphoprotein and the matrix protein. Comparison of the 3′ and 5′ end sequences of the Mokola genome with those of other members of the Lyssavirus genus showed a high homology and led us to propose a genus-specific consensus sequence. However, the latter appeared to vary widely throughout the Rhabdoviridae family.
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The Anterograde Transport of Rabies Virus in Rat Sensory Dorsal Root Ganglia Neurons
More LessSUMMARYWe have previously described the capacity of neurites extending from cultured rat sensory dorsal root ganglia (DRG) neurons to transport rabies virus through axoplasm in the retrograde direction. Here we report the infection of cultured neurons derived from the DRG and the subsequent anterograde transport of rabies virus from the infected cell somas through the extending neurites to its release into the culture supernatant. Viral transport was monitored by titration of the virus yield in the external compartment. Both early and late transport mechanisms of rabies virions were identified. The first one occurred a few hours post-infection and was undetectable 6 h later, before the initiation of viral replication. The velocity of this first wave of infective virions was in the range of 100 to 400 mm/day. The early viral transport was probably the result of a direct translocation of infective virions from the somatic site of entry to the neuritic extensions and subsequent release into the culture medium without replication in the cellular perikaryon. The second virus transport peak was detected 48 h post-infection. In this case, the virions detected in the neuritic compartment were presumably the progeny of the inoculated virus which had replicated in the perikaryon before the viral transport occurs. Using a four-compartment culture device we were able to demonstrate, simultaneously, retrograde and anterograde transport of the virus. The presence of antirabies serum in contact with the exposed neurites did not inhibit either the retrograde or the anterograde transport mechanisms. The viral release from the neuritic extensions after the fast anterograde transport was evaluated to be in the range of 150 to 300 infectious virions per bundle of neurites per day.
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Protection of Woodchucks from Infection with Woodchuck Hepatitis Virus by Immunization with Recombinant Core Protein
S. Roos, K. Fuchs and M. RoggendorfSUMMARYWoodchucks were immunized with recombinant woodchuck hepatitis virus (WHV) core antigen (WHcAg) to investigate whether such immunization protects against WHV infection. The C gene was cloned into a pUC12 vector and expressed in Escherichia coli. Core particles purified by sucrose and CsC1 gradient centrifugation had a buoyant density of 1·37 g/ml which corresponded to the density of WHcAg particles present in chronically infected liver. Two animals immunized with the recombinant antigen developed high antibody titres and were protected against infection after challenge with WHV. The surface antigen (WHsAg) and WHV DNA were not detected in the sera of immunized animals after challenge and these animals did not develop anti-WHs. Three control animals developed a typical WHV infection. The protection from WHV infection may depend not on the presence of antibodies against the core protein but on a cellular immune response to WHcAg.
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IgG-neutralized Influenza Virus Undergoes Primary, but Not Secondary Uncoating in vivo
More LessSUMMARYEven when neutralized by saturating amounts of monoclonal IgG directed against the haemagglutinin, influenza virus attaches to cells with kinetics similar to those of infectious virus. It then enters those cells and is uncoated; its RNA becomes localized within the nucleus and its lipid envelope and associated proteins remain in the cytoplasm. In this report we show that despite the apparent normality of these early stages of virus-cell interaction, neutralized virus underwent no detectable primary transcription. In contrast, there was only a slight inhibition of transcription by neutralized virus in vitro which was insufficient to account for the loss in infectivity, despite using mRNA to measure the production of capped oligonucleotides or to prime the elongation step. To test whether the absence of primary transcription in vivo resulted from non-accessibility of the genome rather than an effect on the transcriptase complex itself, we examined the susceptibility to RNase of virion RNA after inoculation of cells with neutralized virus. Data clearly show that, unlike RNA of infectious virus, RNA of neutralized virus did not become sensitive to RNase and we conclude that neutralization of influenza virus by IgG results in failure of virus to undergo a secondary uncoating process which is necessary for the activity of the virion transcriptase complex. Finally we show that by treatment of virions in vitro with detergent it is possible to produce a core structure which is stable and has some of the properties expected of a structure resulting from primary uncoating.
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Biological and Genetic Evolution of the Nucleoprotein Gene of Human Influenza A Viruses
More LessSUMMARYThere is a significant difference in the ability of human influenza A virus H1N1 strains isolated up to 1977 and those isolated later to rescue temperature-sensitive mutants of fowl plague virus with a defect in the nucleoprotein (NP) gene. Therefore the NP genes of five human H1N1 and H3N2 influenza A virus strains, isolated between 1950 and 1978, have been sequenced. By comparison with previous and more recent isolates, an evolutionary pathway has been established. Three amino acid replacements were found which might be responsible for the functional difference between the USSR (1977) and the Brazil (1978) strains. The California (H1N1) strain isolated in 1978 had acquired by reassortment the NP gene of a human H3N2 virus circulating at about 1977 as had been previously suggested by investigations involving RNase fingerprint or hybridization techniques.
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A Herpes Simplex Virus Type 2 Variant in which a Deletion across the L-S Junction Is Replaced by Single or Multiple Reiterations of Extraneous DNA
More LessSUMMARYWe have isolated and characterized a novel variant of herpes simplex virus type 2 (HSV-2) strain HG52 which has a deletion of 13·5 kb across the L–S junction of the genome, resulting in all of the IRL region, half of the IRS region and the intervening L-S junction ‘a’ sequence(s) being removed. The deleted DNA has been replaced by single or multiple (up to 14) reiterations of a DNA sequence approximately 1 kb in length. Individual genomes within the population range in size from approximately 12 kb smaller than unit length to unit length or marginally larger. The L component of the genome is fixed in the prototype orientation while the S component inverts inefficiently. The variant is viable in tissue culture, is not temperature-sensitive, demonstrates impaired single-cycle growth characteristics and, apart from altered mobility of a single species (29.5K), its polypeptide profile in infected cells appears normal. Southern blot analysis has failed to identify the inserted sequences as being derived from HSV-2.
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Differential Sensitivity of Macrophages from Herpes Simplex Virus-resistant and -susceptible Mice to Respiratory Burst Priming by Interferon-α/β
More LessSUMMARYHerpes simplex virus primes mouse macrophages for a genetically determined respiratory burst mediated in an autocrine manner by interferon (IFN)-α/β. We have analysed the effect of IFN-α/β on the respiratory burst capacity of mouse peritoneal macrophages by luminol-dependent chemiluminescence using phorbol myristate acetate as trigger. Crude macrophage-produced IFN-α/β as well as purified IFN-α and -β regularly augmented the respiratory burst capacity of peritoneal cells in a concentration-dependent manner. The augmented response was exclusively mediated by macrophages and was manifest after 4 h incubation with IFN-α/β, peaked after 8 h and gradually declined to near background levels after 24 h. The effect of macrophage-produced IFN-α/β was completely abolished by preincubation of IFN with antiserum to IFN-α/β. The data obtained with this antiserum indicated that endogenous IFN, undetectable by a standard cytopathic effect-inhibition assay, was sometimes spontaneously produced by the peritoneal cells. Furthermore, the crude macrophage preparation seemed to contain a macrophage deactivating factor counteracting the effect of IFN-α/β. Genetic analysis of the sensitivity of macrophages for the respiratory burst-priming effect of IFN-α/β revealed that the trait is inherited as a co-dominant autosomal feature. Macrophages from herpes simplex virus-resistant C57BL/6 mice were more sensitive than macrophages from virus-susceptible BALB/c mice and cells from mice of the reciprocal crosses showed an equal sensitivity intermediate between those of the parental strains. A physiological role of differential IFN sensitivity in the context of resistance to virus infections is suggested.
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Antibody Responses and Protection in Mice Immunized with Herpes Simplex Virus Type 1 Antigen Immune-stimulating Complex Preparations
More LessSUMMARYThe formation of immune-stimulating complexes (iscoms) obtained by mixing the glycoside Quil A with an antigen preparation derived from herpes simplex virus type 1 (HSV-1)-infected cell cultures using a zwitterionic detergent is described. The HSV-1 antigen preparation incorporated into iscoms elicited significantly greater antibody responses in mice than the same preparation administered together with aluminium hydroxide gel, and provided complete protection against HSV-1 or HSV-2 lethal, systemic challenge infection in animals given a single dose containing 5 μg of protein. The HSV-1 iscom preparation also provided significant protection in mice against local reactions following challenge with HSV-1 by skin scarification.
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A Role for Herpes Simplex Virus Type 1 Glycoprotein E in Induction of Cell Fusion
More LessSUMMARYThe role of herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) in the induction of multinucleate cell (syncytium) formation was investigated using monoclonal antibodies and agE deletion mutant, R7023. We found that monoclonal antibodies directed against gE blocked HSV-1-induced syncytium formation in human cells. R7023 also failed to induce syncytium formation in tissue culture cells. The results indicate that gE, in addition to glycoproteins B, D, H and the gene sequence located between 0·732 and 0·745 map units, is involved in cell fusion. Thus, this important biological property appears to be regulated by several HSV-1 gene products.
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In vitro Promoter Activity Associated with the Latency-associated Transcript Gene of Herpes Simplex Virus Type 1
More LessSUMMARYThe herpes simplex virus latency-associated transcript (LAT) gene is the only viral gene that shows substantial transcriptional activity during neuronal latency. The LAT RNA produced is antisense to the mRNA of the immediate early gene ICP0, partially overlaps the ICP0 mRNA, and is suspected of playing some role in latency. Sequence analysis of the region 5′ to the reported transcription start site has not revealed any high consensus RNA polymerase II promoter elements. Nonetheless, LAT RNA is transcribed in low abundance during acute infection in tissue culture. As the initial step in mapping the promoter for this latency-associated gene, we analysed the ability of different regions of the LAT gene to drive the transcription of an indicator gene in vitro. Using chloramphenicol acetyltransferase (CAT) assays, we found that the genomic region between –940 and –662 nucleotides upstream of the transcription start site of the LAT gene was most efficient at directing transcription of the indicator CAT gene in Vero cells. This suggests that the LAT promoter, or at least the promoter controlling transcription of this gene during acute infection in tissue culture, may have an unusual location of more than 662 nucleotides upstream from the reported start of RNA transcription.
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Retention and Expression of the Left End Subfragment of the Herpes Simplex Virus Type 2 BglII N DNA Fragment Do Not Correlate with Tumorigenic Conversion of NIH 3T3 Cells
More LessSUMMARYCotransfection experiments have been carried out using recombinant plasmids pAG60, conferring resistance to antibiotic G418, and pXho3 which contains the left end subfragment (map coordinates 0·583 to 0·596) of the transforming herpes simplex virus type 2 BglII N DNA fragment and encodes the 36K polypeptide associated with the viral ribonucleotide reductase activity. Several NIH 3T3 cell clones resistant to G418 and having morphological changes commonly observed for transformed NIH 3T3 cells were isolated and examined for the presence and stable retention of the viral sequences. Seven of the clones that retained the transfected viral sequences were analysed for the expression of the 36K polypeptide and the tumorigenic phenotype. The results gathered from these studies show that neither the retention of the viral DNA nor the expression of the 36K polypeptide correlated with tumorigenic conversion of these cells.
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