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Volume 42,
Issue 1,
1979
Volume 42, Issue 1, 1979
- Review Article
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- Articles
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Adenosine Triphosphate Content in Lactobacillus casei and the Blender-resistant Phage-Cell Complex-forming Ability of Cells on Infection with PL-1 Phage
More LessSUMMARYThe intracellular ATP content of Lactobacillus casei ATCC 27092 grown in a glucose-containing medium was almost constant (2 to 3 µg/mg dry wt. cells) through the early to middle stage of logarithmic phase, but it was lowered to less than 0.1 µg/mg after cessation of growth owing to the exhaustion of available glucose. All the cells in the early stage of stationary phase were still viable and thus considered to be in a starved state. When such starved cells were infected with PL-1 phages in a tris-maleate buffer of pH 6.0, the process of forming blender-resistant phage-cell complexes signifying the complete infection of phage genomes into the cells was much inhibited. There was a good correlation between the ATP content of cells and the extent of the formation of blender-resistant phage-cell complexes and the correlation coefficient between them was 0.89 ± 0.09 at the 95% confidence limit. On the other hand, the process of forming both the phage-adsorbed cells and the anti-phage serum-resistant phage-cell complexes were not affected by the ATP content of cells. Feeding of glucose to such starved cell cultures caused the cells to restore both the ATP content and the ability to form blender-resistant phage-cell complexes. Such restoration was also observed when the starved cells collected by centrifugation were incubated in a glucose-containing medium.
The significance of the intracellular level of high energy compounds such as ATP for the mechanism of the injection of phage genomes into the cells is discussed.
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Structural Polypeptides of the Murine Coronavirus JHM
More LessSUMMARYAnalysis by SDS-polyacrylamide gel electrophoresis shows that the purified coronavirus JHM contains six polypeptides. The apparent mol. wt. of the polypeptides (GP1, GP2, GP3, VP4, GP5 and VP6) are 170000; 125000; 97500; 60800; 24800 and 22700, respectively. Four polypeptides are glycosylated (GP1, GP2, GP3 and GP5). The analysis of particles obtained after limited proteolysis with pronase suggests that GP2 and GP3 are protruding from the lipid envelope and, together with GP1, form the spike layer. Protein VP6 and a part of GP5 are located within the lipid bilayer. Protein VP4 is susceptible to digestion at a concentration of pronase which changes the morphology of the virus particles making the interior of the virus accessible. Subviral particles produced after treatment with the detergent Nonidet P40 banded at a higher density than the virus and contained only VP4, GP5 and VP6.
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Detection of Polyoma Virus DNA in PML-Brain Tissue by (in situ) Hybridization
More LessSUMMARYThe human papova (JC) virus was extracted from brain of a patient with progressive multifocal leukoencephalopathy. A single band of virus was obtained at a density of 1.345 g/ml CsCl. JC virus DNA was purified and a highly specific cRNA was generated in vitro. In situ hybridization with JC virus cRNA and autoradiography on sections of the same brain revealed silver grains over oligodendrocytes, astrocytes and possible vascular endothelial cells, indicating the presence of JC virus DNA in these different cell classes.
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Transcription of Double-stranded RNA in Virions of Aspergillus foetidus Virus S
G. Ratti and K. W. BuckSUMMARYIt is shown that the virion RNA polymerase of Aspergillus foetidus virus S, which has a genome consisting of three double-stranded (ds)RNA species, is a transcriptase. Synthesis of single-stranded RNA (ssRNA) in vitro continues for up to 48 h, during which time 6 to 8 full length transcripts are produced, on average, per molecule of dsRNA2, i.e. re-initiation of transcription occurs in this in vitro system. Full length ssRNA transcripts of dsRNA1 are also produced in smaller quantity, but no transcripts of dsRNA3 could be detected. In reactions containing 3H-UTP, label is also incorporated into dsRNA2 reaching maximum levels after 6 h, but no incorporation of labelled precursor into dsRNA1 or dsRNA3 could be detected. The ssRNA transcripts produced were released into the medium, but labelled dsRNA remained within the virions. The locations, in the AfV-S multicomponent system, of RNA polymerase activities promoting incorporation of 3H-UMP into the two ssRNA transcripts and into dsRNA2 have been determined.
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Biosynthesis of the Influenza Virus Envelope in Abortive Infection
More LessSUMMARYSynthesis and processing of the envelope proteins of influenza A virus (fowl plague virus) have been analysed in BHK, HeLa and L cells, in which the virus undergoes abortive replication and does not form virus particles, and in the productive chick embryo fibroblast system. In abortive infection, synthesis of the M protein is specifically inhibited. The extent of this defect varies depending on the host cell and the amount of virus particles formed closely reflects the amount of M synthesized. Cell fractionation experiments demonstrated that the haemagglutinin glycoprotein HA is synthesized in abortive as well as in productive cells at the rough endoplasmic reticulum, that it migrates via smooth internal membranes to the plasma membrane and that it is cleaved by proteolysis into fragments HA1 and HA2 in the course of migration. Immune electron microscopy using monospecific antibodies against haemagglutinin and neuraminidase showed that both glycoproteins are exposed at the cell surface. Thus, synthesis and processing of the virus glycoproteins does not depend on the formation of the M protein. However, the M protein appears to be necessary for budding and thus for particle formation.
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Comparison of Some Biophysical Properties of the Nudaurelia β and ε Viruses
More LessSUMMARYA study of the physical properties of the Nudaurelia β and ε viruses showed the ε virus to be slightly larger but less dense in CsCl than the β virus even though no difference in size or shape could be detected by electron microscopy. Comparative values for the physical properties of the β and ε viruses respectively were: sedimentation coefficient 213 and 217S, diffusion coefficient 0.97 and 0.79 × 107 cm2/s, buoyant density in CsCl 1.30 and 1.28 g/ml, and diam. in the electron microscope 39 to 40 nm for both viruses. All preparations of β virus included small amounts of β virus antigen with a buoyant density of 1.33 g/ml.
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A Small Iridescent Virus (Type 29) Isolated from Tenebrio molitor: a Comparison of its Proteins and Antigens with Six Other Iridescent Viruses
More LessSUMMARYA small iridescent virus (type 29) has been isolated from the meal worm Tenebrio molitor. The virus is distinct from a number of previous isolates of small iridescent viruses (types 2, 6, 21, 22, 23 and 28) judged by polyacrylamide gel electrophoresis of its structural polypeptides. Immunodiffusion and immunoprecipitation tests showed that iridescent virus type 29 is related to types 22 and 23 but not types 2, 6, 21 and 28. The relationships between iridescent virus types 22, 23 and 29 were further studied by complement fixation and kinetic neutralization. Complement fixation tests confirmed that these viruses were related and showed that type 29 is distantly related to types 22 and 23 (which were more closely related to each other) though type 23 shares a closer relationship with type 29 than type 22. Kinetic neutralization experiments suggested a close relationship between types 29 and 23, and that both these viruses were remotely related to type 22. Low neutralization rate constants were obtained with these sera.
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Pathogenesis of Scrapie: Agent Multiplication in Brain at the First and Second Passage of Hamster Scrapie in Mice
More LessSUMMARYThe intracerebral (i.c.) injection of mice with a particular source of hamster passaged scrapie produced disease after an incubation period of 325 ± 6 days (mean ± s.e.). The incubation period at the second i.c. passage in mice was reduced to 149 ± 2 days. Studies were made of the dynamics of agent replication at 1st and 2nd passages in mice. At first passage, there was a ‘zero phase’ lasting about 175 days, when no infectious agent was detected in brain (or spleen), followed by a period of agent replication which lasted 150 days. At second passage, there was no significant ‘zero phase’ and agent replication occupied the whole of the incubation period. The occurrence of a ‘zero phase’ on interspecies passage of scrapie is discussed in relation to other reports of a ‘zero phase’ in mouse passaged scrapie.
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Antigenicity and Polypeptide Composition of Native and Heated Echovirus Type 7 Procapsids
A. Hasegawa and S. InouyeSUMMARYThe native procapsid (naturally occurring empty capsid) of echovirus type 7 (E7) possesses N-antigenicity, which is as highly strain-specific as the native virion. When the native procapsid is heated, its antigenicity is converted to H-antigenicity which is common among strains of E7 and thus type-specific. However, no difference was detected in the sedimentation rate (80S) and polypeptide composition (VP0, 1 and 3) of the native and heated procapsids.
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Ultrastructural Development of Guinea Pig Cytomegalovirus in Cultured Guinea Pig Embryo Cells
More LessSUMMARYThe ultrastructural development of guinea pig cytomegalovirus (GPCMV) in guinea pig embryo cells was studied using electron microscopy. Tubular structures were found in nuclei of virus infected cells, followed by the appearance of intranuclear inclusions containing virus nucleocapsids. While some nucleocapsids were enveloped at the inner nuclear membrane, others were released into the cytoplasm where they were associated with, or within, dense matrix which was subsequently enveloped by cytoplasmic membranes to form enveloped dense virions. Dense bodies without virus capsids were formed in the cytoplasm and enveloped in a similar manner. An involvement of the nuclear pores in the release of unenveloped virus capsids from the nucleus to the cytoplasm was postulated. Evidence that the enveloped dense virions and dense bodies shared common envelope antigen(s) was obtained by immunoelectron microscopy. The similarities and differences in the ultrastructural development of GPCMV and other cytomegaloviruses are discussed.
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Pseudotypes of Vesicular Stomatitis Virus and Pichinde Virus
More LessSUMMARYSuper-infection of Pichinde virus-infected cells with vesicular stomatitis virus (VSV) resulted in the production of pseudotype virus which was not neutralized by antiserum to VSV but which was neutralized by antiserum to Pichinde virus. Analysis of pseudotype virus production in relation to the kinetics of replication of Pichinde virus demonstrated that pseudotype virus production occurred when super-infection with VSV was initiated 8 h or more after infecting the cells with Pichinde virus. The quantities of pseudotype virus produced correlated with the quantities of Pichinde virus antigen detected on the surface of the cells both during acute infection and in cells chronically infected with Pichinde virus. The observations indicate that pseudotype of VSV and Pichinde virus are readily formed and that the formation of pseudotype virus may be used to examine the Pichinde virus antigens expressed on the surface of infected cells.
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Characterization of Human TK− Cell Lines Transformed to a TK+ Phenotype by Herpes Simplex Virus Type 2 DNA
More LessSUMMARYHuman TK− cells carrying the HSV-2 TK gene as a result of transformation with virus DNA express a TK activity of virus origin and maintain the TK+ phenotype when grown in HAT medium. Under non-selective conditions, however, reversion to a TK− phenotype occurs with a significant frequency characteristic of each transformed line. Once reversion has occurred the TK− phenotype appears to be stable, since only very rare instances of TK− to TK+ reversion have been observed. TK− revertants were susceptible to re-transformation by virus DNA, but no reactivation of a silent virus TK gene could be obtained by superinfecting them with a TK− virus mutant. The data presented are consistent with the hypothesis that acquisition of the TK− phenotype is brought about by loss of the virus sequences coding for TK.
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Murine Cytomegalovirus-induced Protein Synthesis
More LessSUMMARYMurine cytomegalovirus (MCMV)-induced protein synthesis in mouse embryo fibroblast (MEF) cells was studied using polyacrylamide gradient SDS gel electrophoresis and autoradiography. Synthesis of at least 14 virus induced proteins (VIPs) was consistently detected in a lytic cycle. They were designated VIPs 132, 118, 99, 98, 88, 81, 76, 74, 58, 56, 51, 38, 36 and 33 on the basis of their mol. wt.
Judging from the pattern of the rate of protein synthesis, VIPs can be classified into three groups: group A VIPs were synthesized actively for a brief period of time and then their synthesis was no longer detectable. This group included two major VIPs, 98 and 88 and three minor VIPs, 58, 56 and 38. Group B VIPs 81, 74, 36 and 33 were similar to group A except that, following a brief period of active synthesis, a low level of synthesis continued during the entire lytic cycle. Group C VIPs 132, 118, 99, 76 and 51 were synthesized at low steady levels at all times after initiation and seemed to accumulate slowly.
According to temporal sequences of initiation of VIP synthesis, these proteins can also be divided into three groups: immediate early, early and late VIPs. The synthesis of the immediate early VIPs 132, 98, 88, 81, 76, 74 and 38 was initiated immediately after virus infection. The early VIPs included 58, 56, 51, 36 and 33 and their synthesis was initiated from 1 to 3 h post-infection. VIPs 118, 99 and several minor VIPs were first synthesized during 12 to 13 h post-infection which corresponded to the time of initiation of virus DNA synthesis and they are classified as late VIPs.
Cycloheximide reversal experiments indicated that the initiation of synthesis of early VIPs must be preceded by the synthesis of immediate early VIPs. In the presence of actinomycin D, the immediate early VIPs (0 to 1 h post-infection) were not synthesized indicating that immediate early VIPs are translated from virus mRNA synthesized after virus infection.
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Varicella-Zoster Virus Immunizes Patas Monkeys against Simian Varicella-like Disease
More LessSUMMARYTo define further the antigenic relationship between human varicella-zoster virus and herpesviruses which produce varicella-like disease in certain simian species, patas monkeys were inoculated with varicella-zoster virus and then challenged with Delta herpesvirus, which uniformly produces severe, clinically apparent disease in susceptible animals. Protection against Delta herpesvirus was conferred both by hyperimmunization with varicella-zoster virus and by a single immunization with a cell-free preparation of varicella-zoster virus. Although the immunological relationship between the human and simian varicella viruses is not completely reciprocal, these studies confirmed that antigens which induce immunity are shared by the human and simian viruses. No clinical symptoms were seen in monkeys inoculated with varicella-zoster virus, but the rapid and marked antibody responses to the virus suggested that subclinical infection had occurred. In contrast, a chimpanzee inoculated with one of the same varicella-zoster virus preparations produced only low levels of antibody.
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Characterization of a Retrovirus Isolated from Normal Mink Cells Co-cultivated with a Dog Mammary Tumour
More LessSUMMARYA retrovirus antigenically distinct from known type C, B and D viruses was isolated from normal mink (Mustela vison) lung cells that had been co-cultivated with 5-iododeoxyuridine- and dexamethasone-treated dog mammary tumour cells. Cytogenetic studies of the virus-releasing co-culture showed mitotic figures identical to the normal mink cell line (MvlLu) with the exception of a low frequency of cells with extensive chromosomal breakage and uncoiling. The new virus bands at a buoyant density of 1.16 g/ml, contains 60S RNA and a reverse transcriptase which prefers Mn2+ over Mg2+ for the synthesis of DNA. This enzyme utilizes poly(rA).oligo(dT) more efficiently than poly(dA).oligo(dT) and is also able to synthesize DNA copies from the endogenous RNA. Morphologically, it is a typical type C virus. Filtered virus readily infects mink, dog and other mammalian cells indicating the amphotropic nature of its cell growth requirement. Hybridization studies showed that normal mink DNA contains multiple copies of proviral sequences of this newly isolated virus. Serological analyses indicate that the mink endogenous virus contains in its core protein, in addition to the interspecies type-C determinant, and antigenic component related to one of the determinants found in the feline leukaemia virus p30 protein. This determinant is not present in the Rauscher leukaemia virus, RD114 virus or simian sarcoma virus.
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Assessment of the Interferon-Like Activity in Preparations of Leukocyte Pyrogen
More LessSUMMARYPreparations of crude leukocytes from rabbit peritoneal exudates contain interferon-like activity which may be separated from pryogen activity both by Sephadex chromatography and by purification of the pyrogen. This indicates that, despite some similarities, leukocyte pyrogen is not an interferon.
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The Matrix Protein Gene Determines Amantadine-Sensitivity of Influenza Viruses
More LessSUMMARYThe genetic composition of several recombinant strains, produced by mixed infection with an amantadine-sensitive and an amantadine-resistant influenza virus, have been compared with their response to amantadine. It is concluded that transfer of resistance or sensitivity to amantadine is determined by a single gene, that coding for the matrix protein.
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Concentration of Human Cytomegalovirus from Large Volumes of Tissue Culture Fluids
C. Hamelin and G. LussierSUMMARYThree methods of pelleting, ultracentrifugation (95000 g for 60 min), precipitation with polyethylene glycol 6000 (5% v/v), and with ammonium sulphate (38% w/v), were used to concentrate human cytomegalovirus (CMV) from tissue culture fluids. Maximum recovery of infectious virus particles was obtained with the polyethylene glycol (PEG) method. The precipitating activity of PEG 6000 and PEG 20000 was then compared at different concentrations. The best results were obtained with PEG 6000 at a final concentration of 5% (v/v). Changes in pH or salt concentration, treatment of the concentrates with Pronase and long periods of time at 4 °C significantly reduced the number of biologically active CMV particles recovered by PEG precipitation.
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Phenotypic Mixing between Two Primate Oncoviruses
More LessSUMMARYPhenotypic mixing between the primate oncoviruses HL23V and BEV has been demonstrated to occur in doubly-infected bat lung (Tb) cells with the production of HL23V(BEV) pseudotype virus. The presence of the HL23V(BEV) pseudotype permitted the host range for replication of HL23V to be extended to murine cells previously ‘resistant’ to HL23V replication due to a block at the level of virus penetration. Expression of BEV genetic information was observed in doubly-infected rat cells and also in mouse and rat MSV-transformed non-producer cell lines co-cultivated with BEV-producing Tb cells. No evidence for genetic recombination between these viruses could be demonstrated.
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