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Volume 50,
Issue 2,
1980
Volume 50, Issue 2, 1980
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Immunological Relationships Between Homologous Structural Polypeptides of Measles and Canine Distemper Virus
More LessSUMMARYThe major structural polypeptides of measles and canine distemper virus (CDV) were isolated by SDS-polyacrylamide slab gel electrophoresis. Rabbit hyperimmune sera directed against the isolated HA, P, NP, F and M polypeptides were prepared. In addition, rabbit hyperimmune sera directed against purified native internal components of measles virus and against purified virions of measles and CDV, untreated or treated with trypsin, were prepared. These sera were used to study the immunological relationships between measles and CDV in immune precipitation tests with 35S-methionine-labelled extracellular virions and intracellular virus polypeptides and by use of complement fixation and mixed haemadsorption tests.
An immunological relationship between the M polypeptides was demonstrated with sera against both native and partially denatured antigens. A reciprocal immunological cross-reactivity between the NP polypeptides was demonstrated by the use of sera directed against native antigens. However, antisera directed against the NP polypeptides isolated from gels did not show any cross-reaction, indicating that they lacked antibodies against the shared antigenic determinants. The immunological cross-reactivity between envelope components of the two viruses was analysed. A close reciprocal immunological relationship between the F polypeptides was demonstrated by immune precipitation of labelled polypeptides. Immunological cross-reactivity between the HA polypeptides of the two viruses could not be demonstrated by use of the same technique neither with sera directed against denatured antigen nor with sera against native antigen when extracellular denatured 35S-methionine-labelled purified virions were used in immune precipitation. A slight cross-reactivity could, however, be demonstrated with sera against native antigens in immune precipitation tests with 35S-methionine-labelled intracellular antigens.
It is concluded that all the major structural polypeptides of measles and CDV show varying degrees of reciprocal immunological cross-reactions. The M polypeptides of the two viruses appear most closely related, whereas the HA polypeptides show the most pronounced differences.
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Biological and Antigenic Characteristics of HEL-12 Virus
More LessSUMMARYThe biological and antigenic properties of HEL-12 virus have been compared with gibbon ape lymphosarcoma virus (GALV) and simian sarcoma and simian sarcoma-associated viruses, SiSV and SSAV, respectively. HEL-12 virus did not transform human or marmoset fibroblasts but rescued SiSV focus-forming activity from non-productively transformed marmoset cells (HF/SiSV-NP). Like SSAV and GALV, HEL-12 virus induced syncytia with XC cells. In addition, HEL-12 cells which did not produce virus but which contained HEL-12 proviral DNA, rescued SiSV from HF/SiSV-NP cells in co-cultivation experiments. Results of neutralization and serum cytotoxicity tests utilizing SiSV rescued by HEL-12 [SiSV-(HEL-12)] indicated that HEL-12 virus envelope proteins are very closely related to those of SSAV but readily distinguished from those of GALV. Antigenic diversity of SiSV(SSAV), SiSV(GALV) and SiSV(HEL-12) envelope glycoproteins (gp70) was shown in competition radioimmunoassays (RIA) designed to detect minor antigenic differences using antiserum monospecific for SiSV(SSAV) gp70 or Friend murine leukaemia virus gp70. Antigenic differences between these gp70s were demonstrated in RIA using purified SSAV gp70 or HEL-12 gp70. These data indicate that HEL-12 virus has biological properties similar to those of SSAV and GALV, is distinguished from GALV in neutralization tests and has both distinct and SSAV-related gp70 antigenic determinants.
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DNA-binding Proteins in the Nuclei of Herpes Simplex Virus-infected, Arginine-deprived, BSC-1 Cells
More LessSUMMARYNine proteins accumulate in nuclei of arginine-deprived herpes simplex virus-infected cells. Of these, eight were found to bind to double-stranded DNA-cellulose and to elute with 0·3 and 0·6 m-KCl, while one of 160000 mol. wt. had no affinity for the DNA. Five of the DNA-binding proteins were identified as virus enzymes: two were DNA polymerases, two were alkaline DNases and one was the thymidine kinase.
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Isolation of the Glycoprotein of Vesicular Stomatitis Virus and its Binding to Cell Surfaces
More LessSUMMARYThe glycoprotein (G) of vesicular stomatitis virus (VSV) was radiolabelled, extracted and purified so that its potential interaction with host cell surfaces could be studied. When BHK-21 cells were incubated with the radiolabelled virus glycoprotein, the virus component rapidly attached to the cell surface. The attachment was shown to be temperature-dependent and saturated at approx. 3 × 105 molecules/cell. The omission of Mg2+ or Ca2+ from the incubation medium had little effect on the glycoprotein binding. Treating the isolated G protein and intact virions with neuraminidase did not significantly decrease their binding to BHK-21 cells. Pre-incubating cells with trypsin did not decrease the attachment of VSV virions nor the binding of purified G protein. Treating cells with phospholipase A or phospholipase C suggested that the binding of the glycoprotein and the intact virion might have been dissimilar. Unlabelled glycoprotein competitively inhibited binding of the labelled molecules although the presence of intact virions did not inhibit attachment of the G protein. Likewise, saturating amounts of the glycoprotein did not decrease binding of VSV to BHK-21 cells. These observations suggested that either the isolated glycoprotein bound to cell surface components that were distinct from the virion receptor or that the manner of the purified glycoprotein attachment differed from the G protein still associated with the intact virion. Chemical crosslinking and diagonal two-dimensional gel electrophoresis were used to identify and to compare the cell surface components responsible for glycoprotein and virion attachment.
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The Influence of the Host Cell on the Inhibition of Virus Protein Synthesis in Cells Doubly Infected with Vesicular Stomatitis Virus and Mengovirus
More LessSUMMARYThe ability of mengovirus to inhibit the synthesis of vesicular stomatitis virus (VSV) proteins and of VSV to inhibit the synthesis of mengovirus proteins during double infection in three different cell lines was investigated. Although cellular protein synthesis was inhibited after infection of cells by each virus, the ability of one virus to decrease translation of the mRNA species of the co-infecting virus varied with the cell type. Superinfection of mengovirus-infected L-929 cells by VSV resulted in essentially no inhibition in the synthesis of either mengovirus or VSV proteins. In HeLa cells and CHO cells the synthesis of both VSV and mengovirus proteins was inhibited under conditions of simultaneous or sequential infection. The inhibition of VSV protein synthesis after infection of HeLa cells by mengovirus was not a result of a modification or inactivation of virus mRNAs. When extracted from doubly infected cells, the VSV mRNAs manifested normal biological activity, as determined by their ability to stimulate the synthesis of VSV proteins in a micrococcal nuclease-treated cell-free system from L cells.
The interference or non-interference of one virus by another in different cell lines was also measured by quantifying the number of infectious particles produced in each cell line. The results were similar to those reported above for protein synthesis inhibition. These experiments suggest that the interference of mengovirus with VSV mRNA translation in HeLa cells is not necessarily reflective of the mechanism by which mengovirus inhibits cellular protein synthesis. Also, the host cell appears to influence the extent or nature of the interference of one virus by the other.
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Characterization of Two Morphology Mutants of Autographa californica Nuclear Polyhedrosis Virus with Large Cuboidal Inclusion Bodies
More LessSUMMARYTwo morphology mutants designated m-5 and m-6, of Autographa californica nuclear polyhedrosis virus (NPV) were isolated from virus grown in the presence of N-methyl-N′-nitro-N-nitrosoguanidine. Infected cells contained single, large cuboidal inclusion bodies with a crystalline lattice ultrastructure and envelope similar to that of wild-type (wt) polyhedra. The inclusion bodies had low infectivity when fed to Trichoplusia ni larvae. The paracrystalline lattice structure of the mutants was similar to wt, but occluded viruses were rarely found within the mutants. The m-5 polyhedrin (mol. wt. 30000) could be distinguished from wt and m-6 polyhedrin on the basis of migration in SDS-PAGE gels. Peptide maps of polyhedrin were obtained following digestion with chymotrypsinogen or Staphylococcus aureus V8 protease. They were identical for m-6 and wt polyhedrin but m-5 polyhedrin gave a different pattern. Thus, the altered morphology may be due to a change in polyhedrin composition not detectable in m-6 polyhedrin by the methods used here, or it may be the result of a mutation affecting a protein not yet identified.
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Replication and Expression of Rubella Virus in Human Lymphocyte Populations
More LessSUMMARYHuman mononuclear cells (lymphocytes and monocytes) from peripheral blood were examined for their ability to support the replication of rubella virus (RV) after infection in vitro. Replication was shown to occur in mixed lymphocytes and to be enhanced by stimulation with phytohaemagglutinin or pokeweed mitogen. A comparison of RV polypeptide synthesis in lymphocytes and RK13 cells showed no major differences in the polypeptides induced by infection. However, cellular translation was inhibited in the lymphocytes facilitating identification of virus polypeptides and eliminating the need for hypertonic labelling conditions used with RK13 cells. RV replication was also shown to occur in sub-populations of T-cells but not in B-cells. However, RV could be rescued from the B and monocyte population by co-cultivation with RK13 cells.
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The Amino Acid Sequence of a Hong Kong Influenza Haemagglutinin Light (HA2) Chain
More LessSUMMARYThe amino acid sequence of the Hong Kong haemagglutinin light chain (HA2; 222 residues) is nearly complete, lacking only the definition of a highly aggregated region near the carboxyl terminal end of the chain. This unsequenced area of approx. 25 residues occurs near the carboxyl terminal end of cyanogen bromide peptide CN-1, whose structure determination is discussed in this paper. All -cystine residues present in HA2 occur in CN-1, as a proximal cluster involving residues 137, 144 and 148, and as a distal cluster involving four other -cystine residues near the carboxyl terminus of HA2, three of which have been placed within peptides. The single glycosylated asparagine in HA2 also occurs in CN-1; the carbohydrate moiety is complex. The structure of HA2 is discussed in terms of its properties, and compared with published data from haemagglutinins from other influenza strains.
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Cellular Quiescence Modulates the Replication of L ts Mutants of Vesicular Stomatitis Virus
More LessSUMMARYInfectious particle production by temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV) was measured in a variety of different host cell types maintained in a state of quiescence or stimulated to proliferate. At permissive temperatures, all ts mutants and the wild-type virus replicated equally well and with the same kinetics in both quiescent and proliferating cells. At semi-permissive temperatures, however, L ts mutants, with temperature-sensitive virion polymerases, showed a delay of about 6 h in infectious particle production relative to wild-type virus in proliferating cells and greater than 16 h in quiescent cells. The effect was specific for the L ts class of mutants and was not seen for representative mutants in any of the four other complementation groups of VSV. Regarding cellular determinants, the effect was correlated only with the growth phase and not with the species of origin, interferon inducibility or with malignant transformation.
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Differentiation of Strains of Yellow Fever Virus in γ-irradiated mice
More LessSUMMARYThe mouse sensitized by optimal, sub-lethal γ-irradiation has been used for the differentiation of strains of yellow fever virus and for the resolution of their immunogenicity and pathogenicity as distinct characteristics. For different strains of yellow fever virus, the patterns of antibody-synthesis, regulatory immunity (pre-challenge) and protective immunity (post-challenge) are differentially sensitive to γ-irradiation. These critical differentiations of strains of yellow fever virus in γ-irradiated mice have been compared with those shown in normal athymic and immature mice in order to elucidate the range of quantifiable in vivo characteristics and the course of the virus-host interaction. This is discussed as a basis for the comparison of the responses of model and principal hosts to vaccines and pathogens.
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Immunological Studies of the Group B Coxsackieviruses by the Sandwich Enzyme-linked Immunosorbent Assay (ELISA) and Immunoprecipitation
More LessSUMMARYA microplate double antibody sandwich ELISA was employed in an immunological study of the group B Coxsackieviruses. The assay, described in detail, detected high dilutions of virion antigen (less than 10 ng) in purified preparations and in crude infected cell extracts. Furthermore, by using a constant amount of antigen, group B virus antibodies in hamster antisera could be quantified with a sensitivity equivalent to the virus neutralization test. Titrations of virus antigens and antibodies were found to be type-specific when purified virions were employed in the assay. Urea disruption of virions exposed antigens common to all six group B viruses. The heterotypic reactivity of disrupted group B virions did not extend to the other viruses tested. Immunoprecipitation and SDS-PAGE analysis revealed that, of the four virion structural polypeptides (VP1 to 4), VP1 contained the major common antigenic determinants shared by members of the group B Coxsackieviruses.
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Serological and Immunological Relationships between the 146S and 12S Particles of Foot-and-Mouth Disease Virus
More LessSUMMARYIntact 146S particles of the seven serotypes of foot-and-mouth disease virus (FMDV) produce type-specific precipitating, complement-fixing and neutralizing antibodies in cattle and guinea-pigs. However, the 12S structural subunit, produced from the virus particle by mild acid treatment (pH 6) or by heating at 56 °C, although stimulating the production of precipitating and complement-fixing antibodies, produces only low levels of neutralizing antibody. Nevertheless, 12S particles were as active as 146S particles in stimulating the production of neutralizing antibody in guinea-pigs primed with a vaccine prepared from 146S particles. Moreover, heterotypic 12S and 146S particles also boosted the neutralizing antibody response to the first virus. These results point to an antigenic similarity between the 146S particles of each type and to a close antigenic relationship between the 146S and 12S particles.
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Restriction of the Rat-specific RNA Sequences of Ki-MSV to the Nucleus of Dibutyryl Cyclic AMP-treated K-A31 Cells
More LessSUMMARYThe treatment of oncogenically transformed cells in culture, with dibutyryl cyclic AMP (cAMP) has, in many cases, resulted in a general phenotypic change towards the normal state. A virus-specific gene product(s) is responsible for the transformation of cells by sarcoma viruses and it has been suggested that the src gene product may act through the alteration of cAMP levels. With these premises we have studied the effects of dibutyryl cAMP on cell growth and virus genome expression in a Kirsten sarcoma virus-transformed mouse cell line. Our results suggest that certain virus-specific RNA sequences are restricted to the nucleus of these cells after several days of growth in medium containing dibutyryl cAMP and that these sequences appear to be those coding for the sarcoma information.
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A Study of the Level of Nucleotide Sequence Conservation between the RNAs of Two Serotypes of Foot-and-Mouth Disease Virus
More LessSUMMARYThe level of nucleotide sequence conservation between the RNAs of type A and type O foot-and-mouth disease virus (FMDV) has been compared by three methods. (1) RNA hybridization of fragments containing either the poly(C) tract at the 5′ end of the RNA or the poly(A) tract at the 3′ end of the RNA indicates that there is a similar level of sequence conservation (65% homology) across the genome. RNase T1 fingerprinting of these fragments did not show the presence of any long regions of completely conserved nucleotides apart from the poly(C) and the poly(A) tracts. (2) RNase T1 fingerprints of the RNA on the 5′ side of the poly(C) tract (the S fragment) show that there is more conservation in this region of the RNA than indicated by the hybridization results. (3) Direct nucleotide sequencing of the poly(C) tract and of the 54 nucleotides at the 5′ end of the two genomes shows that there is considerable sequence conservation at the extreme 5′ end of the RNA.
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Influenza Virus Haemagglutinin: Estimation of Tryptophan and Tyrosine Content and Localization of Tryptophan Residues
More LessSUMMARYThe tryptophan and tyrosine content of the bromelain-released subtype H3 haemagglutinin (H3 BHA) of influenza virus were measured by u.v. absorption and fluorescence techniques. The values obtained (8 and 18 residues, respectively) are in close agreement with those derived from amino acid analysis. Essentially all of the tryptophan residues are demonstrated to be localized on the surface of the BHA molecule.
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Biochemical Characterization of the Virus Causing Leukoencephalitis and Arthritis in Goats
More LessSUMMARYGoat leukoencephalitis-arthritis virus (GLV) has the density of a retrovirus in sucrose and contains an endogenous RNA-dependent DNA polymerase (reverse transcriptase). The virion reverse transcriptase utilizes the synthetic RNA template poly(rA).(dT)12 but not the synthetic DNA template poly(dA).(dT)12. A high mol. wt. RNA similar in size to visna virus RNA was isolated from 3H-uridine-labelled virions. The major structural protein of GLV has the same mol. wt. as that of visna virus. From these data the GLV appears to be a retrovirus.
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Antiviral Effects of Atropine and Caffeine
Z. Yamazaki and I. TagayaSUMMARYThe antiviral activities of atropine and caffeine were investigated. Atropine inhibited the multiplication of enveloped viruses and caffeine suppressed the growth of polio, influenza, herpes simplex and vaccinia viruses but not Japanese encephalitis virus, Newcastle disease virus and type 2 adenovirus.
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A Model in Mice for the Study of the Early Death Phenomenon after Vaccination and Challenge with Rabies Virus
J. Blancou, B. Andral and L. AndralSUMMARYThe reactions of mice were studied for a period of 10 days after their vaccination with an inactivated rabies vaccine. The kinetics of their resistance to an intracerebral challenge and neutralizing antibody activity of their serum were determined daily. Protection began on the fourth day after vaccination and was approximately correlated with virus-neutralizing antibody titres from the sixth to the tenth day. However, in vaccinated mice still unprotected, death following the intracerebral challenge occurred earlier than in unvaccinated control mice. This ‘early death phenomenon’ is proposed as a model for immuno-pathological study of reaction to rabies vaccines.
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Production of Chick Interferon by Reactivating Chick Erythrocytes
More LessSUMMARYWhen chick erythrocytes were fused with mouse L929 or A9 cells and the heterokaryons induced for interferon production on consecutive days, considerable amounts of mouse interferon were produced every day. Small amounts of chick interferon were produced 2 to 3 days after fusion, coincident with the appearance of a chick enzyme, appearance of nucleoli and increase in chick cell nuclear diameter.
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SDS-Polyacrylamide Gel Electrophoresis of Purified Human Leucocyte Interferon and the Antiviral and Anticellular Activities of the Different Interferon Species
More LessSUMMARYHuman leucocyte interferon (HuLeIF) was purified by a series of techniques involving precipitation, gel filtration, Cu-chelate-, blue dextran- and antibody-affinity chromatography. The two major species of HuLeIF were identified in SDS-PAGE as two clearly separable and stainable proteins representing 85% of the biological activity. Three more species of HuLeIF representing 15% of the biological activity were also demonstrated. The specific activity of pure interferon proteins was approx. 109 IFU/mg protein. Recovery was about 50% and the purification factor exceeded 350000. All five species of HuLeIF had definite anticellular activities when tested with Daudi cells (inhibition of thymidine uptake).
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