- Volume 68, Issue 4, 1987
Volume 68, Issue 4, 1987
- Bacterial
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Characterization of Mycobacteriophage I8 and its Unrelatedness to Mycobacteriophages I1, I3 and I5
More LessSUMMARYHomology among the genomes of mycobacteriophages I1, I3, I5 and I8 has been studied. Based on restriction endonuclease cleavage patterns, dot blot hybridization and Southern blot hybridization analysis, the DNAs of phages I1, I3 and I5 have been shown to be homologous and indistinguishable, but entirely different from phage I8. Unlike the others, the I8 genome does not harbour any single-strand interruptions. The DNA is 43 kb in length with limited cyclic permutations and has a G + C content of 54%. The presence of 5-methylcytosine in I8 DNA was indicated from the restriction patterns of MspI and HpaII. The number of sites and fragment sizes for several restriction enzymes on I8 DNA has been determined. Phage I8 has a replication cycle of 300 min, with a latent period of 180 min, a rise period of 120 min and a burst size of 100. The viability of phage I8 is significantly reduced by treatment with organic solvents.
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The Head-Tail Linker Protein of Bacteriophage T5: Genetic and Immunological Studies
More LessSUMMARYWe investigated the properties of amber mutants of coliphage T5 defective in a late stage of phage assembly. The non-functional heads synthesized by mutants am25 or aw 158 were unable to combine with functional tails to produce viable phage particles. These mutants were shown to lack a single protein, designated the head-tail linker protein (HTLp), which was identified by polyacrylamide gel electrophoresis and Western blot analysis and had an M r of 18000. An extract containing the HTLp, when added to the HTLp-deficient heads, restored their ability to combine with functional tails.
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Four Serotypes of Haemorrhagic Fever with Renal Syndrome Viruses Identified by Polyclonal and Monoclonal Antibodies
SUMMARYAntigenic relationships among 20 strains of haemorrhagic fever with renal syndrome (HFRS) viruses isolated in Korea, China, U.S.S.R., Finland, Japan and U.S.A. were examined with rat immune sera, patient sera, eight monoclonal antibodies against the SR-11 strain and 10 monoclonal antibodies against the 76–118 strain. Antigen analyses by indirect immunofluorescent antibody and immune adherence haemagglutination tests using polyclonal and monoclonal antibodies demonstrated that HRFS viruses may be divided into four serotypes, i.e. Apodemus (Type 1), Rattus (Type 2), Clethrionomys (Type 3) and Microtus (Type 4). Further, it was demonstrated that Type 1 could be divided into three subtypes and Type 3 into two subtypes. The two sets of monoclonal antibodies were useful for identification of the antigenic types of viruses isolated from patients in endemic areas.
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Preliminary Studies on Antigenic Variation of Poliovirus Using Neutralizing Monoclonal Antibodies
SummaryCross-neutralization assays were done using 85 strains of poliovirus type 1 with five groups of monoclonal antibodies. These strains were classified into 10 subgroups which had marked differences in antigenicity. Subgroups P1-2 (28%) and P1-5 (43%) were dominant and have been epidemic in China in recent years. These two subgroups were antigenically different from the Sabin-1 strain, but according to their responses to one group of monoclonal antibodies they had antigenic epitopes in common with the Mahoney and Brunhilde strains. Similarly, 91 strains of type 3 poliovirus were classified into six subgroups with another five groups of monoclonal antibodies. The results showed that strain P3/Yunnan/2/84, which was isolated from cases of poliomyelitis in a local epidemic in the Yunnan province of China in 1984, and strain P3/Finland/23127/84, which was isolated in Finland in 1984, were both antigenically different from the Sabin-3 strain and the reference virulent strain.
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Intracellular RNAs of the Feline Infectious Peritonitis Coronavirus Strain 79-1146
More LessSUMMARYIn Felis catus whole foetus D cells infected with feline infectious peritonitis virus (FIPV), strain 79–1146, six virus-specific, poly(A)-containing RNA species of about 20, 9·6, 5·2, 3·8, 2·8 and 1·6 kb were found. By translation in vitro the 3·8 and 2·8 kb RNAs were shown to encode the 25K envelope protein and the 45K nucleocapsid protein, respectively. The partial map of the FIPV genome was compared with genomic maps of porcine, murine and avian coronaviruses. Differences in these maps suggest that transcription units have been lost or gained during coronavirus divergence.
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Nucleotide Sequence of the Coding and Flanking Regions of the Human Parainfluenza Virus Type 3 Fusion Glycoprotein Gene
More LessSUMMARYThe complete nucleotide sequence of the human parainfluenza virus type 3 (HPIV3) fusion (F) protein gene has been determined. The HPIV3 F gene is 1851 nucleotides long including six U residues in the genomic RNA, which probably direct synthesis of the first few nucleotides in the F mRNA polyadenylate tail. The HPIV3 F gene contains a single long open reading frame coding for 539 amino acids. The predicted molecular weight of the unglycosylated precursor F0 protein was 60031. Four potential carbohydrate acceptor sites were identified. Comparison of the HPIV3 F protein sequence with the F gene sequences of two other paramyxoviruses, Sendai virus and simian virus 5, indicated a very close evolutionary relationship between HPIV3 and Sendai virus. Sequence analysis of HPIV3 F gene flanking regions identified signals which appear to be responsible for polymerase recognition and polyadenylation.
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Ultrastructure and Immunoelectron Microscopy of Human T-cell Leukaemia Virus Type I-producing Lymphoid and Non-lymphoid Human Tumour Cells
More LessSUMMARYThe ultrastructure and distribution of some viral and cellular antigens was examined in lymphoid C91/PL and fibroblastic HOS/PL cells infected with human T-cell leukaemia virus type I. Tubuloreticular structures in the endoplasmic reticulum, characteristic of virus-infected or interferon-treated cells, were found in both cell types. Virions were observed particularly in the HOS/PL cells in budding and mature forms and in coated pits, coated vesicles and lysosomes, indicative of receptor-mediated endocytosis. Using immunogold indirect labelling, viral antigens, recognized by sera of infected patients and by monoclonal antibody, were detected in patches on the cell surface and in material loosely attached to the envelope of some virions. Beta2- microglobulin was associated with virion envelopes from C91/PL and HOS/PL cells, but major histocompatibility complex class 1 (HLA) antigens were not associated with virions even when produced by HLA-positive HOS/PL cells.
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The Assay, Purification and Properties of Vaccinia Virus-induced Uncoating Protein
More LessSummaryVaccinia virus cores prepared in vitro can be uncoated by incubation with extracts of cells infected with vaccinia virus, as determined by the conversion of the genome to DNase susceptibility. This uncoating activity had all the characteristics of the corresponding in vivo activity and of the agent responsible for non-genetic reactivation. Thus, it was not induced by heat-inactivated virus, nor was it produced in the presence of inhibitors of RNA or protein synthesis. The uncoating protein induced by cowpox virus will uncoat vaccinia virus cores. The uncoating protein was concentrated from infected cell extracts by ultrafiltration and purified by gel filtration and ion-exchange and affinity chromatography. It was characterized as a trypsin-like protease with a mol. wt. of 23050. Cores treated with the purified uncoating protein had an altered sedimentation rate but no differences between treated and untreated cores were detected by electron microscopy or polyacrylamide gel electrophoresis.
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Maintenance of Woodchuck Hepatitis Virus Activity in Woodchuck Hepatocyte Primary Culture
Primary cultures of non-proliferating hepatocytes isolated by the two-step collagenase perfusion method from woodchuck naturally infected with hepatitis virus (WHV) were used to study WHV propagation in vitro. Hepatocytes carrying WHV DNA exhibited a very high level of survival and retained their morphological characteristics for 2 to 3 months. Over this time, they were found to produce virus-specific proteins and release viral particles with DNA polymerase activity into the medium. Using Southern blot analysis and a recombinant hepatitis B virus DNA plasmid probe, intracellular and extracellular viral DNA was consistently detected. Only extrachromosomal forms of WHV DNA were observed and no integration could be demonstrated in the DNA of the cells. The WHV DNA patterns were repeatedly identical with a characteristic smear starting from 3·3 kb associated with other smaller DNA fragments which presumably represented intermediate replicative forms of viral DNA. Furthermore, dot blot hybridization of the total RNA revealed the presence of WHV-specific transcripts in cells after 3 weeks of culture. All these results are compatible with the maintenance of active WHV replication in vitro although it was somewhat reduced after the first day of culture. This provides a mammalian model for hepadnavirus replication studies in stable primary hepatocyte cultures.
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Lymphotropic Strain SL3 of Aleutian Disease Virus: Identification of Replicative Form DNA, Molecular Cloning and Expression of Capsid-specific Proteins
More LessSummaryReplicative form (RF) DNA of the lymphotropic strain SL3 of Aleutian disease virus was isolated from infected cell cultures. A novel intermediate of about 7·6 kilobases was demonstrated in Hirt lysates in addition to single-stranded viral, double-stranded monomer and dimer RF DNA. The monomer RF DNA exhibited a length heterogeneity of 70 bp and 160 bp at its 3′ and 5′ termini. The two major monomer RF DNA species each contained hairpins in the extended or the foldback configurations.A central fragment between map units 0′15 and 0′88 was cloned into plasmid pUC18. The recombinant clone expressed virus-specific proteins ranging from 32000 to 74000 mol. wt.
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Organization of Terminal Reiterations in the Virion DNA of Herpesvirus Saimiri
SummaryThe population of herpesvirus saimiri (HVS) genomes extracted from extracellular virions are double-stranded, linear DNA molecules of about 160 kilobase pairs (kbp) each composed of a central segment of 110 to 112 kbp and 36% (G + C) (i.e. ‘light’ or L-DNA) linked to direct reiterations of a 1·44 kbp repeat unit of 71 % (G + C) (i.e. ‘heavy’ or H-DNA) at each terminus. In this paper, we show that the population of HVS DNA molecules contains approximately equal concentrations of genomes with all possible integral numbers of complete repeat units (i.e. from > 30 to 1) at either ‘left’ or ‘right’ ends but that all molecular ends are derived by a unique cleavage at a site close to the single Apal restriction endonuclease site of the H-DNA repeat unit. Junctions of proximal H-DNA repeat units with L-DNA occur at, or very close to, the sequence present at the molecular ends. The transition from L- to H-DNA occurs abruptly at this site at the ‘right’ end of the L-DNA component but some rearranged restriction enzyme cleavage sites typical of H-DNA are found within the first 0·8 kbp of the L-DNA sequences at the ‘left’ H-L DNA junction. HVS appears to provide an extreme example of the general process whereby herpesvirus DNAs are matured from concatemeric intermediates by a site-specific cleavage/recombination process involving random choice between equivalent sites for the initiation of the process and with choices between alternative termination sites being limited by a headful packaging mechanism.
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Genetic Relations between Varicella-Zoster Virus and Epstein-Barr Virus
More LessSummaryVaricella-zoster virus (VZV) and Epstein-Barr virus (EBV) are important human pathogens which belong to different subfamilies of the herpesviruses: the Alpha- and Gammaherpesvirinae, respectively. Computer comparisons of the amino acid sequences of proteins predicted from the published complete VZV and EBV DNA sequences resulted in the detection of EBV counterparts to 29 of the 67 unique VZV genes. Conserved genes were detected only in the UL component of each genome, and are located in three major regions, within which conserved genes are generally colinear. However, the three regions are arranged differently in the two genomes. These results make it possible in principle to propose the functions of EBV genes on the basis of the functions of their VZV counterparts. The data also allow identification of the types of events which may have occurred during divergence of VZV and EBV, as representatives of the Alpha- and Gammaherpesvirinae, from a common ancestor.
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Epstein-Barr Virus-specific Transcription in Normal and Malignant Nasopharyngeal Biopsies and in Lymphocytes from Healthy Donors and Infectious Mononucleosis Patients
SummaryCytoplasmic RNA was prepared from biopsy material obtained from the nasopharynx of normal individuals and nasopharyngeal carcinoma (NPC) patients. Similar RNA preparations were prepared from lymphocytes of healthy donors and infectious mononucleosis patients. RNA was radioactively labelled in vitro and hybridized to cloned fragments of B95-8 Epstein-Barr virus (EBV). In all seropositive cases the minimum pattern of EBV-specific RNA expression was like that observed previously in latently infected, EBV-positive lymphoblastoid cell lines or Burkitt’s lymphoma biopsies. A novel observation was that all of the control biopsies from normal nasopharynxes expressed EBV-specific RNA, some of which appeared to be associated with a more active state of EBV infection. The pattern of expression in NPC patients was similar to that of the normal donors but showed some variations in complexity. In general the virus-specified small RNAs were not present in normal nasopharyngeal tissue but were expressed in the NPC biopsies.
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Suppression of Delayed Type Hypersensitivity to Herpes Simplex Virus Type 1 Following Immunization with Anti-idiotypic Antibody: an Example of Split Tolerance
More LessSUMMARYIntraperitoneal (i.p.) immunization with herpes simplex virus type 1 (HSV-1) or an anti-idiotypic antibody (anti-id C) prepared against a monoclonal antibody specific for glycoprotein C of HSV-1, tolerizes mice for an HSV-1 delayed type hypersensitivity (DTH) response. This tolerization could be adoptively transferred to naive X- irradiated mice by splenic T cells, and was specific for HSV-1 DTH. Thus, DTH- tolerized mice responded to vaccinia virus or HSV-2 challenge, while remaining tolerized for HSV-1 DTH. In addition, these animals demonstrated a form of split tolerance such that HSV antibody, cytotoxic T cell and lymphoproliferative responses were detected in vitro. Thus, anti-id C induced a T cell population capable of specifically suppressing the HSV-1 DTH response, mimicking the effect of i.p. and intravenous immunization with HSV-1 found previously.
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Specificity of the Immune Response of Mice to Herpes Simplex Virus Glycoproteins B and D Constitutively Expressed on L Cell Lines
More LessSUMMARYMouse L cell lines have been developed which constitutively express glycoproteins B (gB) and D (gD) of herpes simplex virus type 1. When used to study the immune response of mice to the viral glycoproteins, it was found that both gB and gD induce a delayed type hypersensitivity response and both also induce an antibody response, but only the cell line expressing gD could stimulate the production of neutralizing antibody. Virus-specific cytotoxic T lymphocytes (CTLs) recognized gB expressed by the cell line and this line could also induce CTLs in mice. Recognition of gD by major histocompatibility complex class I restricted CTLs was never seen. Vaccination of mice with the cell lines provided protection from viral challenge and inhibited the establishment of a latent infection, although gD proved to be the better protective immunogen.
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Murine T Cell Clones Directed to Rabies Virus: Isolation and Some of Their Properties
More LessSUMMARYSeventeen Thy-1+ cell clones were induced in A/J mice immunized with the HEP- Flury strain of rabies virus after repeated stimulations with antigens in vitro. Ten clones with cell surface phenotypes Thy-1+, Lyt-1−,2+ were cytotoxic T lymphocytes (CTL) which lysed the virus-infected target cells under H-2 restriction. Target cells expressed the G and M2 structural proteins of rabies virus on their surface; however, target lysis by CTL clones was not blocked by anti-rabies antibody or by monoclonal antibodies to these proteins. All of the CTL clones efficiently and equally lysed target cells infected with three different strains of rabies virus and were cross-reactive for target cells infected with one (Duvenhage virus) of three different rabies serogroup viruses. Another five clones having phenotype Thy-1+, Lyt-1+,2− did not show any cytotoxic activity. The proliferation response of these clones to antigen stimulation was virus- specific and H-2-restricted. These clones were able to grow in culture medium without any or with the addition of low concentrations of T cell growth factor, in contrast to CTL clones, and were considered to be helper T lymphocytes (HTL). Both CTL and HTL clones produced y-interferon in response to antigen stimulation. The remaining two clones were Thy-1+, Lyt-1−,2−, asialo-GM1+, and were not cytotoxic to target cells even in the presence of anti-rabies antibody but were cytotoxic to YAC-1 cells. Further studies with these clones should allow us to investigate more closely the role of T cells in the pathogenesis of rabies.
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Requirement for Endocytosis of Poly(rI).Poly(rC) to Generate Toxicity on Interferon-treated LM Cells
More LessSUMMARYPoly(rI). poly(rC) induces a lytic reaction in interferon-treated mouse LM cells. We have attempted to determine whether the intracellular penetration of poly(rI). poly(rC) is a prerequisite for cell lysis and to gain some insight into the pathway followed. We found that poly(rI). poly(rC) coupled to Sepharose beads was unable to generate lysis of interferon-treated cells whereas the cells underwent lysis after microinjection of poly(rI).poly(rC). Some inhibitors of endocytosis were found to inhibit the development of the lytic reaction. Lysosomotropic amines or a low temperature (19 °C) blocked endocytosis of poly(rI). poly(rC) but did not prevent its uptake. The internalization of poly(rI). poly(rC) was energy-dependent and was blocked when sodium azide and 2-deoxyglucose were added simultaneously. We conclude that poly(rI). poly(rC) is internalized and reaches an acidic compartment before triggering the lytic reaction in the cell.
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Inactivation of Influenza B Virus by Normal Guinea-pig Serum
More LessSUMMARYNormal guinea-pig serum (GPS) lacking detectable antiviral antibody efficiently neutralized the infectivity of influenza B virus grown in chick embryos or MDCK cells. The inhibitor was heat-labile and sensitive to trypsin digestion. This β-like inhibitor required Ca2+ and the complement components C1 and C4 for its activity. In contrast, GPS did not inactivate influenza A virus. Influenza B virus from which the neuraminidase activity of the spikes on the viral envelope had been eliminated by trypsin digestion was also inactivated to a level comparable to untreated virus. Complement component C1 alone bound directly to influenza B virus and inhibited its haemagglutinin activity. We suggest that the β like inhibitor in GPS is a component of the classical complement pathway which is triggered by the protein moiety of influenza B virus haemagglutinin, leading to virus neutralization.
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Inhibitory Effect of Papaverine on HVJ (Sendai Virus) Replication in Rat Glioma C6 Cells
More LessSUMMARYThe replication of HVJ (Sendai virus) in C6 rat glial cells was found to be inhibited by treatment of the cells with papaverine, an inhibitor of cAMP phosphodiesterase, but not with cAMP or dibutyryl cAMP. In addition, cyclic GMP which often manifests a reciprocal relationship to cAMP did not counteract the inhibition of HVJ yield by papaverine. Both viral genome replication and transcription were suppressed slightly by treatment of the cells with papaverine. In the cells cultured in the presence of papaverine, the synthesis of viral proteins and their phosphorylation occurred at normal rates. Membrane immunofluorescence and cell surface immunoprecipitation showed that the viral glycoproteins HN and F0 were expressed on the cell surface of the papaverine-treated cells. Moreover, all the viral structural proteins were associated with plasma membrane isolated from the treated cells. These results indicate that papaverine treatment suppresses some part of the process of virus budding at the plasma membrane.
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PGJ2, A New Antiviral Prostaglandin: Inhibition of Sendai Virus Replication and Alteration of Virus Protein Synthesis
More LessSUMMARYProstaglandin J2 (PGJ2) was found to suppress dramatically Sendai virus replication in African green monkey kidney cells in culture. PGJ2 was not toxic at the active dose to uninfected cells and did not significantly inhibit macromolecular synthesis, but it specifically stimulated the synthesis of a polypeptide of 74000 mol. wt. In Sendai virus-infected cells, PGJ2 partially inhibited virus protein synthesis and caused an alteration in the mobility of the virus glycoprotein HN in SDS-PAGE, corresponding to a decrease of about 4000 in its mol. wt. We propose that the PGJ2-induced alteration in the molecular structure of the HN protein prevents the insertion of this protein into the cell membrane thereby blocking virus maturation. The α, β-unsaturated carbonyl group in the cyclopentane ring of the PGJ2 molecule may be necessary for antiviral activity.
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A Major Phosphoprotein of Cells Infected with Pseudorabies Virus Is Phosphorylated by Cellular Casein Kinase II
More LessSUMMARYEndogenous protein phosphorylation was studied in extracts of hamster fibroblasts infected with pseudorabies virus. The major phosphorylation was detected quite late in infection and involved an acidic protein of Mr 62000. It was catalysed by an enzyme activity with the properties of cellular casein kinase II. Two-dimensional gel analysis was used to demonstrate that this same protein was also phosphorylated in vivo. The phosphoprotein was detected in mature virions and is most likely viral in origin.
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Generation of a Herpes Simplex Virus Type 1 Variant Devoid of XbaI Sites
More LessSUMMARYUsing both selection enrichment and site-directed mutagenesis, a herpes simplex virus type 1 (HSV-1) strain 17 genome lacking all four XbaI sites has been generated. The site at 0·45 map units which lies within the gene coding for a polypeptide of 28 000 molecular weight was removed by selection enrichment, while the site at 0·29 map units which lies within the gene coding for glycoprotein H was removed by site-directed mutagenesis. The parental virus from which these two XbaI sites were deleted had previously had the sites at 0·07 and 0·63 map units removed through selection enrichment. The variant devoid of XbaI sites (X4) showed normal growth characteristics; its phenotype was normal apart from the absence of the thymidine kinase protein, which is believed to be unrelated to the loss of XbaI sites.
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Transmission of Mouse Thymic Virus
More LessSUMMARYMouse thymic virus (MTV) is a naturally occurring herpesvirus of mice which produces persistent infection in salivary glands. No transmission study has been reported previously. In the present work, transmissibility of MTV has been studied by close contact between cage-mates, by the transplacental route from experimentally infected pregnant mice to their foetuses at term or delivered by Caesarean section and by nursing mothers to their sucklings. Transmission of MTV was detected between cage-mates after a long period of contact. The virus was also recovered from newborns nursed by infected mothers inoculated 1 day post-delivery. However, no transmission was detected in the foetuses following infection of mothers at different stages of pregnancy.
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Susceptibility of Human Peripheral Blood Dendritic Cells to Infection by Human Immunodeficiency Virus
More LessSUMMARYPreparations of human peripheral blood dendritic cells have been infected with human immunodeficiency virus (HIV). After 5 days in culture they were examined by electron microscopy. Virus was observed budding from the plasma membrane of dendritic cells and mature virions were observed on the cell surface. In addition, a second cell type, similar in morphology to ‘classical’ dendritic cells but containing numerous cytoplasmic granules, was also found to support replication of the virus. We speculate that the growth of HIV in dendritic cells could cause immunosuppression by impairing antigen presentation.
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Enhanced Production of a Human Spumavirus (Retroviridae) in Semi-permissive Cell Cultures after Treatment with 5-Azacytidine
More LessSUMMARYInfection by a human spumavirus of human foetal diploid lung (HFDL) cells was found to be productive with virus titres ranging from 103 to 105 p.f.u./ml. In contrast, infection of recovered amnion (RA) aneuploid cells resulted in a persistent infection with less than 100 p.f.u./ml infectious virus produced. The decreased sensitivity of RA cells to the spumavirus was not due to the failure of virus to penetrate into the cell since infectious virus was not produced even after transfection of infectious proviral DNA. The effect of 5-azacytidine, an inhibitor of DNA methylation, on virus replication was examined. Whereas virus production in HFDL cells was not affected, there was a 100fold increase in virus yield in RA cells treated with the drug for at least 48 h and maximum virus yields were obtained 4 days post-infection.
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Lack of Effect of Thymus and Spleen on the Incubation Period of Creutzfeldt-Jakob Disease in Mice
More LessSUMMARYGenetically athymic and asplenic (Lasat), athymic (Nude), asplenic (Dh) or normal littermate (Hetero) mice with a BALB/c genetic background were injected either intracerebrally or intraperitoneally with a 1% or 10% homogenate of mouse brains infected with the Fukuoka 1 strain of the Creutzfeldt-Jakob disease (CJD) agent. As there were no significant differences in incubation periods among the five groups (Lasat, Nude, Dh, Hetero and BALB/c) inoculated with the same dilution, via the same route, it was concluded that cell-mediated immunity dependent on the thymus plays no significant role in host defence against the CJD agent, and the spleen, a critical site of agent replication, is apparently not an obligatory source from which infection spreads to the central nervous system.
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Isolation and Characterization of a Cold-sensitive Strain of Coxsackievirus A10
More LessSUMMARYA coxsackievirus A10 strain, isolated from a clinical specimen from a patient with pharyngitis, was characterized with respect to its growth properties in dilferent cultured cells and at different incubation temperatures. This virus multiplied within cultured cells and produced cytopathogenic effects, whereas a prototype strain of coxsackievirus A10 did not. The isolate multiplied efficiently in cultured cells at 37 °C but its replication was markedly restricted at 32 °C. Temperature shift experiments indicated that the cold-sensitive event affected the late function(s) of the virus.
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Dependence of Guanidine Sensitivity of Poliovirus Replication on the Concentration of Monovalent Cations in the Culture Medium
More LessSUMMARYAn earlier suggestion that guanidine may inhibit picornavirus replication by interfering with a monovalent cation-mediated event was tested by determining the effect of varying monovalent cation concentration in isotonic medium on the sensitivity of poliovirus replication in HeLa cells to 0·2 mm-guanidine. Lowering [Na+] in the medium to 50 mm had no effect on virus replication. It was found that the degree of inhibition of virus replication by 0·2 mm-guanidine was inversely related to [Na+] in the medium: 99·8 %, 99·1 %, 38% and 0% inhibition in the presence of 50, 75, 100 and 145 mm-Na+ respectively. Likewise, guanidine uptake by HeLa cells was also inversely related to [Na+] in the medium. On the other hand, lowering medium [Na+] to 50 or 75 mm resulted in reduced intracellular [Na+] and [K+]. The increased sensitivity of virus replication to guanidine in the presence of low Na+ medium could be abolished with excess K+ in such medium. Excess K+ in low Na+ medium restored intracellular [Na+] and reduced guanidine uptake. Thus, the increased sensitivity of poliovirus replication to guanidine in the presence of low Na+ medium correlated with reduced intracellular [Na+] and [K+] and elevated guanidine uptake.
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Genetic Relatedness of Colorado Tick Fever Virus Isolates by RNA-RNA Blot Hybridization
More LessSUMMARYThe sequence relatedness of ten isolates of the Colorado tick fever (CTF) serogroup of orbiviruses was examined by RNA-RNA blot hybridization. The 12 dsRNA genome segments of each of the isolates were electrophoresed in a 10% polyacrylamide gel, the segments were transferred electrophoretically to membranes and hybridized to radiolabelled genomic RNA from CTF Florio mouse-adapted strain (CTF FMA) or CTF SS-18. All genome segments of the ten CTF viruses exhibited cross-hybridization signals with either CTF FMA or CTF SS-18, under conditions in which ≥74% sequence homology was required to form stable hybrids. Although the dsRNA polyacrylamide gel profiles were unique for each isolate examined, the CTF genes did not exhibit sequence divergence as has been seen among other Orbivirus serogroups. These hybridization analyses suggest that the CTF gene pool is relatively homogeneous which may be a reflection of the lack of multiple serotypes of CTF strains in neutralization tests. Nevertheless, the hybridization signals of segments 4 and 6 were lighter than those of the other genes, indicating that these two genes exhibited the highest degree of sequence variability among these isolates. These data are compared with hybridization data on other Orbivirus serogroups.
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Sequence Analysis of the Haemagglutinin of A/Taiwan/1/86, a New Variant of Human Influenza A(H1N1) Virus
More LessSUMMARYA/Taiwan/1/86 is representative of newly emerged antigenic variants of influenza A(H1N1) viruses which are readily distinguishable from all previous A(H1N1) isolates. Nucleotide sequence analysis of the haemagglutinin HA1 coding region of A/Taiwan/1/86 suggests that this virus has evolved from viruses circulating in the Hong Kong region in 1982 to 1983. The considerable alteration in antigenicity of this new isolate is likely to have arisen from five amino acid substitutions in a nine amino acid stretch, situated on the outer surface of a short oc-helix on the tip of the HA molecule in antigenic site Sb.
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Heterogeneity of the Respiratory Syncytial Virus 22K Protein Revealed by Western Blotting with Monoclonal Antibodies
More LessSUMMARYRespiratory syncytial (RS) virus-infected HeLa, HEp-2, Vero and BS-C-1 cell lysates were electrophoresed on SDS-polyacrylamide gels under reducing conditions and analysed by Western blotting and immunoperoxidase using monoclonal antibodies specific for the 22K protein (relative mol. wt. of 23000 in our gel system). Three novel polypeptides with mol. wt. of 24000, 21000 and 17000 were stained in addition to the 23000 polypeptide which was present in the greatest amount in all three virus strains tested regardless of host cell line. When samples were electrophoresed under nonreducing conditions each of the three higher mol. wt. polypeptides seen in reducing gels migrated as two bands (total of six bands) with altered electrophoretic mobilities. In experiments using the alkylating agent iodoacetamide under conditions where the novel 24000, 21000 and 17000 polypeptides were not visible, the number of mobility variants of the 23000 polypeptide which could be detected in non-reducing conditions was increased from two to four. At least one, and possibly three, of these variants was the result of conformational variation in the 23000 polypeptide caused by the generation or rearrangement of intrachain disulphide bonds after the infected cells were lysed in SDS-PAGE sample buffer. Post-lysis conformational changes were minimized by treatment of the infected cells with iodoacetamide before solubilization or by decreasing the SDS concentration or using milder detergents in the lysis buffer.
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Expression of the Respiratory Syncytial Virus 22K Protein on the Surface of Infected HeLa Cells
More LessSUMMARYImmunofluorescent staining of unfixed respiratory syncytial virus-infected HeLa cells with monoclonal antibodies (MAbs) demonstrated that the 22K protein is expressed on the cell membrane along with the fusion (F) protein and large glycoprotein (G). All three proteins were detected in the cytoplasm at 17 h postinfection and in the case of the F and G proteins this coincided with their appearance on the cell surface. However, the 22K protein could not be detected on the surface until approximately 16 h after its detection in the cytoplasm, when cytopathic effect was extensive. No evidence for the surface expression of the phosphoprotein (P), matrix (M) or nucleocapsid (N) proteins was found. Trypsin treatment of infected cells prior to unfixed immunofluorescent staining and Western blot analysis indicated that, unlike the G protein, the quantity of 22K protein detected on the cell surface constituted only a small proportion of the total present in the cell. A comparison of the patterns of immunofluorescent staining produced by MAbs on acetone-fixed infected cells suggested that the N, P and 22K proteins, but not the M protein, may be associated with the same intracellular structures.
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Some Properties of Isometric Virus Particles which Contain the Satellite RNA of Tobacco Mosaic Virus
More LessSUMMARYA ssRNA, which has a satellite-like relationship with tobacco mosaic virus (TMV), had an estimated mol. wt. of 0·38 × 106 and was found to be encapsidated in isometric particles with a diameter of 17 nm. The capsid protein had a mol. wt. of 18 × 103. This satellite virus of TMV (STMV) is highly immunogenic and no serological relatedness was detected between it and either TMV or satellite tobacco necrosis virus (STNV) or satellite panicum mosaic virus (SPMV) in immunodiffusion tests with antibodies specific for STMV, TMV, STNV and SPMV. Randomly primed STMV cDNA hybridized with STMV-specific dsRNA and ssRNA but did not hybridize to ssRNA of TMV, SPMV, panicum mosaic or cucumber mosaic viruses. The dsRNAs of different isolates of STMV differed in their electrophoretic mobilities, but they all shared nucleotide sequence homology. Particles of STMV tended to aggregate and crystallize upon purification. Replication of STMV in tobacco plants was supported by TMV but not by cucumber mosaic virus. This is the first report of a satellite virus of a rod-shaped plant virus.
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Conservation of the 3′-Terminal Sequences of the Genome RNA Species of Tomato Black Ring Virus
More LessSUMMARYSequences of more than 100 residues adjacent to the 3′-terminal poly(A) tract were determined for each of the two genome RNA species of tomato black ring nepovirus (TBRV) strains A and C; in addition a shorter portion of sequence was determined for RNA-2 of strain G. Comparison of these results and the sequence previously determined for RNA-2 of strain S showed that in this region the sequences of RNA-1 and RNA-2 are almost identical and that considerable sequence homology exists between strains. In contrast, a segment of sequence near the 3′ end of RNA-2 of tobacco ringspot virus, another nepovirus, showed no similarity with the corresponding region of TBRV RNA.
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Invasion of Non-phloem Tissue in Nicotiana clevelandii by Potato Leafroll Luteovirus Is Enhanced in Plants also Infected with Potato Y Potyvirus
More LessSUMMARYNicotiana clevelandii plants doubly infected with potato leafroll luteovirus (PLRV) and potato Y potyvirus (PVY) developed more severe symptoms than plants infected with either virus alone. Compared with singly infected plants, the PLRV concentration was increased up to eightfold by the double infection and the proportion of leaf parenchyma protoplasts that could be stained with fluorescent antibody to PLRV was increased from 0·2 to 1·4%. The concentration of PVY was unaffected by the double infection. In potato plants, in contrast, double infection with PVY did not increase PLRV concentration and none of the parenchyma protoplasts obtained from singly or doubly infected leaves could be stained with fluorescent antibody to PLRV. PLRV seems restricted to phloem tissue in potato but also invades a few parenchyma cells in N. clevelandii, a process that is accentuated in plants also infected with PVY.
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A Comparison between the Predicted Secondary Structures of Potato Virus X and Papaya Mosaic Virus Coat Proteins
More LessSUMMARYComparison of the predicted secondary structures of the coat proteins of the potexviruses potato X and papaya mosaic, on the basis of a combination of prediction methods, shows that although the proteins differ at their amino termini, and possibly at their carboxyl termini, they are similar in the distribution of α-helical segments in the central regions of the protein chains. There is an approximate twofold symmetry in the distribution of predicted α-helical segments between the amino and carboxyl halves of the molecules.
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