- Volume 61, Issue 2, 1982
Volume 61, Issue 2, 1982
- Animal
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Characterization of Theiler's Murine Encephalomyelitis Virus RNA
More LessSUMMARYTheiler’s murine encephalomyelitis viruses are usually included in the enterovirus genus of the family Picornaviridae, although there is little physicochemical evidence to support this classification. In this report, the size of the RNA of highly virulent and less virulent representatives of the Theiler's group of viruses has been determined by sucrose gradient centrifugation and electrophoresis in agarose to be the same as that of other enteroviruses. The absence of a poly(C) residue provides evidence that these viruses are not cardioviruses or aphthoviruses. The base composition of the two members are similar to each other but differ from those of other enteroviruses. However the one- and two-dimensional maps of the ribonuclease T1 hydrolysates of the two virus RNAs show considerable differences despite their close serological similarity. Virus-specified RNA synthesis in cells infected with the more virulent strain of the virus was almost 10 times greater than that induced by the less virulent strain, in accord with the yields of virus particles.
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Genetic and Antigenic Variation in Type 3 Polioviruses: Characterization of Strains by Monoclonal Antibodies and T1 Oligonucleotide Mapping
SUMMARYConsiderable genetic and antigenic heterogeneity was detected among a collection of 17 poliovirus type 3 strains isolated between 1939 and 1958 in studies using monoclonal antibodies and by T1 oligonucleotide mapping. Heterogeneity was detected even amongst a collection of nine viruses designated Saukett and assumed to originate from the same prototype virus. The monoclonal antibodies were found to differ in their strain specificities for poliovirus type 3 strains in virus neutralization or single-radial-immunodiffusion tests. Relationships between strains detected in this way were in general consistent with those detected by oligonucleotide mapping. One of the monoclonal antibodies (NIBp 56) was able to distinguish between certain Saukett virus strains which differed by as little as a single specific oligonucleotide. The heterogeneity detected amongst Saukett viruses is of potential practical importance since these strains are used widely in the manufacture of inactivated poliovirus vaccine.
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Interferon-mediated Persistent Infection of Saint Louis Encephalitis Virus in a Reptilian Cell Line
More LessSUMMARYA persistent infection with Saint Louis encephalitis (SLE) virus in a poikilothermic cell line TH-1 (turtle heart cells) was studied. Infected TH-1 cells were subcultured weekly at 31 °C for 1 year and continued to produce low levels (102 to 103 p.f.u./ml) of virus without obvious cytopathic effects or marked cyclic events. Indirect fluorescent antibody and infectious centre assays indicated that < 1 % of the cells were producing detectable virus proteins or infectious virus. Defective-interfering particles, temperature-sensitive mutants and DNA provirus were not detected. Interferon (IFN) mediation of the persistent infection was considered since the persistently infected cells (PIC) and normal TH-1 cells were resistant to heterologous virus challenge after treatment with virus-free culture fluid from PIC. A direct relationship was found between the m.o.i. and the amount of IFN produced, plateauing at an m.o.i. of approx. 10. The reptilian IFN was physically and chemically similar to mammalian and avian IFN. Certain biological markers of the SLE virus changed during the persistent infection. It was less virulent for mice, showed distinct differences in cell culture host range and had increased thermal lability.
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Development of Protease Activation Mutants of HVJ (Sendai Virus) in Persistently Infected Cell Cultures
More LessSUMMARYHVJ wild-type virus, in which the F protein is activated by trypsin but not by elastase, was spontaneously converted to a mutant with an F protein characterized by being activated by elastase alone. This spontaneous mutation generally occurred during serial passages of cells persistently infected with HVJ, even though the cells were first established by infection with plaque-purified wild-type virus. Multiple-cycle replication, plaque formation, haemolysis and SDS-polyacrylamide gel electrophoretic (SDS-PAGE) analysis showed that all the elastase-activated mutants isolated from HVJ carrier cells no longer required trypsin for F protein activation. At early passages, these protease activation mutants did not show temperature-sensitive (ts) growth, while at a later stage the mutants, together with the ts mutation, appeared dominant. The frequency of such a protease activation mutation during passage in the HVJ carrier cells seemed to depend on the cell species, but was increased when compared to lytic infections.
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Polymorphism of the NS1 Proteins of Type A Influenza Virus
More LessSUMMARYThe type-specific non-structural protein 1 (NS1) of influenza A viruses was found to be heterogeneous with respect to charge, varying in pI by more than two orders of magnitude, and to phosphorylation. Phosphorylation was strain-specific, variable in extent between strains, and in some strains NS1 proteins were not detectably phosphorylated. Phosphorylation was not responsible for the major variations in charge as, paradoxically, the most acidic NS1 proteins were not phosphorylated. Cytoplasmic inclusions, which are formed between NS1 and cellular RNA in infections with a number of human strains, were absent from A/FP/Rostock-infected cells and do not, therefore, appear to be essential in virus multiplication. We suggest that the acidic nature of the NS1 of A/FP/Rostock may prevent it from binding RNA and hence from forming inclusions. The variation in charge of NS1 proteins which we determined experimentally correlates with the overall differences in charge adduced from published amino acid sequences and implications of this variability to the biological role of NS1 are discussed.
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Replication and Release of Epizootic Haemorrhagic Disease Virus in BHK-21 Cells
More LessSUMMARYEpizootic haemorrhagic disease virus (EHDV) was seen by light and electron microscopy to replicate in perinuclear locations. Tubules, paracrystals and virus matrices were associated with replication sites. As infection proceeded, aggregates of virus migrated towards the cell periphery, resulting in cell membrane rupture near the virus aggregate with the subsequent release of the virus aggregates. Virus release, as seen by light microscopy, gave the appearance of occurring by a ‘budding’ process whereby part of the cell would swell and subsequently rupture or break away. Infectivity studies indicated that approx. 80% of newly replicated virus was released extracellularly in aggregates which required disruption to maximize infectious virus yield. Trypsin did not enhance virus infectivity. Of the six EHDV isolates used in this study each isolate was characterized by its own maximum yield obtained after several serial passages in cell culture.
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The Isolation and Characterization of Mutants of Herpes Simplex Virus Type 1 that Induce Cell Fusion
More LessSUMMARYSix cell fusion-causing syn mutants were isolated from the KOS (syn-101 to syn-106) and three from the HFEM (syn-107 to syn-109) strains of herpes simplex virus type 1 (HSV-1). The mutants were studied by complementation and recombination with syn-20 (a syncytial mutant of KOS) and ts-B5 (a syncytial mutant of HFEM). Some studies also employed MP, a syncytium-inducing strain isolated from the non-syncytial parent, mP. Complementation and recombination of syn-20 and ts-B5 indicated that these two mutants were altered in two different virus genes. The recombination frequency between syn-20 and ts-B5 was very similar to that observed between MP and ts-B5, indicating that syn-20 and MP may represent alterations in the same virus gene, syn-101, syn-103, syn-104 and syn-105 were tentatively assigned to the syn-20 complementation group, while syn-107 and syn-109 were tentatively assigned to the fs-B5 complementation group, syn-106 and syn-108 were excluded from the ts-B5 group, syn-102 could not be excluded from either complementation group, syn-101 induced markedly less fusion at 38 °C relative to 34 °C. At 34 °C the patterns of syn-101-infected cell peptides and glycopeptides, examined by SDS-gel electrophoresis, were normal, but at 38 °C the amount of glycopeptide gC was particularly reduced, syn-102 produced decreased amounts of glycoproteins, and a non-glycosylated peptide, probably ICP6, was absent from extracts infected with syn-106.
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Polypeptides of Varicella-Zoster Virus (VZV) and Immunological Relationship of VZV and Herpes Simplex Virus (HSV)
More LessSUMMARYVaricella-zoster virus (VZV), labelled with [35S]methionine or [14C]glucosamine, was purified by centrifugation through sucrose gradients followed by equilibrium centrifugation in CsCl, and analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). At least 32 polypeptides ranging in mol. wt. from approx. 280 × 103 to 21.5 × 103 (280K to 21.5K) and six glycopeptides ranging in mol. wt. from approx. 115K to 45K (gpl to gp6) were found in the virion. The immunological relationship of VZV and herpes simplex virus (HSV) was investigated. In neutralization (NT) tests, no cross-neutralization was observed between VZV and HSV-1 or -2. In fluorescent antibody staining, however, a cross-reaction was observed between VZV- and HSV-1-infected human embryonic lung (HEL) cells and heterologous antiserum. When cross-reactions were investigated by immunoprécipitation followed by SDS-PAGE, several cross-reacting polypeptides were discovered. Cross-reacting glycopeptides of 64K (gp3) and 55K (gp5) were isolated from VZV-infected cell lysates by affinity column chromatography to immobilized HSV-1 antibodies.
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Virus Particles and Glycoproteins Excreted from Cultured Cells Infected with Varicella-Zoster Virus (VZV)
More LessSUMMARYVirus particles and virus proteins excreted from cultured human embryonic lung (HEL) cells infected with varicella-zoster virus (VZV) were examined by electron microscopy and affinity column chromatography using an antibody to VZV followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Approximately 1 × 109 to 2 × 109 virus particles/ml with no detectable infectivity, of which 30 to 80% were enveloped, were observed in the culture fluid 48 to 72 h after infection, when cytopathic effect (c.p.e.) appeared. In the sonicated infected cell suspension, 1 × 109 to 2 × 109 virus particles/ml, of which 30 to 50% were enveloped, were observed and the virus particle/infectivity ratio was approx. 106:1. The culture fluid of infected HEL cells labelled with [35S]methionine or [3H]glucosamine was centrifuged at 100000 g for 2 h to remove virus particles and the supernatant was examined for excreted virus proteins. Affinity column chromatography of the supernatant using immobilized human zoster convalescent serum, led to the isolation of virus antigens which were analysed by SDS-PAGE. Polypeptides with mol. wt. of approx. 115K and 45K, both of which were glycosylated, were detected, suggesting that these VZV glycoproteins were excreted from the infected cells.
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Neuronal Function Impairment in Rabies-infected Rat Brain
More LessSUMMARYIn homogenates of rabies-infected rat brain the specific binding of a tritium-labelled antagonist of muscarinic acetylcholine (mACh) to mACh receptors (mAChR) varied during the course of infection. A small increase in the binding of the antagonist, quinuclidinyl benzylate (QNB), during the first days after infection was followed by a marked decrease as the symptoms of rabies appeared. Measurements of 3H-labelled QNB binding in dissected brain regions, i.e. brain stem, caudatus nucleus, cortex, hippocampus, cerebellum and medulla, showed that the decrease in binding was greater in the hippocampus than in any other brain region. We conclude that in the central nervous system (CNS) of the rabies-infected rat neuronal impairment may be one manifestation of rabies pathogenesis.
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The 3' Terminal RNA Sequences of Bunyaviruses and Nairoviruses (Bunyaviridae): Evidence of End Sequence Generic Differences within the Virus Family
SUMMARYThe 3' terminal nucleotide sequences of the three virus RNA species of viruses representing eight serogroups of bunyaviruses (genus Bunyavirus, Bunyaviridae) and six serogroups of nairoviruses (genus Nairovirus, Bunyaviridae) have been characterized. Members of the Bunyavirus genus have conserved 3' end sequences (generally, 3' UCAUCACAUGA…) that differ from the conserved 3' end sequences of members of the Nairovirus genus (generally, 3' AGAGUUUCU…).
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Multiplication of Lymphocytic Choriomeningitis Virus in Thymocytes During its Persistence in Mice
More LessSUMMARYPersistence of lymphocytic choriomeningitis (LCM) virus in mice infected in utero or neonatally is due to impairment of the specific subsets of thymus-dependent lymphocytes which, in the adult normal mouse, are involved in elimination of LCM virus. Virus-thymocyte interactions were studied since it was likely that this impairment takes place in the thymus. Using an infectious centre assay, we found that about 1% of the thymocytes from foetal and neonatal mice were productively infected by the virus while thymocytes from older mice were refractory to infection. The infected cells were Thy 1-positive and agglutinated by peanut lectin together with immature lymphocytes. Later, when virus persistence was established, the number of infected thymocytes declined to about 0.1% and these cells were not agglutinated by lectin. The results are compatible with the assumption that thymic precursor T-cells capable of eliminating LCM virus are chronically infected by the virus and rendered non-functional.
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Characterization of Rice Stripe Virus: a Heavy Component Carrying Infectivity
More LessSUMMARYRice stripe virus (RSV) preparations contain a previously unreported nucleopro- tein component (nB) which sediments faster in sucrose gradients than middle (M) and bottom (B) components; the latter two components are 20-fold more concentrated than nB. Electron microscopy of purified nB preparations revealed filamentous particles (8 nm wide), and other structures which might be derived from these particles. The nB preparations contained four RNA species of mol. wt. 0.9 × 106, 1.0 × 106, 1.4 × 106 and 1.9 × 106 (2700, 3000, 4100 and 5600 nucleotides respectively); the three smaller RNAs may be derived from M and B components contaminating nB. A single protein (mol. wt. 3.2 × 104) was present in M, B and nB preparations. The nB component appears to be required for infectivity because the planthopper vector (Laodelphax striatellus) became viruliferous after injection with nB but not after injection with M or B.
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Distribution of Determinants for Symptom Production and Host Range on the Three RNA Components of Cucumber Mosaic Virus
More LessSUMMARYEighteen pseudorecombinants were constructed in vitro by exchanging the three genomic RNA segments between pairs of three strains of cucumber mosaic virus (CMV). Inoculation of the CMV strains and the pseudorecombinants to 10 selected host plant species revealed that infection and symptom expression in a plant can be a complex interaction of the genetic material of the virus with that of the host genome. Whereas some host reactions were determined only by either RNA-2 and some by RNA-3, others were the results of the interaction of both these RNA species. Furthermore, some host reactions resulted from the interactions of any two, or perhaps even all three RNA segments of the virus.
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Effect of Temperature on the Multiplication of Cauliflower Mosaic Virus
More LessSUMMARYThe synthesis of cauliflower mosaic virus (CaMV) was greatly affected by temperature. The optimum temperature for virus multiplication in turnip plants and their protoplasts was about 20 °C but that for the formation of viroplasm matrix was higher than 20 °C. CaMV multiplication ceased around 33 °C but was restored by reverting to 20 °C. The intensity of vein-clearing symptoms and of formation of viroplasm in turnip plants did not directly relate to the yield of the virus. In protoplasts, normal viroplasm did not seem to be formed and only accumulations of virus particles were observed. This result suggests that the viroplasm matrix was not required for CaMV multiplication.
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