Detection of Lassa virus antigens and Lassa virus-specific immunoglobulins G and M by enzyme-linked immunosorbent assay.
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Rapid diagnosis of Lassa fever is desirable for the timely therapeutic intervention and implementation of strict quarantine procedures both in West Africa field hospitals where the disease is endemic and at international crossroads. An enzyme-linked immunosorbent assay (ELISA) to measure Lassa virus antigens in viremic sera was developed in which experimentally infected monkeys were used as a model for the human disease. In this test, Lassa virus antigens in test sera were captured in wells of microtiter plates by monkey anti-Lassa virus immunoglobulin. Guinea pig anti-Lassa virus immunoglobulin was then added, and binding of specific immunoglobulin was quantitated by the addition of rabbit anti-guinea pig immunoglobulin followed by alkaline phosphatase-labeled anti-rabbit immunoglobulin. This test detected viremia titers as low as 2.1 log10 PFU/ml in experimentally infected monkey sera, a titer often exceeded in patients with Lassa fever. Inactivation of infectious virus by beta-propiolactone or gamma-irradiation did not diminish reactivity. Antigen-ELISA concentrations increased with infectivity for the first 10 days after infection but then declined while infectivity titers remained high, suggesting that the presence of humoral antibody in viremic sera diminishes the sensitivity of the antigen ELISA. Lassa virus-specific immunoglobulin M (IgM) titers measured in an IgM capture ELISA were detectable within 10 days of infection and peaked after 36 days but remained detectable for 1.5 years. The Lassa virus-specific IgG ELISA response was slightly delayed, peaking on day 73 but declining only slightly thereafter. These studies in a realistic primate model suggest that the antigen detection ELISA or the IgM capture ELISA described, in which beta-propiolactone-inactivated sera are used, should be useful for the rapid diagnosis of human Lassa fever. (+info)
Structural and cell-associated proteins of Lassa virus.
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Lassa virus was purified from culture fluids of infected CV-1 monkey kidney cells and its structural proteins analysed by polyacrylamide gel electrophoresis. Stained gels showed a typical arenavirus profile, with a prominent protein of molecular weight 60000, corresponding to the nucleocapsid protein N, and two faint broad bands with molecular weights of 45000 and 38000, the envelope glycoproteins G1 and G2. G1 and G2 were shown to be glycosylated by their ability to bind concanavalin A to nitrocellulose transfers of the separated proteins ('Western blots'). N and G2 bound antibody from guinea-pig or human convalescent sera but G1 was inactive, presumably as a result of denaturation. This technique also revealed other apparently virus-specific minor bands with molecular weights of 76000 and 68000. When Western blots of proteins of infected cells which had been lysed in SDS were probed with anti-Lassa virus serum or stained for glycoproteins, four virus-specific bands were apparent: the N, G1 and G2 proteins seen in purified virus, and a glycoprotein of molecular weight 72000 which probably corresponds to the envelope protein precursor (GPC) seen in other arenavirus systems. Immunoprecipitates from infected CV-1 cells labelled with [35S]methionine contained three major virus-specific proteins: the nucleocapsid protein N and proteins of 36000 and 24000 molecular weight (designated fN1 and fN2). Similar immunoprecipitates from Vero cells contained fN1 and fN2 and only very low levels of N. The polypeptides fN1 and fN2 are most probably fragments of N, since Western blots probed with anti-Lassa virus serum showed that lysis of cells in non-ionic detergent rather than SDS results in the appearance of fN2 with concomitant reduction or disappearance of N. These fragments do not exist in the intact cell, but are found as a consequence of rather specific proteolysis upon disruption under non-denaturing conditions. The proteolytic activity responsible was refractory to inhibition by phenylmethylsulphonyl fluoride, aprotinin, pepstatin A or sodium bisulphite, and was more active in Vero than in CV-1 cells. (+info)
Sequencing studies of pichinde arenavirus S RNA indicate a novel coding strategy, an ambisense viral S RNA.
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Analyses of the complete sequence of the 1.1 X 10(6)-dalton, small (S) RNA of the arenavirus Pichinde and virus-induced cellular RNA species have revealed that the viral nucleoprotein, N, is coded in a subgenomic, non-polyadenylated, virus-complementary mRNA corresponding to the 3' half of the viral RNA (Auperin et al., Virology 134:208-219, 1984). By contrast, a second S-coded product, presumably the viral glycoprotein precursor (GPC), is coded in a subgenomic, virus-sense mRNA corresponding to the 5' half of the RNA. Between the two genes is a unique RNA sequence that can be arranged in a hairpin configuration and may function as a transcription terminator for both genes. The term ambisense RNA is coined to describe this novel coding strategy of a viral RNA. The unique feature of the strategy is that the presumptive GPC mRNA and its translation product cannot be made until viral RNA replication has commenced. In addition, it allows the two subgenomic mRNA species to be regulated independently from each other or from other viral mRNA species. The implications of this strategy on possible mechanisms for the induction and maintenance of viral persistence, an important attribute of arenavirus infections, are discussed. (+info)
Virion RNA species of the arenaviruses Pichinde, Tacaribe, and Tamiami.
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The principal RNA species isolated from labeled preparations of the arenavirus Pichinde usually include a large viral RNA species L (apparent molecular weight = 3.2 X 10(6)), and a smaller viral RNA species S (apparent molecular weight = 1.6 X 10(6)). In addition, either little or considerable quantities of 28S rRNA as well as 18S rRNA can also be obtained in virus extracts, depending on the virus stock and growth conditions used to generate virus preparations. Similar RNA species have been identified in RNA extracted from Tacaribe and Tamiami arenavirus preparations. Oligonucleotide fingerprint analyses have confirmed the host ribosomal origin of the 28S and 18S species. Such analyses have also indicated that the Pichinde viral L and S RNA species each contain unique nucleotide sequences. Viral RNA preparations isolated by conventional phenol-sodium dodecyl sulfate extraction often have much of their L and S RNA species in the form of aggregates as visualized by either electron microscopy or oligonucleotide fingerprinting of material recovered from the top of gels (run by using undenatured RNA preparations). Circular and linear RNA forms have also been seen in electron micrographs of undenatured RNA preparations, although denatured viral RNA preparations have yielded mostly linear RNA species with few RNA aggregates or circular forms. (+info)
Fine structure analysis of Pichinde virus nucleocapsids.
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The structure and organization of the ribonucleoprotein (RNP) complex of an arenavirus, Pichinde virus, was investigated. The basic configuration of the RNP was found to be a linear array of globular subunits or nucleosomes, 4 to 5 nm in diameter, that represent individual molecules of the major N polypeptide. This filament appears to fold progressively through a number of intermediate helical structures, 12 to 15 nm in diameter, that reveal an increasing number of nucleosomes associated with each turn of the helix. They range from a fragile configuration of two or three nucleosomes per turn to a more stable fibre in which the nucleosomes cannot be resolved. The strands were shown to form closed circles and it appeared that the twisting of these circular forms resulted in the formation of 20 nm-thick fibres which were seen in isolated viral core structures. The association of these RNP structures with other viral components is discussed. (+info)
Inhibition of mouse peritoneal macrophage DNA synthesis by infection with the arenavirus Pichinde.
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Macrophage DNA synthesis and proliferation occur during the development of cell-mediated immunity and in the early nonspecific reaction to infection. Arenaviruses have a predilection for infection of cells of the reticuloendothelial system, and in this study we have examined the effect of the arenavirus Pichinde on macrophage DNA synthesis. We have found that infection of mouse peritoneal macrophages with Pichinde caused a profound dose-dependent inhibition of the DNA synthesis induced by macrophage growth factor-colony stimulating factor. At a multiplicity of inoculum of 5, there is a 75 to 95% inhibition of DNA synthesis. Viable virus is necessary for inhibition since Pichinde inactivated by heat or cobalt irradiation had no effect. Similarly, virus pretreated with an antiserum to Pichinde was without inhibitory effect. Inhibition was demonstrated by measuring DNA synthesis spectrofluorometrically as well as by [3H]thymidine incorporation. The inhibition of DNA synthesis was not associated with any cytopathology. There was no evidence that the inhibition was due to soluble factors, such as prostaglandins or interferon, released by infected cells. These studies demonstrate, for the first time in vitro, a significant alteration in macrophage function caused by infection with an arenavirus. It is possible that inhibition of macrophage proliferation represents a mechanism by which some microorganisms interfere with host resistance. (+info)
Passive antibody therapy of Lassa fever in cynomolgus monkeys: importance of neutralizing antibody and Lassa virus strain.
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Lassa virus-infected cynomolgus monkeys were passively immunized with immune plasma of primate or human origin to gain insight into criteria for plasma selection and administration to human Lassa fever patients. Protective efficacy was correlated with neutralizing antibody concentrations, expressed as a log10 neutralization index (LNI). Convalescent Lassa-immune monkey plasma was titrated for protective efficacy in monkeys by intravenous inoculation with dilutions of plasma on the day of subcutaneous Lassa virus inoculation (day 0) and again on days 3 and 6. Monkeys that received undiluted plasma (LNI = 4.1) (1 ml/kg per treatment) survived a lethal viral dose, whereas those given a 1:3 dilution (LNI = 2.6) of this same plasma (1 ml/kg per treatment) died. Protection was restored when the volume of the 1:3 plasma dilution was increased to 3 ml/kg per treatment. Plasma diluted 1:9 or more (LNI = 1.5 or less) delayed onset and suppressed the magnitude of viremia but failed to confer protection at 3 ml/kg per treatment. Immunological enhancement, defined as increased viremia or accelerated death, did not occur following inadequate treatment. Human convalescent plasma also protected recipient monkeys; reductions in mortality and viremia were accurately predicted by the LNI of the plasma. Plasma of Liberian origin neutralized a Liberian Lassa strain more effectively than a Sierra Leone strain in vitro (LNI = 2.8 and 1.6, respectively) and protected monkeys more effectively against the Liberian strain. Geographic origin is thus a factor in the selection of optimal plasma for treatment of human Lassa fever, since geographically matched plasma is more likely to contain adequate LNI titers against homologous Lassa virus strains.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)
Effect of storage temperature on the stability of Lassa virus complement-fixing antigen.
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Inactivated Lassa virus complement-fixing antigen is normally prepared as a wet frozen antigen, and shipped in dry ice from the Center for Disease Control, Atlanta, Georgia, USA, to other laboratories not equipped for handling live Lassa virus. However, with this antigen there is a considerable loss of potency during shipping. In the search for a more stable antigen, inactivated Lassa antigen was lyophilized and subjected to different storage temperatures. Lyophilization did not significantly affect the CF titre, and the lyophilized antigen remained stable for 15 and 28 days at 25 degrees C and 4 degrees C, respectively. (+info)