RESUMO
BACKGROUND: Junin virus (JV), a member of the Arenaviridae family, is the etiological agent of Argentine hemorrhagic fever (AHF). A low pH-pulse, induces fusion of Vero cells infected with JV to form syncytia, whose production can be inhibited by neutralizing antibodies against the JV major glycoprotein. OBJECTIVES: To characterize the existence of an antifusogenic activity present in sera obtained from natural infections of AHF over a 20-year period and to study both the fusogenic activity of one pathogenic and two attenuated strains of JV in Vero cells, at different pH. The study sample consisted of sera obtained from two provinces in the Argentine Republic. Vero cells grown in monolayers, were infected with different strains of JV and a 2 h pulse, at different pH, was performed. Syncytium production was evaluated 12 h later, after staining with Giemsa. Neutralization tests against the attenuated strain XJCl3 were carried out and the antifusogenic activity of immunosera was studied by incubating serum with JV-infected Vero cells. Also the fusion activity in Vero cells infected with three JV strains was assayed. RESULTS AND CONCLUSIONS: A pathogenic strain XJ exhibited the highest fusogenic activity at pH 5. Syncytium formation was prevented by patients' sera obtained from different geographical locations, independently of time of infection. However, when Vero cells were infected with XJ, a significant reduction of syncytium production was observed, though the level of inhibition was lower than that detected in other JV strains-infected cells. These results could be explained by the existence of a conserved domain on JV proteins and also antigenic heterogeneity among strains.
Assuntos
Fusão Celular , Febre Hemorrágica Americana/imunologia , Soros Imunes/farmacologia , Vírus Junin/fisiologia , Animais , Especificidade de Anticorpos , Linhagem Celular Tumoral , Chlorocebus aethiops , Células Gigantes/efeitos dos fármacos , Células Gigantes/virologia , Febre Hemorrágica Americana/sangue , Humanos , Concentração de Íons de Hidrogênio , Vírus Junin/imunologia , Vírus Junin/patogenicidade , Células VeroRESUMO
C167, a mutant derived from the XJC13 strain of Junin virus, is highly attenuated in its pathogenic properties for newborn mice. Whereas 10(2).PFU of XJC13 injected intracerebrally killed 100% of two-day-old mice, the mutant showed no detectable lethality. Survival of mice infected with C167 was associated with a reduced and delayed virus replication in brain and a defective spread of virus from the site of inoculation to the other tissues, including spleen, kidney, thymus, liver, peritoneal cells and serum. As an apparent consequence of the restricted replication of C167 in mice, no detectable interferon induction and low levels of neutralizing antibodies were observed. Analysis of multiplication kinetics of C167 and XJC13 in different cell cultures in vitro has confirmed that the attenuated phenotype of C167 was related to a specific inefficient replication in murine cells. This host-range restriction was due to a combination of adsorption and penetration blockage.
Assuntos
Arenaviridae/patogenicidade , Arenavirus do Novo Mundo/patogenicidade , Animais , Animais Recém-Nascidos , Arenavirus do Novo Mundo/genética , Arenavirus do Novo Mundo/fisiologia , Febre Hemorrágica Americana/microbiologia , Interferons/sangue , Camundongos , Mutação , Virulência , Replicação ViralRESUMO
The influence of glycoprotein processing, cleavage and transport on Junin virus (JV) infectivity was investigated using monensin combined with lectin binding assays. Yields of extracellular virus were more significantly reduced than cell-associated virus, indicating that monensin inhibited the transport of infectious virus to the extracellular space on a late stage of the replicative cycle. Shown by lectin reactivity and immunoprecipitation, the intracellular processing of JV glycoproteins involved first the maturation of GPC oligosaccharides to a complex form and then the precursor cleavage which might occur late in transit through or exit from the Golgi cisternae. Cleavage of GPC to yield the mature GP38 as well as cell surface immunofluorescence were blocked by monensin. Thus, GP38 production together with glycoprotein transport to the cell membrane seemed to be required for the release of infectious virus from JV-infected cells.
Assuntos
Glicoproteínas/metabolismo , Vírus Junin/patogenicidade , Proteínas Virais/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Chlorocebus aethiops , Imunofluorescência , Vírus Junin/efeitos dos fármacos , Vírus Junin/fisiologia , Lectinas/farmacologia , Monensin/farmacologia , Peptídeo Hidrolases , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Células Vero , Proteínas do Envelope Viral , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Human influenza infections are a significant cause of morbidity worldwide. Though damage to the respiratory epithelium and has been related to apoptosis, which occurs subsequent to influenza virus infection, little information is available regarding cell cytotoxicity of human strains. OBJECTIVE: To study cytotoxicity performed in vitro by various circulating strains in Argentina. The study sample consisted of three vaccine strains (H1N1, H3N2, and B) administered during 1999-2000 in South America and three strains isolated from clinical samples, one, NAC (H1N1) obtained from an adult inpatient with human pneumonia; and the other two (T) and (T2) (H3N2) with influenza syndrome. Viral antigen was detected by an immunofluorescence test, conducted prior to viral isolation in MDCK cells. Strains were subtyped by the hemmaglutination inhibition test. Cytotoxic properties were determined by lactate dehydrogenase reaction (LDH), crystal violet staining and Hoechst staining. Caspase-3 activity, morphological changes of apoptosis, and viral yields were measured in MDCK infected cells. RESULTS AND CONCLUSIONS: Cells infected by each of the strains exhibited apoptosis morphology by Hoechst staining and caspase-3 activity was high for both H1N1 strains. Further, high levels of LDH activity were detected for NAC and H3N2 strains tested, indicating the possible role of different viral proteins or functions on cell cytotoxicity. The NAC strain, isolated from human pneumonia and antigenically related to A/New Caledonia /20/99 (H1N1), was the highest cytotoxic strain and an excellent inducer of caspase-3 activity. In turn, no parameter was related to different viral yields. We conclude that human strains studied in this paper may be useful tools in the characterization of molecular determinants involved in viral cytopathogenicity.
Assuntos
Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/patogenicidade , Vírus da Influenza B/isolamento & purificação , Vírus da Influenza B/patogenicidade , Influenza Humana/virologia , Adulto , Animais , Argentina , Caspase 3 , Caspases/biossíntese , Linhagem Celular , Citotoxicidade Imunológica , Cães , Indução Enzimática , Humanos , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Especificidade da Espécie , Cultura de VírusRESUMO
Meliacine, a peptide isolated from leaves of Melia azedarach L. inhibited the multiplication of Junin virus in Vero cells treated with the compound before infection (pre-treatment) or immediately after virus adsorption. Analysis of early events following infection demonstrated that meliacine blocks virus penetration by preventing the uncoating step. The addition of meliacine at different times after infection indicated that meliacine also interferes with the release of infectious particles to the extracellular medium and inhibits the low-pH-induced fusion of infected cells. Intracellular transport of viral glycoproteins to the cell membrane was not affected by meliacine, as revealed by immunofluorescence staining. Taken together, these results suggest that meliacine affects two events of the virus replicative cycle that require membrane fusion: uncoating and budding.
Assuntos
Antivirais/farmacologia , Vírus Junin/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Extratos Vegetais , Animais , Antivirais/isolamento & purificação , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Técnica Indireta de Fluorescência para Anticorpo , Células Gigantes/efeitos dos fármacos , Humanos , Vírus Junin/isolamento & purificação , Vírus Junin/fisiologia , Fusão de Membrana/efeitos dos fármacos , Peptídeos Cíclicos/isolamento & purificação , Folhas de Planta , Fatores de Tempo , Células VeroRESUMO
A number of lysosomotropic compounds of diverse chemical structure, ammonium chloride, procaine and chlorpheniramine, were found to inhibit the infection of Vero cells by Junín virus. Viral replication was almost totally inhibited by 15 mM ammonium chloride, when added either before or within the first hour of infection and a significant inhibition (97.8%) was observed when it was added 8 hours after infection. These results agree with a model which postulates that arenavirus entry occurs by receptor-mediated endocytosis.
Assuntos
Cloreto de Amônio/farmacologia , Arenavirus do Novo Mundo/efeitos dos fármacos , Clorfeniramina/farmacologia , Procaína/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Arenavirus do Novo Mundo/fisiologia , Depressão Química , Endocitose/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Receptores Virais/efeitos dos fármacos , Células VeroRESUMO
The virulence in neonatal mice of a temperature-sensitive mutant of Junin virus, named C167, was studied. The thermosensitive properties of this mutant were tested by titration on Vero cells at 37 and 40 degrees C. The ratio of infectivity 40/37 was approximately 100-fold lower for C167 with respect to XJC13 (Table 1). The attenuation of C167 was determined by measurement of mean survival time and 50% lethal dose after intracerebral injection of 2 and 11 day old mice. For C167 the lethality index (expressed ad the ratio TCID50/LD50) was greater than 580, while for XJC13 the index was 4.4 (Table 2). The lack of virulence of C167 was correlated with a restricted ability to replicate in suckling mouse brains. By contrast, the mutant and the parental virus grew to a similar titre in Vero cells (Figure 2). Both viruses were indistinguishable in cross-neutralization tests using hyperimmune antisera.
Assuntos
Arenaviridae/patogenicidade , Arenavirus do Novo Mundo/patogenicidade , Mutação , Fatores Etários , Animais , Anticorpos Antivirais/análise , Arenavirus do Novo Mundo/genética , Arenavirus do Novo Mundo/imunologia , Arenavirus do Novo Mundo/fisiologia , Cricetinae , Febre Hemorrágica Americana/mortalidade , Camundongos , Temperatura , Células Vero , Replicação ViralRESUMO
Different immunomodulatory activities present in Trichilia glabra (TG) leaf extracts have already been described. Particularly, chloroform-methanol extracts were responsible for an in-vivo anti-inflammatory effect. The effect of such extracts on the infectivity of enveloped and naked viruses were investigated. Methanolic fraction extracts were active against herpes simplex virus type 1 (HSV-1) and vesicular stomatitis virus (VSV), while no activity against poliovirus type 3 was observed. VSV was slightly more affected than HSV-1: 2.8 log10 reduction in VSV titer against 2.4 log10 reduction in HSV-1 titer when 0.25 mg/ml F2 fraction was tested and a reduction of 2.7 log10 in VSV virus titer and of 1.5 log10 in HSV-1 virus titer was observed when 0.25 mg/ml F3 fraction was tested. Results obtained in this work suggest a potential pharmaceutical use of TG extract components.
Assuntos
Antivirais/isolamento & purificação , Meliaceae/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Animais , Antivirais/farmacologia , Fracionamento Químico , Chlorocebus aethiops , Clorofórmio , Herpesvirus Humano 1/efeitos dos fármacos , Metanol , Extratos Vegetais/isolamento & purificação , Poliovirus/efeitos dos fármacos , Células Vero , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacosRESUMO
The chemical nature of cellular structures involved in the attachment of Junin virus (wild type XJC13 and host range mutant Cl 67) to Vero cells was investigated. Enzyme treatment of cells before virus infection indicated that whereas lipids are not directly involved in virus attachment, cellular proteins play a significant role in early interaction with JV. Moreover aromatic residues, leucine and basic amino acid seem to actively participate in this interaction with different affinity for the assayed strains.
Assuntos
Enzimas/farmacologia , Vírus Junin/efeitos dos fármacos , Células Vero/efeitos dos fármacos , Animais , Células Cultivadas , Chlorocebus aethiops , Feminino , Vírus Junin/fisiologia , Masculino , Células Vero/fisiologia , Células Vero/virologiaRESUMO
Since the appearance of resistance to antiretroviral treatment is unavoidable, the host cell's transcription factor NF-kappaB is a novel HIV target. The goal of this study was to characterize the effect of two immunomodulators, curcumin (Cur) and sulfasalazine (Sul), with a protease inhibitor, indinavir (IDV), on HIV-1 persistently infected CD4+ T-cells. Viral p24 antigen production, viral infectivity (tested on MAGI cells) and viral relative infectivity (viral infectivity/p24) were analysed. When used alone, both immunomodulators were able to reduce viral infectivity. When in combination, both 10 microM IDV plus 10 microM Cur and 10 microM IDV plus 250 microM Sul showed a significant reduction in viral infectivity and viral relative infectivity when compared to the reduction produced by IDV alone. Thus, the use of immunomodulators with IDV could help to reduce HIV-1 production in persistently infected cells.
Assuntos
Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/virologia , Curcumina/farmacologia , HIV-1/efeitos dos fármacos , Indinavir/farmacologia , Sulfassalazina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Proteína do Núcleo p24 do HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/fisiologia , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Quinase Induzida por NF-kappaBRESUMO
Junin virus (JV) infected Vero cells were used to investigate virus capacity to induce cell-cell fusion. Polykaryocyte formation due to JV was found to be pH and temperature-dependent. A reduced fusion activity was detected on BHK-21 cells. Different JV-strains exhibited a similar extent and pH dependence of their fusion activity. Neutralizing antibodies against the main viral glycoprotein (GP38) inhibited syncytium production and GP38 conformational changes in response to acid treatment were detected by an immunoprecipitation assay.
Assuntos
Vírus Junin/fisiologia , Animais , Fusão Celular , Chlorocebus aethiops , Glicoproteínas/química , Concentração de Íons de Hidrogênio , Conformação Proteica , Temperatura , Células Vero , Proteínas do Envelope Viral/fisiologiaRESUMO
We studied the binding of Junin virus (Arenaviridae) glycoproteins, G1 and G2, to two insolubilized lectins. The results showed that mannose, N-acetyl-glucosamine and galactose residues were exposed on G2, while only the latter predominated on G1. Heterogeneity of carbohydrate chains was found in G2, the only glycoprotein that was iodinated by the lactoperoxidase method.
Assuntos
Arenaviridae/metabolismo , Arenavirus do Novo Mundo/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Cromatografia de Afinidade , Lactoperoxidase/metabolismo , Lectinas/metabolismo , Células VeroRESUMO
Experimental infection of three mouse strains with a non-pathogenic mutant of Junin virus named Cl67 was compared with respect to the parental XJCl3 strain. After intracerebral (ic) or intraperitoneal inoculation, XJCl3 was highly virulent for 2 day-old C3H/HeJ, OF1, and BALB/cJ mouse strains, whereas its derivative Cl67 was attenuated. Survival of the Cl67-infected mouse was associated with a restricted replication at the site of inoculation which would impair spread of virus. Thus, the reduced virulence of Cl67 for suckling mice is independent of the mouse strain and the route of viral entry. When Cl67 was preinoculated ic 10 days before the challenge inoculation with XJCl3 by the same route, mice were partially protected from lethal infection. Since neutralizing antibodies were first detected at 30 days post-infection, an interference mechanism is postulated as a mechanism of protection of the mice.
Assuntos
Arenavirus do Novo Mundo/patogenicidade , Febre Hemorrágica Americana/microbiologia , Animais , Anticorpos Antivirais/sangue , Arenavirus do Novo Mundo/genética , Arenavirus do Novo Mundo/crescimento & desenvolvimento , Arenavirus do Novo Mundo/imunologia , Modelos Animais de Doenças , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/prevenção & controle , Cinética , Camundongos , Camundongos Endogâmicos , Mutação/genética , Testes de Neutralização , Células Vero , Ensaio de Placa Viral , Virulência/genéticaRESUMO
The XJC13 strain of Junin virus (JV) and the mouse-attenuated mutant C167 showed different GP38 peptide mapping after limited proteolysis with ficin and papain; viral infectivity of both viruses also exhibited a different susceptibility to protease treatment. A correlation between envelope glycoprotein alteration and JV virulence in neonatal mice is proposed.
Assuntos
Arenaviridae/genética , Arenavirus do Novo Mundo/genética , Mutação , Proteínas do Envelope Viral/genética , Animais , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/genética , Camundongos , Mapeamento de Peptídeos , Replicação Viral/genéticaRESUMO
Vero cell cultures persistently infected with the arenaviruses Junin, Pichinde, Tacaribe, and Tamiami were established and designated Vero-Jun, Vero-Pic, Vero-Tac, and Vero-Tam, respectively. Two types of carrier cultures could be easily distinguished: Vero-Jun and Vero-Tac systems were characterized by a lack of infectious virus production after a few cell transfers, whereas a more productive state with continuous release of virus was observed in Vero-Pic and Vero-Tam cultures. These differences appeared to be related to resistance of the culture to viral superinfection. In fact, Vero-Jun and Vero-Tac cultures totally excluded only the replication of the serologically more closely related arenaviruses Amapari, Junin, or Tacaribe, while the refractoriness of Vero-Pic and Vero-Tam cultures was extended to most of the virus group members. The resistance of Vero-Jun cells to superinfection by Junin or Tacaribe virus could be ascribed to the production of specific uv-resistant Junin interfering particles, which showed a specific range of interference against Junin and Tacaribe viruses. Interfering particles against homotypic and heterotypic arenaviruses were isolated from Vero-Pic cultures. However, the degree of interference developed by these Pic-interfering particles was not enough to fully explain reinfecting virus exclusion from Vero-Pic cultures. Viral susceptibility of persistent cultures is proposed as a useful tool to examine relationships of members of the arenavirus group.
Assuntos
Arenaviridae/fisiologia , Arenavirus do Novo Mundo/fisiologia , Interferência Viral , Animais , Arenaviridae/efeitos da radiação , Arenavirus do Novo Mundo/efeitos da radiação , Linhagem Celular , Chlorocebus aethiops , Raios Ultravioleta , Ensaio de Placa ViralRESUMO
Nuclear involvement of the host cell in Junin virus (JV) replication was studied. alpha-Amanitin was shown to inhibit JV yield, and RNA synthesis in cell nuclei was found to be augmented during the eclipse period. JV infection exerted a selective effect on cellular RNA polymerase activities, when measured in purified infected nuclei. RNA polymerase I was inhibited, but RNA polymerase II was stimulated. These data support the hypothesis that RNA polymerase II is involved in arenavirus replication.
Assuntos
Arenaviridae/crescimento & desenvolvimento , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerase II/metabolismo , Amanitinas/farmacologia , Animais , Linhagem Celular , Chlorocebus aethiops , Indução Enzimática , Rim , RNA/biossíntese , RNA Polimerase I/metabolismo , RNA Nuclear Heterogêneo/biossíntese , Vírus da Estomatite Vesicular Indiana/crescimento & desenvolvimentoRESUMO
The immunological response to rubella virus structural proteins was analyzed by immunoprecipitation assay in sera collected in Cordoba, Argentina, during early convalescence and after remote natural infections. All of the viral structural proteins, the two glycoproteins E1 and E2, and the nucleocapsid C protein, were precipitated by sera obtained from patients soon after infection. By contrast, C reactive antibodies were not detected in sera from remote infections. These results suggest that E1 and E2 play a major role in the long-lasting protective immunity to rubella virus.
Assuntos
Anticorpos Antivirais/biossíntese , Vírus da Rubéola/imunologia , Rubéola (Sarampo Alemão)/imunologia , Proteínas Estruturais Virais/imunologia , Adulto , Animais , Antígenos Virais/imunologia , Argentina , Capsídeo/imunologia , Chlorocebus aethiops , Glicoproteínas/imunologia , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Lactente , Células Vero , Proteínas do Envelope Viral/imunologiaRESUMO
Two temperature sensitive (ts) mutants of Junin virus, a member of Arenaviridae, have been partially characterized. Both mutants, named ts-32 and ts-40, had a relative plating efficiency 40 degrees C/34 degrees C lower than 10(-3) an exhibited a leak yield below 10(-4). Standard growth curves showed that at 34 degrees C the viral mutants multiplied slower than wt virus and at high multiplicity did not display autointerference. No differences in thermolability were observed between wt and ts mutants. By contrast, when the pathogenic properties of the mutants were investigated they were significantly attenuated for mice. At the restrictive temperature both mutants were unable to synthesize viral-specific polypeptides, while at the permissive temperature the pattern was similar to wt virus. Shift-up and down experiments suggested that ts defect is expressed between 2 and 4 hours post-infection. It is concluded that ts-32 and ts-40 are early function mutants. The possible nature of their defect is discussed.
Assuntos
Arenaviridae/genética , Arenavirus do Novo Mundo/genética , Animais , Animais Recém-Nascidos , Arenavirus do Novo Mundo/crescimento & desenvolvimento , Arenavirus do Novo Mundo/metabolismo , Arenavirus do Novo Mundo/patogenicidade , Genes Virais , Camundongos , Mutação , Temperatura , Ensaio de Placa Viral , Proteínas Virais/biossínteseRESUMO
The action of reducing, oxidizing and thiol-alkylating agents on early steps of Junin virus (JV) multiplication in Vero cells was investigated. The presence of reducing agents during virus adsorption as well as incubation of viral particles with these compounds before infection enhanced JV infectivity. On the contrary, the thiol-alkylating agent 5,5' dithiobis (2-nitrobenzoic acid) and the oxidizing compound potassium periodate showed an inhibitory effect, suggesting that sulfhydryl groups, and certain sugar moieties of viral glycoproteins play an important role in the first steps of JV infection. Also enzymatic treatment of cell monolayers and addition of concanavalin A to cultures prior to infection suggest that cellular glycoproteins are involved in virus attachment.
Assuntos
Alquilantes/farmacologia , Vírus Junin/efeitos dos fármacos , Vírus Junin/fisiologia , Oxidantes/farmacologia , Substâncias Redutoras/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Chlorocebus aethiops , Concanavalina A/farmacologia , Ácido Ditionitrobenzoico/farmacologia , Ditiotreitol/farmacologia , Ficina/farmacologia , Glicoproteínas de Membrana/fisiologia , Mercaptoetanol/farmacologia , Ácido Periódico/farmacologia , Compostos de Potássio/farmacologia , Receptores Virais/efeitos dos fármacos , Receptores Virais/fisiologia , Reagentes de Sulfidrila/farmacologia , Fosfolipases Tipo C/farmacologia , Células Vero , Virulência/efeitos dos fármacos , Virulência/fisiologiaRESUMO
The entry mechanism of Junin virus (JV) into Vero cells was studied analyzing the effect of lysosomotropic compounds and acid pH on JV infection. Ammonium chloride, amantadine, chlorpheniramine and procaine inhibited JV production. The action of ammonium chloride was exerted at early times of infection. Virus internalization was inhibited and viral protein expression was not detected. When the extracellular medium was buffered at low pH, the ammonium chloride induced block on JV infection was overcome. Furthermore, JV was able to induce fusion of infected cells at pH 5.5 leading to polykaryocyte formation. Taken together, these results demonstrate that JV entry occurs through an endocytic mechanism requiring a low pH dependent membrane fusion.