Two immunomodulators, curcumin and sulfasalazine, enhance IDV antiretroviral activity in HIV-1 persistently infected cells.

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.

Inhibition of cell fusion in Junin virus-infected cells by sera from Argentine hemorrhagic fever patients.

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.

Virucidal activity presence in Trichilia glabra leaves.

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.

Influenza circulating strains in Argentina exhibit differential induction of cytotoxicity and caspase-3 in vitro.

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.

Virucidal activity presence in Trichilia glabra leaves / Presencia de actividad antiviral en hojas de Trichilia glabra

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 log10reduction in HSV-1 titer when 0.25 mg/ml F2 fraction was tested and a reduction of 2.7 log10in VSV virus titer and of 1.5 log10in 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.

Previamente se han descripto distintas actividades inmunomoduladoras, presentes en extractos de hojas de Trichilia glabra (TG). En particular, se ha demostrado una actividad antiinflamatoria presente en extractos metanólicos. En este trabajo se investigó la actividad virucida de dichos extractos sobre virus envueltos y desnudos. Distintos extractos metanólicos han inactivado en forma moderada los virus herpes simplex tipo 1 (HSV-1) y el virus de la estomatitis vesicular (VSV), mientras no evidenciaron actividad sobre poliovirus tipo 3. VSV resultó algo mas afectado que HSV-1: se observó una reducción en el título viral de 2,8 log10para VSV y de 2,4 log10para HSV-1 cuando se uso una concentración de 0,25 mg/ml de la fracción F2 y una reducción de 2,7 log10para VSV y de 1,5 log 10para HSV-1 cuando se usó una concentración de 0,25 mg/ml de la fracción F3. Los resultados obtenidos en este trabajo, sugieren un potencial uso farmacéutico de los componentes presentes en los extractos de TG.

Virucidal activity presence in Trichilia glabra leaves.

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.

Effects of reducing, oxidizing and alkylating agents on early steps of Junin virus multiplication.

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.

In vitro anti-Junin virus activity of a peptide isolated from Melia azedarach L. leaves.

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.

Low-pH-induced fusion of Vero cells infected with Junin virus.

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.

Influencia del tratamiento enzimático sobre la interacción virus Junín-células Vero / Influence of enzyme treatment on the interaction of Junin virus with Vero cells

Se investigó la naturaleza bioquímica de las estructuras presentes en la superficie de células Vero, implicadas en la interacción con el virus Junín (cepa XJ C13) y una mutante de rango de huésped denominada Cl 67. Los tratamientos enzimáticos realizados a las células antes de la infección indican que las primeras moléculas que interaccionan con el virus Junín serían proteínas o grupos proteícos con aminoácidos básicos o aromáticos expuestos, existiendo diferencias en la afinidad hacia las cepas estudiadas

Influencia del tratamiento enzimático sobre la interacción virus Junín-células Vero / Influence of enzyme treatment on the interaction of Junin virus with Vero cells

Se investigó la naturaleza bioquímica de las estructuras presentes en la superficie de células Vero, implicadas en la interacción con el virus Junín (cepa XJ C13) y una mutante de rango de huésped denominada Cl 67. Los tratamientos enzimáticos realizados a las células antes de la infección indican que las primeras moléculas que interaccionan con el virus Junín serían proteínas o grupos proteícos con aminoácidos básicos o aromáticos expuestos, existiendo diferencias en la afinidad hacia las cepas estudiadas (AU)

[Influence] of enzymatic treatment on Junin virus--Vero cells interaction]. / Influencia del tratamiento enzimático sobre la interacción virus Junín-células Vero.

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.

Influencia del tratamiento enzimático sobre la interacción virus Junín-células Vero. / [Influence] of enzymatic treatment on Junin virus--Vero cells interaction]

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.

Intracellular processing and transport of Junin virus glycoproteins influences virion infectivity.

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.

The entry of Junin virus into Vero cells.

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.

Experimental infection of suckling mice with a host range mutant of Junin virus.

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.

[Lysosomotropic compounds inhibiting the multiplication of Junin virus]. / Compuestos lisosomotrópicos inhibidores de la multiplicación del virus Junín.

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.

Compuestos lisosomotrópicos inhibidores de la multiplicación del virus Junín. / [Lysosomotropic compounds inhibiting the multiplication of Junin virus]

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.

A comparison of Junin virus strains: growth characteristics, cytopathogenicity and viral polypeptides.

The growth characteristics, cytopathogenicity and viral polypeptides of the virulent strain XJ of Junin virus (JV), its attenuated derivative XJC13 and another naturally attenuated JV strain, IV4454, were comparatively studied. IV4454 and XJC13 viruses showed the highest and lowest cytopathology for Vero cells, respectively, as measured by plaque morphology, cell viability and inhibition of host cell protein synthesis. The kinetics and electrophoretic patterns of viral polypeptides in infected cell extracts were very similar among the three strains, whereas differences were detected in the surface glycoprotein GP38 by peptide mapping after limited proteolysis.

A mouse attenuated mutant of Junin virus with an altered envelope glycoprotein.

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.