RESUMEN

Herpes simplex virus-derived amplicon vectors simultaneously expressing the open reading frame encoding NR1 subunit of the NMDA receptor, either in sense or antisense orientation, as well as the open reading frame encoding the green fluorescent protein (GFP), as distinct transcription units, were constructed. Vector expression in cells was demonstrated by GFP-fluorescence, immunofluorescence, Western blots and RT-PCR. The vectors were inoculated into the dorsal hippocampus of adult male rats, which were then trained for habituation to an open field and for inhibitory avoidance to a foot-shock. Those animals injected with vectors expressing NR1 protein showed habituation to a new environment, and achieved the criteria for a step-down inhibitory avoidance to a foot-shock. In contrast, animals injected with vectors carrying the NR1 open reading frame in antisense position, showed neither habituation nor appropriate performance in the inhibitory avoidance task. There was no evidence for motor impairment or motivational disturbance, since all the animals exhibit similar behavior and performance in the training sessions. Hence, the impaired performance might be due to either amnesia or disability to record events. Transgene expression in brain, as revealed by GFP fluorescence, was mainly observed in pyramidal cells of CA1, but also in CA3. Therefore, our results strongly support the participation of hippocampal NR1 subunit in habituation to a new environment, but also in recording events for the inhibitory avoidance task. Hence, amplicon vectors appear to be useful tools to modify endogenous gene expression at a defined period, in restricted brain regions, and should allow investigating in vivo functions of genes.

Asunto(s)

Conducta Animal/fisiología , Técnicas de Transferencia de Gen , Vectores Genéticos , Herpesvirus Humano 1/genética , Hipocampo/virología , Oligonucleótidos Antisentido/genética , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Línea Celular , Cricetinae , Expresión Génica , Haplorrinos , Masculino , Aprendizaje por Laberinto/fisiología , Plásmidos , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/genética , Transgenes

RESUMEN

1. The aim is to study some roles of the hippocampal NMDA receptor, by modifying the expression of the essential NR1 subunit, with temporal and spatial restrictions in the central nervous system (CNS) of the rat. 2. Due to their neurotropism and the size of inserts they can accomodate, herpes simplex virus type-1 (HSV-1) derived amplicon vectors were used to transfer sequences, either in sense (+) or antisense (-) orientations, of the NR1 subunit gene, or of the green fluorescent protein (GFP) gene, into the CNS. 3. Vector expression in cell lines was followed by GFP autofluorescence, immunofluorescence and western blot. 4. The vectors were inoculated into the dorsal hippocampus of adult male Wistar rats, which were evaluated for habituation to an open field, and then, for expression of the transgenes, by autofluorescence and western blot; the expression mainly happened in pyramidal cells of CA1. 5. The animals injected with vectors carrying the NR1(+) transgene showed habituation to the new environment, as also happened with rats injected with vectors carrying only the GFP transgene. 6. In contrast, animals injected with vectors carrying NR1(-) sequence, did not show habituation. This might be retrograde amnesia or disability to record the trace, suggesting that the NR1 subunit in the dorsal hippocampus, is involved in habituation to a new environment. 7. HSV-1 derived amplicon vectors appear to be useful tools to modify endogenous gene expression, at a defined period, in restricted regions of the CNS.

Asunto(s)

Habituación Psicofisiológica/genética , Hipocampo/metabolismo , Trastornos de la Memoria/genética , Terminales Presinápticos/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Transmisión Sináptica/genética , Animales , Conducta Animal/fisiología , Cricetinae , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes , Hipocampo/fisiopatología , Inmunohistoquímica , Aprendizaje/fisiología , Proteínas Luminiscentes , Masculino , Memoria/fisiología , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Oligonucleótidos Antisentido , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Simplexvirus/genética

RESUMEN

Herpetic stromal keratitis is caused by ocular infection with herpes simplex virus type 1 (HSV-1) and constitutes a leading cause of human blindness. The effect of meliacine, an antiviral compound isolated from leaves of Melia azedarach L. that inhibits HSV-1 replication in vitro, was examined on experimental corneal HSV-1 inoculation in Balb/c mice. Mice were inoculated with HSV-1 strain KOS at their corneas after abrasion. Meliacine was administered topically 3 times a day for 4 days beginning 1 day before inoculation. Infected animals treated or not with meliacine were observed carefully for the development of stromal keratitis and the clinical scoring was done 14 days post-infection. Histological examination of corneas and viral isolation from eyes from HSV-1 infected mice treated or not with meliacine were also carried out. It was found that the treatment of HSV-1-induced ocular disease in Balb/c mice with meliacine reduced significantly the development of clinical disease, as well as the histological damage in corneas. The viral titers detected in eyes of infected and treated mice were 2-orders-of-magnitude lower than those corresponding to HSV-1 infected control animals. Mock-infected and treated mice did not reveal any corneal alteration due to the administration of the compound. Meliacine was found to exert a strong antiviral action on HSV-1-induced ocular disease in mice with no evidence of toxic effects.

Asunto(s)

Antivirales/uso terapéutico , Herpesvirus Humano 1 , Queratitis Herpética/prevención & control , Plantas Medicinales , Animales , Chlorocebus aethiops , Córnea/patología , Córnea/virología , Femenino , Herpesvirus Humano 1/aislamiento & purificación , Humanos , Queratitis Herpética/patología , Queratitis Herpética/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Extractos Vegetales/uso terapéutico , Factores de Tiempo , Células Vero , Ensayo de Placa Viral , Replicación Viral/efectos de los fármacos

RESUMEN

The anti-HSV-1 activity of meliacine (MA), a peptide isolated from leaves of Melia azedarach L., alone and in combination with acyclovir (ACV), was assayed against thymidine kinase-deficient (TK(-)) virus yields in vitro. MA alone proved to inhibit significantly TK(-) viral replication, whereas ACV was more potent than MA as an inhibitor of TK(+) replication. TK(-) and TK(+) synergistic inhibition by the combination of both agents was observed at concentrations that did not alter cell viability. The interaction between MA and ACV was quantitatively determined by calculating the combination index and plotting the data by the isobologram method. Besides, MA and ACV were able to suppress synergistically the antigen expression on HSV-I infected cells processed by an immunofluorescence assay. These in vitro findings suggest that combinations of MA and ACV at appropriate doses may provide an increased efficacy in inhibiting both TK(-) and TK(+) HSV-1 multiplication.

RESUMEN

The establishment of an experimental persistent infection with Junin virus, the aetiological agent of argentine hemorrhagic fever, involves the emergence of antigenic variants in brain and blood of the cricetid Calomys musculinus. We demonstrate that antigenic variants can also be isolated in vitro under the selective pressure of polyclonal antibodies and from a long-term infected C. musculinus primary embryo fibroblast culture. The participation of neutralizing antibodies and host cells in the appearance of viral variants in vivo is discussed.

Asunto(s)

Variación Antigénica , Antígenos Virales/inmunología , Arenavirus del Nuevo Mundo/inmunología , Animales , Antígenos Virales/genética , Arenavirus del Nuevo Mundo/genética , Arenavirus del Nuevo Mundo/aislamiento & purificación , Arvicolinae , Células Cultivadas , Relación Dosis-Respuesta Inmunológica , Fiebre Hemorrágica Americana/inmunología , Sueros Inmunes/inmunología , Células Vero

RESUMEN

The major natural reservoir of Junin virus, the aetiological agent of Argentine haemorrhagic fever, is the cricetid Calomys musculinus. Neonatal animals experimentally infected with Junin virus (XJCl3 strain) developed typical disease and approximately 80% of them died. Most survivors become persistently infected. Antigenically variant viruses were isolated from the blood and brain of infected cricetids during the acute and chronic stages of the disease. These variants could be distinguished from the parental strain by kinetic neutralization assays using polyclonal antibodies. Some biological properties were shared with the parental virus strain including its virulence for newborn C. musculinus. These variant viruses may play a major role in chronic disease since we have shown that a viral isolate from an infected brain was poorly neutralized by serum obtained from the same animal.

Asunto(s)

Antígenos Virales/análisis , Arenaviridae/aislamiento & purificación , Arenavirus del Nuevo Mundo/aislamiento & purificación , Fiebre Hemorrágica Americana/microbiología , Animales , Anticuerpos Antivirales/inmunología , Variación Antigénica , Arenavirus del Nuevo Mundo/inmunología , Arvicolinae , Cinética , Pruebas de Neutralización

RESUMEN

Neonatal Calomys musculinus experimental infection with Junín virus (JV) XJCl3 strain causes either death or a persistent infection in the major part of surviving animals. JV can be isolated from peritoneal macrophages early during infection, and from brain and salivary glands during the chronic state of disease. It was of interest to investigate the appearance of virus in blood of infected animals. For this purpose, we decided to study the development of viremia in inoculated cricetids. A high frequency of viremia was registered during the acute state of disease (figure 1), which became sporadic approximately from 20 days post-infection onwards. Considering the results above mentioned, the characteristic lymphotropism of arenaviruses and the well-known JV replication in human mononuclear cells, cultures of lympho-monocytes obtained from blood of infected C. musculinus were done in order to investigate the eventual detection of infectious virus in the supernatants. JV was isolated, although in low titres, from cultures established with mononuclear cells belonging to animals in the acute state of disease (table 1); in the case of chronically infected cricetids, attempts to isolate JV were negative. These results show that viremia is currently detected in experimentally infected C. musculinus and that circulating mononuclear cells are permissive for JV multiplication, during the acute state of disease.

Asunto(s)

Arenaviridae/aislamiento & purificación , Arenavirus del Nuevo Mundo/aislamiento & purificación , Arvicolinae/microbiología , Animales , Animales Recién Nacidos , Células Cultivadas , Enfermedad Crónica , Fiebre Hemorrágica Americana/sangre , Fiebre Hemorrágica Americana/microbiología , Linfocitos/microbiología , Viremia/microbiología , Replicación Viral

RESUMEN

Neonatal Calomys musculinus experimental infection with Junín virus (JV) XJCl3 strain causes either death or a persistent infection in the major part of surviving animals. JV can be isolated from peritoneal macrophages early during infection, and from brain and salivary glands during the chronic state of disease. It was of interest to investigate the appearance of virus in blood of infected animals. For this purpose, we decided to study the development of viremia in inoculated cricetids. A high frequency of viremia was registered during the acute state of disease (figure 1), which became sporadic approximately from 20 days post-infection onwards. Considering the results above mentioned, the characteristic lymphotropism of arenaviruses and the well-known JV replication in human mononuclear cells, cultures of lympho-monocytes obtained from blood of infected C. musculinus were done in order to investigate the eventual detection of infectious virus in the supernatants. JV was isolated, although in low titres, from cultures established with mononuclear cells belonging to animals in the acute state of disease (table 1); in the case of chronically infected cricetids, attempts to isolate JV were negative. These results show that viremia is currently detected in experimentally infected C. musculinus and that circulating mononuclear cells are permissive for JV multiplication, during the acute state of disease.