Treatment with meliacine, a plant derived antiviral, prevents the development of herpetic stromal keratitis in mice.

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.

Antiviral activity of meliacine on the replication of a thymidine kinase-deficient mutant of Herpes simplex virus type 1 alone and in combination with acyclovir.

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.

In vitro selection of Junin virus antigenic variants.

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.

Differentiation of Junin virus and antigenic variants isolated in vivo by kinetic neutralization assays.

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.

Role of Calomys musculinus peritoneal macrophages in age-related resistance to Junin virus infection.

In nature, the cricetid Calomys musculinus is the principal host of Junin virus, the etiological agent of Argentine hemorrhagic fever. In the experimental infection, adult C. musculinus survived whereas newborns died after intraperitoneal inoculation with the XJ.Cl3 strain of Junin virus. The role of peritoneal macrophages in this age-related resistance was studied. Junin virus multiplied in cultivated macrophages from either neonatal or adult animals and, therefore, it was not possible to correlate the susceptibility of peritoneal macrophages to Junin virus infection with the age-dependent resistance. When adult and neonatal animals were treated with silica prior to Junin virus infection, deaths occurred in the adults, while a delay and decrease in the mortality rate were observed in neonatals. These results suggest that in neonatal C. musculinus macrophages could be permissive cells for Junin virus multiplication, whereas in adult cricetids, these cells would act as a barrier against viral infection by means of an extrinsic antiviral activity.

Antigenic variants of Junín virus isolated from infected Calomys musculinus. Brief report.

Junín virus establishes a long-term persistent infection in its natural host, Calomys musculinus. Virus recovered from blood of infected animals from 14 to 61 days p.i. behaved antigenically distinct to parental virus, as shown by cross neutralization assays. The emergence of antigenic viral variants occurs during both the acute and persistent state of infection.

[Isolation of Junin virus from blood and peripheral lymphocytes of infected Calomys musculinus]. / Aislamiento de virus Junín a partir de sangre y linfo-monocitos periféricos de Calomys musculinus infectados.

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.

Aislamiento de virus Junín a partir de sangre y linfo-monocitos periféricos de Calomys musculinus infectados. / [Isolation of Junin virus from blood and peripheral lymphocytes of infected Calomys musculinus]

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.