Immunoperoxidase tracing of Junin virus neural route after footpad inoculation.

To determine the pathway adopted by peripherally inoculated Junin virus (JV) to reach the CNS, rat tissues were serially harvested to trace the sequence of viral progression from right hind footpad to brain. Immunoperoxidase (PAP) labeling of viral antigen, concomitantly with infectivity assays and histological examination of each selected sample, were carried out. As from the 2nd week post-infection (pi), neurological disease inducing 100% mortality at 1 month was evident. At day 5 pi, viral antigen was first detected at footpad level in epidermic and dermic cells, as well as in neighbouring myocytes; labeled macrophages infiltrating small nerve branches were also disclosed. As from 10-15 days pi, viral antigen became apparent along ipsilateral sciatic nerve structures and within lumbar spinal ganglion neurons, followed by a fast viral spread throughout CNS neurons that involved spinal cord and brain. Concurrent histopathology featured minimal inflammatory reaction together with generalized astrocytic activation. Hematogenous viral transport was negligible, since JV was isolated much earlier and in higher infectivity titers in neural tissues than in blood. It may be concluded that after viral replication in footpad, JV neural route was demonstrated by its PAP labeling from peripheral nerves to cerebral cortex.

[Protection against encephalitis in rats caused by a pathogenic strain of the Junin virus, using peripheral inoculation of an attenuated strain]. / Protección contra la encefalitis producida en ratas por una cepa patógena de virus Junín por inoculación periférica con una cepa atenuada.

Argentine Hemorrhagic Fever manifests itself in man either subclinically or in hemorrhagic or neurological forms, mortality reaching 20%. Although Candid 1 strain is undergoing pilot trials, current therapy still resorts to convalescent serum administration. A neurological model was used to evaluate protection conferred by the attenuated XJC13 Junin virus strain. Newborn rats inoculated intraperitoneally (ip) prove resistant, whereas 8-12 day-old animals infected by intracerebral route with the XJ prototype strain suffer 100% mortality with neurological signs. The aim of this study was to achieve protection in this model and attempt to elucidate the mechanisms involved in resistance. It was observed that the longer the inoculation challenge interval, the greater was the survival percentage. In protected animals, brain viral titres were 3 log lower than in challenged controls, while XJC13 infected unchallenged controls presented low CNS values throughout. Neutralizing antibody levels were not significantly different in experimental versus challenged control groups, ruling out any secondary booster effect on protected rats. Neither the transfer of immunoserum nor of endogenous or exogenous interferon altered mortality. However, when splenocytes from rats infected 10 days previously were transferred prior to XJ challenge, survival was increased to 50%, but there was no gain in protection when cells were treated with antithymocyte serum plus complement. Consequently, protection in this neurological model can be attributed to a cellular immune response.

Protección contra la encefalitis producida en ratas por una cepa patógena de virus Junín por inoculación periférica con una cepa atenuada. / [Protection against encephalitis in rats caused by a pathogenic strain of the Junin virus, using peripheral inoculation of an attenuated strain]

Argentine Hemorrhagic Fever manifests itself in man either subclinically or in hemorrhagic or neurological forms, mortality reaching 20

. Although Candid 1 strain is undergoing pilot trials, current therapy still resorts to convalescent serum administration. A neurological model was used to evaluate protection conferred by the attenuated XJC13 Junin virus strain. Newborn rats inoculated intraperitoneally (ip) prove resistant, whereas 8-12 day-old animals infected by intracerebral route with the XJ prototype strain suffer 100

mortality with neurological signs. The aim of this study was to achieve protection in this model and attempt to elucidate the mechanisms involved in resistance. It was observed that the longer the inoculation challenge interval, the greater was the survival percentage. In protected animals, brain viral titres were 3 log lower than in challenged controls, while XJC13 infected unchallenged controls presented low CNS values throughout. Neutralizing antibody levels were not significantly different in experimental versus challenged control groups, ruling out any secondary booster effect on protected rats. Neither the transfer of immunoserum nor of endogenous or exogenous interferon altered mortality. However, when splenocytes from rats infected 10 days previously were transferred prior to XJ challenge, survival was increased to 50

, but there was no gain in protection when cells were treated with antithymocyte serum plus complement. Consequently, protection in this neurological model can be attributed to a cellular immune response.

Macrophage maturity and modulation of response to Junín virus in infected rats.

Replication of Junín virus in peritoneal macrophages from newborn rats was greater for prototype strain XJ than for strain XJC13, whereas in cells from adult animals viral multiplication proved minimal. Transfer of peritoneal adherent cells from normal adult to strain XJ-infected newborn rats lowered mortality significantly. Silica blockade of macrophages protected two-day-old strain XJ-infected animals and depressed brain titers of virus significantly, whereas treatment had no effect on strain XJC13-infected rats. Macrophages from infected two-day-old rats caused illness and death in recipient animals. Silica blockade rendered macrophage-mature 11-day-old rats partially susceptible to infection with Junín virus. Thus pathogenicity of strain XJ in the two-day-old rat by the intraperitoneal route depends on the ability to replicate in peritoneal macrophages, whereas strain XJC13 is nonlethal because it fails to multiply efficiently in these cells. Macrophage maturity seems essential to inhibit viral replication and thus confer protection on the adult host.

Junin virus access to CNS by extraneural rat inoculation.

This study was carried out to determine the pathways along which two strains of Junin virus (JV), the pathogenic XJV and the attenuated XJC13V, reach the CNS following IP inoculation of 2-day-old rats. A sequential study of infectivity and antigen distribution in peritoneal macrophages, spleen, and brain was performed. Mortality was 85% with the former strain, but only 15% with the latter. At 4-7 days PI, XJV-infected animals had viral antigen in 10% of peritoneal macrophages. Viremia and spleen virus lasted for 10-15 days. Low brain titers were detected at day 7, with a peak at day 15. Brain antigen correlated with virus titers. In contrast, XJC13V-infected rats, macrophage antigen appeared later and to a lesser degree (1% of cells). Viremia and spleen virus were transient, while both the titer of brain virus and the viral antigen proved lower. Antibody titers were over twofold higher for XJ-infected animals. It is suggested that the different replication rate at the inoculation site could account for the greater ability of the XJV strain to reach the CNS. A greater antigen mass and/or more numerous antigenic determinants presented by the macrophage could explain the higher antibody titers found in XJ-injected rats, which were unable, however, to prevent viral spread.

[Protection conferred by a hyperimmune serum and its fractions on rats infected with the Junin virus]. / Protección conferida por un suero hiperinmune y sus fracciones a ratas infectadas con virus Junín.

Suckling rats infected by ic route with 10(3)LD50 of the XJC13 strain of JV were passively immunized with homologous hyperimmune serum (HIS). Animals treated at 2 days pi with HIS showed a significant increase in survival vs. non-treated infected controls (82% vs 5%). However, at 4 days pi, transfer failed to modify survival. By means of DEAE Sephadex A25 column chromatography, the presence of neutralizing immunoglobulin closely correlated with protective antibodies, but were not restricted to the IgG-containing fraction. Employing Sephadex G200, chromatography demonstrated the absence of neutralizing and protective activity in the high molecular weight protein fraction. Results show that the success of HIS treatment depends on early administration. Besides, it was found that fractions capable of conferring protection exhibited high neutralizing antibody titers.

Protección conferida por un suero hiperinmune y sus fracciones a ratas infectadas con virus Junín. / [Protection conferred by a hyperimmune serum and its fractions on rats infected with the Junin virus]

Suckling rats infected by ic route with 10(3)LD50 of the XJC13 strain of JV were passively immunized with homologous hyperimmune serum (HIS). Animals treated at 2 days pi with HIS showed a significant increase in survival vs. non-treated infected controls (82

vs 5

). However, at 4 days pi, transfer failed to modify survival. By means of DEAE Sephadex A25 column chromatography, the presence of neutralizing immunoglobulin closely correlated with protective antibodies, but were not restricted to the IgG-containing fraction. Employing Sephadex G200, chromatography demonstrated the absence of neutralizing and protective activity in the high molecular weight protein fraction. Results show that the success of HIS treatment depends on early administration. Besides, it was found that fractions capable of conferring protection exhibited high neutralizing antibody titers.

Attenuation parameters for Junin virus in the newborn rat.

To characterize a virus strain as attenuated, both biologic and biochemical criteria are necessary. In the case of Junin virus, the 2-day-old rat has proved to be a biologic attenuation marker as regards mortality. Here we studied the behaviour of the prototype XJ vs the attenuated XJC13 strain inoculated by either ic or ip route to determine differential hematologic and splenic parameters. Humoral immune response against SRBC was also investigated. By either route XJ caused significant leucocytosis, while the other hematologic parameters remained unchanged. No alterations were found following XJC13 infection. XJ produced significant splenomegaly whereas XJC13 had no effect. Similarly PFC anti-SRBC count was decreased during XJ infection but not after XJC13 infection. These differences between the pathogenic XJ and the attenuated XJC13 strain may be attributed to the former's greater spread. The drop in PFC could be due to spleen dysfunction and/or viral effects on the cell subpopulation involved.

Attenuation parameters for Junin virus in the newborn rat.

To characterize a virus strain as attenuated, both biologic and biochemical criteria are necessary. In the case of Junin virus, the 2-day-old rat has proved to be a biologic attenuation marker as regards mortality. Here we studied the behaviour of the prototype XJ vs the attenuated XJC13 strain inoculated by either ic or ip route to determine differential hematologic and splenic parameters. Humoral immune response against SRBC was also investigated. By either route XJ caused significant leucocytosis, while the other hematologic parameters remained unchanged. No alterations were found following XJC13 infection. XJ produced significant splenomegaly whereas XJC13 had no effect. Similarly PFC anti-SRBC count was decreased during XJ infection but not after XJC13 infection. These differences between the pathogenic XJ and the attenuated XJC13 strain may be attributed to the formers greater spread. The drop in PFC could be due to spleen dysfunction and/or viral effects on the cell subpopulation involved.

[Differentiation among strains of Junín virus by intraperitoneal infection in the rat]. / Diferenciación de cepas de virus Junín por la infección intraperitoneal en la rata.

The 2-day-old rat is known to resist intracerebral infection with the XJ prototype strain of Junin virus, but 95-100% mortality results when infected with the attenuated XJC13 strain. When this animal was inoculated by intraperitoneal route, behaviour was diametrically opposite: the XJ strain proved lethal, while de XJC13 led to low mortality. Studies on mortality, virus titer in different organs, and anti-viral humoral response in 2-day-old rats infected with Junin virus strains were carried out in order to use this system as a new attenuation marker. Mortality rates recorded for rats inoculated with either strain, were markedly different, being 84% in the XJ-infected group and barely reaching 17% in the XJC13 group. Brain viral titers were higher in the former group than the latter (10(5.26) PFU/ml vs. 10(3) PFU/ml at day 17 pi). For this reason, viral replication may be used as a virulence marker in this experimental model. Antibody levels were also higher in the XJ group most likely due to greater viral replication. The above findings support the use of the 2-day-old rat as a biologic attenuation marker since susceptibility to infection is strain-dependent.

Diferenciación de cepas de virus Junín por la infección intraperitoneal en la rata. / [Differentiation among strains of Junín virus by intraperitoneal infection in the rat]

The 2-day-old rat is known to resist intracerebral infection with the XJ prototype strain of Junin virus, but 95-100

mortality results when infected with the attenuated XJC13 strain. When this animal was inoculated by intraperitoneal route, behaviour was diametrically opposite: the XJ strain proved lethal, while de XJC13 led to low mortality. Studies on mortality, virus titer in different organs, and anti-viral humoral response in 2-day-old rats infected with Junin virus strains were carried out in order to use this system as a new attenuation marker. Mortality rates recorded for rats inoculated with either strain, were markedly different, being 84

in the XJ-infected group and barely reaching 17

in the XJC13 group. Brain viral titers were higher in the former group than the latter (10(5.26) PFU/ml vs. 10(3) PFU/ml at day 17 pi). For this reason, viral replication may be used as a virulence marker in this experimental model. Antibody levels were also higher in the XJ group most likely due to greater viral replication. The above findings support the use of the 2-day-old rat as a biologic attenuation marker since susceptibility to infection is strain-dependent.