Scientist tests the public trust.

An unreported accident in a virus laboratory at Yale is but one of many factors that challenge society's trust in scientists' promises to do hazardous research.

Pathological and virological features of arenavirus disease in guinea pigs. Comparison of two Pichinde virus strains.

A guinea pig passage-adapted strain of the arena-virus Pichinde (adPIC) is highly virulent in inbred guinea pigs, whereas the related strain PIC3739 is attenuated. Both viruses were macrophage tropic and infected peritoneal, splenic, liver, and alveolar macrophages during experimental Pichinde virus infection. Infection with the virulent strain was associated with unlimited viral replication in the face of exaggerated delayed-type hypersensitivity response, manifested by the macrophage disappearance reaction. Histopathological lesions unique to adPIC-infected guinea pigs included intestinal villus blunting with mucosal infiltration by pyknotic debris-laden macrophages and apoptosis of crypt epithelial cells. Splenic red pulp necrosis was also significantly associated with adPIC infection but not PIC3739 infection. These findings may provide clues to the pathogenesis of a group of poorly understood human viral hemorrhagic fevers.

Description of Guanarito virus (Arenaviridae: Arenavirus), the etiologic agent of Venezuelan hemorrhagic fever.

This paper characterizes Guanarito virus, the etiologic agent of Venezuelan hemorrhagic fever. Based on its morphology and antigenic properties, Guanarito virus appears to be a new member of the Tacaribe complex of the genus Arenavirus, family Arenaviridae. Complement fixation and indirect fluorescent antibody tests showed that Guanarito virus and its antiserum are broadly cross-reactive with other members of the Tacaribe complex, but it can be differentiated from other members of the complex by neutralization test. Guanarito virus causes mortality in suckling mice and adult guinea pigs, but not in adult mice. Inoculated rhesus monkeys developed viremia and became ill; however, they subsequently recovered and responded with production of antibody. To date, all isolates of Guanarito virus have come from sick persons or wild rodents living within a single geographic focus in the central plains of Venezuela.

New arenavirus isolated in Brazil.

A new arenavirus, called Sabiá, was isolated in Brazil from a fatal case of haemorrhagic fever initially thought to be yellow fever. Antigenic and molecular characterisation indicated that Sabiá virus is a new member of the Tacaribe complex. A laboratory technician working with the agent was also infected and developed a prolonged, non-fatal influenza-like illness. Sabiá virus is yet another arenavirus causing human disease in South America.

Experimental neuroinvasiveness of wild and laboratory Junin virus strains.

The neuroinvasiveness of Candid 1 and XJCL3 laboratory strains and CbalV4454 and CbaFHA5069 wild strains of Junin virus was studied in albino mice, guinea pigs, and a South American wild rodent, Calomys musculinus (Cm), of different ages inoculated by a non-neural route. Infectivity in brain, blood and organs, as well as lethality, were determined. The results with the 3 hosts indicate that Junin virus neuroinvasiveness is virus-strain-dependent, host species- and age-dependent, with the Candid 1 strain proving to be the least neuroinvasive of the strains studied. The lethal efficiency index (log PFU/LD50) in 2-day old albino mice and the neuroinvasiveness index (Log PFU/ND50) in 6 +/- 1 day-old Cm of the various strains using the intraperitoneal (ip) route could therefore be useful markers of Junin virus neuroinvasiveness. Moreover, different patterns of infection were established using the results of the presence of infectious virus in brain and viraemia in the 3 hosts. In nearly all cases, virus neuroinvasion was present without detectable viraemia (virus in plasma). Current evidence leads to the assumption that virus might reach the brain associated with the white cells in blood (undetectable by conventional isolation methods) or by another possible mechanism of neuroinvasion which is not haematogenous.

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.

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.

Rapid vascular clearance of two strains of Junin virus in Calomys musculinus: selective macrophage clearance.

Clearance of Junin (JUN) virus strains with different virulence for Calomys musculinus (Cm) was followed using the Candid #1 virulent and CbaFHA 5069 attenuated strains. In addition, virulent virus albino mice (AM) were included as control host and Venezuelan equine encephalitis (VEE-VI) virus as control virus. The virus inoculum (Vo) and the blood samples (Vt) obtained at different times post-inoculation (p.i.) were titrated on Vero cells and the cleared plaque forming-units (PFU) were calculated as the log Vt/Vo. In Cm both JUN virus strains were cleared rapidly (within 5 min the Candid #1 strain and within 10 min the CbaFHA 5069 strain); meanwhile, VEE-VI virus could be recovered from blood until 30 min p. i. Furthermore, JUN and VEE-VI viruses showed the same behaviour in Am as in Cm. We conclude that the JUN virus strains of different virulence for Cm did not show differences in their clearance from the blood of these animals. Moreover, the rapid clearance observed was independent of the animal host and viral dose.

Neurovirulence of wild and laboratory Junin virus strains in animal hosts.

The neurovirulence of Candid #1 and XJCL3 laboratory strains and CbalV4454 and CbaFHA5069 wild strains of Junin virus was studied in albino mice, guinea pigs, and a South American wild rodent, Calomys musculinus, of different ages inoculated by the intracerebral route. Infectivity in brain and organs, lethality, and neuropathological lesions were determined. The laboratory and wild strains showed similar neurovirulence only in 2-day-old mice. The neurovirulence of laboratory strains decreased with the age of the animal, and the Candid #1 strain affected only 2-day-old mice. In guinea pigs, the 2 wild strains and XJCL3 laboratory strain were neurovirulent for 11-day-old and adult animals giving moderate lymphocytic infiltration in the brain and mild lesions in the spinal cord. Virus titres from the brain and the spinal cord were lower with the XJCL3 and CbalV4454 strains than with the CbaFHA5069 strain; with the latter, virus was recovered only from the lymph nodes, the lung, kidney, liver, and spleen. The Candid #1 strain was not neurovirulent even for 11-day-old animals. In contrast, the laboratory strains were neurovirulent for Calomys musculinus, depending on the age of the animal. Virus was recovered from the brains showing lymphocyte infiltration but not from other organs. The CbaFHA5069 strain was not neurovirulent, although virus was recovered from the brain, spleen, liver, lymph nodes, and salivary glands. These results with the 3 hosts indicate that Junin virus neurovirulence is virus strain-dependent, and host species and age-dependent, with the Candid #1 strain proving the least neurovirulent of the strains studied.

Variacion de parametros sanguineos en Calomys musculinus infectados con virus Junin, cepa XJCL3 / Blood parameters variation in Calomys musculinus infected with Junin virus, strain XJCL3

Se investigó la eolución virológica, hematológica y de los nveles de glucemia de Calomys musculinus neonatos infectados con virus Junin, cepa XJCi3 por vía intraperitoneal. Como controles se utilizaron animales sanos y cricétidos inoculados con un homogeneizado de cerebro de ratón blanco no infectado. Los cricétidos que recibieron virus Junin desarrollaron el cuadro característico de la infección aguda, muriendo entre 50-70%. Se aisló virus del cerebro de los animales infectados, durante la fase aguda de la enfemedad, a partir del 6ª día post infectión, siendo el título de alrededor de 10***8 DL50/ml a los 12 días post infección. A partir del día 11 pi. todos los animales infectados presentaron anticuerpos neutralizantes que perduiraron durante el estado crónico de la infección, iniciado después del día 24 pi. Del análisis de los valores hematolóficos obtendos durante la fase aguda, se concluye que aproximadamente el 40% de los animales inoculados presentan linfopenia y neutrofilia, mientras que en la fase crónica nose encontraron modificaciones. Los estudios de glucemia realizados durante la infección aguda demostraron hipoglucemia (X=3,52mmol/VI) en el lote de animales infectados respecto del grupo control (X = 6,15 mmol/l) con una p < 0,01)

Variacion de parametros sanguineos en Calomys musculinus infectados con virus Junin, cepa XJCL3 / Blood parameters variation in Calomys musculinus infected with Junin virus, strain XJCL3

Se investigó la eolución virológica, hematológica y de los nveles de glucemia de Calomys musculinus neonatos infectados con virus Junin, cepa XJCi3 por vía intraperitoneal. Como controles se utilizaron animales sanos y cricétidos inoculados con un homogeneizado de cerebro de ratón blanco no infectado. Los cricétidos que recibieron virus Junin desarrollaron el cuadro característico de la infección aguda, muriendo entre 50-70%. Se aisló virus del cerebro de los animales infectados, durante la fase aguda de la enfemedad, a partir del 6¬ día post infectión, siendo el título de alrededor de 10***8 DL50/ml a los 12 días post infección. A partir del día 11 pi. todos los animales infectados presentaron anticuerpos neutralizantes que perduiraron durante el estado crónico de la infección, iniciado después del día 24 pi. Del análisis de los valores hematolóficos obtendos durante la fase aguda, se concluye que aproximadamente el 40% de los animales inoculados presentan linfopenia y neutrofilia, mientras que en la fase crónica nose encontraron modificaciones. Los estudios de glucemia realizados durante la infección aguda demostraron hipoglucemia (X=3,52mmol/VI) en el lote de animales infectados respecto del grupo control (X = 6,15 mmol/l) con una p < 0,01) (AU)

Junin virus replication in peripheral blood mononuclear cells of patients with Argentine haemorrhagic fever.

To study the relationship of Junin virus (JV) to populations of peripheral blood mononuclear cells (PBMC) from patients with Argentine Haemorrhagic Fever (AHF), blood samples were obtained during the acute period of disease and cultured as total, adherent, and non-adherent cell populations. JV was sequentially sought in these cell populations by using an Infectious Centre (IC) assay, whereas free JV in the supernatants was evaluated by plaque formation. IC were obtained in cultures of total PBMC from 8 out of 19 patients. Maximum numbers of IC showed high variation among patients, ranging from 3 to 410 IC per 10(6) viable PBMC. In contrast, IC were sporadically demonstrated in the non-adherent cell population. The release of JV into culture supernatants was detected only in total PBMC cultures, thus in the presence of macrophages. These results demonstrate that circulating monocytes (macrophages) are targets for JV replication contributing to the viral spread in the acute phase of AHF.

Vertical transmission of Junin virus in experimentally infected adult Calomys musculinus.

The response to infection with Junin virus, wild strain Cba An 9446, and the antenatal and postnatal transmission of the pathogen in its natural host, Calomys musculinus, were studied. Intranasal infection in adult animals (90-120 days) did not produce mortality or illness during the 150-day period of observation. From day 21 to 150 after infection, 50% of the animals showed viral persistence with shedding of virus in both urine and saliva. The remaining half became seropositive, and no infectious virus was recovered from them. Although the virus did not infect fetuses during gestation, 50% of weaned pups nursed by viremic mothers were infected. Neither persistence nor immunologic response altered the reproductive pattern of the animals. The absence of reproductive failure in the infected host and the efficiency of postnatal transfer of Junin virus indicate that vertical transmission could contribute to the viral maintenance over time.

[Blood parameters variation in Calomys musculinus infected with Junin virus, strain XJCl3]. / Variación de parámetros sanguíneos en Calomys musculinus infectados con virus Junin, cepa XJCl3.

The aim of this study was to analyze the alterations in homeostasis induced by Junin virus during acute and persistent infection of C. musculinus. Virus presence in brain, hematological response and glycemia levels were evaluated. Newborn C. musculinus inoculated with 4000 DL50 of Junin virus, strain XJCl3 by intraperitoneal route developed a typical acute disease, with 50-70% mortality. Virus was isolated from brain starting day 6 post-infection (Fig. 1) and the peak titer (10(8) DL50/ml) was reached at 12 days post-infection. Neutralizing anti-Junin virus antibodies were detected from day 11 post-infection and all chronically infected animals developed persistent levels of neutralizing antibodies. In the acute stage of infection, 40% of the animals developed lymphopenia and neutrophilia (Fig. 2) while a slight variation was observed in the monocyte population. An important hypoglycemia was also seen in the acute infection (mean = 3.52 mmol/l) in comparison with control values (mean = 6.21 mmol/l), p less than 0.01 (Fig. 3). By contrast during the chronic stage of infection, neither hematological parameters (Table 2) varied between infected and control animals.

Reduced virulence of a Junin virus mutant is associated with restricted multiplication in murine cells.

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.

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.

Long-term protection against Argentine hemorrhagic fever in Tacaribe virus infected marmosets: virologic and histopathologic findings.

Tacaribe virus may represent a better alternative than attenuated strains of Junin virus (JV) for immunization against Argentine hemorrhagic fever (AHF) because of possible risk of persistent infection of disease associated with live, attenuated strains. Callithrix jacchus marmosets, which suffer 100% mortality if inoculated with the pathogenic XJ strain of JV, were used to evaluate possible Tacaribe virus persistence, subclinical, or long-term disease and the duration of protection against challenge with JV. Histologic studies did not show pathogenic changes due to Tacaribe virus in primates sacrificed from 7 to 480 days postinoculation (pi). No virus was recovered in tissue samples after primary culture or cocultures with sensitive cells. The presence of anti-Tacaribe neutralizing serum antibodies and protection against pathogenic JV were detected up to 480 days after a single dose of Tacaribe virus. However, anti-Junin antibodies were detected only after challenge. In other experiments, protection against JV was evaluated histologically and virologically. Two primates were immunized with Tacaribe virus, challenged with JV, and sacrificed 18 or 21 days later. Subclinical histopathologic findings were associated with recovery of JV only by the sensitive primary culture-coculture techniques. The immunogenicity, degree of protection, and safety of Tacaribe virus indicate its potential as a vaccine against human AHF.

Mortality induced by adoptive immunity in Junin virus-infected athymic mice.

The effect of normal or sensitized spleen cell transfer from syngeneic euthymic mice to Junin virus-infected suckling athymic mice was studied. Transfer was performed 1 or 7 days after infection. In both cases, an acute lethal disease developed 6-11 days after transfer. The mortality reached 100% in all infected groups receiving normal or sensitized splenocytes, while it was negligible for different control groups of athymic mice. Transfer of normal or sensitized splenocytes was unable to significantly modify brain viral titers, as compared with infected nontransferred athymic mice killed after a 25-day observation period. Brain lesions were demonstrated in about half of the infected athymic mice transferred with sensitized splenocytes and in all euthymic infected mice. These results show that splenocyte transfer from immunocompetent donors is able to change the normal course of persistent Junin virus infection in nude mice to a lethal acute disease, thus pointing to a main role for T cells in its pathogenesis.

Viral strain dependent differences in experimental Argentine hemorrhagic fever (Junin virus) infection of guinea pigs.

Guinea pigs infected with low-passage Junin virus of human origin showed viral strain dependent differences in mortality, LD50, time to death, and in viral spread and distribution. Different Junin strains appeared to cause at least two broad patterns of Argentine hemorrhagic fever in guinea pigs. A number of strains of Junin virus caused a viscerotropic type of illness in which virus replicated predominantly in lymph nodes, spleen, and bone marrow. With the most severe visceral forms of Argentine hemorrhagic fever, the guinea pigs became viremic, developed necrosis of spleen, lymph nodes, and bone marrow, showed gastric hemorrhages, and all animals died within 13-15 days. Other Junin strains induced a neurological type of illness with transient viral replication in and lymphocyte depletion of spleen and lymph nodes, with no detectable viremia or viral replication in bone marrow. Subsequently, virus was found in the brain with varying severities of polioencephalitis, and the guinea pigs frequently showed rear leg paralysis before death occurred 28-34 days after inoculation. Not all animals infected with a neurotropic strain developed all these signs. One viral strain induced some signs characteristic of both patterns of illness. Although the disease forms in the guinea pig model did not strictly correlate with those observed in the humans from which these strains were obtained, the different strains of Junin virus consistently caused very different patterns of illness in infected guinea pigs.

Effect of polymorphonuclear depletion on experimental Argentine hemorrhagic fever in guinea pigs.

The role that polymorphonuclear leukocytes (PMN) may play in Argentine hemorrhagic fever (AHF), an endemo-epidemic disease caused by Junín virus (JV), was investigated in experimentally infected guinea pigs depleted of PMN by means of specific antiserum. In leucopenic animals the evolution of the infection with a highly pathogenic strain of JV was more severe, with earlier mortality and higher virus yields in blood and viscera. The pathological study showed similar lesions in both the control and PMN-depleted animals with the exception of the lung, which showed the pathological picture of the human "pulmonary distress syndrome of the adult" in nontreated guinea pigs and appeared histologically unaltered in the PMN-depleted animals. On the basis of these results it is suggested that in AHF, PMN play a dual role. In the first stage of infection they display a defensive antiviral action, but later on they participate in the pathogenesis of tissue damage.