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The effects of cyclophosphamide (CY), anti-mouse thymocyte sera, oxisuran, and cytosine arabinoside were evaluated in adult and newborn mice infected with Junin virus. Treatment with CY was the most effective, Adult mice, which are not normally susceptible to intracerebral infection with 10(3) LD50 of Junin virus, were rendered susceptible by treatment with an adequate dose and schedule of CY. However, CY-treated newborn mice were protected from death. This animal model may be suitable for use in pathogenesis and protection studies.

Junin virus-induced encephalitis in suckling mouse is a delayed-type hypersensitivity reaction, whose immunopathologic nature has been proven by suppressing the thymus-dependent response. Cyclophosphamide (CY) given at day +6 post-infection (p.i.) has been shown to modulate infection, presumably by TDTH lymphocyte inactivation. To determine critical timing and i.p. drug dose, brain histology and survival were studied in 3-day-old Balb/c mice, inoculated i.c. with Junin virus. Optimal protection was achieved with a non-toxic, 50 mg/kg CY dose at day 6 p.i. (+6): no brain tissue damage was detected in animals killed at day +12, when the necropsied controls exhibited widespread lesions. Other timings (day +3, +4, +5) proved less effective. As regards alternative dosage at day +6, 30 mg was useless, and severe leptomeningitis was evident, whereas 40 mg significantly lowered mortality, and lesions were much milder and less constant. It seems that the 50 mg/kg CY dose must be administered at a critical time p.i. to inactivate sensitized TDTH lymphocytes and to reduce mortality and CNS pathology significantly.

The CY-enhancing effect on DTH response of mice against SRBC was studied by administering to sensitized animals graded amounts of drug at various times during the immune response. The use of a staggered schedule for CY administration made it clear that this enhancing effect could be augmented even further by lowering the standard 200 mg/kg CY dose. Animals immunized on day 0 with 1 X 10(8) SRBC receiving 50 mg/kg doses on days -1, +1, and +4 showed higher DTH responses on day +7 than those similarly sensitized 1 day after the administration of 200 mg/kg body weight. In addition, we wanted to demonstrate that the TDTH effector cell is sensitive to CY in vivo, because a single 50 mg/kg dose inoculated on day +6 can lower by 50% an already established DTH response. This effect was not due to an effect of CY treatment on bone marrow-derived cells recruited to DTH responses; inhibition of DTH responses were transferred with spleen cells of CY-treated recipients. The action of CY is not dose-dependent; the administration on day +6 of a single dose of 200 mg/kg results in no further depression of the DTH reaction. We conclude that CY affects not only T supp cells, but also cell type(s) involved in the cell-mediated response of mice against SRBC, and that the DTH-enhancing effect of the drug is a blend of its action upon all these cells.

The data presented confirm previous evidence of delayed hypersensitivity in mice following infection with Junin virus. Adaptive transfer of sensitized cells from adult mice which had received 5 Junin virus injections into preinfected newborn mice shortened their survival by 72 hours. It was clear, however, that the development of the immunological mechanism in adult mice occurred under certain conditions. This evidence was confirmed by the observations that the induction of cell-mediated immunity and the response of sensitized lymphocytes to viral antigens appeared to be related to multiple injections of the virus. On the other hand, the sensitized cells were present in adult mice by day 9 and vanished 60 days after the infection. These results revealed the difference between the lymphoid cells from adult mice in the early and the late stages of immunity. The implications of these results in the development of the fatal neurological disease induced by the Junin virus in newborn mice are discussed.

Several aspects of the appearance and development of delayed hypersensitivity in mice infected with Junin virus are described. The results obtained showed that the development of the immunological mechanisms occurs irrespective of age. Spleen cells of donor mice inoculated with one i.p. dose of Junin virus had a poor cytotoxic activity, as demonstrated by 51Cr release and adoptive immunity procedures. Spleen cells treated with anti-theta serum and complement did not strikingly affect the development of Junin virus disease in mice. This demonstrated the capacity of T lymphocytes to influence the course of the viral infection of newborn mice adoptively transferred with immune spleen cells. No difference were detected in the virus titer in the brains of transferred and control animals, this fact suggests that immune T lymphocytes are not involved in Junín virus clearance.

The object of this paper was to determine the influence of cyclophosphamide immunosuppression on the mortality of 40-45 day old Balb/c mice infected intracerebrally with a pathogenic strain of Junin virus, using different administration schedules. Up to 200 mg/kg of cyclophosphamide were not toxic. Results show that, unlike in other experimental models, three or four 50 mg/kg cyclophosphamide doses given both before and after viral infection were required to break-down resistance to Junin virus (90-96.5% mortality vs. 8% in controls). Taking into account the effect of cyclophosphamide on the cell populations involved in the immune response, causes likely to lead to the greater susceptibility of the suppressed adult mouse to Junin virus are discussed.

Delayed-type-hypersensitivity (DTH) response "in vivo" is commonly evaluated by the footpad swelling test (FPST). High doses of Sheep Red Blood Cells (SRBC) are known to produce negligible DTH, while low doses lead to optimal sensitization. As expected FPST values obtained in Balb/c mice using 10(6) or 10(8) SRBC as sensitizing doses, showed that in 9 out of 10 batches from individual rams, the former dose resulted in higher values than the latter. However, only 3 out of the above 9 exhibited statistically significant differences between immunizing doses (Table 1). Therefore, in our hands, the accuracy of FPST is highly dependent on the SRBC source. We suggest the need of testing individual SRBC batches at both dilutions before use.

The immune response of the Buffalo/Sim rat to heterologous sheep red blood cells (SRBC) was studied here. The earliest time of response to 10(9) SRBC, the most suitable inoculation route and the behavior to challenge were determined. The intraperitoneal (ip) proved more effective than the subcutaneous (sc) route, since serum agglutinins became detectable in low titers in animals inoculated at 6-7 days of life by the former route and at 12 days by the latter. No splenic plaque-forming cells (PFC) were found in rats immunized ip at 2 days of age, and strong inhibition developed on challenge at day 14 post-inoculation (pi) (agglutinin titers at day 7 pi: 0.71 +/- 0.47 vs 4.6 +/- 0.51 in unprimed controls; PFC/10(7) cells at day 5: 122.21 +/- 36.17 vs 3,977.38 +/- 777.5 in unprimed controls). Serum agglutinin formation was also decreased, though to a lesser degree, when: a) animals were challenged at 30 or 60 days of age; b) both priming and challenge took place by sc route; or c) antigen dose was lowered to 10(7) or 10(5) SRBC. Mechanisms interpreting observed behavior are discussed.

Rabbit anti-mouse thymocyte serum (ATS) administrated as late as 7 days after infection suppressed host cell-mediated responsiveness to intracerebrally injected Junín virus, thereby diminishing the morbility and mortality of this infection. It did not affect either the humoral antibody response or the virus titer in brain. This findings suggest that: a) in mouse brain cells, Junín virus infection is basically non-cytopathic: b) cell-mediated immunity is responsible for morbility and mortality and does not clear up virus from brain as in other viral encephalitides; and c) ATS may be of therapeutic interest by suppressing or diminishing the cell-mediated response to Junín virus.

Different parameters of specific immune response involved in the resistance to intracerebral Junin virus (JV) infection were studied in adult BALB/c mice. The relationship of virus replication to production of antiviral antibodies, to occurrence of cytotoxic T cells and to development of delayed-type hypersensitivity response was evaluated. Spleen cytotoxic T cells were assayed by 51Cr-release method on virus-infected H2 compatible targets. Effector T cells were detected on day 2, reached peak concentrations by day 6 and declined on day 10. These cells seemed responsible, at least in part, for virus clearance from the infected target organ, since virus could not be recovered from the brain in any sample taken on days 2, 5, 6, 8, 10, 15 and 20 post infection (p.i.). All three main antibody classes common in viral infection were present. Serum antibodies appeared later than the T cell cytotoxic response. Neutralizing antibodies and those detected by immunofluorescence prevailed in the IgG fraction, whereas the IgM antibody class was reactive in complement fixation assay. Challenge of infected mice with JV did not result in production of delayed-type hypersensitivity as measured by footpad swelling irrespective of the route of sensitization. The possible interpretations of these findings are discussed in connection with the resistance of adult mice to JV infection.

Tacaribe virus is the member most closely related to Junín virus within the Tacaribe complex. It has been demonstrated that both viruses are indistinguishable by complement-fixation, due to the high cross-reactivity. However, adult guinea pigs are highly sensitive to infection with the XJ pathogenic strain of Junín virus whereas Tacaribe virus is nonpathogenic for this species. Furthermore this last virus protects them against Junín virus. The XJ strain reduces the immune response of guinea pigs to many antigens. Both the humoral response and the hypersensitivity of the Arthus type have been reduced in infected animals. Considering that Tacaribe virus could be used as vaccine antigen, the purpose of the present study was to investigate the effect of Tacaribe infection on the immune system of guinea pigs. The data reported here supports earlier findings showing that the XJ strain of Junín virus suppresses humoral immune response as indicated by lower precipitating antibody titers to ovoalbumin (which contributed to milder Arthus cutaneous reactivity) and a significant depression of plaque-forming cells to sheep erythrocytes. In contrast, Tacaribe-infected guinea pigs did not show detectable immunosuppression employing the same models. Similar results were found when the cell-mediated immunity was investigated. Tacaribe-infected guinea pigs had a normal immune response to contact sensitivity to 2-4 dinitro-1-fluorobenzene as demonstrated by measuring ear swelling and unmodified tuberculin reaction, after injection with complete Freund's adjuvant. Our results and those of previous investigations justify the consideration of Tacaribe immunization as an approach to the prophylaxis of Argentine Hemorrhagic Fever.

Passive immunity, naturally acquired from immune mothers or artificially induced by the administration of homologous hyperimmune serum, conferred on suckling mice a high degree of resistance against infection with Junín virus. Maternal antibodies in the circulating blood of the young were not detectable in the first days after birth, but rised rapidly from the 8th to the 20th day of lactation. By cross-foster nursing experiments it was shown that the greater part of the transmission of passive immunity occurred after birth, although there was transmission of a significant, though small part, before birth. The virus passage from mothers to offspring was excluded, since Junín virus was not recovered from brains, livers, spleens and kidneys of uninfected young, born from infected mothers.

As previously postulated, the pathological changes which develop in 1-4 day old mice after intracerebral inoculation of 1-1000 DL50 of Junín virus prototype strain (XJ), was designated as experimental hemorrhagic fever of the mouse EHFm. In this paper, virus distribution, hematological alterations, interferon and circulating antibody responses are described. A mortality of 93.45% occurred between 9 and 20 days post-infection (p.i.), with 81.6% of death occurring between 11 and 18 days p.i. This last period can be considered to be the critical period of the disease. The study of virus distribution shows that the brain, where the virus was inoculated, was the only place where virus could be detected 48 hs, p.i. Four days p.i., the titer in the brain increased remarkably and virus was detected in the blood. Virus, within the same values, could be isolated up to the 10th. day. Invasion of liver and spleen occurred on the 10th. and 15th. days p.i., respectively (Fig. 1). The onset of clinical symptoms coincided with widespread disemination of the virus. CF antibodies were found only 15 days p.i., with a titer of 1/64. Neutralizing antibodies remained below detection levels during the whole experiment (Fig. 1). Surviving mice (6.3%) had high circulating antibody titers 40 days p.i. This result would indicate that the morobidity of EHFm is aproximately 100% (Table 1). Poor interferon response was registered in all the organs examined, indicating a low intereron producing ability for Junin virus6. Total leukocytes and lymphocyte counts showed a slight tendecy to drop, although the values were within normal range during the first ten days (Table 2, Fig 1). On day 14 p.i., a statistically significant decrease (p less than 0.001) was found. This leuko-lymphopaenia continued until death of the animals. It is expected that the data presente here would contribute to a better understanding of the Junin virus infection in the newborn mouse, the experimental animal used.

Otherwise resistant adult mice were rendered susceptible to intracerebral Junin virus (JV) infection only when a staggered cyclophosphamide (CY) schedule was used. Forty-five-day old Balb/c mice, intracerebrally JV-infected and immunosuppressed with four 50 mg/kg body weight CY doses at days -1, +1, +4, +6 (day 0: viral infection) developed a lethal disease (86.6 per cent mortality) with high CNS viral titers and brain lesions. Neutralizing antibodies were absent throughout, while immunofluorescent antibody levels were considerably diminished. The transfer of hyperimmune serum conferred partial though significant protection on CY-treated animals but no correlation was found between CNS viral titers and mortality since in both infected CY-treated and untreated mice similar brain viral content was found. This was also confirmed by immune spleen cell transfer at day 0 where the clearance achieved was unable to modify the time course of the disease. Feasible mechanisms explaining recovery from JV infection by means of the protective effect of antibodies and the cell-mediated clearance are discussed.

Junín virus is uniformly lethal in newborn mice, but fails to kill adult animals. But there is direct evidence that in older mice the immunological mechanism occurs when certain conditions are fulfilled. The present results showed that in Junín virus infection of mice, the development of the immunological mechanism occurs irrespectively of age. The induction of a regular cell-mediated immunity appears to be intimately related to multiple injections of the virus. These results were confirmed by the 51Cr release assay and by the effect of the transplanted sensitized cells. The extent of infection in the brain is another variable to be considered. Only when the virus has reached high titres and has been concentrated in the brain, the damage done by the immune attack is lethal. On the other hand the data obtained suggest that Junín virus-sensitized spleen cells do not possess the ability to transfer significant antiviral effects to recipient preinfected newborn mice.

A new attenuation marker to distinguish a virulent strain (XJJV) from an attenuated strain (XJC13JV or XJOJV) of Junin virus by means of the humoral and cellular responses to unrelated antigens was studied in guinea pigs. Strain XJJV suppressed the humoral immune response, as shown by the lower titers of precipitating antibody to ovalbumin. The concomitant decrease in serum complement level contributed to a milder Arthus cutaneous reactivity. In contrast, the attenuated strains did not decrease the humoral response. The pathogenic strain suppressed cell-mediated immunity, as demonstrated by decreased contact sensitivity to 2,4-dinitro-1-fluorobenzene and by depression of delayed skin reactions to tuberculin purified protein derivative. When attenuated strains were used, such suppressive effects were not observed. For virulent strain XJJV, virus replication in lymphoid organs and immunosuppressive effects were correlated. These findings provide a further means to differentiate between virulent and attenuated strains of Junin virus for the purpose of vaccine control of Argentine hemorrhagic fever.