Two simultaneous hepatitis B virus epidemics among injecting drug users and men who have sex with men in Buenos Aires, Argentina: characterization of the first D/A recombinant from the American continent.

Previous studies have revealed that hepatitis B virus (HBV)/D and HBV/F predominate among blood donors from Buenos Aires, Argentina. In the present study, blood samples from two high-risk groups were analysed: 160 corresponding to street- and hospital-recruited injecting drug users [81.2% showing the 'anti-hepatitis B core antigen (anti-HBc) only' serological pattern] and 20 to hepatitis B surface antigen (HBsAg)(+)/anti-HBc(+) men who have sex with men. HBV genotypes were assigned by polymerase chain reaction amplification followed by restriction fragment length polymorphism and confirmed by nucleotide sequencing of two different coding regions. HBV DNA was detected in 27 injecting drug users (16.9%, occult infection prevalence: 7.7%), and 14 men who have sex with men (70%). HBV/A prevailed among injecting drug users (81.8%) while HBV/F was predominant among men who have sex with men (57.1%). The high predominance of HBV/A among injecting drug users is in sharp contrast to its low prevalence among blood donors (P = 0.0006) and men who have sex with men (P = 0.0137). Interestingly, all HBV/A S gene sequences obtained from street-recruited injecting drug users encoded the rare serotype ayw1 and failed to cluster within any of the known A subgenotypes. Moreover, one of the HBV strains from a hospital-recruited injecting drug user was fully sequenced and found to be the first completely characterized D/A recombinant genome from the American continent. Data suggest that two simultaneous and independent HBV epidemics took place in Buenos Aires: one spreading among injecting drug users and another one sexually transmitted among the homosexual and heterosexual population.

Protection against a pathogenic strain of Junin virus by mucosal infection with an attenuated strain.

In order to determine the degree of mucosal infectivity of the attenuated XJCl3 strain of Junin virus, guinea pigs were orally or nasally inoculated. Infectivity was 85% for the oral and 100% for the nasal route, as detected by death or serum antibody development. The presence of serum antibodies was closely associated with resistance to challenge with the XJ pathogenic strain, which killed 100% of controls when inoculated by the parenteral or nasal route. However, mortality rates after mucosal infection were low, depending on the dose. Guinea pigs which survived nasal inoculation developed serum neutralizing antibodies, and were fully resistant to challenge with the XJ pathogenic strain.

Junin virus infection of Callithrix jacchus: pathologic features.

Infection of Callithrix jacchus, a New World primate, with the prototype strain of Junin virus produced a severe disease. The animals developed multifocal hemorrhages and characteristic microscopic lesions such as meningoencephalitis, interstitial pneumonia, lymphocytic depletion of lymphatic tissue, hepatocytic necrosis, and a variable decrease in bone marrow cellularity. High virus concentrations correlated with lesions, and with the presence of viral antigenic determinants as revealed by immunofluorescent methods. With the exception of central nervous system damage, the morphological features and immunohistochemical and viral findings were similar to those recorded in human Argentine hemorrhagic fever.

Passive immunity against Junín virus in mice.

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.

Junin virus infection of Calithrix jacchus: haematological findings.

Haematological changes produced by experimental Junin virus infection of a platyrrhine monkey, Callithrix jacchus were studied. Normocytic and normochromic anaemia appeared after 7 days post infection (p. i.), and increased steadily until day 21 p. i. Reticulocytes and circulating erythroblasts were elevated during the anaemia, reached a peak on day 7 p. i., and disappeared later. Platelets and leukocytes showed a significant decrease from days 14 and 18 p. i., respectively. These alterations could be attributed to the damage of bone marrow and lymphatic tissue.

Nasal and intrathalamic inoculations of primates with Tacaribe virus: protection against Argentine hemorrhagic fever and absence of neurovirulence.

Callithrix jacchus marmosets were inoculated by different routes with two stocks of Tacaribe virus, one from suckling mouse brain and another from human diploid MRC5 cells. All 12 primates inoculated by nasal route developed neutralizing serum antibodies without any clinical signs. All 6 primates receiving the mouse brain-Tacaribe virus were protected against lethal challenge with pathogenic XJ strain of Junin virus, while protection was also conferred in 4 out of 6 primates receiving the diploid cell-Tacaribe virus stock. Intramuscular (i.m.) inoculation also elicited antibodies and conferred protection to 4 primates receiving the diploid cell-virus stock. Intrathalamic (i.t.) inoculation of mouse brain-virus stock caused no clinical signs or histopathologic changes in groups of 3 primates each examined on days 33 and 90 post-infection (p.i.). All primates developed antibody response, but no virus could be detected in their brain. Thus, Tacaribe virus proved harmless and immunogenic in Callithrix jacchus and protected most marmosets against challenge with the lethal XJ strain of Junin virus.

[Human immunodeficiency virus infection associated with tuberculosis]. / Asociación de la infección por el virus de la inmunodeficiencia humana (HIV) y tuberculosis.

In order to detect an association between HIV infection and tuberculosis (TB), 130 TB inpatients were studied one of whom presented a pulmonary disease due to Mycobacterium avium intracellulare. All had advanced TB, 95.4%, with pulmonary localization. Serum anti-HIV antibodies were detected by ELISA and their presence confirmed by immunoblotting in 4 (3.1%) individuals, three males and one female, with different degrees of pulmonary TB. Of the males, 1 was bisexual, 2 were promiscuous, and the female was the sexual partner of a non symptomatic HIV-infected man. No immunological disturbances or other AIDS related alterations were observed. There was one case of miliary TB, but neither atypical X-ray abnormalities nor extrapulmonary involvement were found. Tuberculin reaction was positive in three of the four HIV infected patients. Clinical, radiological and bacteriological evolution were favorable. Adverse drug reaction occurred in two cases, one of them presenting serious toxidermia caused by isoniazid. Of the 130 individuals, 12 presented risk factors for HIV infection so that the prevalence of anti-HIV antibodies presented here, 4 cases out of 12, is consistent with data from previous reports for high risk populations.

Hantavirus infection in laboratory and wild rodents in Argentina.

Serum samples from urban and laboratory rats, laboratory mice and wild and laboratory cricetids in Argentina were tested by immunofluorescence and plaque reduction neutralization tests to investigate prevalence of anti-Hantavirus antibodies. A total of 102 sera were obtained from laboratory rodents in 4 different animal-rooms, 31 from harbor rats and 30 from wild cricetids in 1985-1987. Anti-Hantavirus antibodies were detected in 22.5% of Rattus norvegicus in 3 of the animal-rooms but harbor rats were found to be free of Hantavirus infection. Previously, the presence of anti-Hantavirus antibodies had been demonstrated in the sera obtained from laboratory workers in these same 3 animal-rooms; it can be concluded that the laboratory rats were the source of this human infection. On the contrary, laboratory mice and cricetids failed to show Hantavirus infection while the wild vesper mouse Calomys musculinus (the main Junin virus reservoir) showed a prevalence of 23.5%. The presence of Hantavirus infection is hereby reported for the first time in wild C. musculinus and in laboratory R. norvegicus in Argentina.

Serological evidence of human Hantavirus infection in Argentina, Bolivia and Uruguay.

A serological survey was conducted in 1985-1987 to determine the presence of infection for Hantavirus in the general population in Argentina, Uruguay, Paraguay and Bolivia, as well as among rodent-exposed laboratory workers in Argentina. Out of 748 individuals tested by immunofluorescence 20 proved positive for Hantaan virus 76/118 strain of whom 16 also reacted against Seoul virus 80/39 strain and 2 against Puumala virus Sotkamo strain. Ten out of 72 Argentine laboratory workers were positive for the first 2 viruses by ELISA, immunofluorescence and/or plaque reduction neutralization test, in 4 of whom recent infection was demonstrated by IgM antibody presence. Inapparent Hantavirus infection was thus demonstrated for the first time in 2.7% of regional inhabitants, together with 13.9% among rodent-exposed laboratory workers. Our data established the existence of human Hantavirus infection nearly 10 years before the recognition of clinical cases of hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome in Argentina.

Hantavirus pulmonary syndrome in Argentina. Possibility of person to person transmission.

In March 1995 the first case of a familiar outbreak of Hantavirus pulmonary syndrome (HPS) was notified in El Bolson, in the South of Argentina. Until December 15, 1996, a total of 77 cases of HPS had been notified with 48% mortality, distributed in three geographical areas of the country, South, North and Center. During 1996, of the 19 cases from El Bolsón, three were local physicians, one of whom -during the prodrome of her illness- travelled to Buenos Aires to be attended. In the hospital, two of the physicians who assisted her, developed HPS 27 and 28 days after the first contact. These data suggest for the first time the possibility of interhuman transmission of the Hantavirus responsible for the pulmonary syndrome.

Antiviral effect of ribavirin on Junin virus replication in vitro.

The effect of Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) on the replication in vitro of Junin virus, the causative agent of Argentine Hemorrhagic Fever (AHF), was examined. A concentration as low as 3.12 micrograms/ml of the drug inhibited the cytopathic effect observed five days post-infection (pi) on Vero cells. On the other hand, a concentration of 25 micrograms/ml reduced the virus yield and specific antigen formation to undetectable levels. This same concentration of Ribavirin prevented plaque formation under methylcellulose. The high sensitivity of Junin virus to Ribavirin in vitro, the low toxicity of the drug for mammals and the therapeutic effect observed on in vivo infections with other arenaviruses, makes Ribavirin a promising antiviral agent that deserves testing on AHF primate models before clinical trials are undertaken.

[Detection of laboratory infections caused by Junín virus using the neutralization and immunofluorescence technics comparatively]. / Detección de infecciones de laboratorio por virus Junín utilizando comparativamente las técnicas de neutralización e inmunofluorescencia.

To study Junin virus infection among laboratory workers and to compare immunofluorescence and neutralization tests, blood samples were taken from 48 individuals, of which 42 were considered high risk personnel. None of the 16 low risk workers exhibited antibodies. Neutralizing antibodies were detected in 15 high risk laboratory workers. Nine of the latter were already known to carry antibodies from a previous survey in 1978. Titers detected were either at previous levels or slightly higher. Of the remaining 6 out of the 15 positive cases, 3 showed mild clinical and subclinical infection, equivalent to a 12% incidence rate over the 1978-1980 period. An adequate correlation was observed between neutralization and immunofluorescence test: 66.6% for both positive tests and 97.1% for both negative tests. Although the immunofluorescence test ies easier to perform the neutralization test appears to be more reliable clinically. The overall prevalence rate of neutralizing antibodies among non-vaccinated personnel was almost 19%, which warns against the health hazard involved in Junin virus handling.

[Association of the infection of the thymus and bone marrow with the establishment of persistent infection with Junin virus in 2 genera of rodents]. / Asociación de la infección del timo y médula ósea con el establecimiento de la infección persistente con virus Junín en dos géneros de roedores.

Junin virus infection of immune system organs was correlated with persistence establishment in the mouse and rat. Rockland mice under 24 or at 72 and 120 h of age received 10(4) pfu of Junin virus XJ strain by ic route. Separately, two groups of mice under 24 h old were infected with the same dose of XJ or XJCl3 strain by the same route respectively. Results showed that higher thymus virus titer correlated with greater survival. In turn, the former also seemed to correlate with decreasing age at inoculation time, although there was considerable strain dependence. In order to correlate replication levels in thymus with clinical progress in mice, animals under 24 h of age were inoculated with XJ. At 14 days pi apparently healthy mice from this batch were separated from those presenting severe neurologic sings. In the asymptomatic mice, thymus titers ranged from 1.7 to 3.2 log, while no virus was found in thymus harvested from obviously ill animals. However brain virus titers in the two groups proved similar. To confirm these findings, 72 h old Wistar rats were inoculated in with 10(4) pfu of either Junin virus strain: with XJ strain (90% survival) virus could be readily isolated from thymus and bone marrow at day 7 pi, whereas with XJCl3 (5% survival) no virus could be rescued from any organ tested. Therefore, our results strongly suggest a close correlation between productive thymic infection and Junin virus persistence establishment in these rodents, depending on immune response regulation rather than on viral variation.

[Optimal conditions for preserving the infectivity of Tacaribe virus under various temperatures]. / Condiciones óptimas para el mantenimiento de la infectividad del virusTacaribe ante las variaciones de temperatura.

The stability of Tacaribe virus at different temperatures and the stabilizing effect of calf serum or human albumin were studied. The viral stock employed in this study was a 10% suckling mouse brain suspension prepared in phosphate buffer saline and fractionated in three aliquots: the first was supplemented with 0.5% human albumin, the second with 5% calf serum and the third was maintained without stabilizing agent. The infectivity of Tacaribe virus kept at -70 degrees C was preserved during several months and the reduction in infectivity was not significant even if the viral sample was frozen and thawed four times. However, the addition of an stabilizing protein to the viral stock was necessary to maintain it at -16 or 4 degrees C. Under these conditions, the viral infectivity was completely lost after 3 days when the stock was prepared in phosphate buffer saline alone, while more than 40 days were necessary to destroy the infectivity in presence of serum or albumin. The viral inactivation was completed after 48 hours at 25 or 37 degrees C or 10 minutes at 56 degrees C.

[Experimental infection of guinea pigs with Tacaribe virus: effect on the functioning of the immunocompetent system]. / Infección experimental del cobayo con virus Tacaribe: acci n sobre lafunción del aparato inmunocompetente.

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.

[Infection of New World primates with Junín virus. IV. Aotus trivirgatus]. / Infección de primates del nuevo mundo con virus Junín. IV. Aotus trivirgatus.

Owl monkeys (Aotus trivirgatus) were inoculated with XJ, a pathogenic strain of Junin virus, seeking new animal models for Argentine Hemorrhagic Fever. Nine monkeys were inoculated intramuscularly with 30 or 300,000 TCID50 of junin virus. Hematological and virological studies showed no alteration in blood elements such as red cell, reticular cell and platelets, up to 28 days after inoculation. Hemoglobin and hematocrit determinations also remained constant. However, significant neutropenia was seen at day 11 and minimal viremia was detected in some animals during the second and third week post-inoculation. No clinical or behavioral modifications were observed during the eighty-days observation period. Non-specific necropsy findings included pyelonephritis, pneumonitis, liver abscess and eosinophilic spleen infiltrate. All of these findings seem to be unrelated to Junin virus inoculation. No virus was present in organs of animals killed 29, 57 or 85 days post-inoculation. All nine owl monkeys developed serum neutralizing antibodies by day 22. It is concluded that the owl monkey suffers a subclinical infection when inoculated with Junin virus, similar to that seen in other primate species (Saimiri sciureus and Alouatta caraya).