Factor VIII: C and factor VIII R: Ag in Argentine hemorrhagic fever.

Factor VIII procoagulant activity (F VIII:C) and factor VIII related antigen (F VIII R: Ag) were investigated in 35 patients with Argentine hemorrhagic fever. Since the results obtained in the three clinical forms of the disease were not significantly different, they were tabulated altogether. F VIII:C was low in early stages of the disease but increased progressively in later days (days 5-6: 0.54 +/- 0.10 I. U/ml; days 13-14: 0.95 +/- 0.13 I.U./ml). In contrast, the levels of F VIII R: Ag were high all along the disease and they returned to normal values during the convalescence period (days 5-6; 2.58 +/- 0.54 I.U./ml; day 30: 1.30 +/- 0.14 I.U./ml). The levels of F VIII R: ag were similar in samples drawn before (11 cases) or after (10 cases) the treatment with immune plasma infusion. Plasma samples from 12 patients were studied by two-dimensional immunoelectrophoresis. The only abnormality found was increased height of the immune precipitation arc.

Early markers of blood coagulation and fibrinolysis activation in Argentine hemorrhagic fever.

Junin virus, an arenaviridae, is the etiological agent of Argentine hemorrhagic fever. In addition to thrombocytopenia, patients present several alterations in both the blood coagulation and the fibrinolytic system, but diffuse intravascular coagulation could not be demonstrated. To investigate further the activation status of the two systems, levels of thrombin-antithrombin complexes (TAT), prothrombin fragment 1 + 2, protein C, total and free protein S, C4bBP, antithrombin III, t-PA, PAI-1 and D-dimer were measured. Fourteen patients with a confirmed diagnosis of Argentine hemorrhagic fever were included in the study, 2 were severe, 3 moderate and 9 mild clinical cases, but hemorrhages were slight throughout. Blood samples were collected for 6 consecutive days on admission and on remission. At admission TAT and F1 + 2 levels were increased in 13/14 patients, reaching 0.33 nM (0.06-0.87) and 2.16 nM (0.96-6.5), respectively. PC was low in 4 cases, fPS in 6 and tPS in 2, whereas C4bBP and ATIII values were within normal range. t-PA and D-dimer levels were high in 11/14 patients, reaching 20 ng/ml (2.7-106) and 1660 ng/ml (877-3780) respectively, while PAI-1 was considerably increased in the 2 severe cases and normal in the remainder. These results suggest low level though persistent process of blood coagulation and fibrinolysis activation in this viral hemorrhagic disease. We believe these abnormalities may lead to the well described bleeding manifestations in these patients.

Antiviral treatment of Argentine hemorrhagic fever.

Argentine hemorrhagic fever is a systemic viral disease caused by Junin virus, with a mortality of 15-30% in untreated individuals. Current specific therapy is highly effective in reducing mortality, and consists of the early administration of immune plasma in defined doses of specific neutralizing antibodies per kg of body weight. However, several reasons suggest the need to investigate alternative therapies. Ribavirin, a broad spectrum antiviral agent, is effective in the treatment of other viral hemorrhagic fevers, and the studies done with Junin virus infections to date indicate that this drug may also have a beneficial effect in Argentine hemorrhagic fever.

Tolerance and antiviral effect of ribavirin in patients with Argentine hemorrhagic fever.

Tolerance and antiviral effect of ribavirin was studied in 6 patients with Argentine hemorrhagic fever (AHF) of more than 8 days of evolution. Administration of ribavirin resulted in a neutralization of viremia and a drop of endogenous interferon titers. The average time of death was delayed. A reversible anemia was the only adverse effect observed. From these results, we conclude that ribavirin has an antiviral effect in advanced cases of AHF, and that anemia, the only secondary reaction observed, can be easily managed. The possible beneficial effect of ribavirin during the initial days of AHF is discussed.

A plasma inhibitor of platelet aggregation in patients with Argentine hemorrhagic fever.

Hemorrhage in patients with Lassa fever is associated with the presence of a circulating plasma inhibitor of platelet aggregation. This study was to determine whether patients with Argentine hemorrhagic fever (AHF) develop a similar inhibitor. Normal platelets showed significantly weaker aggregation responses to a sub-maximal dose of adenosine diphosphate (ADP) when mixed with plasma from 10 patients with AHF (mean percent of control +/- 1 SE = 57.2 +/- 6.7%) compared to those mixed with plasma from 9 viral control patients (79.5 +/- 4.1%; P less than 0.05) and 9 febrile patients with septicemia (103.8 +/- 3%; P less than 0.001). Plasma from 3 patients with severe AHF inhibited in a dose-dependent fashion the aggregation responses of normal platelets to collagen, sodium arachidonate, a calcium ionophore (A23187), and ristocetin; none of 4 samples from convalescent AHF patients showed this inhibitory activity. The platelet inhibition was sudden in onset and unaffected by a 30 min pre-incubation, not neutralized by convalescent plasma with high titer antibody to Junin virus, and abolished after heating plasma from an AHF patient at 56 degrees C for 30 min. Hemorrhage in AHF is associated with the presence of a circulating inhibitor of platelet aggregation, and disturbed hemostasis in arenavirus-induced hemorrhagic fevers may have a common basis.

Prevalence of lymphocytic choriomeningitis virus infection in a human population of Argentina.

The activity of lymphocytic choriomeningitis virus (LCMV) in the endemic area of Argentine hemorrhagic fever has been previously reported and represents the first evidence of the coexistence of two arenaviruses pathogenic for humans, Junin and LCMV, in the same geographic area. Data are presented on the prevalence of LCMV human infection in a 10,000-km2 area located in Santa Fe Province, Argentina. Study subjects were males, 15-65 years old, living and/or working in the rural area of 41 localities. One serum sample was obtained from each 7,227 volunteers from a total population of 21,340 individuals with the described features. Antibodies to LCMV were assessed by means of an indirect immunofluorescence assay. These antibodies were found in 172 serum samples, with titers ranging from 1:8 to 1:128 (geometric mean titer = 15.03), and a mean percentage of infection of 2.38%. A significantly different distribution of positive individuals was found between the eastern (1.54%) and western (3.07%) borders of the region studied (P < 0.0003). The higher percentage of infection on the western side was due to the existence of two clusters of counties with a mean percentage of 6.06% that was significantly different from the 1.67% obtained in the rest of the study area (P < 0.0003). These results provide new information on the LCMV activity in Argentina, and update the evidence on the coexistence of two arenaviruses in the same region of Argentina. This circumstance increases the probability of generation of viral reassortants with changes that could determine the need for new therapeutic and/or preventive strategies for arenaviral diseases.

Isolation of a Hantaan-related virus from Brazilian rats and serologic evidence of its widespread distribution in South America.

A serosurvey of domestic rats was conducted in several South American cities between September 1982 and March 1983 for evidence of hantavirus infection. Antibody-positive rats were found in Belem, São Paulo and Recife-Olinda, Brazil and in Buenos Aires, Argentina, with the highest antibody prevalence rate detected in Belem (30 positive of 54 tested, 56%). A virus isolated from tissues of a Rattus norvegicus captured in Belem, was shown to be antigenically similar to Girard Point viruses isolated from domestic rats captured in the United States and clearly distinct from prototype Hantaan virus, causative agent of Korean hemorrhagic fever in Asia. This represents the first isolation of a virus of the genus Hantavirus from South America and supports previous observations that indicate a widespread distribution of urban rat-associated hantaviruses. The abundance of domestic rats and their regionally high antibody rates suggest that risk of human hantavirus infection in some locations of South America may be significant.

Prevalence of infection with Junin virus in rodent populations in the epidemic area of Argentine hemorrhagic fever.

We report the results of indirect fluorescent antibody screening for antibody to Junin virus in 1,101 sera from small mammals captured on two mark-recapture grids in the epidemic area of Argentine hemorrhagic fever. Twenty-six of 29 seropositive animals were the cricetid rodent Calomys musculinus, for a 30-month prevalence of 7.9% in that species. Combining these data with previously published data on antigen detection provided an estimated total prevalence of infection of 10.9% for this, the principal reservoir species. Other infected species included two cricetids, C. laucha and Bolomys obscurus, and a predatory carnivore, Galictis cuja. Approximately half of infected animals simultaneously carried serum antibody and antigen in blood and saliva, some for 29-61 days. Except for C. laucha, which was associated with crop habitats, seropositive animals were strongly associated with the relatively rare roadside and fence-line habitats. Seropositive C. musculinus were predominantly males in the oldest age and heaviest body mass classes, and seropositive males were twice as likely to have body scars as seronegative males. These observations suggest that most infections were acquired through horizontal transmission and that aggressive encounters among adult, male C. musculinus in relatively densely populated roadside and fence-line habitats are an important mechanism of transmission of Junin virus within reservoir populations.

Proinflammatory cytokines and elastase-alpha-1-antitrypsin in Argentine hemorrhagic fever.

Argentine hemorrhagic fever (AHF) is a disease caused by Junin virus. In the acute phase, patients present hematologic and neurologic involvement with high levels of interferon-alpha and tumor necrosis factor-alpha (TNF-alpha. Nineteen patients with a confirmed diagnosis of AHF were studied: six severe, four moderate and nine mild cases. Serum levels of interleukin-6 (IL-6), IL-6 soluble receptor (IL-6sR), IL-8, IL-10, and elastase-alpha1-antitrypsin complex (E-alpha 1AT) were assayed by ELISAs. Levels of IL-6, IL-8, and IL-10 were high in nine, 12, and 13 patients, respectively, while levels of IL-6sR were high in two patients and low in one patient. Seven patients had increased levels of E-alpha1AT. Significant correlations were found between levels of both IL-8 and IL-10 with those of TNF-alpha as well as between IL-8 and E-alpha 1AT. These data demonstrate activation of pro-inflammatory and anti-inflammatory cytokine pathways, and statistical analysis showed differences among the clinical forms of illness. This study shows that IL-8 plays an essential role in neutrophil activation in AHF patients as demonstrated in other infectious diseases.

A longitudinal study of Junin virus activity in the rodent reservoir of Argentine hemorrhagic fever.

We monitored Junin virus (JV) activity in rodent populations for 30 months at seven mark-recapture grids located in agricultural fields and adjacent roadsides and fence lines in endemic and nonendemic areas of Argentine hemorrhagic fever. Blood and oral swabs taken from rodents captured at five-week intervals were analyzed by enzyme-linked immunosorbent assay for JV antigen (Ag). Calomys laucha and C. musculinus were the most frequently captured rodents, making up 47% and 22% of captures, respectively. Of 41 Ag-positive captures, 37 were C. musculinus and four were C. laucha; 34 were from two trapping grids in the same locality. Antigen-positive Calomys were more frequently male (76%), and were found significantly more frequently among the oldest animals and the largest body mass classes. These patterns, combined with the greater mobility and higher frequencies of wounds among males than females, implicated horizontal transmission as the primary route of JV transmission between rodents. Seasonal maximum levels in JV prevalence (up to 25% of captured Ag-positive C. musculinus) occurred during periods of maximal population densities of Calomys. Spatial distribution of Ag-positive rodents reflected habitat preferences; most Ag-positive C. musculinus were captured from border habitats (roadsides and fence lines), and all Ag-positive C. laucha were captured in crop fields. These distinct, but previously undocumented, habitat preferences suggest that the disease in humans may be related to exposures to the primary reservoir species, C. musculinus, in border habitats rather than in crop fields.

Junin virus activity in rodents from endemic and nonendemic loci in central Argentina.

Small mammals were trapped during a 21-month period at 27 farm sites in 15 localities within and beyond the known endemic area for Argentine hemorrhagic fever (AHF). Prevalence of Junin virus (JV) was assessed by antigen-capture enzyme immunoassay (ELISA) on samples of body fluids and/or organs from 3, 282 captured rodents. Infection in rodent populations was variable (0-3.7%) among localities but, in all cases, was lower than previously reported rates. Overall prevalence was 1.4% in the AHF epidemic area, 0.6% in the historic (currently low incidence of AHF) area, and 0.4% in two localities beyond the previously defined endemic area. These low values underestimate the actual prevalence of JV, as ELISA validation by virus isolation indicated a sensitivity of 30% and a specificity of 99%. Of 37 positive rodents, 28 (76%) were of two species: Calomys musculinus (23 animals) and C. laucha (5 animals). Antigen also was found in three Akodon azarae, four Bolomys obscurus, one Mus musculus, and one Oxymycterus rufus, and JV was isolated from two Oligoryzomys flavescens. Three of these rodent species (B. obscurus, O. flavescens, and O. rufus) have heretofore not been implicated in JV maintenance in the field. Evidence suggests that the AHF endemic area may continue to expand northward.

Evaluation of an enzyme-linked immunosorbent assay for quantitation of antibodies to Junin virus in human sera.

An enzyme-linked immunosorbent assay (ELISA) was evaluated for the quantitation of anti-Junin virus (JV) antibodies, in 83 selected cases of Argentine haemorrhagic fever (AHF). Serum samples were studied in two groups to facilitate comparative analysis; the first group was ELISA with indirect immunofluorescence (IF) test, in the second ELISA with plaque reduction neutralization test (PRINT). From the results obtained by using ELISA and IF on the same serum samples, a clear tendency of ELISA to demonstrate seroconversion for JV earlier and at higher frequency than IF test was noted. Simultaneous titration of specific antibodies by ELISA and PRNT tests rendered significantly correlated titers (r = 0.81), both methods being equivalently specific (100%). The demonstration of specific antibodies by ELISA in two cases that were undetected by the PRNT test resulted in a higher sensitivity index for ELISA than for PRNT (100% vs 97%). It is concluded that ELISA could efficiently replace IF and PRNT tests for the diagnosis of AHF.

A refined complement-enhanced neutralization test for detecting antibodies to Junin virus.

A refined, complement-enhanced, plaque-reduction neutralization test was developed for measuring neutralizing antibodies against Junin (Argentine hemorrhagic fever) virus. The assay measured neutralizing antibodies after natural as well as vaccine-induced Junin virus infections. Among vaccinated individuals, titers were 2-4-fold higher than those obtained with conventional assays, without loss of specificity. Enhanced sensitivity was achieved by using a standardized complement source (vs human or animal serum) for virus dilution, incubation of virus-serum mixtures at 36 degrees C for 2 h (vs overnight at 4 degrees C) prior to plaque assay, control of age and density of cell monolayers, and variation in overlay conditions.

Plasminogen abnormalities in patients with Argentine hemorrhagic fever.

Plasminogen, alpha 2-antiplasmin, alpha 2-macroglobulin, alpha 1-antitrypsin and fibrinogen degradation products (FDP) were studied in 45 patients with Argentine hemorrhagic fever. Patients were grouped into: 17 mild, 14 moderate and 14 severe cases. Plasminogen antigen level and functional activity were found to be reduced in the moderate and severe groups, when compared to the results obtained at recovery. The functional activity of alpha 2-antiplasmin was within the normal range, except for a slight decrease on days 10-11, alpha 2-macroglobulin remained normal during the course of illness. alpha 1-antitrypsin also remained normal except on days 10-11, when an increase in the antigen level was noted. FDP titre was normal (less than 10 micrograms/ml) in all patients during the course of disease. Plasminogen decrease was not attributable to liver insufficiency neither to a primary nor secondary fibrinolysis. The decreased antigen and reduced functionality of plasminogen in these patients we believe is related to proteolytic degradation by leukocyte enzymes.

Lymphatic tissue in Argentine hemorrhagic fever. Pathologic features.

Spleen and lymph nodes from seven fatal cases of Argentine hemorrhagic fever were studied by light and electron microscopy and by immunofluorescent techniques. In addition, Junin virus infectivity was determined. Destruction of splenic white pulp and of lymph node cortex was observed. The ultrastructural study showed a characteristic cytopathic effect on the macrophages with presence of intracisternal virus-like particles and occasional virions budding from the plasma membrane. The immunofluorescent techniques indicated the presence of Junin virus antigenic determinants in the cytoplasm of macrophages in the examined organs. Junin virus titers were three times higher than in peripheral blood. These results suggest that lymphatic tissue is one of the main sites of viral replication and that the macrophages are the targets of Junin virus.

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.

[New findings on Junin virus infection in rodents inside and outside the endemic area of hemorrhagic fever in Argentina]. / Nuevas observaciones de la infección de roedores por el virus Junin dentro y fuera de la zona endemica de la Fiebre Hemorragica Argentina.

In conjunction with field trials for a vaccine against Argentine Hemorrhagic Fever (AHF), small mammals were trapped during a 28-month period (1 November 1987 to 13 March 1990) in 3 epidemiologically defined areas of the central Argentine pampas: northern and central Buenos Aires provinces were included in the AHF "historic" area, where the disease was common 15-20 years ago, but case rates are currently low; southern Santa Fe province is the current high-incidence area for AHF; the nonendemic area was represented by two localities 60-90 km beyond the northernmost extension of human disease. Animals were live-trapped for 3 days per month in permanent "mark-recapture" grids in each of the 3 areas. Samples of blood, sera, and oral swabs were taken from these animals before they were marked and released at the site of capture. In addition, "removal" traplines provided animals from 16 localities in these 3 areas which were sacrificed to obtain samples of organs in addition to the aforementioned samples. Samples were tested for the presence of Junin virus (JV) antigen by enzyme immunoassay (ELISA). In this assay, a pool of 13 mouse anti-JV glycoprotein and nucleocapsid monoclonal antibodies adsorbed to the surface of microtiter plates was used to capture JV antigen in sample suspensions. A polyclonal rabbit anti-JV antiserum was added as a detector antibody, and an anti-rabbit antibody conjugated to horseradish peroxidase applied with substrate to complete the sandwich.(ABSTRACT TRUNCATED AT 250 WORDS)