Argentine Hemorrhagic Fever Research Articles

On the relationship between the environmental history and the epidemiological situation of Argentine hemorrhagic fever

AbstractThe aim of this work was to establish the relationship between different Argentine hemorrhagic fever (AHF) epidemiological situations found at different sites and the related large‐scale environmental conditions. Large‐scale environmental records (vegetation index, temperature, precipitation and elevation) were obtained from a series of monthly NOAA satellite images and global databases considered suitable for modeling climatic and other environmental determinants of large‐scale biogeographical regions. The temporal variation in vegetation for cycles of winter‐summer showed a greater variation in the nonendemic region than in the other two regions. On the other hand, the average of the temporal variation in precipitation in cycles of spring–autumn was more different in the historic region than in the other two regions, and land surface temperatures in cycles of spring–autumn showed differences between the epidemic region and the other two regions. We found good separation among the epidemic, historic and nonendemic sites, with the greatest difference found between epidemic and nonendemic sites. The classification of sites showed a tendency for grouping according to the epidemiological situation, but there was some variation. It seems possible to establish a close relationship between the state of AHF incidence and the environmental history of sites suggesting the possibility of predicting epidemiological behavior using environmental conditions derived from satellite data.

Spatial variation in abundance of the junin virus hosts in endemic and nonendemic Argentine haemorrhagic fever zones

Abstract Argentine haemorrhagic fever (AHF) is caused by Junin (JUN) virus, which is hosted by the drylands vesper mouse (Calomys musculinus). In this work we monitored population abundance of C. musculinus and rodent assemblages for 3 years in and outside the AHF endemic zones (northern Buenos Aires, southern Córdoba and Santa Fe Provinces, Argentina). The study area was divided into endemic and nonendemic zones. In the endemic zone epidemic sites were recognized, characterized by recent emergence and maintenance of AHF cases, and also historical sites, characterized by decreased incidence or disappearance of AHF human cases. In the nonendemic zone AHF has never been recognized. Although differences were statistically significant only during some periods, population abundance of C. musculinus was usually lower in the nonendemic sites. The pattern and magnitude of seasonal fluctuations in C. musculinus populations were also distinct in the nonendemic sites as compared to endemic sites. The relative abundance of C. musculinus in rodent assemblage was lower in nonendemic sites than in the endemic sites. The lower population densities and dampened seasonal dynamics may be at least partly responsible for the absence of AHF cases in the nonendemic zone. It is suggested that the balance between intra and interspecific interactions might be the cause of the pattern of incidence and prevalence of pathogens in the host species.

TEMPORAL FLUCTUATION OF EFFECTIVE SIZE IN POPULATIONS OF CALOMYS MUSCULINUS (MURIDAE: SIGMODONTINAE)

Calomys musculinus is the natural reservoir of Junin virus, the etiological agent of Argentine hemorrhagic fever. In this paper we measure the effective size of 2 populations of the rodent over a 2-year period. Twenty enzyme-coding loci were analyzed using vertical starch gel electrophoresis. Effective population sizes (Nes) were estimated by the pseudolikelihood method in 2 populations 280 km apart in central Argentina. Both populations experienced marked seasonal changes in relative density and in Ne (between 19.8 and infinity). Changes in percentage of polymorphic loci and mean number of alleles per locus were statistically significant and were roughly correlated with density and Ne. Observed changes in heterozygosity, in contrast, were not significant. After low-density periods, mixing of surviving individuals coming from different demes may play an important role in the maintenance of variability and recovery of Ne in populations of C. musculinus.

Immune Response to Vaccination Against Argentine Hemorrhagic Fever in an Area Where Different Arenaviruses Coexist

Neutralizing antibody (NT Ab) titers to Candid #1 (C#1) vaccine against Argentine hemorrhagic fever were studied for 2 years post-vaccination in 330 volunteers, to assess whether the kinetics and/or magnitude of this immune response is modified by previous infection with the arena viruses Junin (JUN) and lymphocytic choriomeningitis (LCM). A total of 160 volunteers received C#1, distributed as follows: without detectable pre-infection with arenaviruses (n = 54); with pre-existing antibodies to JUN (n = 55); with pre-existing antibodies to LCM (n = 51). The remaining 170 individuals received placebo. Levels of anti-JUN NT Ab displayed a trend in which titers increased with the virulence of the infecting strain, from C#1 (X = 49), through subclinical JUN infection (X = 229), vaccination following subclinical infection (X = 367) to JUN clinical infection (X =773). It was also found that the mean titer of NT Ab to C#1 did not vary significantly during 2 years of study and was: a) significantly lower than that elicited by wild strains of JUN, both clinical and subclinical infections (p < 0.01); b) significantly increased the titers of pre-existing anti-JUN Ab (p < 0.01); and c) was not modified by pre-existing anti-LCM Ab (p > 0.05). These data indicate that the immune response to C#1 boosts pre-existing immunity to JUN virus and is not changed by previous experience with LCM virus.

Genomic Features of Attenuated Junín Virus Vaccine Strain Candidate

Junin virus strain Candid #1 was developed as a live attenuated vaccine for Argentine haemorrhagic fever. In this paper, we report the nucleotide sequences of L RNA of Candid #1 and examine the relationship to its more virulent ancestors Junin virus XJ#44 and XJ 13 (prototype) and other closely and distantly related arenaviruses. Comparisons of the nucleotide and amino acid sequences of L and Z genes of Candid #1 and its progenitor strains revealed twelve point mutations in the L polypeptide that are unique to the vaccine strain. These changes could be provisionally associated with the attenuated phenotype. In contrast, Z ORF was completely conserved among all strains.

A Survey of Antiviral Drugs for Bioweapons

Smallpox (variola major), and the haemorrhagic fever viruses (filoviruses and arenaviruses) are classified as Category A biowarfare agents by the Centers for Disease Control. Category A agents pose a significant risk to public health and national security because they can be easily disseminated by aerosol, although with the exception of variola, they are not easily transmitted from person to person. An attack with these viruses would result in high morbidity and mortality and cause widespread panic. With the exception of smallpox and Argentine haemorrhagic fever virus, there are no vaccines or approved treatments to protect against these diseases. In this review we focus on promising prophylactic, therapeutic and disease modulating drugs (see Figure 1 for select chemical structures).

Análisis de la utilidad de la vacuna Candid 1 en la prevención de la fiebre hemorrágica argentina en niños

To determine the most rational strategy of vaccination with Candid 1 vaccine in order to prevent Argentine hemorrhagic fever among children under 15 years old living an endemic area. To analyze the estimated effectiveness, a decision tree model was designed, with two possible options: vaccinate all the children under age 15 in the endemic area ("expanded vaccination") or vaccinate only the children at greater risk ("selective vaccination"). These two options were compared with the alternative of not vaccinating. The evaluation was complemented with a sensitivity analysis to identify the threshold values of the critical variables that could change the decision. The probabilities that were used were taken from earlier clinical and epidemiological trials. According to the model that we used, the expanded vaccination strategy was the best option, with a total expected utility of 9.99998 (out of a maximum possible 10.0). The sensitivity analysis showed that selective vaccination would be the best strategy if the incidence in the population with low risk drops to less than 3 per 1,000,000 population or if the rate of serious adverse reactions to the vaccine reaches more than 9 per 100,000 inhabitants. No variation in the parameters used in the model supported the option of not vaccinating. Given the risk and benefit parameters that we used, we recommend vaccinating with Candid 1 all the children under age 15 who live in the area endemic for Argentine hemorrhagic fever. The proposed model can be fitted to future needs, and it can help in decision-making by incorporating prospective disease surveillance data. These results can be used as a basis for cost and efficacy studies and for other quantitative analyses.

Isolation and characterization of microsatellite markers in Calomys musculinus (Muridae, Sigmodontinae, Phyllotini), the natural reservoir of Junin virus

Abstract Calomys musculinus, one of the most abundant rodent species in central Argentina, is the reservoir of Junin virus, the aetiological agent of Argentine hemorrhagic fever. We isolated six polymorphic microsatellite loci for microgeographical studies of population structure in this species. Amplification of these loci in 36 individuals from three natural populations revealed five to 14 alleles per locus and expected heterozygosities from 0.426 to 0.868. Cross‐species amplifications suggest that primers designed for these loci may be useful in other closely related species of the tribe Phyllotini, but not in species of other more distantly related tribes of the subfamily Sigmodontinae.

Polarized entry and release of Junín virus, a New World arenavirus

Junin virus (JUNV), the causative agent of Argentine haemorrhagic fever, is a human pathogen that naturally enters the body through the epithelial cells of the respiratory and digestive tracts. The interaction of JUNV with two types of polarized epithelial cultures, Vero C1008 and A549, was investigated. Radioactive virus-binding assays showed that JUNV infects polarized lines preferentially through the apical surface. High-level expression of viral nucleoprotein was detected in polarized cell lines infected through the apical domain. Virus production from apical media was about 100-fold higher than that found into the basolateral medium. Confocal-immunofluorescence analysis revealed high-level expression of glycoprotein at the apical-membrane surface. Disruption of the microtubule network by colchicine impaired JUNV vectorial release. This is the first study to analyse the interaction between a member of the virus family Arenaviridae and polarized epithelial cells, showing preferential entry and release from the apical plasma membrane.

Serological study of the lymphochoriomeningitis virus (LCMV) in an inner city of Argentina

Lymphocytic choriomeningitis virus (LCMV) is the prototype of the family Arenaviridae and is associated with the natural reservoir, Mus domesticus (Md). It causes meningitis and a flu-like illness characterized by malaise, myalgia, retrorbital headache, and photophobia. This study presents the data obtained in a rodent and human serological study during 6 years (1998-2003) in the city of Rio Cuarto, Argentina. Antibodies anti-LCMV were sought by ELISA in rodents and humans. LCMV was found only in Md species in 9.4% of animals. The results also show some seasonal, no significant variations in the prevalence of the infection. Distribution of positive mice was not modified significantly by trapping sites, sex, or age of the animals. The prevalence of LCMV positive urban residents was found to be consistently low (1-3.6%) along the study period, with overage prevalence of 3.3% and values in males (4.6%) significantly higher than in females (2.6%) (P < 0.05). Seven of 432 pregnant women were found to be LCMV positive, but the absence of LCMV antibodies in the newborns demonstrated that the mothers were infected before pregnancy. This study is the first evidence on endemic LCMV in an Argentine city located outside the endemic area of Argentine hemorrhagic fever (AHF) and described the need to study other areas and increase awareness of this viral infection.

The use of satellite data in modeling population dynamics and prevalence of infection in the rodent reservoir of Junin virus

We present the first results of a simple numerical model of rodent population dynamics and viral infection for Junin virus, etiologic agent of Argentine hemorrhagic fever (AHF), in its host, Calomys musculinus. In contrast to the more common statistical approach, the model incorporates satellite-derived environmental data in a causal approach. In addition, the model incorporates specific biological characteristics of the host, such as birth rate and longevity. Theoretical and actual rodent population densities are compared with several years of capture data at locations on the Argentine pampas. The model appears to be a good tool for simulating dynamics of populations using remotely sensed data. Results are in agreement with field data showing maximum population densities during the autumn in most localities. The differences between simulated population densities and field observed values indicate that, although computer simulation is useful to obtain some aspects of population dynamics, improvements should be introduced in order to generate more robust results.

Inhibition of cell fusion in Junin virus-infected cells by sera from Argentine hemorrhagic fever patients

Junin virus (JV), a member of the Arenaviridae family, is the etiological agent of Argentine hemorrhagic fever (AHF). A low pH-pulse, induces fusion of Vero cells infected with JV to form syncytia, whose production can be inhibited by neutralizing antibodies against the JV major glycoprotein. To characterize the existence of an antifusogenic activity present in sera obtained from natural infections of AHF over a 20-year period and to study both the fusogenic activity of one pathogenic and two attenuated strains of JV in Vero cells, at different pH. The study sample consisted of sera obtained from two provinces in the Argentine Republic. Vero cells grown in monolayers, were infected with different strains of JV and a 2 h pulse, at different pH, was performed. Syncytium production was evaluated 12 h later, after staining with Giemsa. Neutralization tests against the attenuated strain XJCl3 were carried out and the antifusogenic activity of immunosera was studied by incubating serum with JV-infected Vero cells. Also the fusion activity in Vero cells infected with three JV strains was assayed. A pathogenic strain XJ exhibited the highest fusogenic activity at pH 5. Syncytium formation was prevented by patients' sera obtained from different geographical locations, independently of time of infection. However, when Vero cells were infected with XJ, a significant reduction of syncytium production was observed, though the level of inhibition was lower than that detected in other JV strains-infected cells. These results could be explained by the existence of a conserved domain on JV proteins and also antigenic heterogeneity among strains.

The signal peptide of the Junín arenavirus envelope glycoprotein is myristoylated and forms an essential subunit of the mature G1-G2 complex.

Arenaviruses comprise a diverse family of rodent-borne viruses that are responsible for recurring and emerging outbreaks of viral hemorrhagic fevers worldwide. The Junín virus, a member of the New World arenaviruses, is endemic to the pampas grasslands of Argentina and is the etiologic agent of Argentine hemorrhagic fever. In this study, we have analyzed the assembly and function of the Junín virus envelope glycoproteins. The mature envelope glycoprotein complex is proteolytically processed from the GP-C precursor polypeptide and consists of three noncovalently associated subunits, G1, G2, and a stable 58-amino-acid signal peptide. This tripartite organization is found both on virions of the attenuated Candid 1 strain and in cells expressing the pathogenic MC2 strain GP-C gene. Replacement of the Junín virus GP-C signal peptide with that of human CD4 has little effect on glycoprotein assembly while abolishing the ability of the G1-G2 complex to mediate pH-dependent cell-cell fusion. In addition, we demonstrate that the Junín virus GP-C signal peptide subunit is myristoylated at its N-terminal glycine. Alanine substitution for the modified glycine residue in the GP-C signal peptide does not affect formation of the tripartite envelope glycoprotein complex but markedly reduces its membrane fusion activity. In contrast to the classical view that signal peptides act primarily in targeting nascent polypeptides to the endoplasmic reticulum, we suggest that the signal peptide of the arenavirus GP-C may serve additional functions in envelope glycoprotein structure and trafficking.

Recent range expansion and low levels of contemporary gene flow in Calomys musculinus: its relationship with the emergence and spread of Argentine haemorrhagic fever.

The geographic distribution of haplotype diversity in the rodent Calomys musculinus, sampled from 16 wild populations of Argentina, was analysed on two geographical scales. The species is the natural reservoir of the Junin virus, the etiological agent of the Argentine haemorrhagic fever (AHF). In all, 24 composite haplotypes were recognised in the mtDNA D-loop region. Haplotypes 1 and 2, internal in the network, were the most frequent and were present in almost all populations. The absence of large genetic gaps between widely distributed haplotypes, the existence of exclusive haplotypes in more than 50% of the sampled populations and the absence of isolation by distance at a macrogeographical scale are in support of the hypothesis of a recent range expansion of the populations of the Humid Pampa, with low to moderate current gene flow. The dispersal of this opportunistic species would have been favoured by the explosive increments in density after agriculture was introduced. When only nearby populations within the endemic area of AHF were considered, a pattern of isolation by distance was detected. At present, genetic drift appears to be the main force acting to randomly differentiate C. musculinus populations, which would also lead to random differentiation of Junin virus strains and a reduction in the virulence of the pathogen in 'historic' AHF areas. The knowledge of migration patterns of the reservoir populations facilitates reliable prediction of the potential spread of the human disease.

Inheritance of random amplified polymorphic DNA (RAPD-PCR) markers and their use in population genetic studies of Calomys musculinus (Rodentia, Muridae), the reservoir of Argentine hemorrhagic fever.

In order to assess the reliability of RAPD markers in the estimation of the genetic structure of natural populations of the murid rodent Calomys musculinus (reservoir of Junin virus, ethiological agent of Argentine Hemorrhagic Fever), we have analyzed the heritability of RAPD bands in 10 parents and their offspring (33 individuals). Fourteen out of a total of 119 bands obtained were absent in the parental patterns, but consistently amplified in offspring from some families. These bands can be eliminated from analyses. Overall degree of band sharing between individuals, including non-parental bands, correctly grouped members of a family in the same cluster in a UPGMA tree, with a high bootstrap percentage. Results support the usefulness of RAPDs as hereditable markers. One hundred polymorphic RAPD loci were identified in three natural populations of C. musculinus. Mean expected heterozygosity in three natural populations ranged from 0.206 to 0.220. Allele frequency based and phenotype based measures of genetic differentiation among natural populations of C. musculinus gave similar results (Weir and Cockerham's theta = 0.133; Excoffier et al.'s phi = 0.127). These values were considerably higher than those found previously using allozymes as genetic markers, and are compatible with moderate to low levels of gene flow among populations.

Restricted gene flow in Calomys musculinus (Rodentia, Muridae), the natural reservoir of Junin virus.

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) markers were used to evaluate the relative contribution of gene flow as a determinant of the population genetic structure of the wild rodent Calomys musculinus (the reservoir of Argentine hemorrhagic fever [AHF]) in central Argentina. One hundred eighty-seven individuals from 13 populations (9 of them from the endemic zone of AHF and 5 from areas outside it) were analyzed using 78 polymorphic RAPD loci. Genetic variation within each population was high; each individual was characterized by a unique RAPD phenotype. C. musculinus populations showed a moderate to high genetic subdivision and a random pattern of differentiation. Populations separated by the same geographic distance showed very different degrees of genetic divergence. The results indicate that populations of C. musculinus have colonized their present ranges relatively recently and differentiation by genetic drift has proceeded faster than homogenization by gene flow at the macrogeographic scale analyzed (10-700 km).

Inhibitory effect of thiosemicarbazone derivatives on Junin virus replication in vitro.

The inhibitory effect of several thiosemicarbazones (TSCs), synthesized from aromatic ketones and terpenones, and their heterocyclic thiadiazoline (TDZ) derivatives, was investigated against Junin virus (JUNV), an arenavirus agent of Argentine haemorrhagic fever. From the 25 compounds tested, six compounds belonging to the TSC group were found to be selective inhibitors of JUNV, with EC50 values determined by a virus yield inhibition assay in the range 3.4-12.5 microM, and selectivity indices greater than 10. By contrast, most of the TDZs obtained by heterocyclization of the TSCs were not active against JUNV. No conclusive structure-activity relationships could be established but systematically higher activity was associated to TSCs derived from aromatic ketones. The mode of action of one of the most active compound, the 3,4-dihydronaphtalen-1(2H)one thiosemicarbazone (tetralone thiosemicarbazone), was studied further. This TSC lacked virucidal effects on JUNV virions. Results from time of addition experiments and viral protein expression assays suggest that tetralone thiosemicarbazone inhibited a late stage in the replicative cycle of JUNV.

IgG subclasses in human immune response to wild and attenuated (vaccine) Junin virus infection.

Different proportions of IgG subclasses have previously been reported to distinguish the immune response elicited by primary and recurrent viral infections, as well as viral vaccines. The goal of this study was to study the IgG subclasses composition in the immune response of patients with Argentine hemorrhagic fever, and vaccinees with Candid #1 strain of Junin virus. Twenty-four individuals inoculated with Candid #1 vaccine and 67 patients with Argentine hemorrhagic fever were studied. Blood samples were drawn at 30, 60, and/or 180 days post-inoculation with Candid #1 and 30, 60, and 90 days after clinical onset of the disease. Specific anti-Junin virus IgG subclasses were titrated with specific human monoclonal antibody fluorescence isothiocyanate conjugate (FITC) by immunofluorescent assay (IFA). IgG(1) anti-Junin virus was found in every subject studied and IgG(3) was also detected in some patients with a severe form of Argentine hemorrhagic fever. IgG(2) and IgG(4) were not detected in any serum sample studied. The mean titer of specific IgG(1) in vaccinees was significantly lower than in patients with Argentine hemorrhagic fever (P < 0.05), but no difference was found between mild and severe cases of the disease (P > 0.05). The results of this study demonstrated a central role of IgG(1) in human recovery from infection with every strain of Junin virus, an observation stressed by the immune response to Candid #1 vaccine, which resulted in no difference in IgG subclasses composition from that found in mild cases of Argentine hemorrhagic fever.

Endothelial cell function alteration after Junin virus infection.

Hematologic involvement is the main feature of Argentine hemorrhagic fever (AHF), an endemo-epidemic disease caused by Junin virus (JV). Since endothelial dysfunction could play a role in AHF-altered hemostasis, we studied human umbilical vein endothelial cell (HUVEC) infection with a virulent (JVv) and a non-virulent (JVa) JV strain. Cells were infected by the two JV variants with no detectable apoptosis or cytopathic effect. Both viral variants up-regulated ICAM-1 and VCAM-1 levels, while von Willebrand factor (VWF) production was decreased. Prostacyclin (PGI2) release and decay accelerating factor (DAF) expression were greater in JVv- than in JVa-infected or control cells. Furthermore, nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) expression was only raised in JVv-infected supernatants. Significant NO and PGI2 values were also detected in AHF patient sera. These data demonstrate that endothelial cell responses are triggered subsequently by JV infection, suggesting that such alterations play a major role in the pathogenesis of AHF and perhaps in other viral-induced hemorrhagic diseases.

Mode of inactivation of arenaviruses by disulfide-based compounds

Several disulfide-based compounds, including intermolecular aromatic disulfides of the type Ph-S-S-Ph and dithianes with the sulfur atoms tethered in a ring structure, have shown effective inhibitory activity against the arenaviruses Junin (JUNV), agent of Argentine hemorrhagic fever, and Tacaribe (TCRV). These compounds showed a strong virucidal effect with inactivating concentration 50% (IC 50) values in the range 0.6–5.0 μM, and also were effective to reduce virus yields from infected cells. The mode of inactivating action of two active compounds, the aromatic bis disulfide NSC20625 and the dithiane NSC624152, was further studied. Both compounds were able to inactivate arenaviruses after a few minutes of direct contact with virions, in a concentration- and time-dependent manner. The ability of drug-treated virus to perform several steps of the replication cycle was analyzed. The killed virus particles were found to bind and enter to Vero cells with the same efficacy as infectious native virions, but the ability of inactivated virions to synthesize viral proteins in Vero cells was abolished. Thus, treatment of JUNV and TCRV with these compounds destroyed virion infectivity, generating particles which entered the host cell but were unable to complete the viral biosynthetic processes.