Clinical case definitions for Argentine hemorrhagic fever.
Argentine hemorrhagic fever (AHF) is a potentially lethal infection in Argentina. The case-fatality ratio is >15%, but treatment reduces the mortality rate to <1%. Diagnosis is based on clinical and laboratory criteria, but no case definition has been validated. A chart review was conducted for patients hospitalized with suspected AHF. Individuals with a fourfold rise in antibody titer were classified as cases. The combination of a platelet count of <100,000/mm3 and a white blood cell (WBC) count of <2,500/mm3 had a sensitivity and specificity of 87% and 88%, respectively, thus suggesting that the use of these criteria in a case definition would be helpful for epidemiological studies of AHF. The combination of a platelet count of <100,000/mm3 and a WBC count of <4,000/mm3 had a sensitivity of 100% and a specificity of 71%; the use of these criteria in a case definition should be helpful for screening patients for therapy with immune plasma in the region where AHF is endemic. (+info)
Homologous and heterologous glycoproteins induce protection against Junin virus challenge in guinea pigs.
Tacaribe virus (TACV) is an arenavirus that is genetically and antigenically closely related to Junin virus (JUNV), the aetiological agent of Argentine haemorrhagic fever (AHF). It is well established that TACV protects experimental animals fully against an otherwise lethal challenge with JUNV. To gain information on the nature of the antigens involved in cross-protection, recombinant vaccinia viruses were constructed that express the glycoprotein precursor (VV-GTac) or the nucleocapsid protein (VV-N) of TACV. TACV proteins expressed by vaccinia virus were indistinguishable from authentic virus proteins by gel electrophoresis. Guinea pigs inoculated with VV-GTac or VV-N elicited antibodies that immunoprecipitated authentic TACV proteins. Antibodies generated by VV-GTac neutralized TACV infectivity. Levels of antibodies after priming and boosting with recombinant vaccinia virus were comparable to those elicited in TACV infection. To evaluate the ability of recombinant vaccinia virus to protect against experimental AHF, guinea pigs were challenged with lethal doses of JUNV. Fifty per cent of the animals immunized with VV-GTac survived, whereas all animals inoculated with VV-N or vaccinia virus died. Having established that the heterologous glycoprotein protects against JUNV challenge, a recombinant vaccinia virus was constructed that expresses JUNV glycoprotein precursor (VV-GJun). The size and reactivity to monoclonal antibodies of the vaccinia virus-expressed and authentic JUNV glycoproteins were indistinguishable. Seventy-two per cent of the animals inoculated with two doses of VV-GJun survived lethal JUNV challenge. Protection with either VV-GJun or VV-GTac occurred in the presence of low or undetectable levels of neutralizing antibodies to JUNV. (+info)
Genetic diversity of the Junin virus in Argentina: geographic and temporal patterns.
RNA was purified from 39 strains of cell-cultured Junin virus (JUN) from central Argentina, which included both human- and rodent-derived isolates (a total of 26 and 13, respectively), as well as from 2 laboratory JUN strains, XJ Cl3 and XJ #44. JUN-specific primers were used to amplify a 511-nucleotide (nt) fragment of the nucleocapsid protein gene and a 495-nt fragment of the glycoprotein 1 (GP1) gene. Genetic diversity among JUN strains studied was up to 13% at the nt level and up to 9% at the amino acid (aa) level for the GP1 gene and up to 9% (nt) and 4% (aa) for the NP gene. Phylogenetic analyses of both genes revealed three distinct clades. The first clade was composed of the JUN strains from the center of the endemic area and included the majority of JUN strains analyzed in the current study. The second clade contained 4 JUN strains isolated between 1963 and 1971 from Cordoba Province, the western-most edge of the known endemic area. The third clade contained 4 JUN strains that originated from Calomys musculinus trapped in Zarate, the northeastern edge of the known endemic area. Certain JUN sequences, which were obtained from GenBank and identified as XJ, XJ #44, and Candid #1 strains, appeared to form a separate clade. Over 400 nt of the GP1 and GP2 genes were additionally sequenced for 7 JUN strains derived from patients with different clinical presentations and outcomes of Argentine hemorrhagic fever. Analysis of the corresponding aa sequences did not allow us to attribute any particular genetic marker to the changing severity or clinical form of the human disease. (+info)
Zinc-binding properties of Junin virus nucleocapsid protein.
The arenavirus nucleocapsid protein (N) is a highly basic 63 kDa protein with a dual function during the virus life-cycle. First, it is involved in essential steps of genome replication, promoting the synthesis of the full-length antigenomic copy of S RNA, and second it associates with the genomic RNA to form the nucleocapsid. We have expressed the N protein of Junin virus in E. coli and shown that it binds zinc in vitro. This property is in agreement with the presence in the carboxy-terminal region of the N protein of the CX(2)HX(23)CX(4)C sequence, which resembles a classical zinc-finger motif. The specificity for zinc binding was demonstrated by competition with other divalent metal ions. The ability of the predicted motif to bind zinc was established by analysis of a series of N mutants, including truncated variants and amino acid substitutions. In addition, alternative zinc-binding sites were found. (+info)
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). (+info)
The signal peptide of the Junin 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 Junin 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 Junin 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 Junin 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 Junin 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. (+info)
Polarized entry and release of Junin 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. (+info)
Genetic analysis of heptad-repeat regions in the G2 fusion subunit of the Junin arenavirus envelope glycoprotein.
The G2 fusion subunit of the Junin virus envelope glycoprotein GP-C contains two hydrophobic heptad-repeat regions that are postulated to form a six-helix bundle structure required for the membrane fusion activity of Class I viral fusion proteins. We have investigated the role of these heptad-repeat regions and, specifically, the importance of the putative interhelical a and d position sidechains by using alanine-scanning mutagenesis. All the mutant glycoproteins were expressed and transported to the cell surface. Proteolytic maturation at the subtilisin kexin isozyme-1/site-1-protease (SKI-1/S1P) cleavage site was observed in all but two of the mutants. Among the adequately cleaved mutant glycoproteins, four positions in the N-terminal region (I333, L336, L347 and L350) and two positions in the C-terminal region (R392 and W395) were shown to be important determinants of cell-cell fusion. Taken together, our results indicate that alpha-helical coiled-coil structures are likely critical in promoting arenavirus membrane fusion. These findings support the inclusion of the arenavirus GP-C among the Class I viral fusion proteins and suggest pharmacologic and immunologic strategies for targeting arenavirus infection and hemorrhagic fever. (+info)