Recent developments on Junin virus, a causative agent for Argentine haemorrhagic fever.

Junin virus consists of ribonucleic acid as the genome and is responsible for a rapidly changing tendency of the virus. The virus is accountable for ailments in the human body and causes Argentine Haemorrhagic Fever (AHF). The infection is may be transmitted through contact between an infected animal/host and a person, and later between person to person. Prevention of outbreaks of AHF in humans can be a tough practice, as their occurrence is infrequent and unpredictable. In this review, recent information from the past 5 years available on the Junin virus including the risk of its emergence, infectious agents, its pathogenesis in humans, available diagnostic and therapeutic approaches, and disease management has been summarised. Altogether, this article would be highly significant in understanding the mechanistic basis behind virus interaction and other processes during the life cycle. Currently, no specific therapeutic options are available to treat the Junin virus infection. The information covered in this review could be important for finding possible treatment options for Junin virus infections.

[Sensitivity and specificity of the ELISA Kit for the detection of antidobies to Junin virus].

The goal of this work was to describe methodological approaches to determination of sensitivity and specificity of the enzyme-linked immunosorbent assay kit (ELISA Kit) for detection of the specific anti-Junin virus (JV) antibody. Comparison of ELISA to plaque reduction neutralization test (PRNT) showed direct relationship between antibody titers in the samples of serum of immunized animals, determined by either PRNT or ELISA methods. The obtained results provided an opportunity to form the panels of positive and negative serum samples to determine the sensitivity and specificity of the ELISA Kit. Sensitivity of the ELISA Kit was at least 98% when studying the samples of serum of immunized guinea pigs and rabbits (determined as positive in PRNT). The sensitivity of the ELISA Kit was at least 68% when studying the samples determined by PNRT as uncertain positive. The specificity was 98%. The specificity of the ELISA Kit was 98%.

[EXPERIENCE OF STUDY AND POSSIBLE WAYS OF ELIMINATION OF FALSE POSITIVE AND FALSE NEGATIVE RESULTS DURING EXECUTION OF POLYMERASE CHAIN REACTION ON AN EXAMPLE OF JUNIN VIRUS RNA DETECTION].

AIM: Experience of study and possible ways of elimination of false positive and false negative results during execution of polymerase chain reaction on an example of Junin virus RNA detection. MATERIALSS AND METHODS: Junin virus--causative agent of Argentine hemorrhagic fever (AHF) strain XJpR37/5787 was obtained from the State collection of pathogenicity group I causative agents of the 48th Central Research Institute. Reagent kit for detection of Junin virus RNA by RT-PCR was developed in the Institute and consists of 4 sets: for isolation of RNA, execution of reverse-transcription reaction, execution of PCR and electrophoretic detection of PCR products. RT-PCR was carried out by a standard technique. Continuous cell cultures of African green monkey Vero B, GMK-AH-1(D) were obtained from the museum of cell culture department of the Centre. RESULTS: An experimental study of the effect of various factors of impact on the sample under investigation ("thawing-freezing", presence of formaldehyde, heparin) on the obtaining of false negative results during Junin virus RNA detection by using RT-PCR was studied. Addition of 0.01% heparin to the samples was shown to completely inhibit PCR. Addition of 0.05% formaldehyde significantly reduces sensitivity of the method. A possibility of reduction of analysis timeframe from 15 to 5 days was shown during detection of the causative agent in samples with low concentration of the latter by growing the samples and subsequent analysis of the material obtained by using RT-PCR. CONCLUSION: During detection of causative agent by using RT-PCR false negative results could appear in the presence of formaldehyde and heparin in the sample. A possibility of elimination of false negative PCR results due to concentration of the causative agent in the sample under investigation at a level below sensitivity threshold was shown on the example of Junin virus RNA detection by using growing of the pathogen in appropriate accumulation system with subsequent analysis of the material obtained using PCR.

[Argentine hemorrhagic fever].

Features of the Argentine hemorrhagic fever are described in the review. Epidemiology, etiology, clinical presentation and pathogenesis of the disease are examined. Special consideration is given to the features of the pathological agent of Argentine hemorrhagic fever--the Junin virus. Features of the disease diagnostics are indicated--virological and serological studies, immunochemical and molecular-biological methods of identification of the pathological agent and antibodies against it. Approaches to etiotropic therapy of this disease and vaccination are examined. Based on the foreign experience perspective guidance for the creation of the system of protection of the population of the Russian Federation against Argentine hemorrhagic fever are presented.

Ocozocoautla de espinosa virus and hemorrhagic fever, Mexico.

Arenavirus RNA was isolated from Mexican deer mice (Peromyscus mexicanus) captured near the site of a 1967 epidemic of hemorrhagic fever in southern Mexico. Analyses of nucleotide and amino acid sequence data indicated that the deer mice were infected with a novel Tacaribe serocomplex virus (proposed name Ocozocoautla de Espinosa virus), which is phylogenetically closely related to Tacaribe serocomplex viruses that cause hemorrhagic fever in humans in South America.

Use of single-cycle infectious lymphocytic choriomeningitis virus to study hemorrhagic fever arenaviruses.

Several arenaviruses, chiefly Lassa virus (LASV) and Junin virus in West Africa and Argentina, respectively, cause hemorrhagic fever (HF) disease in humans that is associated with high morbidity and significant mortality. The investigation of antiviral strategies to combat HF arenaviruses is hampered by the requirement of biosafety level 4 (BSL-4) facilities to work with these viruses. These biosafety hurdles could be overcome by the use of recombinant single-cycle infectious arenaviruses. To explore this concept, we have developed a recombinant lymphocytic choriomeningitis virus (LCMV) (rLCMVΔGP/GFP) where we replaced the viral glycoprotein (GP) with the green fluorescent protein (GFP). We generated high titers of GP-pseudotyped rLCMVΔGP/GFP via genetic trans complementation using stable cell lines that constitutively express LCMV or LASV GPs. Replication of these GP-pseudotyped rLCMVΔGP/GFP viruses was restricted to GP-expressing cell lines. This system allowed us to rapidly and reliably characterize and quantify the neutralization activities of serum antibodies against LCMV and LASV within a BSL-2 facility. The sensitivity of the GFP-based microneutralization assay we developed was similar to that obtained with a conventionally used focus reduction neutralization (FRNT) assay. Using GP-pseudotyped rLCMVΔGP/GFP, we have also obtained evidence supporting the feasibility of this approach to identify and evaluate candidate antiviral drugs against HF arenaviruses without the need of BSL-4 laboratories.

Characterization of monoclonal antibodies to Junin virus nucleocapsid protein and application to the diagnosis of hemorrhagic fever caused by South American arenaviruses.

Junin virus (JUNV), Machupo virus, Guanarito virus, Sabia virus, and Chapare virus are members of New World arenavirus clade B and are the etiological agents of viral hemorrhagic fevers that occur in South America. In this study, we produced three monoclonal antibodies (MAbs) to the recombinant nucleocapsid protein of JUNV, designated C6-9, C11-12, and E4-2. The specificity of these MAbs was examined by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay, and an epitope-mapping method. Using these MAbs, we developed antigen (Ag) capture ELISA systems. We showed that by using MAb C6-9, JUNV Ag was specifically detected. On the other hand, by using MAb C11-12 or E-4-2, the Ags of all human pathogenic South American arenaviruses were detected. The combined use of these Ag capture ELISA systems in the present study may be useful for the diagnosis of acute-phase viral hemorrhagic fever due to infection by a South American arenavirus.

Argentine hemorrhagic fever diagnostic test based on recombinant Junín virus N protein.

Junín arenavirus is the etiologic agent of Argentine hemorrhagic fever. Due to its morbidity and high mortality rate in untreated cases, this endemic disease is of mandatory report in Argentina. Secure and accurate diagnostic methods are needed for the epidemiological surveillance of the disease. Current assays rely on antigens prepared from lysates of virus infected mammalian cells. The bio-safety issue related to the manipulation of large quantities of virus restricts such antigen production to laboratories with the appropriate containment facilities. In this report, we describe the development of an enzyme linked immunosorbent assay for the etiologic confirmation of the disease, based on recombinant antigens expressed in insect cells. Eight different variables of the assay were optimized with the Taguchi approach for experimental design (L18 design, seven three-level factors and one two-level factor). The area under the receiver operating characteristics (ROC) curve was 0.966, showing the high accuracy of the test discriminating positive from negative samples. Taking into account the biosafety benefits, the high yields of antigen in cell culture, and the general performance of the assay, it is expected that it will be a useful alternative to the current ELISA for the detection of antibodies in sera from convalescent patients.

Chapare virus, a newly discovered arenavirus isolated from a fatal hemorrhagic fever case in Bolivia.

A small focus of hemorrhagic fever (HF) cases occurred near Cochabamba, Bolivia, in December 2003 and January 2004. Specimens were available from only one fatal case, which had a clinical course that included fever, headache, arthralgia, myalgia, and vomiting with subsequent deterioration and multiple hemorrhagic signs. A non-cytopathic virus was isolated from two of the patient serum samples, and identified as an arenavirus by IFA staining with a rabbit polyvalent antiserum raised against South American arenaviruses known to be associated with HF (Guanarito, Machupo, and Sabiá). RT-PCR analysis and subsequent analysis of the complete virus S and L RNA segment sequences identified the virus as a member of the New World Clade B arenaviruses, which includes all the pathogenic South American arenaviruses. The virus was shown to be most closely related to Sabiá virus, but with 26% and 30% nucleotide difference in the S and L segments, and 26%, 28%, 15% and 22% amino acid differences for the L, Z, N, and GP proteins, respectively, indicating the virus represents a newly discovered arenavirus, for which we propose the name Chapare virus. In conclusion, two different arenaviruses, Machupo and Chapare, can be associated with severe HF cases in Bolivia.

High-throughput molecular detection of hemorrhagic fever virus threats with applications for outbreak settings.

Within the past dozen years, outbreaks of filoviral hemorrhagic fever within the human population have been occurring with increasing frequency, with an average of 1 epidemic now occurring every 1-2 years. Many of the outbreaks have been large (involving >150 cases), necessitating rapid responses from the international community to help implement infection control and surveillance. This increased activity, combined with today's climate of bioterrorism threats, has heightened the need for high-throughput methodologies for specific detection of these high-hazard viruses in sophisticated laboratory setups and mobile field laboratory situations. Using Zaire Ebola virus as an example, we describe here the development of a high-throughput protocol for RNA extraction and quantitative reverse-transcription polymerase chain reaction analysis that is safe, fast, and reliable. Furthermore, the applicability of this method to an outbreak setting was demonstrated by correct analysis of >500 specimens at a field laboratory established during a recent outbreak of Marburg hemorrhagic fever in Angola.

[Viral hemorrhagic fevers in Brazil]. / Febres hemorrágicas por vírus no Brasil.

To call attention to viral hemorrhagic fevers, diseases that are mostly underdivulged and, probably, underreported, we present here case reports of the 4 diseases of this kind that occur in Brazil: yellow fever, dengue haemorrhagic fever/dengue shock syndrome, arenavirus haemorrhagic fever and hantavirus cardiopulmonary syndrome. Relevant clinical, epidemiological and laboratorial diagnostic aspects of these viral haemorrhagic fevers are also shown here. These diseases have a high case fatality rate, induce capillary leaking and blood coagulation disturbances that are evidenced by hemoconcentration and thrombocytopenia. An early clinical diagnosis and treatment is fundamental for patient survival.

Febres hemorrágicas por vírus no Brasil / Viral hemorrhagic fevers in Brazil

Chamando a atencão para as febres hemorrágicas por vírus, que em sua maioria tem escassa informacão divulgada e provavelmente são subnotificadas, mostra-se neste artigo casos clínicos das 4 doencas deste tipo que ocorrem no Brasil: febre amarela, dengue hemorrágico/síndrome de choque do dengue, febre hemorrágica por arenavírus e síndrome pulmonar e cardiovascular por hantavírus. Também, relevantes aspectos clínicos, laboratoriais e epidemiológicos destas viroses são aqui abordados. São doencas que têm alta letalidade e induzem extravasamento capilar e coagulopatia, que podem ser evidenciados pela elevacão do hematócrito e plaquetopenia. A suspeita clínica e o tratamento precoce são fundamentais à sobrevida dos pacientes.

[Neutralization test for lymphocytic choriomeningitis virus for distinguishing between two arenavirus infections in Argentina]. / Prueba de neutralización del virus de la coriomeningitis linfocítica para diferenciar infecciones con dos arenavirus en Argentina.

The active coexistence of two pathogenic arenaviruses, Junin (JUNV) and lymphocytic choriomeningitis (LCMV), in the same region of Argentina, has been known since the early 70's, and records of clinical and subclinical human infections by one and/or the other agent have been continuously produced for the last 25 years. Anti-LCMV antibody is currently searched only by indirect immunofluorescence, a test that shows cross reactions among a number of arenaviruses yielding, in the cases of LCMV and JUNV consecutive infections, a concomitant seroconversion for both viruses, as an inconclusive diagnostic result. In contrast, neutralization (NT) tests reveal arenavirus antibodies directed to unique epitopes on these virus envelopes, thus allowing to disclose the sequence in the cases of consecutive infections. In this paper, the characteristics of neutralization (NT) test for LCMV in cell cultures are described, as well as its performance in the field diagnosis of LCMV human infections. The native LCMV strain Cba An 13065 was inoculated on L-929 cell (ATCC CCL 1), and procedures were followed to perform a constant virus-variable serum NT test. Final points of sera titrations were expressed as the maximal serum dilution that yielded 75% of pfu inhibition. This NT test was assayed on paired serum samples of 36 patients with confirmed Argentine hemorrhagic fever (AHF) (a disease caused by JUNV), who had had a known previous contact with LCMV through IFI. The use of this one test led to confusing diagnosis of the disease due to concomitant seroconversion for JUNV and LCMV. By using NT test, it was shown that: some of them were possibly not infected by LCMV, and that 30/36 cases (83.3%) had a pre-existing level of LCMV antibody, with titers in the range of 5 to 640, remaining unchanged 60 days after the clinical AHF. This shows that NT antibodies to LCMV are not influenced by the outcome of the immune response to JUNV, thus confirming the efficiency of NT test as identificator among arenaviruses. To assess the performance of this NT test in individuals having only IFI antibodies to LCMV, 126 serum samples obtained through serological surveillance in a rural area of Argentina, were used. It was found that NT had improved coincidence with IFI as IFI titers increased. Interpretations were based on the pan-arenavirus antibody response obtained by using IFI as the only test. Results presented herein prove that the described NT test is a valuable tool for the detection of LCMV infections, particularly when a previous infection with LCMV has to be demonstrated during the acute phase of Argentine hemorrhagic fever.

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.

Viral hemorrhagic fevers and hantavirus infections in the Americas.

Several arenaviruses and hantaviruses have been isolated in the Americas during the last 4 decades. These are rodent-borne viruses responsible for the South American hemorrhagic fevers (SAHF) and hantavirus pulmonary syndrome (HPS). Although rare, SAHF and HPS are serious illnesses with high mortality rates. Most viral isolates found in the Americas represent New World lineages of their respective viral families. Their presence in the Western hemisphere is likely ancient, their relationship with their rodent hosts is likely coevolutionary, and their recent detection forebodes the likelihood of detecting additional arena- and hantaviral species in the Americas.

Protective efficacy of a live attenuated vaccine against Argentine hemorrhagic fever. AHF Study Group.

Argentine hemorrhagic fever (AHF), caused by the arenavirus Junin, is a major public health problem among agricultural workers in Argentina. A prospective, randomized, double-blind, placebo-controlled, efficacy trial of Candid 1, a live attenuated Junin virus vaccine, was conducted over two consecutive epidemic seasons among 6500 male agricultural workers in the AHF-endemic region. Twenty-three men developed laboratory-confirmed AHF during the study; 22 received placebo and 1 received vaccine (vaccine efficacy 95%; 95% confidence interval [CI], 82%-99%). Three additional subjects in each group developed laboratory-confirmed Junin virus infection associated with mild illnesses that did not fulfill the clinical case definition for AHF, yielding a protective efficacy for prevention of any illness associated with Junin virus infection of 84% (95% CI, 60%-94%). No serious adverse events were attributed to vaccination. Candid 1, the first vaccine for the prevention of illness caused by an arenavirus, is safe and highly efficacious.

Evaluation of an enzyme immunosorbent assay for the diagnosis of Argentine haemorrhagic fever.

To elaborate a set of serological tests for the diagnosis of Argentine haemorrhagic fever (AHF), an enzyme-linked immunosorbent assay (ELISA) for detection of specific anti-Junin virus (JV) IgG is described, and its performance is compared with that of the plaque reduction neutralization test (PRNT). The reproducibility, sensitivity, specificity, and confidence limits for positive and negative results for ELISA were statistically analysed. The value of 800 was demonstrated as the lowest positive titer. Titers > or = 800 varied within one (two-fold) dilution in 95.6% of the tests, while the sensitivity and specificity were 99.2% and 98.8%, respectively. The assay yielded 1% of false positives and 0.05% of false negatives. A comparison of ELISA to PRNT in detecting the seroconversion for JV was studied by the chi square test (comparison of proportions in paired samples) and the K parameter for agreement proportion. Comparison of ELISA to PRNT showed no significant difference in the proportions of positive and negative results of these assays (P < 0.01), demonstrating an equivalent performance (K = 0.98) in the diagnosis of AHF. In addition, the simplicity and safety of the procedures involved make this ELISA the most suitable test to detect natural human JV infections.

Detección retrospectiva de infecciones clínicas por hantavirus en la Argentina / Retrospective detection of hantavirus clinical infections in Argentina

La actividad de los hantavirus en la Argentina en roedores y humanos ya era conocida desde la década de 1980. En este trabajo, se investigó retrospectivamente la infección por hantavirus entre los pacientes notificados para Fiebre Hemorrágica Argentina (FHA) entre 1987 y 1994, sin confirmación virológica. La detección de anticuerpos IgG e IgM antihantavirus se realizó por técnica de ELISA. Entre 1028 pacientes estudiados se encontraron 13 infecciones recientes (1,26 por ciento) y 13 remotas (l,26 por ciento). Entre 745 personas sanas procedentes de las mismas localidades de los casos de infección reciente, sólo se encontraron anticuerpos IgG en una (O,13 por ciento). De las 13 infecciones recientes, 9 se presentaron como FHSR y 4 como SPH. Se realizó una comparación clínica y epidemiológica entre los 9 pacientes con FHSR y dos grupos control apareados: uno de casos de FHA y otros considerados Síndrome febril de etiología indeterminada (SFEI), que fueron negativos para Hantaan, Junín y Linfocoriomeningits (LCM). No se encontraron diferencias entre los signos y sintomas clínicos. Para el diagnóstico diferencial resulto sugerente la presencia de leucocitos normales o aumentados, con plaquetopenia, hemoconcentración, creatininemia aumentada y proteinuria en las FHSR. Estos resultados muestran la coexistência dentro del área endémica de FHA de virus Junín y hantavirus, e indican la necesidad de incluir a las infecciones por estos virus en el diagnóstico diferencial de las Fiebres Hemorrágicas y de los Síndromes de Distrés Respiratorio de etiología desconocida. La variabilidad clínica encontrada sugiere la presencia de más de un serotipo de hantavirus en nuestro país.

Detección retrospectiva de infecciones clínicas por hantavirus en la Argentina / Retrospective detection of hantavirus clinical infections in Argentina

La actividad de los hantavirus en la Argentina en roedores y humanos ya era conocida desde la década de 1980. En este trabajo, se investigó retrospectivamente la infección por hantavirus entre los pacientes notificados para Fiebre Hemorrágica Argentina (FHA) entre 1987 y 1994, sin confirmación virológica. La detección de anticuerpos IgG e IgM antihantavirus se realizó por técnica de ELISA. Entre 1028 pacientes estudiados se encontraron 13 infecciones recientes (1,26 por ciento) y 13 remotas (l,26 por ciento). Entre 745 personas sanas procedentes de las mismas localidades de los casos de infección reciente, sólo se encontraron anticuerpos IgG en una (O,13 por ciento). De las 13 infecciones recientes, 9 se presentaron como FHSR y 4 como SPH. Se realizó una comparación clínica y epidemiológica entre los 9 pacientes con FHSR y dos grupos control apareados: uno de casos de FHA y otros considerados Síndrome febril de etiología indeterminada (SFEI), que fueron negativos para Hantaan, Junín y Linfocoriomeningits (LCM). No se encontraron diferencias entre los signos y sintomas clínicos. Para el diagnóstico diferencial resulto sugerente la presencia de leucocitos normales o aumentados, con plaquetopenia, hemoconcentración, creatininemia aumentada y proteinuria en las FHSR. Estos resultados muestran la coexistÛncia dentro del área endémica de FHA de virus Junín y hantavirus, e indican la necesidad de incluir a las infecciones por estos virus en el diagnóstico diferencial de las Fiebres Hemorrágicas y de los Síndromes de Distrés Respiratorio de etiología desconocida. La variabilidad clínica encontrada sugiere la presencia de más de un serotipo de hantavirus en nuestro país. (AU)