[Spatial difference in the incidence of Argentine Hemorrhagic Fever and composition and abundance of rodents in the assemblage]. / Diferencia espacial de la incidencia de fiebre hemorrágica argentina y la composición y abundancia de roedores en el ensamble.

BACKGROUND: The Argentine Hemorrhagic Fever (AHF) is a zoonotic disease endemic in a wide area of the humid pampa of Argentina. The etiologic agent is the Junin virus that is maintained in the wild by the rodent Calomys musculinus and transmitted to humans, mainly, through aerosols generated from secretions and excretions. AIMS: To characterize and compare the assemblages of small rodent composition and diversity inside the epidemic, historic and non-endemic zone of AHF and to register C. musculinus abundance in each zone and in each area within each zone, registering the prevalence of infection in rodent populations. METHOD: One central and two peripheral areas were delimited to sample rodents in each zone with different incidence of AHF. Thus, 18 localities were selected to do the sampling in two years. Host abundance between zones and among areas inside each zone and among nearby areas between zones were compared applying nested ANOVA's. RESULTS: In each zone, the rodent assemblage showed differences in composition, diversity and numeric representation of C. musculinus. The epidemic zone was the richest of the three, registering also great host abundance; meanwhile in the historic zone, A. azarae was the dominant numeric species with less number of other species. Regarding the non-endemic zone, the assemblage composition and C. musculinus abundance varied respect the sampled year. Junin virus infection was only detected in C. musculinus individuals corresponding to the epidemic zone, with a prevalence of 2.7 and 1.1% for the years 2007 and 2008, respectively. CONCLUSION: In this system, the abundance of C. musculinus could be impacting over the pathogen dynamic, rather than the assemblage diversity or the A. azarae presence.

Diferencia espacial de la incidencia de fiebre hemorrágica argentina y la composición y abundancia de roedores en el ensamble / Spatial difference in the incidence of Argentine Hemorrhagic Fever and composition and abundance of rodents in the assemblage

Resumen La fiebre hemorrágica argentina (FHA) es una enfermedad zoonótica endémica en una amplia zona de la pampa húmeda de Argentina. El agente etiológico es el virus Junin que es mantenido en la naturaleza por el roedor Calomys musculinus y transmitido, principalmente, al humano a través de aerosoles generados de las secreciones y excreciones. Objetivos: Caracterizar la composición y diversidad de los ensambles de pequeños roedores, determinar la abundancia del hospedador C. musculinus y la prevalencia del virus de la FHA en las zonas epidémica, histórica y no endémica de dicha enfermedad en Argentina. Métodos: Para el muestreo de roedores en cada una de las zonas se demarcaron un área central y dos periféricas para 18 localidades de la región central de Argentina (incluyendo las provincias de Córdoba, Buenos Aires y Santa Fe) muestreadas en dos años. Se comparó la abundancia de C. musculinus entre zonas y entre las áreas dentro de cada zona y áreas cercanas entre zonas, utilizando modelos de análisis de varianza anidados. Resultados. Dentro de cada zona, el ensamble de roedores mostró diferencia espacial en la composición específica, diversidad y abundancia de C. musculinus. La zona epidémica registró mayor número de especies y mayor abundancia del hospedador. En zona histórica se capturó el menor número de especies (de roedores) y Akodon azarae fue la más abundante. En zona no endémica la composición del ensamble y la abundancia de C. musculinus variaron entre los dos años. Sólo se detectó infección por virus Junin en C. musculinus correspondientes a la zona epidémica con una prevalencia de 2,7 y 1,1% para los años 2007 y 2008, respectivamente. Conclusión: En este sistema, la abundancia del hospedador estaría afectando la dinámica espacial de este virus, más que la diversidad del ensamble o la presencia de A. azarae.

Background. The Argentine Hemorrhagic Fever (AHF) is a zoonotic disease endemic in a wide area of the humid pampa of Argentina. The etiologic agent is the Junin virus that is maintained in the wild by the rodent Calomys musculinus and transmitted to humans, mainly, through aerosols generated from secretions and excretions. Aims: To characterize and compare the assemblages of small rodent composition and diversity inside the epidemic, historic and non-endemic zone of AHF and to register C. musculinus abundance in each zone and in each area within each zone, registering the prevalence of infection in rodent populations. Method: One central and two peripheral areas were delimited to sample rodents in each zone with different incidence of AHF. Thus, 18 localities were selected to do the sampling in two years. Host abundance between zones and among areas inside each zone and among nearby areas between zones were compared applying nested ANOVA's. Results: In each zone, the rodent assemblage showed differences in composition, diversity and numeric representation of C. musculinus. The epidemic zone was the richest of the three, registering also great host abundance; meanwhile in the historic zone, A. azarae was the dominant numeric species with less number of other species. Regarding the non-endemic zone, the assemblage composition and C. musculinus abundance varied respect the sampled year. Junin virus infection was only detected in C. musculinus individuals corresponding to the epidemic zone, with a prevalence of 2.7 and 1.1% for the years 2007 and 2008, respectively. Conclusion: In this system, the abundance of C. musculinus could be impacting over the pathogen dynamic, rather than the assemblage diversity or the A. azarae presence.

Effective Population Size Differences in Calomys musculinus, the Host of Junín Virus: Their Relationship with the Epidemiological History of Argentine Hemorrhagic Fever.

Argentine hemorrhagic fever (AHF) is a serious endemic disease in Argentina, produced by Junín virus, whose host is the Sigmodontinae rodent Calomys musculinus. Within the endemic area, human incidence and proportion of infected rodents remains high for 5-10 years after the first appearance of the disease (epidemic [E] zone) and then gradually declines to sporadic cases (historic [H] zone). We tested the hypothesis that host populations within the E zone are large and well connected by gene flow, facilitating the transmission and maintenance of the virus, whereas those in the H and nonendemic (NE) zones are small and isolated, with the opposite effect. We estimated parameters affected by levels of gene flow and population size in 14 populations of C. musculinus: population effective size (Ne), genetic variability, and mean relatedness. Our hypothesis was not supported: the lowest levels of variability and of Ne and the highest genetic relatedness among individuals were found in the H zone. Populations from the NE zone displayed opposite results, whereas those in the E zone showed intermediate values. If we consider that populations are first NE, then E, and finally H, a correlative decrease in Ne was observed. Chronically infected females have a low reproductive success. We propose that this would lower Ne because each cohort would originate from a fraction of females of the previous generation, and affect other factors such as proportion of individuals that develop acute infection, probability of viral transmission, and evolution of virulence, which would explain, at least partly, the changing incidence of AHF.

A Proteomics Survey of Junín Virus Interactions with Human Proteins Reveals Host Factors Required for Arenavirus Replication.

Arenaviruses are negative-strand, enveloped RNA viruses that cause significant human disease. In particular, Junín mammarenavirus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. At present, little is known about the cellular proteins that the arenavirus matrix protein (Z) hijacks to accomplish its various functions, including driving the process of virus release. Furthermore, there is little knowledge regarding host proteins incorporated into arenavirus particles and their importance for virion function. To address these deficiencies, we used mass spectrometry to identify human proteins that (i) interact with the JUNV matrix protein inside cells or within virus-like particles (VLPs) and/or (ii) are incorporated into bona fide JUNV strain Candid#1 particles. Bioinformatics analyses revealed that multiple classes of human proteins were overrepresented in the data sets, including ribosomal proteins, Ras superfamily proteins, and endosomal sorting complex required for transport (ESCRT) proteins. Several of these proteins were required for the propagation of JUNV (ADP ribosylation factor 1 [ARF1], ATPase, H+ transporting, lysosomal 38-kDa, V0 subunit d1 [ATP6V0D1], and peroxiredoxin 3 [PRDX3]), lymphocytic choriomeningitis mammarenavirus (LCMV) (Rab5c), or both viruses (ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide [ATP5B] and IMP dehydrogenase 2 [IMPDH2]). Furthermore, we show that the release of infectious JUNV particles, but not LCMV particles, requires a functional ESCRT pathway and that ATP5B and IMPDH2 are required for JUNV budding. In summary, we have provided a large-scale map of host machinery that associates with JUNV and identified key human proteins required for its propagation. This data set provides a resource for the field to guide antiviral target discovery and to better understand the biology of the arenavirus matrix protein and the importance of host proteins for virion function.IMPORTANCE Arenaviruses are deadly human pathogens for which there are no U.S. Food and Drug Administration-approved vaccines and only limited treatment options. Little is known about the host proteins that are incorporated into arenavirus particles or that associate with its multifunctional matrix protein. Using Junín mammarenavirus (JUNV), the causative agent of Argentine hemorrhagic fever, as a model organism, we mapped the human proteins that are incorporated into JUNV particles or that associate with the JUNV matrix protein. Functional analysis revealed host machinery that is required for JUNV propagation, including the cellular ESCRT pathway. This study improves our understanding of critical arenavirus-host interactions and provides a data set that will guide future studies to better understand arenavirus pathogenesis and identify novel host proteins that can be therapeutically targeted.

Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics.

BACKGROUND: We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics. METHODOLOGY: An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients. PRINCIPAL FINDINGS: The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1-10 genome equivalents) and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours. CONCLUSIONS: Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring.

[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.

[Phenotypic markers of attenuation in Junin virus strains recently isolated from individuals vaccinated with Junin Candid#1 strain]. / Marcadores fenotípicos de atenuación en cepas de virus Junín recuperadas de individuos vacunados con la cepa Junín Candid#1.

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.

Marcadores fenotípicos de atenuación en cepas de virus Junín recuperadas de individuos vacunados con la cepa Junín Candid#1 / Phenotypic markers of attenuation in Junin virus strains recently isolated from individuals vaccinated with Junin Candid#1 strain

La Fiebre Hemorrágica Argentina es una enfermedad producida por el virus Junín. Para la prevención de esta enfermedad se obtuvo una vacuna efectiva denominada Candid#1. Durante un ensayo clínico realizado en el INEVH, dos cepas de virus Junín fueron aisladas de sangre periférica de dos voluntarios mediante co-cultivo de células mononucleares. El objetivo de este trabajo fue comparar las características fenotípicas de atenuación de esas dos cepas recuperadas de humanos con las de la vacuna Candid#1 utilizando los indicadores de atenuación desarrollados por Contigiani y Sabattini en 1977. A tal fin se midieron los índices de letalidad, infección y protección en cobayos y ratones de diferentes edades. Las tres cepas investigadas resultaron letales para ratones recién nacidos pero no para ratones de 10 a 12 días, ratones adultos ni cobayos, aun a la más baja dilución inoculada. Los cobayos inoculados con las cepas recuperadas de humanos y con la cepa Candid#1 no presentaron síntomas de enfermedad y mostraron estar protegidos cuando fueron desafiados con una cepa patógena. Los índices de infección y de protección hallados indican que estas cepas poseen elevada capacidad infectante y protectora en las especies animales aquí estudiadas. Estos resultados demuestran que las cepas de virus Junín aisladas de voluntarios inmunizados con Candid#1 mantienen el mismo fenotipo atenuado de la vacuna Candid#1 después de un pasaje por humanos.

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.

Marcadores fenotípicos de atenuación en cepas de virus Junín recuperadas de individuos vacunados con la cepa Junín Candid#1 / Phenotypic markers of attenuation in Junin virus strains recently isolated from individuals vaccinated with Junin Candid#1 strain

La Fiebre Hemorrágica Argentina es una enfermedad producida por el virus Junín. Para la prevención de esta enfermedad se obtuvo una vacuna efectiva denominada Candid#1. Durante un ensayo clínico realizado en el INEVH, dos cepas de virus Junín fueron aisladas de sangre periférica de dos voluntarios mediante co-cultivo de células mononucleares. El objetivo de este trabajo fue comparar las características fenotípicas de atenuación de esas dos cepas recuperadas de humanos con las de la vacuna Candid#1 utilizando los indicadores de atenuación desarrollados por Contigiani y Sabattini en 1977. A tal fin se midieron los índices de letalidad, infección y protección en cobayos y ratones de diferentes edades. Las tres cepas investigadas resultaron letales para ratones recién nacidos pero no para ratones de 10 a 12 días, ratones adultos ni cobayos, aun a la más baja dilución inoculada. Los cobayos inoculados con las cepas recuperadas de humanos y con la cepa Candid#1 no presentaron síntomas de enfermedad y mostraron estar protegidos cuando fueron desafiados con una cepa patógena. Los índices de infección y de protección hallados indican que estas cepas poseen elevada capacidad infectante y protectora en las especies animales aquí estudiadas. Estos resultados demuestran que las cepas de virus Junín aisladas de voluntarios inmunizados con Candid#1 mantienen el mismo fenotipo atenuado de la vacuna Candid#1 después de un pasaje por humanos.(AU)

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.(AU)

[Optimization of conditions of quantitative evaluation of argentine hemorrhagic fever causative agent].

AIM: Optimization of conditions of quantitative evaluation of Argentine hemorrhagic fever causative agent. MATERIALS AND METHODS: Junin virus (XJ P37 strain) was obtained from National collection of viral hemorrhagic fever causative agents of the 1st pathogenicity group of the 33rd Central Research Testing Institute. Junin virus (XJ P37 strain) culture with biological activity of 5.2 lg PFUxm(-1) was used in the experiments. Vero B, 6619-1(D) and GMK-AH-1(D) were obtained from collection of cell culture of the Research Scientific Centre of the 33rd Central Research Testing Institute. Calculation of biological activity of Junin virus during titration in cell cultures was carried out by Kerber method with modification by I.P. Ashmarin. RESULTS: During incubation for 4 - 7 days after the infection of cell monolayer the determined biological activity was 4.4 - 6.4 lg PFUxml(-1); the size of the formed negative colonies--(1.5 +/- 0.5) mm. CONCLUSION: The conditions of quantitative evaluation of Argentine hemorrhagic fever were optimized by negative colonies method (using 5 - 7 day Vero B cell culture monolayer with staining of monolayer on day 5 of secondary incubation, recording of results at day 7 after the infection).

Marcadores fenotípicos de atenuación en cepas de virus Junín recuperadas de individuos vacunados con la cepa Junín Candid#1. / [Phenotypic markers of attenuation in Junin virus strains recently isolated from individuals vaccinated with Junin Candid#1 strain].

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.

Effect of ribavirin on junin virus infection in guinea pigs.

Junin virus (JUNV) is the aetiological agent of Argentine haemorrhagic fever. The pathogenesis of the infection is not well understood, no licensed vaccines exist and no specific antiviral therapy is available. Previous studies have demonstrated the ability of ribavirin to delay and reduce JUNV disease and virus burden in guinea pigs without preventing death. Based on available data, we performed three different studies to determine the efficacy of ribavirin against JUNV in the guinea pig model with a focus on survival. Different doses and treatment schedules of ribavirin were tested in a lethal model of JUNV infection. Our results show that prolonged treatment with high doses of ribavirin significantly reduces the mortality in guinea pigs infected with JUNV. These results may be useful in future experimental studies or clinical testing.

Viral diversity of Junín virus field strains.

The Argentine Hemorrhagic Fever, an endemic disease present in a much of Argentina, is caused by the Junín virus (JUNV). Currently, there are sequences available from several strains of this virus, like those belonging to the vaccine lineage (XJ13, XJ#44 and Candid#1), as well as MC2 (rodent isolate) and IV4454 (human isolate). In this article, we report sequence information on two fragments of genomic segment S of viral isolates from the endemic area. A Nested-RT-PCR was used to amplify discrete genomic regions of 13 isolates of rodent and human origin. The bioinformatics studies revealed a great homogeneity of sequences among the JUNV isolates. The phylogenetic classification showed greater evolutionary distance between the old world arenaviruses (Lassa and LCM virus) than between the new world arenaviruses (JUNV and Machupo virus).

Superinfection exclusion in BHK-21 cells persistently infected with Junín virus.

We characterized a persistently Junín virus (JUNV)-infected BHK-21 cell line obtained by experimental infection with the XJCl3 strain. This cell line, named K3, produced low levels of virus in supernatants which were not influenced by the presence of defective interfering (DI) particles after the first year of infection. K3 cells were able to exclude superinfection of the homologous JUNV and the antigenically related Tacaribe virus (TCRV), whereas the non-related arenaviruses lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PICV) could replicate normally. Although superinfecting virus binding and internalization to persistently infected cells were slightly reduced, earlier biosynthesis of antigenomic RNA was observed in comparison with BHK-21 cells. Despite the fact that superinfection did not increase the number of cells expressing viral antigens, de novo synthesis of superinfecting virus proteins was detected. The virus produced by JUNV-superinfected K3 cells remained mostly cell-associated in the form of particles tethered to the plasma membrane and aberrant tubular structures. JUNV restriction was correlated with an overexpression of cellular protein TSG101 in K3 cells, which has been pointed out as involved in the budding of several RNA viruses. This correlation was also observed in a cell clone isolated from K3. Reduction of TSG101 expression favoured the release of infectious virus to the supernatant of JUNV-superinfected K3 cells. Our data suggest that overexpression of TSG101 in K3 cells is a novel mechanism that may contribute, along with a diminished synthesis of superinfecting virus proteins, to explain superinfection exclusion in persistently arenavirus-infected cells.

Arenaviruses other than Lassa virus.

The family Arenaviridae includes 23 viral species, of which 5 can cause viral hemorrhagic fevers with a case fatality rate of about 20%. These five viruses are Junin, Machupo, Guanarito, Sabia and Lassa virus, the manipulation of which requires biosafety level 4 facilities. They are included in the Category A Pathogen List established by the Center for Disease Control and Prevention that groups agents with the greatest potential for adverse public health impact and mass casualties whether a situation characterized by a ill-intentioned abuse of natural or engineered arenavirus would be encountered. The aims of this article are to (i) summarize the current situation; (ii) provide information to help anticipating the effects to be expected in such a situation; and to (iii) emphasize the need for fundamental research to allow the development of diagnostic, prevention and therapeutic tools as countermeasures to weaponized arenaviruses.

[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.

Analysis of viral glycoproteins by glycosidic digestion inside a polyacrylamide gel.

We adapted the method described by Cleveland et al. (1977); (Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102-1106) to study the glycosidic residues linked to the viral glycoproteins of two enveloped viruses: Junin virus (JV) and rubella virus (RV). Radioiodinated glycoproteins were obtained from purified virions, isolated from SDS-polyacrylamide gels and then hydrolysed by specific glycosidases inside a second gel. N-linked oligosaccharides, mannose and galactose were found as terminal residues in the JV-G1 glycoprotein. Mannose and N-glycans of complex hybrid type were present on RV glycoproteins.

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.

In vitro anti-Junin virus activity of a peptide isolated from Melia azedarach L. leaves.

Meliacine, a peptide isolated from leaves of Melia azedarach L. inhibited the multiplication of Junin virus in Vero cells treated with the compound before infection (pre-treatment) or immediately after virus adsorption. Analysis of early events following infection demonstrated that meliacine blocks virus penetration by preventing the uncoating step. The addition of meliacine at different times after infection indicated that meliacine also interferes with the release of infectious particles to the extracellular medium and inhibits the low-pH-induced fusion of infected cells. Intracellular transport of viral glycoproteins to the cell membrane was not affected by meliacine, as revealed by immunofluorescence staining. Taken together, these results suggest that meliacine affects two events of the virus replicative cycle that require membrane fusion: uncoating and budding.

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.