Reemergencia de la encefalitis equina del oeste (WEEV) en la Argentina: una revisión de aspectos epidemiológicos, virológicos y clínicos de relevancia / Reemergence of western equine encephalitis in Argentina

La encefalitis equina del oeste (WEEV, por su sigla en inglés, Western Equine Encephalitis) es una enfermedad reemergente en Argentina a partir del año 2023. La co-municación inicial fue en 1933, las últimas epizootias ocurrieron en 1983 y el último caso humano se registró en 1996. Se revisan las características del agente causal, la ecología con especial referencia a los vectores iden-tificados en el país, su competencia en la transmisión y el ciclo así como los factores de riesgo para adquirir la enfermedad. La situación epidemiológica en equinos y humanos desde noviembre 2023 hasta marzo 2024 es analizada. Se describen las formas clínicas de presen-tación de la enfermedad humana, las posibilidades evo-lutivas, los datos disponibles en los casos confirmados y el tratamiento. La metodología y algoritmo empleados para el diagnóstico etiológico en el Centro Nacional de Referencia son detallados. Las estrategias para la pre-vención y el control se basan en la vacunación de los equinos, el saneamiento ambiental y el control del foco ante la presentación de la enfermedad animal (vigilancia epidemiológica activa)

Western equine encephalitis (WEE) is a re-emerging dis-ease in Argentina starting in 2023. Since the initial notifi-cation in 1933, the last epizootics occurred in 1983, and the last human case was recorded in 1996.The charac-teristics of the causative agent, the ecology with special reference to vectors identified in the country, their compe-tence in transmission, and the cycle as well as the risks factors for acquiring the disease, are reviewed.The epidemiological situation in horses and humans from November 2023 to March 2024 is analyzed. The clinical presentation of the human disease, its evolutionary po-tential, available data in confirmed cases, and the treat-ment are described.The methodology and algorithm used for the etiological diagnosis at the National Reference Center are detailed. Strategies for prevention and control are based on vaccination of horses, environmental sani-tation and outbreak control in the presence of the animal disease (active epidemiological surveillance)

Superinfection exclusion studies using West Nile virus and Culex flavivirus strains from Argentina.

In Argentina, many Flavivirus were recognised including West Nile virus (WNV). During 2009 several strains of Culex Flavivirus (CxFV), an insect-specific flavivirus, were isolated in the same region where circulation of WNV was detected. Hence, the objective of this study was to analyse the effect of co-infection in vitro assays using CxFV and WNV Argentinean strains in order to evaluate if CxFV could affect WNV replication. Our results showed that WNV replication was suppressed when multiplicity of infection (MOI) for CxFV was 10 or 100 times higher than WNV. Nevertheless, in vivo assays are necessary in order to evaluate the superinfection exclusion potential.

Superinfection exclusion studies using West Nile virus and Culex flavivirus strains from Argentina

In Argentina, many Flavivirus were recognised including West Nile virus (WNV). During 2009 several strains of Culex Flavivirus (CxFV), an insect-specific flavivirus, were isolated in the same region where circulation of WNV was detected. Hence, the objective of this study was to analyse the effect of co-infection in vitro assays using CxFV and WNV Argentinean strains in order to evaluate if CxFV could affect WNV replication. Our results showed that WNV replication was suppressed when multiplicity of infection (MOI) for CxFV was 10 or 100 times higher than WNV. Nevertheless, in vivo assays are necessary in order to evaluate the superinfection exclusion potential.

Vector competence of Aedes aegypti for different strains of Zika virus in Argentina.

The importance of Zika virus (ZIKV) has increased noticeably since the outbreak in the Americas in 2015, when the illness was associated with congenital disorders. Although there is evidence of sexual transmission of the virus, the mosquito Aedes aegypti is believed to be the main vector for transmission to humans. This species of mosquito has not only been found naturally infected with ZIKV, but also has been the subject of study in many vector competence assays that employ different strains of ZIKV around the world. In Argentina, the first case was reported in February 2016 and a total of 278 autochthonous cases have since been confirmed, however, ZIKV virus has not been isolated from any mosquito species yet in Argentina. In order to elucidate if Argentinian Ae. aegypti populations could be a possible vector of ZIKV, we conducted vector competence studies that involved a local strain of ZIKV from Chaco province, and a Venezuelan strain obtained from an imported case. For this purpose, Ae. aegypti adults from the temperate area of Argentina (Buenos Aires province) were fed with infected blood. Body, legs and saliva were harvested and tested by plaque titration on plates of Vero cells for ZIKV at 7, 11 and 14 days post infection (DPI) in order to calculate infection, transmission, and dissemination rates, respectively. Both strains were able to infect mosquitoes at all DPIs, whereas dissemination and transmission were observed at all DPIs for the Argentinian strain but only at 14 DPI for the Venezuelan strain. This study proves the ability of Ae. aegypti mosquitoes from Argentina to become infected with two different strains of ZIKV, both belonging to the Asian lineage, and that the virus can disseminate to the legs and salivary glands.

Assessing cross-reactivity of Junín virus-directed neutralizing antibodies.

Arenaviruses cause several viral hemorrhagic fevers endemic to Africa and South America. The respective causative agents are classified as biosafety level (BSL) 4 pathogens. Unlike for most other BSL4 agents, for the New World arenavirus Junín virus (JUNV) both a highly effective vaccination (Candid#1) and a post-exposure treatment, based on convalescent plasma transfer, are available. In particular, neutralizing antibodies (nAbs) represent a key protective determinant in JUNV infection, which is supported by the correlation between successful passive antibody therapy and the levels of nAbs administered. Unfortunately, comparable resources for the management of other closely related arenavirus infections are not available. Given the significant challenges inherent in studying BSL4 pathogens, our goal was to first assess the suitability of a JUNV transcription and replication-competent virus-like particle (trVLP) system for measuring virus neutralization under BSL1/2 conditions. Indeed, we could show that infection with JUNV trVLPs is glycoprotein (GP) dependent, that trVLP input has a direct correlation to reporter readout, and that these trVLPs can be neutralized by human serum with kinetics similar to those obtained using authentic virus. These properties make trVLPs suitable for use as a proxy for virus in neutralization assays. Using this platform we then evaluated the potential of JUNV nAbs to cross-neutralize entry mediated by GPs from other arenaviruses using JUNV (strain Romero)-based trVLPs bearing GPs either from other JUNV strains, other closely related New World arenaviruses (e.g. Tacaribe, Machupo, Sabiá), or the distantly related Lassa virus. While nAbs against the JUNV vaccine strain are also active against a range of other JUNV strains, they appear to have little or no capacity to neutralize other arenavirus species, suggesting that therapy with whole plasma directed against another species is unlikely to be successful and that the targeted development of cross-specific monoclonal antibody-based resources is likely needed. Such efforts will be supported by the availability of this BSL1/2 screening platform which provides a rapid and easy means to characterize the potency and reactivity of anti-arenavirus neutralizing antibodies against a range of arenavirus species.

[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.

[Outbreak of hantavirus pulmonary syndrome in Tucumán, Argentina]. / Brote de síndrome pulmonar por hantavirus, Tucumán, Argentina.

We describe an outbreak of hantavirus pulmonary syndrome in the Burruyacú Department, Province of Tucumán. The detection in 2016 of a case of hantavirosis affecting a 23-year-old woman, considered at that time to be the first case occurred in that province, promoted a thorough epidemiological study. The investigation allowed the retrospective detection of another case occurred one month earlier in a 5-year-old child in the same Department. In both cases, the infection was confirmed by serology (case 1 at days 4 and 7 of disease onset, case 2 at day 4) and the viral genotype was characterized as HU39694. The contacts of both cases were serologically negative for hantavirus. The rodents captured in the area belonged to genus Akodon, genus Calomys and species Mus musculus. Oligoryzomys, the known reservoir for this viral genotype, was not found. Specific anti-hantavirus antibodies were not detected in the captured rodents. Given that the patients had not visited hantavirus endemic areas and their contacts were negative for hantavirus, we infer that the patients were locally exposed to fluids of infected rodents during their usual social or recreational outdoor activities. In conclusion, we demonstrate that hantavirus HU39694 -a genotype until now considered to be restricted to the Central Pampas of the country- is circulating in the North Western province of Tucumán. The endemic area of hantavirosis is thus expanded to this province but the viral reservoir in the area has not yet been identified.

Brote de síndrome pulmonar por hantavirus, Tucumán, Argentina / Outbreak of hantavirus pulmonary syndrome in Tucumán, Argentina

Se describe un brote de síndrome pulmonar por hantavirus en el departamento de Burruyacú, provincia de Tucumán. La detección en 2016 de un caso de hantavirosis en una joven de 23 años -en ese momento considerado el primero ocurrido en dicha provincia- promovió un estudio epidemiológico exhaustivo, que permitió detectar retrospectivamente otro caso ocurrido en un niño de 5 años, un mes antes, en el mismo departamento. La infección fue confirmada por serología en ambos casos (caso 1 en muestras de 4 y 7 días de evolución, caso 2 en muestra a los 4 días). En ambos casos el genotipo viral fue caracterizado como HU39694 y los contactos fueron serológicamente negativos. En las áreas fueron identificados roedores pertenecientes a los géneros Akodon y Calomys y a la especie Mus musculus, pero no a Oligoryzomys, el reservorio habitual del genotipo HU39694. Tampoco se detectaron anticuerpos anti-hantavirus en suero de los roedores capturados. La ausencia de registro de viajes a área endémica de este genotipo y los hábitos recreacionales de los pacientes, sumados a los resultados serológicos negativos para hantavirus en los contactos, permiten inferir la posible exposición de los pacientes a fluidos de roedores infectados durante actividades recreativas o sociales al aire libre en sus respectivas áreas de residencia. En conclusión, se demuestra la circulación en Tucumán del genotipo viral HU39694, hasta ahora considerado restringido a la región pampeana central. Se extiende así a Tucumán el área endémica de hantavirosis, pero no se identificó el reservorio en el área.

We describe an outbreak of hantavirus pulmonary syndrome in the Burruyacú Department, Province of Tucumán. The detection in 2016 of a case of hantavirosis affecting a 23-year-old woman, considered at that time to be the first case occurred in that province, promoted a thorough epidemiological study. The investigation allowed the retrospective detection of another case occurred one month earlier in a 5-year-old child in the same Department. In both cases, the infection was confirmed by serology (case 1 at days 4 and 7 of disease onset, case 2 at day 4) and the viral genotype was characterized as HU39694. The contacts of both cases were serologically negative for hantavirus. The rodents captured in the area belonged to genus Akodon, genus Calomys and species Mus musculus. Oligoryzomys, the known reservoir for this viral genotype, was not found. Specific anti-hantavirus antibodies were not detected in the captured rodents. Given that the patients had not visited hantavirus endemic areas and their contacts were negative for hantavirus, we infer that the patients were locally exposed to fluids of infected rodents during their usual social or recreational outdoor activities. In conclusion, we demonstrate that hantavirus HU39694 -a genotype until now considered to be restricted to the Central Pampas of the country- is circulating in the North Western province of Tucumán. The endemic area of hantavirosis is thus expanded to this province but the viral reservoir in the area has not yet been identified.

Vaccine-elicited receptor-binding site antibodies neutralize two New World hemorrhagic fever arenaviruses.

While five arenaviruses cause human hemorrhagic fevers in the Western Hemisphere, only Junin virus (JUNV) has a vaccine. The GP1 subunit of their envelope glycoprotein binds transferrin receptor 1 (TfR1) using a surface that substantially varies in sequence among the viruses. As such, receptor-mimicking antibodies described to date are type-specific and lack the usual breadth associated with this mode of neutralization. Here we isolate, from the blood of a recipient of the live attenuated JUNV vaccine, two antibodies that cross-neutralize Machupo virus with varying efficiency. Structures of GP1-Fab complexes explain the basis for efficient cross-neutralization, which involves avoiding receptor mimicry and targeting a conserved epitope within the receptor-binding site (RBS). The viral RBS, despite its extensive sequence diversity, is therefore a target for cross-reactive antibodies with activity against New World arenaviruses of public health concern.

Detection of the mosquito-borne flaviviruses, West Nile, Dengue, Saint Louis Encephalitis, Ilheus, Bussuquara, and Yellow Fever in free-ranging black howlers (Alouatta caraya) of Northeastern Argentina.

Several medically important mosquito-borne flaviviruses have been detected in Argentina in recent years: Dengue (DENV), St. Louis encephalitis (SLEV), West Nile (WNV) and Yellow Fever (YFV) viruses. Evidence of Bussuquara virus (BSQV) and Ilheus virus (ILHV) activity were found, but they have not been associated with human disease. Non-human primates can act as important hosts in the natural cycle of flaviviruses and serological studies can lead to improved understanding of virus circulation dynamics and host susceptibility. From July-August 2010, we conducted serological and molecular surveys in free-ranging black howlers (Alouatta caraya) captured in northeastern Argentina. We used 90% plaque-reduction neutralization tests (PRNT90) to analyze 108 serum samples for antibodies to WNV, SLEV, YFV, DENV (serotypes 1and 3), ILHV, and BSQV. Virus genome detection was performed using generic reverse transcription (RT)-nested PCR to identify flaviviruses in 51 antibody-negative animals. Seventy animals had antibodies for one or more flaviviruses for a total antibody prevalence of 64.8% (70/108). Monotypic (13/70, 19%) and heterotypic (27/70, 39%) patterns were differentiated. Specific neutralizing antibodies against WNV, SLEV, DENV-1, DENV-3, ILHV, and BSQV were found. Unexpectedly, the highest flavivirus antibody prevalence detected was to WNV with 9 (8.33%) monotypic responses. All samples tested by (RT)-nested PCR were negative for viral genome. This is the first detection of WNV-specific antibodies in black howlers from Argentina and the first report in free-ranging non-human primates from Latin-American countries. Given that no animals had specific neutralizing antibodies to YFV, our results suggest that the study population remains susceptible to YFV. Monitoring of these agents should be strengthened to detect the establishment of sylvatic cycles of flaviviruses in America and evaluate risks to wildlife and human health.

Monoclonal antibody therapy for Junin virus infection.

Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic.

Molecular Basis for Antibody-Mediated Neutralization of New World Hemorrhagic Fever Mammarenaviruses.

In the Western hemisphere, at least five mammarenaviruses cause human viral hemorrhagic fevers with high case fatality rates. Junín virus (JUNV) is the only hemorrhagic fever virus for which transfusion of survivor immune plasma that contains neutralizing antibodies ("passive immunity") is an established treatment. Here, we report the structure of the JUNV surface glycoprotein receptor-binding subunit (GP1) bound to a neutralizing monoclonal antibody. The antibody engages the GP1 site that binds transferrin receptor 1 (TfR1)-the host cell surface receptor for all New World hemorrhagic fever mammarenaviruses-and mimics an important receptor contact. We show that survivor immune plasma contains antibodies that bind the same epitope. We propose that viral receptor-binding site accessibility explains the success of passive immunity against JUNV and that this functionally conserved epitope is a potential target for therapeutics and vaccines to limit infection by all New World hemorrhagic fever mammarenaviruses.

Potential for Co-Infection of a Mosquito-Specific Flavivirus, Nhumirim Virus, to Block West Nile Virus Transmission in Mosquitoes.

Nhumirim virus (NHUV) is an insect-specific virus that phylogenetically affiliates with dual-host mosquito-borne flaviviruses. Previous in vitro co-infection experiments demonstrated prior or concurrent infection of Aedes albopictus C6/36 mosquito cells with NHUV resulted in a 10,000-fold reduction in viral production of West Nile virus (WNV). This interference between WNV and NHUV was observed herein in an additional Ae. albopictus mosquito cell line, C7-10. A WNV 2K peptide (V9M) mutant capable of superinfection with a pre-established WNV infection demonstrated a comparable level of interference from NHUV as the parental WNV strain in C6/36 and C7-10 cells. Culex quinquefasciatus and Culex pipiens mosquitoes intrathoracically inoculated with NHUV and WNV, or solely with WNV as a control, were allowed to extrinsically incubate the viruses up to nine and 14 days, respectively, and transmissibility and replication of WNV was determined. The proportion of Cx. quinquefasciatus mosquitoes capable of transmitting WNV was significantly lower for the WNV/NHUV group than the WNV control at seven and nine days post inoculation (dpi), while no differences were observed in the Cx. pipiens inoculation group. By dpi nine, a 40% reduction in transmissibility in mosquitoes from the dual inoculation group was observed compared to the WNV-only control. These data indicate the potential that infection of some Culex spp. vectors with NHUV could serve as a barrier for efficient transmissibility of flaviviruses associated with human disease.

[New transmission scenarios of the Argentine hemorrhagic fever since the introduction of the live attenuated junin virus vaccine (Candid #1): an experience in migrant workers]. / Los nuevos escenarios de transmisión de la fiebre hemorrágica Argentina desde la introducción de la vacuna a virus junín vivo atenuado (Candid#1): una experiencia en trabajadores golondrinas.

The Argentine hemorrhagic fever (AHF) is a severe acute viral disease caused by the Junin virus of the Arenaviridae family. The AHF endemic area coincides geographically with the largest grain export agro-industrial complex of the country [Argentina]. Since the implementation of vaccination with the Candid #1 vaccine, a significant reduction in incidence was achieved and risk patterns were modified. A previous study allowed characterizing these changes and identifying three transmission scenarios: classic, emergent-reemergent, and traveler. The latter scenario includes seasonal migrant workers who move each year, mainly from the province of Santiago del Estero, the endemic area to work in the detasseling of maize. With the objective of protecting this group of workers, a prevention campaign was initiated which included: capacity building of health personnel in the province, health education, and immunization with the vaccine Candid #1. 3,021 workers were vaccinated. Prior to vaccination, serum samples were taken from a group of 104 volunteers. Tests for neutralizing antibodies specific to the Junin virus were performed and 6 (5.76%) tested positive. The unexpected finding of a high percentage of workers with antibodies suggests the need to evaluate several hypotheses: a) that the result is the product of non-probabilistic sampling; b) that it could be people who fell ill in previous travels, c) or who were vaccinated in previous travels; or d) consider this region as an emerging scenario.