ABSTRACT Flaviviruses are viruses transmitted by arthropods, mainly mosquitoes. They can cause diseases in humans and animals, they also include specific insect viruses that only infect and replicate in insects, not in vertebrates. In Paraguay, dengue, yellow fever, and Zika viruses were detected in human infections, but studies of flaviviruses in mosquitoes are still scarce. Therefore, the objective of the present study was the implementation of a flavivirus detection system in mosquitoes at IICS-UNA. First, trainings on collection, pool preparation and processing by conventional RT-PCR techniques were organized by international experts for local professionals (biochemists and biologists). In addition, data log sheets and sample transport control forms from the collection sites to the IICS were implemented. A total of 201 pools of 1 to 35 mosquitoes were prepared, each grouped by species, locality, among others. To ensure the integrity of the extracted RNA, an internal control (Actin-1) detection was performed, all pools being positive for it; 91/201 pools were positive for flaviviruses. The sequencing of 19/91 pools positive for flavivirus was carried out, identifying flavivirus in all cases of insects (mainly detecting Culex Flavivirus, cell fusing agents Flavivirus and Kamiti river virus), evidencing the high distribution of these viruses. These results demonstrate that it was feasible to implement the flavivirus detection system in mosquitoes, which could contribute to strengthen the detection, surveillance and control of these viruses, as well as, the knowledge about the ecological importance of insect flaviviruses.

RESUMEN Los Flavivirus constituyen virus transmitidos por artrópodos, principalmente mosquitos. Pueden producir enfermedades en humanos y animales, también incluyen virus específicos de insectos que solo infectan y se replican en los insectos, no así en vertebrados. En Paraguay los virus dengue, fiebre amarilla y Zika fueron detectados en infecciones humanas, pero los estudios de flavivirus en mosquitos son aún escasos. Por ello, el objetivo del presente estudio fue implementar un sistema de detección de flavivirus en mosquitos en el IICS-UNA. Primero, se organizaron capacitaciones en colecta, preparación de pools y procesamiento por técnicas de RT-PCRs convencionales realizadas por expertos internacionales a profesionales locales (bioquímicos y biólogos). Además, se implementaron planillas de registro de datos y de control de transporte de muestras de los lugares de colectas hasta el IICS-UNA. Se prepararon en total 201 pools de 1 a 35 mosquitos cada uno agrupados por especie, localidad, entre otros criterios. Para asegurar la integridad del RNA extraído se realizó la detección de un control interno (Actina-1), siendo todos los pools positivos para el mismo, 91/201 pools fueron positivos para flavivirus. Se realizó la secuenciación de 19/91 pools positivos para flavivirus identificándose flavivirus de insectos (detectándose principalmente Culex Flavivirus, cell fusing agents Flavivirus y Kamiti river virus), evidenciando la elevada distribución de estos virus. Estos resultados demuestran que fue factible implementar el sistema de detección de flavivirus en mosquitos, lo cual podría contribuir a fortalecer la vigilancia y control de estas virosis, así como el conocimiento sobre la importancia ecológica de flavivirus de insectos.

Os Flavivírus transmitidos por mosquitos são uma ameaça atual e emergente no mundo todo. Nesse gênero, o vírus Encefalite Saint Louis (VESL) causa uma forma grave de doença neuroinvasiva onde a resposta imune é um componente crucial da defesa do hospedeiro. Neste trabalho nos investigamos a interação entre VESL e células de imunidade inata em um modelo de infecção in vitro de monócitos humanos (células U937) com estirpe de diferentes virulências e condições epidemiológicas de isolamento (CbaAr-4005 e 78V-6507). Foi avaliada a capacidade do vírus de infectar e replicar , assim como o efeito citopático e a viabilidade cinética dos monócitos durante a infecção. Os resultados demonstraram a suscetibilidade dos monócitos à infecção, replicação e morte por ambas as estirpes virais. No entanto, foram detectadas diferenças significativas entre eles. A estirpe epidémica e de maior virulenta CbaAr-4005 teve uma maior taxa de infecção e replicação do que a estirpe endémica e menos virulenta 78V-6507. Foi comprovado também que o VESL induziu a morte de monócitos humanos, dependendo do tempo pós-infecção (pi) e da estirpe. Assim, a CbaAr-4005 causou a partir do dia 3 pi o dobro da mortalidade celular o que a 78V- 6507. Além disso, alterações nos parâmetros morfológicos foram observadas nos monócitos infectados que poderiam estar relacionadas ao tipo de mecanismo de morte celular associado à infecção pelo VESL.

Mosquitoes borne Flavivirus infections are an actual and emergent worldwide threat to human health. Within this genus, Saint Louis Encephalitis Virus (SLEV) causes a severe neuroinvasive disease where immune response is crucial for host survival. In this study the interaction between SLEV and innate immune cells was evaluated. An in vitro infection model with human monocytes (U937 cells) and strains with variations in virulence and isolation conditions (CbaAr-4005 and 78V-6507) were used. Infection capacity, replication capacity, cytopathic effect and monocyte viability kinetics were measured. The results showed susceptibility to infection and replication to both strains. However, significant differences were found among them. CbaAr-4005, the epidemic and more virulent strain, showed higher infection and replication ratios compared to 78V-6507. SLEV infection that induces cell death of human monocytes was also found in a post-infection time and in a strain dependent manner. Since day 3 post-infection, twice the mortality in CbaAr-4005 infected cells was observed. Furthermore, infected monocytes showed alterations in morphologic parameters that could be related with apoptosis mechanisms associated to SLEV infections.

Los flavivirus transmitidos por mosquitos son una amenaza actual y emergente en todo el mundo. Dentro de este género, el virus Encefalitis San Luis (VESL) causa una forma severa de enfermedad neuroinvasiva donde la respuesta inmune es un componente crucial de la defensa del huésped. En este trabajo se investigó la interacción entre VESL y células de la inmunidad innata, en un modelo de infección in vitro de monocitos humanos (células U937) con cepas de distinta virulencia y condiciones epidemiológicas de aislamiento (CbaAr-4005 y 78V-6507). Se evaluó la capacidad de infectar y replicar del virus, como también el efecto citopático y la cinética de viabilidad de monocitos durante la infección. Los resultados demostraron la susceptibilidad de los monocitos a la infección, replicación y muerte por ambas cepas virales. Sin embargo, se hallaron diferencias significativas entre ellas. La cepa epidémica y de mayor virulencia CbaAr-4005 registró una tasa de infección y replicación superior a la de la cepa endémica y de menor virulencia 78V-6507. Se comprobó también que el VESL indujo la muerte de monocitos humanos, dependiendo del tiempo post-infección (pi) y de la cepa. Así, CbaAr-4005 provocó a partir del día 3 pi el doble de mortalidad celular que 78V-6507. Además, en los monocitos infectados se observaron alteraciones de parámetros morfológicos que podrían relacionarse con el tipo de mecanismo de muerte celular asociado a la infección por VESL.

ABSTRACT Achyrocline satureioides (Lam.) DC. Asteraceae, ‘marcela del campo', possess several pharmacological properties. Previously we reported antiviral activity of an aqueous extract of A. satureioides against an alphavirus, Western Equine Encephalitis virus. Alphaviruses are highly virulent pathogens which cause encephalitis in humans and equines. There are no effective antiviral to treat its infections. The aim of this study was to evaluate in vitro cytotoxic and antiviral activities against Western Equine Encephalitis virus of five water extract chromatographic fractions from A. satureioides and identify the main compounds of the bioactive fraction. Also, it was to assess in vivo cytogenotoxic ability of the active fraction. Cytotoxicity studies revealed low toxicity of the most of fractions in Vero and in equine peripheral blood mononuclear cells. Antiviral studies showed that the water crude extract – Sephadex LH 20 – fraction 3 MeOH–H2O (Fraction 3) was active against Western Equine Encephalitis virus with Effective Concentration 50% = 5 µg/ml. Selectivity Indices were 126.0 on Vero and 133.6 on peripheral blood mononuclear cells, four times higher than aqueous extract selectivity index. Regarding the mechanism of action we demonstrated that F3 exerted its action in intracellular replication stages. Further, fraction 3 showed important virucidal action. Fraction 3 contains, in order of highest to lowest: chlorogenic acid, luteolin, 5,7,8-trimethoxyflavone, 3-O-methylquercetin and caffeic acid. Fraction 3 did not induce in vivo toxic nor mutagenic effect. Therefore, it is safe its application as antiviral potential. Further studies of antiviral activity in vivo will be developed using a murine model.

Our goal was to determine the presence of neutralizing antibodies against St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) in sera of wild and domestic birds from Buenos Aires City, Argentina. From October 2012 to April 2013, 180 samples were collected and processed by the microneutralization technique. A 7.2 % of the sampled birds were seropositive for SLEV, while no seropositive birds for WNV were detected.

Saint Louis encephalitis virus caused an outbreak of febrile illness and encephalitis cases in Córdoba, Argentina, in 2005. During this outbreak, the strain CbaAr-4005 was isolated from Culex quinquefasciatus mosquitoes. We hypothesised that this epidemic variant would be more virulent in a mouse model than two other non-epidemic strains (78V-6507 and CorAn-9275) isolated under different epidemiological conditions. To test this hypothesis, we performed a biological characterisation in a murine model, including mortality, morbidity and infection percentages and lethal infection indices using the three strains. Mice were separated into age groups (7, 10 and 21-day-old mice) and analysed after infection. The strain CbaAr-4005 was the most infective and lethal of the three variants, whereas the other two strains exhibited a decreasing mortality percentage with increasing animal age. The strain CbaAr-4005 produced the highest morbidity percentages and no significant differences among age groups were observed. The epidemic strain caused signs of illness in all inoculated animals and showed narrower ranges from the onset of symptoms than the other strains. CbaAr-4005 was the most virulent for Swiss albino mice. Our results highlight the importance of performing biological characterisations of arbovirus strains likely to be responsible for emerging or reemerging human diseases.

INTRODUCTION: The aim of the present study was to analyze the larvicidal activity of different crude extracts of Larrea cuneifolia and its most abundant lignan, nordihydroguaiaretic acid (NDGA), against Culex quinquefasciatus. METHODS: Chloroform, methanol, and aqueous extracts from L. cuneifolia and NDGA were tested against larvae of Cx. quinquefasciatus under laboratory conditions. RESULTS: The chloroform extract showed the highest larvicidal effect, with an estimated LC50 of 0.062 mg/ml. NDGA also demonstrated significant larvicidal activity with an estimated LC50 of 0.092 mg/ml. CONCLUSIONS: These results indicate that the chloroform extract of L. cuneifolia and NDGA are promising insecticides of botanical origin that could be useful for controlling Cx. quinquefasciatus.

St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) present ecological and antigenic similarities and are responsible for serious human diseases. In addition, WNV is a significant pathogen in terms of equine health. The purpose of our study was to analyse the seroprevalence of SLEV and WNV in equine sera collected in Santa Fe Province, Argentina. The seroprevalence determined using the plaque reduction neutralisation test was 12.2% for SLEV, 16.2% for WNV and 48.6% for a combination of both viruses. These results provide evidence of the co-circulation of SLEV and WNV in equines in Santa Fe.

Rio Negro virus (RNV) (Venezuelan equine encephalitis subtype VI) circulates only in Argentina; in northern provinces, isolates have been obtained from mosquitoes and rodents since 1980 and have been associated with acute febrile illness in humans. However, no studies of RNV have been performed in the central area of the country. We carried out molecular and serological detection of RNV in Córdoba, a province of the central part of the country, in mosquitoes and humans, respectively. One mosquito pool tested positive for alphavirus RNA by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR). Subsequent sequencing determined that this alphavirus grouped with RNV. Serological studies detected antibodies to RNV in one human serum sample, which was obtained during the same period that RNV was detected using the aforementioned molecular methods. This is the first report of RNV circulation in the central area of Argentina, indicating an expansion of its original distribution. These results highlight the importance of strengthening surveillance procedures in endemic areas, as well as in new regions where RNV may emerge.

In Argentina, most information on hepatitis C virus (HCV) genotype distribution comes from studies carried out in Buenos Aires (east province). In order to identify HCV subtypes in central Argentina, nucleotide sequencing of core region was performed in samples from 36 patients living in Córdoba, the second most populated province of Argentina. The sequence analysis identified subtype 2c as the most prevalent (50%), followed by subtype 1b (33%) and to a lesser extent by subtypes 1a (11%), 3a (3%) and 4a (3%). This is the first report of circulation of HCV subtype 2c in this region of Argentina and also such high prevalence has never been found before in the genotype distribution of South America.

This study was conducted to compare among the most recent generation of five screening tests licensed in Argentina, in order to evaluate which of the tests has the best sensitivity for detection of antibodies against hepatitis C virus (HCV). The tests analyzed were: Detect-HCV™ (3.0) Biochem ImmunoSystems, Canada; Hepatitis C EIA Wiener Lab., Argentina; Equipar HCV Ab, Italy; Murex HCV 4.0, UK and Serodia-HCV particles agglutination test, Japan. The results obtained showed high discrepancy between the different kits used and show that some of the tests assessed have a low sensitivity for anti-HCV detection in both chronic infections and early seroconversion, and indicate that among the commercially available kits in Argentina, Murex HCV 4.0 (UK) and Serodia-HCV particles agglutination test (Japan) have the best sensitivity for HCV screening. Although the sensitivity of the assays is the first parameter to be considered for blood screening, more studies should be carried out to assess the specificity of such assays.

Este trabalho apresenta os resultados da detecção de anticorpos (imunoglobulina G) para os subtipos I e VI dos vírus do complexo EEV (Familia Togaviridae) em população da Ilha General Belgrano, província de Formosa (Argentina). A prevalência de anticorpos neutralizantes para o subtipo VI foi de 30% a 70% na primeira e segunda pesquisa respectivamente, e por inibição de hemaglutinação (IH) de 0%. Para o subtipo IAB, a prevalência foi de 13% a 3,6% por NT, enquanto anticorpos IH não foram detectados nas duas pesquisas. Observou-se que ambos subtipos circularam simultaneamente, mas enquanto o subtipo VI manteve-se constante com alguns picos de maior presença, o subtipo IAB foi encontrado em nível baixo.

This work presents the results of the detection of antibodies (immunoglobulin G) for subtypes I and VI of VEE viruses complex (Togaviridae family) in people from the General Belgrano island, Formosa province (Argentina). The prevalence of neutralizing (NT) antibodies for subtype VI was from 30% to 70% and the prevalence of antibodies inhibitory of hemagglutination (HI) was of 0% in the first and second inquiry respectively. For the subtype IAB the prevalence of NT antibodies was from 13% to 3.6%, similar to the prevalence total for both subtypes. HI antibodies were not detected in any inquiries for any subtype. It was observed that both subtypes circulate simultaneously, while subtype VI remains constant with some peaks, subtype I was found in low level.

O virus Una (Togaviridae) tem ampla distribuição na América do Sul, detectando-se infecções até hoje em mosquitos e hospedeiros vertebrados (humanos, aves e cavalos). Mediante a realização do teste de neutralização em soros humanos provenientes de indivíduos entre 44 e 89 anos, da cidade de Córdoba, foi detectada uma prevalência de 3,8% (3/79) de anticorpos para o vírus UNA. Nenhum soro apresentou anticorpos para o vírus Mayaro. Os títulos foram baixos demonstrando-se a presença de infecção passada. Dados epidemiológicos indicam que a infecção ocorreu em áreas rurais da província de Córdoba (centro da Argentina). Os dados aqui expostos representam o primeiro registro de infecção de humanos por vírus Una na República Argentina.

Una virus (UNAV), Togaviridae family, is widely distributed in South America, where infections have been detected in mosquitoes and vertebrate hosts (humans, birds and horses). We analyzed human sera from Córdoba inhabitants aged 44 to 89 years and using a neutralization test, we found a prevalence of UNAV antibodies of 3.8% (3/79). The low titers detected suggest past infections probably acquired in rural areas of the Province of Córdoba (central Argentina). None sera were found positive for MAYV neutralizing antibodies. This is the first report of human infections by UNAV in Argentina.

Com a finalidade de estudar a relação entre prevalência de anticorpo para o vírus da encefalite St. Louis (ESL) e a idade, foram analisados 615 soros de indivíduos entre 0-87 anos de idade, morando na cidade de Córdoba, Argentina. Detectou-se uma prevalência de anticorpos neutralizantes de 13,98% e observou-se uma relação com a idade estatisticamente significante (p = 0,045). A soroprevalência mais alta foi encontrada em indivíduos de mais de 60 anos (> 20%), enquanto nenhum com menos de 10 anos foi soropositivo para este vírus. Nossos resultados confirmam que o agente é endêmico nesta área. Por esse motivo os autores sugerem que deveríam se realizar estudos de patologias neurológicas nesses indivíduos de 60 anos de idade dado que eles representam o grupo mais suscetível ao vírus ESL.

A serological screening was performed in 615 individuals aged 0-87 years, living in the city of Cordoba, Argentina to study the relationship between antibody prevalence for the SLE virus and age. A 13.98% prevalence of neutralizing antibodies was obtained and its relation to age was significantly high (p = 0.045). The highest seroprevalence was noted on individuals over 60 years old (>20%), whereas no subject under 10 was seropositive for this virus. Our results confirm that the agent is endemic in this area and neurological pathology studies should be performed on those individuals aged 60 since they represent the most susceptible group to SLE virus.

Com a finalidade de dispor de um método rápido e de baixo custo para realizar estudos séricos dirigidos ao virus da encefalite St.Louis (ESL), avaliamos a técnica de imunofluorescência indireta (IFI) artesanal em relação ao teste referência de neutralização (NT). Examinaram-se 213 soros de doadores de sangue. A prevalência dos anticorpos obtidas na técnica de IFI (30,98%) foi mais baixa do que a pesquisada com NT (41,78%). A taxa de especificidade relativa da IFI foi 96,77%, enquanto sua taxa de sensibilidade relativa foi 69,66%. O índice Kappa demonstrou boa concordância entre os dois testes. Os resultados achados revelam que a NT continua sendo a técnica sorológica de maior taxa de especificidade e sensibilidade relativa para a detecção de anticorpos IgG para o vírus ESL. Contudo, a IFI poderia ser o teste de escolha em estudos da circulação desta arbovirose na população humana.

An in house indirect immmunofluorescence assay ( IFA ) in relation to neutralization (NT) reference test, was assessed as a fast and cheap method to carry out serological surveys for St. Louis Encephalitis virus (SLE). Sera obtained from 213 blood donors were analyzed by both tests. The prevalence of seropositivity obtained with IFA was lower than (30.98%) that observed on NT (41.78%). The relative specificity rate of IFA was 96.77% whereas its relative sensitivity rate was 69.66%. Kappa index showed a good correlation between both tests. The results indicate that neutralization assay is still the serological test with the highest sensitivity and specificity relative rates for detecting antibodies against SLE virus. Nevertheless, the IFA could be useful as an alternative test in order to learn the circulation of the Flavivirus genus in a certain area.