Production of recombinant NS1 protein and its possible use in encephalitic flavivirus differential diagnosis.

Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of arboviral encephalitis in the Americas. The co-circulation of related flaviviruses in the Americas and prior vaccination against flaviviruses pose problems to the diagnostic specificity of serological assays due to the development of cross-reactive antibodies. An accurate diagnosis method capable of differentiating these related viruses is needed. NS1 is a glycosylated, nonstructural protein, of about 46 kDa which has a highly conserved structure. Anti-NS1 antibodies can be detected within 4-8 days after the initial exposure and NS1 is the least cross-reactive of the flaviviral antigens. This study was aimed to generate SLEV and WNV NS1 recombinants proteins for the development of a flavivirus diagnostic test. Local Argentinian isolates were used as the source of NS1 gene cloning, expression, and purification. The protein was expressed in Escherichia coli as inclusion bodies and further purified by metal-chelating affinity chromatography (IMAC) under denaturing conditions. Human sera from SLEV and WNV positive cases showed reactivity to the recombinant NS1 proteins by western blot. The unfolded NS1 proteins were also used as immunogens. The polyclonal antibodies elicited in immunized mice recognized the two recombinant proteins with differential reactivity.

First detection of Rio Negro virus (Venezuelan equine encephalitis complex subtype VI) in Córdoba, Argentina.

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.

Immunoglobulin G subclasses in antibody responses to St. Louis encephalitis virus infections.

The aim of this study was to recognize the specific antiviral response patterns of IgG1, IgG2, IgG3 and IgG4 subclasses, elicited during St. Louis encephalitis virus (SLEV) infection in humans. Eighty-five samples of human sera from 44 patients with SLEV infection were obtained between days 1 and 365 or later, after onset of the disease. These samples were processed by immunofluorescence assay for detection of IgG1-, IgG2-, IgG3- and IgG4-specific antibodies. We demonstrate the presence of all isotypes of IgG for more than a year in patients infected with SLEV. However; isotype IgG1 was present at the highest titers, with a peak between days 8 and 30 after onset of the disease.

Caracterización biológica de cepas del virus Encefalitis San Luis en células de la inmunidad innata / Biological characterization of St. Louis Encephalitis virus strains in cells of innate immunity

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.

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.

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.

UNA virus: first report of human infection in 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.

Age-related seroprevalence study for St. Louis encephalitis in a population from Cordoba, Argentina.

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.

Landscape determinants of Saint Louis encephalitis human infections in Córdoba city, Argentina during 2010.

Saint Louis encephalitis virus (SLEV) is endemic in Argentina. During 2005 an outbreak occurred in Córdoba. From January to April of 2010 a new outbreak occurred in Córdoba city with a lower magnitude than the one reported in 2005. Understanding the association of different landscape elements related to SLEV hosts and vectors in urban environments is important for identifying high risk areas for human infections, which was here evaluated. The current study uses a case-control approach at a household geographical location, considering symptomatic and asymptomatic human infections produced by SLEV during 2010 in Córdoba city. Geographical information systems and logistic regression analysis were used to study the distribution of infected human cases and their proximity to water bodies, vegetation abundance, agricultural fields and housing density classified as high/low density urban constructions. Population density at a neighborhood level was also analyzed as a demographic variable. Logistic regression analysis revealed vegetation abundance was significantly (p<0.01) associated with the presence of human infections by SLEV. A map of probability of human infections in Córdoba city was derived from the logistic model. The model highlights areas that are more likely to experience SLEV infections. Landscape variables contributing to the outbreak were the proximity to places with vegetation abundance (parks, squares, riversides) and the presence of low density urban constructions, like residential areas. The population density analysis shows that SLEV infections are more likely to occur when population density by neighborhood is lower. These findings and the predictive map developed could be useful for public health surveillance and to improve prevention of vector-borne diseases.

First detection of Rio Negro virus (Venezuelan equine encephalitis complex subtype VI) in Córdoba, Argentina

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.

Age-related seroprevalence study for St. Louis encephalitis in a population from Córdoba, Argentina

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 percent 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 percent), 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

UNA virus: first report of human infection in 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 percent (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