Replication of Rocio virus in primary cultures of mouse neural cells

ABSTRACT Objective This study verified the replication efficiency of the Rocio virus in a primary culture of mouse neural cells. Methods Mixed primary cultures (neurons/glia) obtained from the brains of newborn isogenic BALB/c mice were inoculated with Rocio virus on the 7 th day of culture, and the development of cytopathogenic effects was monitored. The infection was confirmed via immunocytochemistry (anti-ROCV), while viral replication was quantified in infected primary cultures. The titration method used depended on the infection period. Results Rocio virus efficiently infected primary cultured neural cells, with the highest viral titer causing cytopathic changes was observed at 2 days post infection. The virus-infected primary culture survived for up to 7 days post infection, and viral load quantitation showed viral replication kinetics compatible with the cell death kinetics of cultures. Conclusion The findings of this study suggest that mouse neural cell primary cultures support Rocio virus replication and could be used as an alternative system for studying Flavivirus infection in the central nervous system.

Predicting Antigenic Peptides from Rocio Virus NS1 Protein for Immunodiagnostic Testing Using Immunoinformatics and Molecular Dynamics Simulation.

The mosquito-borne disease caused by the Rocio virus is a neglected threat, and new immune inputs for serological testing are urgently required for diagnosis in low-resource settings and epidemiological surveillance. We used in silico approaches to identify a specific antigenic peptide (p_ROCV2) in the NS1 protein of the Rocio virus that was theoretically predicted to be stable and exposed on its surface, where it demonstrated key properties allowing it to interact with antibodies. These findings related to the molecular dynamics of this peptide provide important insights for advancing diagnostic platforms and investigating therapeutic alternatives.

Rocio Virus: An Updated View on an Elusive Flavivirus.

Rocio virus (ROCV) is a mosquito-borne flavivirus and human pathogen. The virus is indigenous to Brazil and was first detected in 1975 in the Sao Paulo State, and over a period of two years was responsible for several epidemics of meningoencephalitis in coastal communities leading to over 100 deaths. The vast majority of ROCV infections are believed to be subclinical and clinical manifestations can range from uncomplicated fever to fatal meningoencephalitis. Birds are the natural reservoir and amplification hosts and ROCV is maintained in nature in a mosquito-bird-mosquito transmission cycle, primarily involving Psorophora ferox mosquitoes. While ROCV has remained mostly undetected since 1976, in 2011 it re-emerged in Goiás State causing a limited outbreak. Control of ROCV outbreaks depends on sustainable vector control measures and public education. To date there is no specific treatment or licensed vaccine available. Here we provide an overview of the ecology, transmission cycles, epidemiology, pathogenesis, and treatment options, aiming to improve our ability to understand, predict, and ideally avert further ROCV emergence.

Detection of Rocio Virus SPH 34675 during Dengue Epidemics, Brazil, 2011-2013.

Recent seroprevalence studies in animals detected Rocio virus in regions of Brazil, indicating risk for re-emergence of this pathogen. We identified Rocio virus RNA in samples from 2 human patients for whom dengue fever was clinically suspected but ruled out by laboratory findings. Testing for infrequent flavivirus infections should expedite diagnoses.

A Saint Louis encephalitis and Rocio virus serosurvey in Brazilian horses

Introduction Arboviruses are an important public health problem in Brazil, in especially flaviviruses, including the Saint Louis encephalitis virus (SLEV) and the Rocio virus (ROCV), are especially problematic. These viruses are transmitted to humans or other vertebrates through arthropod bites and may cause diseases with clinical manifestations that range from asymptomatic infection, viral hemorrhagic fever to encephalitis. Methods A serological survey of horses from various regions of Brazil using an enzyme-linked immunosorbent assay (ELISA) with recombinant SLEV domain III peptides and ROCV E protein as antigens. Results Overall, 415 (55.1%) of the 753 horses that were screened were seropositive for flavivirus and, among them, monotypic reactions were observed to SLEV in 93 (12.3%) and to ROCV in 46 (6.1%). These results suggested that these viruses, or other closely related viruses, are infecting horses in Brazil. However, none of the studied horses presented central nervous system infection symptoms. Conclusions Our results suggest that SLEV and ROCV previously circulated among horses in northeast, west-central and southeast Brazil. .

Sharing clinical decisions for multimorbidity case management using social network and open-source tools.

INTRODUCTION: Social networks applied through Web 2.0 tools have gained importance in health domain, because they produce improvements on the communication and coordination capabilities among health professionals. This is highly relevant for multimorbidity patients care because there is a large number of health professionals in charge of patient care, and this requires to obtain clinical consensus in their decisions. Our objective is to develop a tool for collaborative work among health professionals for multimorbidity patient care. We describe the architecture to incorporate decision support functionalities in a social network tool to enable the adoption of shared decisions among health professionals from different care levels. As part of the first stage of the project, this paper describes the results obtained in a pilot study about acceptance and use of the social network component in our healthcare setting. METHODS: At Virgen del Rocío University Hospital we have designed and developed the Shared Care Platform (SCP) to provide support in the continuity of care for multimorbidity patients. The SCP has two consecutively developed components: social network component, called Clinical Wall, and Clinical Decision Support (CDS) system. The Clinical Wall contains a record where health professionals are able to debate and define shared decisions. We conducted a pilot study to assess the use and acceptance of the SCP by healthcare professionals through questionnaire based on the theory of the Technology Acceptance Model. RESULTS: In March 2012 we released and deployed the SCP, but only with the social network component. The pilot project lasted 6 months in the hospital and 2 primary care centers. From March to September 2012 we created 16 records in the Clinical Wall, all with a high priority. A total of 10 professionals took part in the exchange of messages: 3 internists and 7 general practitioners generated 33 messages. 12 of the 16 record (75%) were answered by the destination health professionals. The professionals valued positively all the items in the questionnaire. As part of the SCP, opensource tools for CDS will be incorporated to provide recommendations for medication and problem interactions, as well as to calculate indexes or scales from validated questionnaires. They will receive the patient summary information provided by the regional Electronic Health Record system through a web service with the information defined according to the virtual Medical Record specification. CONCLUSIONS: Clinical Wall has been developed to allow communication and coordination between the healthcare professionals involved in multimorbidity patient care. Agreed decisions were about coordination for appointment changing, patient conditions, diagnosis tests, and prescription changes and renewal. The application of interoperability standards and open source software can bridge the gap between knowledge and clinical practice, while enabling interoperability and scalability. Open source with the social network encourages adoption and facilitates collaboration. Although the results obtained for use indicators are still not as high as it was expected, based on the promising results obtained in the acceptance questionnaire of SMP, we expect that the new CDS tools will increase the use by the health professionals.

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.

Encefalite no litoral paulista: a emergência da epidemia e a reação da mídia impressa / Encephalitis on the south coast of the State of São Paulo: the emergence of the epidemic and the response of the written media

No litoral sul do estado de São Paulo, ocorreu uma epidemia de encefalite pelo arbovírus Rocio de 1975 a 1978. As altas taxas de morbidade e mortalidade causaram impacto social. Neste trabalho, o objetivo foi apresentar um estudo sobre como a mídia impressa relatou os acontecimentos sociais relacionados ao surgimento da epidemia no primeiro semestre de 1975. Reportagens sobre a epidemia no litoral sul foram obtidas do banco de dados dos jornais A Tribuna, Folha de S.Paulo e Jornal da Tarde. Foram analisadas as notícias até o mês de julho de 1975, fase inicial e de maior impacto da epidemia. Com a identificação de casos de encefalite, de causa desconhecida, a Secretaria de Estado da Saúde desaconselhou a ida de turistas para o litoral, utilizando a mídia como veículo de divulgação. Diante das notícias, ocorreu a fuga dos turistas e, consequentemente, a crise do comércio. Observou-se a revolta dos comerciantes, que geraram embates contra a mídia, no que tange à forma de divulgação da epidemia. Alguns prefeitos alegaram inveracidade de notícias publicadas. A proibição feita pelas autoridades sanitárias foi relatada pela mídia de forma abrangente, englobando sujeitos envolvidos nesse discurso. Assim, foram reveladas ao público as tensões geradas entre os detentores do conhecimento científico e o poder econômico local. Os jornais realizaram cobertura abrangente, abordando vários temas, entretanto disseminaram incertezas e fizeram uso de imagens sensacionalistas, além de desarticular acontecimentos biológicos e sociais. Os temas chegaram aos leitores de forma fragmentada e com sentidos sociais comprometidos.

On the south coast of the State of São Paulo, there was an encephalitis epidemic due to the arbovirus Rocio from 1975 to 1978. High rates of morbidity and mortality caused a social impact. There was a decrease in tourism and a commercial crisis, which caused turmoil among traders and reactions against the media concerning the communication of the epidemic. This work aimed to present a documental study of the epidemic with the purpose of analyzing how the written press communicated the social facts related to the epidemic in the first half of 1975. Articles about the epidemic were obtained in the database of the newspapers A Tribuna, Folha de S.Paulo and Jornal da Tarde. The pieces of news published up to the month of July of 1975 were analyzed. With the identification of encephalitis cases of unknown causes, the State Department of Health advised tourists, through the media, not to go to the coast. In face of the news, a flight of tourists occurred, and the discomfort of traders was observed. Some mayors alleged that the published news were not true. The prohibition made by the sanitary authorities was reported by the media in an interdisciplinary way, encompassing the researchers and society. Thus, the media revealed to the public tensions between the scientific knowledge holders and the local economic power. The newspapers covered many topics, but they spread uncertainty and used sensationalist images, besides disarticulating biological and social events. The subjects reached the readers in a fragmented way and with damaged social meanings.

Molecular characterization of two rocio flavivirus strains isolated during the encephalitis epidemic in são paulo state, brazil and the development of a one-step rt-pcr assay for diagnosis

Rocio virus (ROCV) was responsible for an explosive encephalitis epidemic in the 1970s affecting about 1,000 residents of 20 coastland counties in São Paulo State, Brazil. ROCV was first isolated in 1975 from the cerebellum of a fatal human case of encephalitis. Clinical manifestations of the illness are similar to those described for St. Louis encephalitis. ROCV shows intense antigenic cross-reactivity with Japanese encephalitis complex (JEC) viruses, particularly with Ilheus (ILHV), St. Louis encephalitis, Murray Valley and West Nile viruses. In this study, we report a specific RT-PCR assay for ROCV diagnosis and the molecular characterization of the SPAn37630 and SPH37623 strains. Partial nucleotide sequences of NS5 and E genes determined from both strains were used in phylogenetic analysis. The results indicated that these strains are closely related to JEC viruses, but forming a distinct subclade together with ILHV, in accordance with results recently reported by Medeiros et al. (2007).

O vírus Rocio (ROCV) foi responsável por uma explosiva epidemia de encefalite que ocorreu nos anos 70 afetando cerca de 1.000 habitantes de 20 municípios litorâneos do Estado de São Paulo, Brasil. ROCV foi isolado em 1975 de cerebelo de caso humano fatal de encefalite. As manifestações clínicas da doença são semelhantes àquelas descritas para encefalite St. Louis. ROCV apresenta intensa reatividade cruzada com os vírus do Complexo da Encefalite Japonesa (JEV), particularmente com o vírus Ilhéus (ILHV) e com os vírus das encefalites St. Louis, Murray Valley e West Nile. Neste estudo, relatamos o desenvolvimento de um teste de RT-PCR específico para diagnóstico de ROCV e a caracterização molecular das cepas SPAn37630 e SPH37623. Foi realizada a análise filogenética das seqüências parciais dos genes NS5 e E, de ambas as cepas. Os resultados indicaram que essas cepas são intimamente relacionadas ao complexo JEV, mas formando um subgrupo com o ILHV, de acordo com os resultados recentemente publicados por MEDEIROS et al. (2007).

Evidências sorológicas da circulação do arbovírus Rocio (Flaviviridae) na Bahia / Serological evidence of the circulation of the Rocio arbovirus (Flaviviridae) in Bahia

Oito casos com anticorpos anti-Rocio são descritos, de quatro cidades do Estado da Bahia, sendo seis portadores de anticorpos IgG (IH e TN) e dois IgM (ELISA e TN). Os autores comentam sobre a circulação deste arbovírus no Estado, e as possibilidades de reações cruzadas com outros vírus antigenicamente relacionados.

Eight antibody anti-Rocio cases, from four distinct cities in the state of Bahia, are described; six of them being carriers of the antibody IgG (HI and NT) and two IgM (ELISA and NT). The authors comment on the circulation of these arboviruses in the state of Bahia and on the possibility of cross reactions with other antigenically related viruses.

Encefalite pelo vírus Rocio / Rocio virus encephalitis

São descritos os achados obtidos pela Seção de Vírus Transmitidos por Artrópodes durante os estudos sobre a epidemia de encefalite ocasionada por um arbovírus, o Rocio, do grupo Flavivírus, novo para a ciência e que significou a emergência de nova doença humana para a região Sul do Brasil, compreendendo os municípios de Peruíbe, Itanhaém, Mongaguá e outros do Vale do Ribeira, no Estado de São Paulo, em 1975. Descreve as características da área endêrnica, os métodos virológicos utilizados, a caracterização do agente etiológico, seus aspectos epidemiológicos e observações a respeito dos ciclos enzoótico e epizoótico do vírus (AU).