Oropouche Virus Infection And Pathogenesis Is Restricted By Mavs, Irf-3, Irf-7, And Type I Ifn Signaling Pathways In Non-myeloid Cells.

Oropouche virus (OROV) is a member of the Orthobunyavirus genus in the Bunyaviridae family and a prominent cause of insect-transmitted viral disease in Central and South America. Despite its clinical relevance, little is known about OROV pathogenesis. To define the host defense pathways that control OROV infection and disease, we evaluated OROV pathogenesis and immune responses in primary cells and mice that were deficient in the RIG-I-like receptor signaling pathway (MDA5, RIG-I, or MAVS), downstream regulatory transcription factors (IRF-3 or IRF-7), IFN-β, or the receptor for type I IFN signaling (IFNAR). OROV replicated to higher levels in primary fibroblasts and dendritic cells lacking MAVS signaling, the transcription factors IRF-3 and IRF-7, or IFNAR. In mice, deletion of IFNAR, MAVS, or IRF-3 and IRF-7 resulted in uncontrolled OROV replication, hypercytokinemia, extensive liver damage, and death whereas wild-type (WT) congenic animals failed to develop disease. Unexpectedly, mice with a selective deletion of IFNAR on myeloid cells (CD11c Cre(+) Ifnar(f/f) or LysM Cre(+) Ifnar(f/f)) did not sustain enhanced disease with OROV or La Crosse virus, a closely related encephalitic orthobunyavirus. In bone marrow chimera studies, recipient irradiated Ifnar(-/-) mice reconstituted with WT hematopoietic cells sustained high levels of OROV replication and liver damage, whereas WT mice reconstituted with Ifnar(-/-) bone marrow were resistant to disease. Collectively, these results establish a dominant protective role for MAVS, IRF-3 and IRF-7, and IFNAR in restricting OROV virus infection and tissue injury, and suggest that IFN signaling in non-myeloid cells contributes to the host defense against orthobunyaviruses. Oropouche virus (OROV) is an emerging arthropod-transmitted orthobunyavirus that causes episodic outbreaks of a debilitating febrile illness in humans in countries of South and Central America. The continued expansion of the range and number of its arthropod vectors increases the likelihood that OROV will spread into new regions. At present, the pathogenesis of OROV in humans or other vertebrate animals remains poorly understood. To define cellular mechanisms of control of OROV infection, we performed infection studies in a series of primary cells and mice that were deficient in key innate immune genes involved in pathogen recognition and control. Our results establish that a MAVS-dependent type I IFN signaling pathway has a dominant role in restricting OROV infection and pathogenesis in vivo.

Apoptosis induced by Oropouche virus infection in HeLa cells is dependent on virus protein expression

Oropouche (OROV) is a single-stranded RNA arbovirus of the family Bunyaviridae, genus Orthobunyavirus, which has caused over half a million cases of febrile illness in Brazil in the past 30 years. OROV fever has been registered almost exclusively in the Amazon region, but global warming, deforestation and redistribution of vectors and animal reservoirs increases the risk of Oropouche virus emergence in other areas. OROV causes a cytolytical infection in cultured cells with characteristic cytopathic effect 48 h post-infection. We have studied the mechanisms of apoptosis induced by OROV in HeLa cells and found that OROV causes DNA fragmentation detectable by gel electrophoresis and by flow cytometric analysis of the Sub-G1 population at 36 h post-infection. Mitochondrial release of cytochrome C and activation of caspases 9 and 3 were also detected by western blot analysis. Lack of apoptosis induced by UV-inactivated OROV reveals that virus-receptor binding is not sufficient to induce cell death. Results obtained in cells treated with chloroquine and cycloheximide indicated that viral uncoating and replication are required for apoptosis induction by OROV. Furthermore, treatment of the cells with pan-caspase inhibitor prevented OROV-induced apoptosis without affecting virus progeny production. The results show that OROV infection in vitro causes apoptosis by an intracellular pathway involving mitochondria, and activated by a mechanism dependent on viral replication and protein synthesis. (C) 2010 Elsevier B.V. All rights reserved.

Oropouche virus entry into HeLa cells involves clathrin and requires endosomal acidification

Oropouche virus (ORO), family Bunyaviridae, is the second most frequent cause of arboviral febrile illness in Brazil. Studies were conducted to understand ORO entry in HeLa cells. Chlorpromazine inhibited early steps of ORO replication cycle, consistent with entry/uncoating. The data indicate that ORO enters HeLa cells by clathrin-coated vesicles, by a mechanism susceptible to endosomal acidification inhibitors. Transmission electron microscopy and immunofluorescence indicated that ORO associates with clathrin-coated pits and can be found in association with late endosomes in a time shorter than 1 h. (C) 2008 Elsevier B.V. All rights reserved.

Oropouche virus experimental infection in the golden hamster (Mesocrisetus auratus)

Oropouche virus (OROV), of the family Bunyaviridae, is the second most frequent arbovirus causing febrile disease in Brazil. In spite of this, little is known about pathogenesis of OROV infection. This report describes an experimental model of OROV in golden hamster (Mesocricetus auratus). Following subcutaneous inoculation of OROV, over 50% of the animals developed disease characterized by lethargy, ruffled fur, shivering, paralysis, and approximately one third died. Animals were sacrificed on days 1, 3, 5, 8 and 11 post-inoculation to collect tissue samples from brain, heart, liver, lung, spleen, muscle and blood for virus titration, histology and OROV immunohistochemistry. OROV was detected in high titers in blood, liver and brain, but not in the other organs. Histopathology revealed meningoencephalitis and hepatitis, with abundant OROV antigen detected in liver and brain. Diffuse galectin-3 immunostaining in brain and liver supports microglial and Kupfer cells activation. This is the first description of an experimental model for OROV infection and should be helpful to study pathogenesis and possibly to test antiviral interventions such as drugs and vaccine candidates. (C) 2010 Elsevier B.V. All rights reserved.

Detection of Oropouche virus segment S in patients and inCulex quinquefasciatus in the state of Mato Grosso, Brazil

This study aimed to investigate the circulation of Orthobunyavirus species in the state of Mato Grosso (MT) Brazil. During a dengue outbreak in 2011/2012, 529 serum samples were collected from patients with acute febrile illness with symptoms for up to five days and 387 pools of female Culex quinquefasciatuscaptured in 2013 were subjected to nested-reverse transcription-polymerase chain reaction for segment S of the Simbu serogroup followed by nucleotide sequencing and virus isolation in Vero cells. Patients (5/529; 0.9%) from Cuiabá (n = 3), Várzea Grande (n = 1) and Nova Mutum (n = 1) municipalities were positive for the S segment of Oropouche virus (OROV). Additionally, eight/387 Cx. quinquefasciatuspools were positive for the segment, with a minimum infection rate of 2.3. Phylogenetic analysis indicated that all the samples belong to the subgenotype Ia, presenting high homology with OROV strains obtained from humans and animals in the Brazilian Amazon. The present paper reports the first detection of an Orthobunyavirus, possibly OROV, in patients and in Cx. quinquefasciatus mosquitoes in MT. This finding reinforces the notion that arboviruses frequently reported in the Amazon Region circulate sporadically in MT during dengue outbreaks.

Experimental infection of suckling mice by subcutaneous inoculation with Oropouche virus

Oropouche virus, of the family Bunyaviridae, genus Orthobunyavirus, serogroup Simbu, is an important causative agent of arboviral febrile illness in Brazil. An estimated 500,000 cases of Oropouche fever have occurred in Brazil in the last 30 years, with recorded cases also in Panama, Peru, Suriname and Trinidad. We have developed an experimental model of Oropouche virus infection in neonatal BALB/c mouse by subcutaneous inoculation. The vast majority of infected animals developed disease on the 5th day post infection, characterized mainly by lethargy and paralysis, progressing to death within 10 days. Viral replication was documented in brain cells by in situ hybridization, immunohistochemistry and virus titration. Multi-step immunohistochemistry indicated neurons as the main target cells of OROV infection. Histopathology revealed glial reaction and astrocyte activation in the brain and spinal cord, with neuronal apoptosis. Spleen hyperplasia and mild meningitis were also found, without viable virus detected in liver and spleen. This is the first report of an experimental mouse model of OROV infection, with severe involvement of the central nervous system, and should become useful in pathogenesis studies, as well as in preclinical testing of therapeutic interventions for this emerging pathogen. (c) 2012 Elsevier B.V. All rights reserved.

Caracterização da resposta imune citocínica na infecção humana pelo vírus oropouche e sua relação com o padrão de soroconversão e a presença de sintomas

A tese aqui apresentada trata-se do primeiro estudo em nível mundial que pesquisa e caracteriza a resposta imune citocínica em infecções humanas pelo Orthobunyavirus Oropuche. Como metodologia para o alcance dos objetivos aqui apresentados foi utilizado um total de 320 amostras de soros humanos, onde 60 destas foram provenientes de Banco de Sangue (Controle negativo) e 260 foram obtidas mediante dois surtos do Vírus Oropouche nos Estados do Pará e Amapá (Brasil), sendo estas últimas divididas em oito subgrupos para obtenção dos dados com exatidão. Nas amostras coletadas foram realizadas análises dos dados clínicos/sintomatologia através dos prontuários, dados sorológicos através da titulação de anticorpos por Inibição da Hemaglutinação (IgM/IgG) e detecção do nível de citocinas plasmáticas por citometria de fluxo a qual permitiu a descrição técnica da dosagem de citocinas possibilitando ainda a análise de frequência de baixos e altos produtores de citocina. Os dados obtidos permitiram observar as variáveis e o comportamento das assinaturas de citocinas expressas pelos pacientes mediante a confirmação sorológica do vírus, bem como o comportamento destes analitos séricos quando da presença de sintomas específicos como febre, calafrios, cefaléia e tontura, permitindo assim que se chegasse à conclusão que a) existe um padrão na síntese de citocinas pró-inflamatórias e reguladoras; b) observa-se um balanço no perfil da resposta imune entre citocinas pró-inflamatórias (Th1) e moduladoras (Th17); c) a infecção pelo Vírus Oropouche altera a produção das citocinas nos indivíduos; d) os resultados mostram também que ao comparar os indivíduos Não respondedores com os Respondedores precoces, houve aumento da IL-1β e diminuição da IL-12; Não respondedores com Respondedores tardios, houve diminuição da IL-8, e aumento da IFN-α, IL-23 e IL-17; Não respondedores comparados com Respondedores precoces ocorreram o aumento de IL-4 e IFN-; Já quando comparado Respondedores precoces e respondedores tardios houve diminuição de IFN-α e IL-6; Respondedores precoces de forma geral apresentaram diminuição da IL-10 e Respondedores tardios apresentaram aumento da IL-5; e) Os resultados mostram ainda a expressão de IL-5 em pacientes que manifestaram os sintomas específicos para a infecção pelo Oropouche (febre, calafrios, cefaléia e tontura), sugerindo este sinal estar associado diretamente à patogênese do vírus; f) há a necessidade da complementação desta pesquisa com mais estudos como àqueles relacionados com a expressão de quimiocinas.

Epidemiologia molecular do Vírus Oropouche (Bunyaviridae: Orthobunyavirus) na Amazônia brasileira

O Virus Oropouche (VORO; Bunyaviridae, Orthobunyavirus) é um dos mais importantes arbovírus que infecta humanos na Amazônia brasileira, e é causador da febre do Oropouche. Entre 1961 e 2009, um grande número de epidemias foi registrado em diferentes centros urbanos dos Estados Brasileiros do Acre, Amapá, Amazonas, Maranhão, Pará, Rondônia e Tocantins, e também no Panamá, Peru e Trinidad & Tobago. Este trabalho teve por objetivo desenvolver um estudo retrospectivo dos aspectos epidemiológicos e moleculares do VORO enfatizando sua distribuição, a dinâmica das epidemias ocorridas no período, bem como a dispersão de diferentes genótipos na América Latina e no Brasil como contribuição à epidemiologia molecular do VORO. Para tanto 66 isolamentos do VORO pertencentes ao acervo do Instituto Evandro Chagas foram propagados em camundongos e em cultura de células VERO, seguida da extração do RNA viral e obtenção do cDNA por RTPCR; os amplicons foram purificados e submetidos ao sequenciamento nucleotídico para análises moleculares e evolução, incluindo o rearranjo genético, estudo de relógio molecular e análise de dispersão viral. Foi demonstrada a presença de quatro linhagens distintas do VORO na Amazônia brasileira (genótipos I, II, III e IV), sendo os genótipos I e II, respectivamente os mais frequentemente encontrados em áreas da Amazônia ocidental e oriental. Esses e o genótipo III estão constantemente evoluindo, mediante o mecanismo “boom and boost” que resulta na emergência seguida de substituição das sublinhagens (subgenótipos) circulantes por outras mais recentes. O genótipo III do VORO, previamente encontrado somente no Panamá, foi descrito na Amazônia e Sudeste do Brasil. Os dados obtidos pela análise filogenética comparativa das topologias para os segmentos PRNA e MRNA sugerem que o VORO utiliza o rearranjo genético como mecanismo de geração de biodiversidade viral, sendo o genótipo I o mais estável e o II o mais instável e, portanto, mais sensível às pressões evolutivas; foi reconhecido um novo genótipo do VORO neste estudo em amostras isoladas em Manaus no ano de 1980, que foi denominado de genótipo IV. O estudo do relógio molecular mostrou que a emergência do VORO se deu no Estado do Pará provavelmente há 223 anos e daí ao longo dos anos se dispersou pela PanAmazônia bem como para o Caribe, sendo que o genótipo I foi o que originou os demais genótipos do VORO.

Short Report: Identification of Oropouche Orthobunyavirus in the Cerebrospinal Fluid of Three Patients in the Amazonas, Brazil

Oropouche fever is the second most frequent arboviral infection in Brazil, surpassed only by dengue. Oropouche virus (OROV) causes large and explosive outbreaks of acute febrile illness in cities and villages in the Amazon and Central-Plateau regions. Cerebrospinal fluid (CSF) samples from 110 meningoencephalitis patients were analyzed. The RNA extracted from fluid was submitted to reverse transcription-polymerase chain reaction and sequencing to identify OROV. Three CSF samples showed the presence of OROV causing infection in the central nervous system (CNS). These patients are adults. Two of the patients had other diseases affecting CNS and immune systems: neurocysticercosis and acquired immunodeficiency syndrome, respectively. Nucleotide sequence analysis showed that the OROV from the CSF of these patients belonged to genotype I. We show here that severe Oropouche disease is occurring during outbreaks of this virus in Brazil

Detection of arboviruses of public health interest in free-living New World primates (Sapajus spp.; Alouatta caraya) captured in Mato Grosso do Sul, Brazil

Introduction A sero-epidemiological survey was undertaken to detect the circulation of arboviruses in free-living non-human primates. Methods Blood samples were obtained from 16 non-human primates (13 Sapajus spp. and three Alouatta caraya) that were captured using terrestrial traps and anesthetic darts in woodland regions in the municipalities of Campo Grande, Aquidauana, Jardim, Miranda and Corumbá in the State of Mato Grosso do Sul, Brazil. The samples were sent to the Instituto Evandro Chagas (IEC) in Ananindeua, Pará, Brazil, to detect antibodies against 19 species of arboviruses using a hemagglutination inhibition test (HI). Results Of the 16 primates investigated in the present study, five (31.2%) were serologically positive for an arbovirus. Of these five, two (12.5%) exhibited antibodies to the Flavivirus genus, one (6.2%) exhibited a monotypic reaction to Cacipacoré virus, one (6.2%) was associated with Mayaro virus, and one (6.2%) was positive for Oropouche virus. Conclusions Based on the positive serology observed in the present study, it was possible to conclude that arboviruses circulate among free-living primates. The viruses in the areas studied might have been introduced by infected humans or by primates from endemic or enzootic areas. Studies of this nature, as well as efficient and continuous surveillance programs, are needed to monitor viral activities in endemic and enzootic regions.

Diversity of biting midges of the genus Culicoides Latreille (Diptera: Ceratopogonidae) in the area of the Yacyretá Dam Lake between Argentina and Paraguay

The Culicoides communities have been analyzed between 1993/1998 in the area influenced by the Yacyretá Dam Lake (Paraná River, Argentina-Paraguay). Adults of Culicoides were collected monthly by using CDC light traps exposed for 24 h in 9 sampling sites located at both margins of the river; 21 species were recorded. Highest values of species richness were recorded during 1993/1994, being Quiteria and Corpus the sites with the higest number of species (10 and 11, respectively). The species diversity was elevated in Quiteria, Zaimán, Candelaria, Santa Tecla, Capitán Meza and Corpus (Shannon's diversity index 1.0-1.9) while Corateí, Ituzaingó and Aguapey showed less richness and diversity. The more abundant species were C. insignis, C. venezuelensis, C. leopoldoi, C. limai, C. flinti, C. debilipalpis, C. paraensis and C. guttatus. C. insignis, potential vector of bluetongue virus (BTV) to domestic and wild rumiants in the Neotropical region, is the predominant species in the area and was the only species widely distributed. C. paraensis, a proven vector of Oropouche virus to humans, is a common and abundant species. C. pusillus and C. lahillei, potential vectors of BTV and a filarial parasite, respectively, were occasionally collected. The taxonomic structure of communities was constant during the study period. The occasional species were not characteristic to one particular site and their presence could be related to non-intrinsic conditions.

In vitro and in vivo studies of the Interferon-alpha action on distinct Orthobunyavirus

Oropouche, Caraparu, Guama, Guaroa and Tacaiuma viruses (Orthobunyavirus genus) cause human febrile illnesses and/or encephalitis. To achieve a therapeutical agent to prevent and/or treat these diseases we evaluated the antiviral action of Interferon-alpha (IFN-alpha) on these orthobunyaviruses. In vitro results showed that all the studied orthobunyaviruses are susceptible to antiviral action of IFN-alpha, but this susceptibility is limited and dependent on both concentration of drug and treatment period. In vivo results demonstrated that IFN-alpha present antiviral action on Oropouche and Guaroa viruses when used as a prophylactic treatment. Moreover, a treatment initiated 3 It after infection prevented the death of Guaroa virus infected-mice. Additionally, mortality of mice was related to the migration and replication of viruses in their brains. Our results suggest that IFN-alpha could be potentially useful in the prevention of diseases caused by Oropouche virus and in the prevention and/or treatment of diseases caused by Guaroa virus. (C) 2007 Elsevier B.V. All rights reserved.

Diagnóstico molecular de arboviroses brasileiras em soros de pacientes com doença febril aguda

Pós-graduação em Microbiologia - IBILCE

Impact Of Pharmacist Interventions On Drug-related Problems And Laboratory Markers In Outpatients With Human Immunodeficiency Virus Infection.

Substantial complexity has been introduced into treatment regimens for patients with human immunodeficiency virus (HIV) infection. Many drug-related problems (DRPs) are detected in these patients, such as low adherence, therapeutic inefficacy, and safety issues. We evaluated the impact of pharmacist interventions on CD4+ T-lymphocyte count, HIV viral load, and DRPs in patients with HIV infection. In this 18-month prospective controlled study, 90 outpatients were selected by convenience sampling from the Hospital Dia-University of Campinas Teaching Hospital (Brazil). Forty-five patients comprised the pharmacist intervention group and 45 the control group; all patients had HIV infection with or without acquired immunodeficiency syndrome. Pharmaceutical appointments were conducted based on the Pharmacotherapy Workup method, although DRPs and pharmacist intervention classifications were modified for applicability to institutional service limitations and research requirements. Pharmacist interventions were performed immediately after detection of DRPs. The main outcome measures were DRPs, CD4+ T-lymphocyte count, and HIV viral load. After pharmacist intervention, DRPs decreased from 5.2 (95% confidence interval [CI] =4.1-6.2) to 4.2 (95% CI =3.3-5.1) per patient (P=0.043). A total of 122 pharmacist interventions were proposed, with an average of 2.7 interventions per patient. All the pharmacist interventions were accepted by physicians, and among patients, the interventions were well accepted during the appointments, but compliance with the interventions was not measured. A statistically significant increase in CD4+ T-lymphocyte count in the intervention group was found (260.7 cells/mm(3) [95% CI =175.8-345.6] to 312.0 cells/mm(3) [95% CI =23.5-40.6], P=0.015), which was not observed in the control group. There was no statistical difference between the groups regarding HIV viral load. This study suggests that pharmacist interventions in patients with HIV infection can cause an increase in CD4+ T-lymphocyte counts and a decrease in DRPs, demonstrating the importance of an optimal pharmaceutical care plan.