As arboviroses acometem centenas de milhares de pessoas anualmente no Brasil, levando, muitas delas, à morte. O Brasil apresenta uma das maiores biodiversidades em arbovírus do planeta, tendo mais de uma centena de espécies já isoladas e documentadas. Estudos prévios revelaram que espécies de arbovírus estão circulando no país de modo velado em áreas urbanas, rurais e florestais. O conhecimento de aspectos da epidemiologia e biologia viral como ciclo silvestre mantenedor, gama de hospedeiros, sazonalidade, disseminação, área de ocorrência, história evolutiva e diversidade genética, são fundamentais para a definição de estratégias de prevenção e controle pelos órgãos públicos de saúde e vigilância epidemiológica. Nesse sentido, tivemos como objetivo pesquisar a ocorrência de flavivírus em pequenos roedores silvestres. Para tal finalidade, capturamos esses animais em fragmentos de Cerrado e propriedades rurais localizadas nas cidades de Ribeirão Preto, Batatais, Cajuru e Luiz Antônio, no nordeste do estado de São Paulo. Amostras de sangue foram coletadas e submetidas ao ensaio sorológico ELISA indireto para detectar anticorpos IgG contra os flavivírus Rocio, Oeste do Nilo e da Encefalite de Saint Louis. Nosso método utilizou peptídeos recombinantes correspondentes ao domínio III da proteína de envelope de cada um desses 3 vírus. Adicionalmente, realizamos RT-PCR com primers gênero-específicos e espécie-específicos para a detecção de genoma de flavivírus. Altas soroprevalências foram encontradas nas diversas espécies de roedores capturadas, para um ou mais dos vírus testados. Esse achado sustenta a hipótese de que esses vírus são capazes de infectar também pequenos mamíferos selvagens, o que, por sua vez, pode indicar a existência de novos atores em um ciclo silvestre mantenedor. / Arboviruses affect hundreds of thousands of people annually in Brazil, leading, many of them, to death. Brazil has one of the largest arbovirus biodiversities in the world, with more than 100 species already isolated and documented. Previous studies have revealed that arbovirus species are circulating in the country veiled in urban, rural and forest areas. The knowledge of aspects of epidemiology and viral biology such as silvatic cycle, range of hosts, seasonality, dissemination, area of occurrence, evolutionary history and genetic diversity, are fundamental for the definition of strategies of prevention and control by public health and epidemiological surveillance agencies. In this sense, we aimed to investigate the occurrence of flavivirus in small wild rodents. For this purpose, we captured these animals in fragments of Cerrado and rural properties located in the cities of Ribeirão Preto, Batatais, Cajuru and Luiz Antônio, in the northeast of the state of São Paulo. Blood samples were collected and subjected to the ELISA-indirect serological assay to detect IgG antibodies against flaviviruses Rocio, West Nile, and Saint Louis Encephalitis. Our method used recombinant peptides corresponding to domain III of the envelope protein of each of these 3 viruses. In addition, we performed RT-PCR with genus-specific and speciesspecific primers for the detection of the flavivirus genome. High seroprevalences were found in several species of rodents captured, for one or more of the viruses tested. This finding supports the hypothesis that these viruses are also capable of infecting small wild mammals, which, in turn, may indicate the existence of new actors in a sustaining silvatic cycle.

Introdução: As arboviroses causam graves problemas de saúde pública no Brasil e em muitos dos países da América Latina. A epidemiologia molecular é um instrumento valioso na compreensão da dispersão, persistência e diversidade desses patógenos virais. Objetivos: Neste projeto, buscamos investigar a dinâmica epidemiológica molecular dos arbovíroses (com especial enfoque aos vírus da dengue-DENV, chikungunya - CHIKV e zika -ZIKV) que circularam no estado do Amapá entre os anos de 2013 e 2016. Métodos: 824 amostras de plasma humano foram coletadas pelos laboratórios de Saúde Publica (LACEN) no estado do Amapá entre os anos de 2013 e 2016; essas amostras foram obtidas de pacientes que apresentavam sintomas consistentes com uma das arboviroses. O material genético viral presente nestas amostras foi extraído e os ensaios de qPCR foram realizados. Todas as amostras foram submetidas inicialmente a um ensaio triplex (ZIKV/DENV/CHIKV), as amostras negativas foram posteriormente submetidas a um ensaio de pan-flavivírus. As amostras positivas para um dos ensaios foram submetidas a NGS (sequenciamento de nova geração). Resultados: Das 824 amostras testadas, 36 foram positivas para DENV, ZIKV ou CHIKV; desses 36 positivos, 24 foram para DENV, 11 para CHIKV e 1 para ZIKV. Foram obtidos 27 genomas completos: 16 de DENV (15 DENV1, genótipo V e 1 DENV2, genótipo III) e 11 de CHIKV (genótipo asiático / caribenho). Das 788 amostras testadas com o ensaio de pan-flavivírus, 22 amostras foram positivas; porem apenas uma amostra produziu genoma completo pela técnica de NGS. Este genoma foi relacionado com um flavivírus com semelhante em 76,81% com o vírus Long Pine Key - LPKV, que anteriormente só tinha sido descrito em mosquitos. Árvores de Maximum likelihood e Maximum clade credibility foram construídas utilizando os genomas do DENV1 obtidos neste estudo. Essas árvores exibiam duas linhagens distintas de DENV1, genótipo V presentes na América Latina. Uma destas linhagens tem um padrão de circulação que inclui países do Caribe, América Central e América do Sul (incluindo Brasil); a outra linhagem distinta circula dentro das fronteiras do Brasil. As árvores também indicam que o DENV1 presente no estado do Amapá é da linhagem que tem o padrão de circulação que inclui o Caribe e as Américas Central e do Sul e que essa linhagem surgiu no Amapá entre 2005 e 2010. Conclusão: Este estudo fornece dados importantes sobre as arboviroses no Amapá e os dados genômicos mais recentes disponíveis para a região, bem como o contexto brasileiro e latino-americano para esses dados. Dados dessa natureza são inestimáveis nos esforços das autoridades de saúde pública para a prevenção e controle de epidemias por estes agentes. / Introduction: Arboviral febrile illnesses plague the nation of Brazil and many of its surrounding Latin America countries. Molecular epidemiology is a growing and increasingly invaluable tool in the field of public health for understanding the dispersal, persistence, and diversity of these impactful viral pathogens. Objectives: In this project, the identities and molecular epidemiological dynamics of arboviruses circulating in the Brazilian state of Amapá between the years 2013 and 2016, with special focus on DENV, CHIKV and ZIKV, were investigated and given Brazilian and Latin American geographical and temporal context via molecular epidemiological analyses. Methods: 824 human blood plasma samples were collected from LACEN laboratories in the state of Amapá between the years 2013 and 2016; these samples originated from patients showing symptoms consistent with any of the common arboviral febrile illnesses. The viral genetic material present in these samples was extracted and qPCR diagnostics assays were performed; all samples first underwent a triplex assay (ZIKV/DENV/CHIKV - ZDC), then the samples yielding negative results for the triplex assay underwent a pan-flavivirus assay. The samples yielding positive results for either assay were submitted for NGS and all whole viral genomes subsequently obtained underwent phylogenetic molecular epidemiological analyses. Results: Of the 824 samples tested, 36 tested positive for the ZDC assay; of those positives, 24 tested positive for DENV, 11 for CHIKV, and 1 for ZIKV. 27 full genomes were obtained from these ZDC positives: 16 of DENV (15 DENV1, genotype V and 1 DENV2, genotype III) and 11 of CHIKV (Asian and Caribbean genotype). Of the 788 samples tested with the pan-flavivirus assay, 22 samples yielded positive results, from only one of which a genome was obtainable. This genome was found to be closely related to a flavivirus previously only found in mosquitoes (76.8% identity with Long Pine Key Virus - LPKV). Maximum likelihood and maximum clade credibility trees were constructed using the DENV1 genomes obtained from this study. These trees displayed two distinct lineages of DENV1, genotype V present in Latin America, one of which has a circulation pattern spanning widely across the Caribbean and Central and South America (including Brazil), while the other circulates within Brazilian borders. The trees also indicate that the DENV1 present in the state of Amapá is of the lineage having the wider circulation pattern and that this lineage emerged in Amapá between 2005 and 2010. Conclusion: This study provides important data concerning the range of the arboviral landscape in Amapá and the most recent genomic data available for the region as well as Brazilian and Latin American context to that data. Data of this nature are invaluable in the efforts of public health officials for the prevention and control of epidemics of these impactful arboviral pathogens.

O vírus Oropouche pertence à família Bunyaviridae, gênero Orthobunyavirus, sorogrupo Simbu, e é segunda causa mais freqüente de arbovirose febril no Brasil. Estima-se que mais de meio milhão de casos de febre do Oropouche tenham ocorrido no Brasil nos últimos 30 anos, havendo também ocorrências no Panamá, Peru, Suriname e Trinidad. Epidemias de febre do Oropouche têm sido registradas quase que exclusivamente na Amazônia. Porém, com o aquecimento global do planeta, desmatamentos e conseqüente redistribuição de insetos vetores e animais reservatórios, há risco de disseminação de vírus Oropouche para outras regiões do Brasil e da América do Sul. A patogenia da infecção por Oropouche não é bem entendida. Modelo em roedores, usando inoculação intracerebral, tem sido descrito, mas não com uso da via subcutânea, que mais se assemelha à rota natural da infecção. O objetivo deste trabalho foi estabelecer e caracterizar um modelo experimental de infecção com Oropouche, usando inoculação subcutânea em hamster, a fim de contribuir para o entendimento da patogenia do mesmo. Oropouche da linhagem BeAn19991, passado em cérebro de camundongo recém-nascido, foi inoculado (106,25 TCID50/100?L) por via subcutânea na coxa de hamsters sírios com 2-3 semanas de idade. Em torno de 3 dias alguns animais desenvolveram doença com sinais clínicos brandos caracterizados por: eriçamento dos pêlos e agressividade, outros com características mais graves: perda de peso, tremores sugestivos de calafrios, letargia e paralisia. Os animais foram sacrificados 1, 3, 5, 8 e 11 dias pós-inoculação ou quando eram encontrados em estado agônico. Baço, cérebro, coração, fígado, músculo e sangue foram colhidos para titulação viral, histologia e imunohistoquímica. Infiltrado inflamatório estava presente no cérebro, principalmente em torno dos vasos, meninges e discretamente no coração. O vírus Oropouche foi detectado no tecido cerebral (107,23 TCID50/g), no fígado (106,67TCID50/g) e no sangue (106 TCID50/g). Antígeno de Oropouche foi encontrado, difusamente no fígado, associado com hepatócitos e em neurônios de diversos locais do cérebro. Este modelo reproduz uma infecção sistêmica por Oropouche e pode tornar-se útil no estudo da patogênese, bem como para testar drogas antivirais e possíveis candidatos à vacina. / Oropouche virus belongs to the family Bunyaviridae, genus Orthobunyavirus, serogroup Simbu and is the second most frequent arboviral febrile illness in Brazil. There are estimates of more than half million cases of Oropouche fever in Brazil in the past 30 years, with cases registered in Panama, Peru, Suriname and Trinidad. Oropouche fever has been registered almost exclusively in the Amazon, but with the global warming, deforestation and the consequent redistribution of vectors and reservoir animals, the risk of Oropouche virus dissemination to others areas of Brazil and South America increases. However, the pathogenesis of Oropouche infection is not well understood. Rodent models using intracerebral inoculation have been described, but no attempts to use a route that more closely resembles the natural route of infection have been published. This study was conducted to establish and characterize an experimental model of infection with Oropouche, using subcutaneous inoculation of hamsters, in order to contribute to the understanding of Oropouche pathogenesis. We have established an experimental model through subcutaneous inoculation of hamsters in order to study pathogenesis. Suckling mouse brain passaged Oropouche strain BeAn19991 was inoculated (106,25 TCID50/100?L) subcutaneously in the thigh of syrian hamsters 2-3 weeks old. Around day 3 animals developed disease characterized by lethargy, paralysis, chill-like shaking and ruffled fur. Animals were sacrificed on days 1, 3, 5, 8 and 11 post-inoculation or whenever found to be agonic. Brain, heart, liver, spleen, muscle and blood were harvested for virus titration, histology and Oropouche immunohistochemistry. Inflammatory infiltrate was present in the brain, mostly as perivascular cuffs, meninges and some in the heart. Oropouche titers were 107,23 TCID50/g of tissue in brain 106,67 TCID50/g in liver, and 106 TCID50/g in blood. Oropouche antigen was detected diffusely distributed in the liver in association with hepatocytes, and in neurons in several regions of the brain. This model reproduces systemic Oropouche infection and may become useful in pathogenesis studies, as well as to test antiviral drugs and possible vaccine candidates.

Arboviruses are blood-borne pathogens that threaten half of the world’s population. The recent outbreak of Zika virus (ZIKV) in Brazil has highlighted the importance of developing new strategies to limit virus spread. While vaccines are in development, one way to immediately suppress viral transmission is through biocontrol of mosquito vector. Novel biocontrol strategies utilize microbe – mosquito interactions to inhibit the transmission of pathogens. A powerful tool under investigation is the intracellular bacteria, Wolbachia pipientis, which are maintained in insect populations through maternal transmission. The Wolbachia strain wMel can be trans-infected into mosquitos limiting ZIKV transmission. However, thermal stress can hinder maternal transmission of the wMel strain of Wolbachia. For Wolbachia-based technologies of vector control, it is important to have additional strains with viral suppression capabilities available. We characterized alternative Wolbachia strains in A. albopictus mosquito cell lines and the underlying mechanisms of these interactions. We identified two novel Wolbachia strains with robust arbovirus repression. wAlbB, native to mosquitos blocked 90% of ZIKV growth. More strikingly, wStri, a nonnative symbiont, ablated ZIKV growth in A. albopictus cells below the limit of detection. After showing that ZIKV growth is rescued in wStri infected A. albopictus cells by the pharmacological removal of Wolbachia, we established these cells as an in vitro model for mechanistic studies. Using novel labeling and reporter techniques, we isolate a block in virus growth by Wolbachia at two stages of viral growth, entry and translation. We further show that cholesterol, which can partially rescue viral growth in Wolbachia wStri infected cells, aids in viral entry but does not promote viral growth post entry. Beyond our Wolbachia studies, we further investigated the limited arbovirus growth observed in many A. aegypti cell lines and identified two insect–specific viruses which interfere with arbovirus growth. To address the limited biocontrol tools in C. pipiens mosquitos, we characterized commensal microbiota that may be used as a direct competitor of viruses or as a tool to genetically enhance an antiviral response in the mosquito gut. Together this work expands our understanding of Wolbachia-mediated biocontrol strategies and offers novel resources to suppress arbovirus transmission. / 2020-07-18T00:00:00Z

The emergence of West Nile virus (WNV) has raised important questions about the capacity of the public health infrastructure to implement surveillance and control programs for WNV and other emerging or re-emerging arboviruses in the United States. Florida's mild climate supports year round enzootic transmission of WNV, St. Louis encephalitis virus (SLEV), and Eastern Equine Encephalitis virus (EEEV). It is unknown what effect the establishment of WNV (in 2001) will have on SLEV transmission in Florida, where these closely related flaviviruses share amplifying hosts, habitats, and vectors. An Arbovirus Isolation Network was formed to obtain and characterize arbovirus strains collected from a large population of naturally exposed birds, including sentinel chickens and wild birds admitted to rehabilitation centers in Florida. Weekly sentinel seroconversion data was used to target sampling of chicken flocks at 37 active sites (17 WNV, 7 EEEV, and 13 SLEV) in eight counties from 224 birds during 2005-2006. Sampling of wild birds occurred following admittance at rehabilitation centers in 2006, based on symptoms and known amplifying host species (n=64), but virus was not detected. We report the isolation of St. Louis encephalitis virus, West Nile virus and detection of Eastern Equine Encephalitis viral RNA from cloacal swabs of naturally exposed adult sentinel chickens. We also report the first known dual infection and isolation of St. Louis encephalitis and West Nile viruses from one chicken. In addition, a novel flavivirus strain was detected in two chickens. Early season transmission of WNV appears to limit subsequent infection and amplification of SLEV late in the year. Phylogenetic analysis revealed that the introduction (and re-introduction) of South American (Brazil) SLEV occurred in 1972 and 2006 in Florida. These strains represent the first reported isolation of South American strains of SLEV in the United States, with placement in Lineage VA and VB, as proposed by Kramer and Chandler (2001). Arbovirus isolation remains an effective tool for surveillance programs and a targeted strategy is most cost-effective to capture arboviruses in their natural settings for molecular epidemiology analysis that can elucidate genetic variations impacting virulence, mosquito infectivity, and disease potential of these pathogens.

Public Health and clinical laboratories occasionally obtain viral isolates that cannot be typed by routine methods. Therefore, the sequence-independent, single primer amplification (SISPA) technique was adapted to rapidly identify and characterize viral isolates of unknown etiology. A panel of known (West Nile virus and St. Louis encephalitis virus) and unknown viral isolates (environmental samples collected in Florida) were used to develop and refine the SISPA technique. Selectivity for viral genomic sequences was obtained through enriching viral particles by centrifugation, removal of cellular debris by filtration and removal of host genomic material by benzonase application. The SISPA method successfully amplified the panel of known viruses and a previously unknown environmental viral isolate. The previously unknown environmental viral isolate was determined to be closely related, if not identical, to Flanders virus, a member of Rhabdoviradae. A Flanders virus specific RT-PCR assay identified a total of five previously unknown environmental viral isolates as Flanders virus. Unidentified viral isolates were obtained during arbovirus surveillance efforts in Florida, either from the Florida Department of Health program (BOL-Tampa) during 2005 – 2009, or collected during an ongoing project at the University of South Florida studying the ecology of arthropod-borne encephalitis viruses at sites located in Florida. In a concurrent study, SISPA was successfully used to characterize an unidentifiable virus isolate related to members of the Bunyaviradae family which was designated as Infirmatus virus. Natural mosquito population (10,557 mosquitoes) collected in Florida was screened for Flanders virus and members of Bunyaviradae to determine infection prevalence. Although Flanders virus was not detected in this population, Infirmatus virus was identified in 14 mosquito pools with the highest infection prevalence in Cx. quinquefasciatus mosquitoes. The SISPA technique was successful for the genetic identification of unknown viral isolates and application of this method to samples with suspected or unidentified viral etiologies may be used to enhance public health surveillance of emerging or re-emerging viruses in Florida.

No description available.

Wolbachia are maternally inherited, obligate, intracellular bacteria inducing a form of sterility known as cytoplasmic incompatibility. Wolbachia based strategies have been proposed for the control of disease vectors. One example is to use a population replacement strategy to drive into natural population a novel Wolbachia that modifies the age structure of a vector population, reducing disease transmission. In this research, the effects of a life-shortening stain of Wolbachia (popcorn Wolbachia) are transferred into the mosquitoes Aedes albopictus (Chapter Two and Three) and A. aegypti (Chapter Four and Five). In Chapter Two, the Wolbachia symbiosis significantly reduced fecundity and egg hatches in A. albopictus, with Wolbachia being highly pathogenic in this mosquito species. In Chapter Three, the relationship between popcorn Wolbachia and its host (in a triple-infected mosquito strain) varied with the mosquito diet. Feeding on mouse blood was associated with the loss of infection, whereas the infection was maintained in human blood-fed mosquito lines. Egg viability of triple infected mosquito was reduced only with mouse blood. In Chapter Four, the reduced competitiveness (e.g., low survival and increased developmental time) of infected A. aegypti immatures was associated with popcorn Wolbachia, relative to uninfected individuals in low food condition. In Chapter Five, the decreased survival of immature A. aegypti was associated with popcorn Wolbachia in the presence of potential predators (i.e., older A. aegypti or A. albopictus larvae). Using a novel behavioral assay, a delayed larval reaction to light avoidance was observed to be associated with the infection, suggesting Wolbachia effects on immature host behaviors. In Chapter Six, popcorn Wolbachia and wAlbB infected A. aegypti showed similar reproduction potential. No reduced level of CI or mating competitiveness was observed in wAlbB infected males. The results suggest the wAlbB infection in A. aegypti can be an additional agent for Wolbachia-based control strategies. In Chapter Seven, a filtering system using commercially available sieves was able to separate immature mosquitoes from water, preventing escape of mosquitoes. In Chapter Eight, an inexpensive artificial blood feeding was designed for feeding multiple mosquito cages. The results support the use of these tools to facilitate mass rearing of mosquitoes.

No description available.

As an outcome of very active arbovirus monitoring programs that began in Australia in the 1950s, some of the most diverse and unusual rhabdoviruses in the world have been isolated from this continent. These novel rhabdoviruses represent an important and valuable pool of highly diverse viruses; however, most of them have remained poorly characterised. In light of the significant disease potential of numerous rhabdoviruses, the characterisation of novel rhabdoviruses is indispensable for threat assessment to livestock, wildlife and humans and preparedness for outbreaks. The genetic characterisation of novel viruses is also an essential step for the development of molecular detection assays for improved monitoring and investigations into unidentified disease cases. In this study, the complete genomes of four novel rhabdoviruses have been sequenced and a fifth is close to completion. The substantial new data generated has significantly extended the understanding of the biology and evolution of the Rhabdoviridae. Wongabel virus (WONV), isolated from the biting midge Culicoides austropalpalis, was found to contain a unique genome structure encoding ten genes, including five novel genes (Chapter 2). Analysis by western blotting suggested that four out of the five novel genes were expressed in infected cell cultures. Ngaingan virus (NGAV), isolated from Culicoides brevitarsis, was found to have the largest genome of any rhabdovirus sequenced to date, and with thirteen genes has the largest number of genes of any (-) ssRNA virus sequenced to date (Chapter 3). Seven of the thirteen genes are novel. Similar to viruses in the genus Ephemerovirus (bovine ephemeral fever virus and Adelaide River virus), NGAV contains a second glycoprotein with an unknown function. Phylogenetic analysis places this virus alongside WONV and the north-American bird and mosquito-associated Flanders virus within the Hart Park group that remains to be classified by the ICTV. Screening of various wildlife and livestock sera collected in northern Australia indicated a strong association of NGAV with macropods. Tibrogargan virus (TIBV) and Coastal Plains virus (CPV) were isolated from cattle and Culicoides brevitarsis (TIBV). Past serological surveys reported both viruses to be highly prevalent in cattle in northern Australia and demonstrated that the two viruses share a relatively close relationship at the antigenic level. The genomic analyses revealed that these two viruses have a unique genome organization, with three additional genes (Chapter 4). These additional genes are highly diverged at the sequence level but the encoded putative proteins share a significant conservation of secondary structure elements. The sequencing of these two related viruses has provided a unique opportunity to gain insights into the characteristics and evolution of novel proteins in two different rhabdoviruses. Phylogenetic analyses showed that TIBV and CPV form an independent cluster which does not appear to belong to any of the current genera, but which is most closely related to the genus Ephemerovirus based on N protein analysis. Although neither virus has been associated with disease, a serological survey of various animal sera collected in northern Australia showed that these viruses are currently highly prevalent in sentinel cattle and buffalo. Oak Vale virus (OVRV) was isolated from mosquitoes, Culex edwardsi and Ochlerotatus vigilax, from two geographically diverse regions of Australia located approximately 3000 km apart. The genome of OVRV was found to contain only one novel gene (Chapter 5). Comparatively, the genome of this virus is much less complex than the others in this study, but this virus displays considerable divergence from all other rhabdoviruses. A high seroprevalence for this virus was found in the feral pig population in northern Australia. The data generated from this study represents a considerable increase in the quantity of genetic data available for this viral family, and has revealed the existence of a large number of previously unidentified genes, highlighting that that the potential for complexity within the prototype genomic model of a rhabdovirus is much greater than previously thought. The novel nature of the additional genes provides grounds for further research into rhabdovirus evolution. Analysis of this new data suggests that these viruses cannot be classified into existing genera under the current criteria and it is clear that the taxonomy of the Rhabdoviridae requires revision. The observation that these viruses are currently circulating in livestock and wildlife in northern Australia accentuates the need for closer monitoring of animals and the need for further study of this diverse and fascinating group of viruses.