ABSTRACT
ABSTRACT Brazil is a great source of arbovirus diversity, mainly in the Amazon region. However, other biomes, especially the Atlantic Forest, may also be a hotspot for emerging viruses, including Bunyaviruses (Negarnaviricota: Bunyavirales). For instance, Vale do Ribeira, located in the Southeastern region, has been widely studied for virus surveillance, where Flavivirus, Alphavirus and Bunyaviruses were isolated during the last decades, including Bruconha virus (BRCV), a member of Orthobunyavirus genus Group C, in 1976. Recently, a new isolate of BRCV named Span321532 was obtained from an adult sentinel mouse placed in Iguape city in 2011, and a full-length genome was generated with nucleotide differences ranging between 1.5%, 5.3% and 5% (L, M and S segments, respectively) from the prototype isolated 35 years earlier. In addition, each segment placed BRCV into different clusters, showing the high variety within Bunyavirales. Although no evidence for reassortants was detected, this finding reiterates the need for new surveillance and genomic studies in the area considering the high mutation rates of arbovirus, and also to identify the hosts capable of supporting the continuous circulation of Orthobunyavirus.
ABSTRACT
Os Flavivirus são transmitidos por mosquitos (Diptera: Culicidae) que se refugiam em remanesctentes de Mata Atlântica. Essas áreas verdes correspondem às Unidades de Conservação e parques urbanos, que estão espalhados pela região metropolitana de São Paulo. Este estudo foi realizado com o intuito de conhecer as espécies de culicídeos que circulam na Área de Proteção Ambiental (APA) Capivari-Monos, na zona Sul do município de São Paulo, e no Parque Estadual da Cantareira (PEC), na zona Norte do mesmo município, e de investigar infecção natural por Flavivirus na fauna de culicídeos amostrada. Também foi proposto relacionar a variedade, a quantidade e identidade dos Flavivirus detectados com os padrões de riqueza, abundância e diversidade das assembleias de mosquitos. Foram realizadas 14 coletas, mensalmente, em quatro pontos de coleta na APA e três no PEC, todos com diferentes níveis de intervenção antrópica, no período de março de 2016 a abril de 2017. Armadilhas automáticas luminosastipo CDC (com atração de CO2 e ácido lático) foram instaladas na copa das árvores e no nível do solo. O esforço amostral foi equivalente para os todos os pontos, sendo que foram instaladas duas armadilhas em cada ponto (uma na copa e outra no solo), com 18 horas de coleta, permitindo amostragem de culicídeos de hábitos diurnos, crepusculares e noturnos. Os espécimes foram transportados com vida para o Laboratório de Saúde Pública da Faculdade de Saúde Pública da Universidade de São Paulo, criopreservados a temperatura -70ºC, identificados morfologicamente e agrupados em pools (com até 10 indivíduos). Os pools foram submetidos à técnica de isolamento viral em cultura de células (C6/36), seguida do teste de imunofluorescência indireta. Os pools positivos foram submetidos à reação de RT-qPCR e, posteriormente, sequenciados. Duas árvores de similaridade foram construídas para confirmação dos Flavivirus. No total, 1216 exemplares de culicídeos foram amostrados (13 gêneros), cuja riqueza foi de 42 táxons. A APA registrou a maior abundância (878 espécimes) e maior riqueza (37 táxons). A Cachoeira foi o ponto de coleta na APA que amostrou a maior riqueza e abundância, contudo, com a mais baixa diversidade. Entretanto, a Borracharia obteve alta riqueza, baixa abundância e a maior diversidade. O PEC amostrou 338 indivíduos e a riqueza foi de 23 táxons. Dentre os pontos do PEC, a Trilha do Pinheirinho amostrou a maior riqueza e abundância. An. (Ker.) cruzii, Cx. (Cux.) sp, Cx. (Mel.) vaxus, Li. durhami, Wy. (Prl.) confusa e Wy. (Pho.) theobaldi foram detectadas com infecção natural por Flavivirus. O sequenciamento revelou infecção por ZIKV em An. (Ker.) cruzii, Li. durhami e Wy. (Prl.) confusa, e infecção por DENV-2 em Cx. (Cux.) sp e Cx. (Mel.) vaxus. Concluiu-se que a riqueza, abundância e diversidade estão relacionadas entre si e, juntas, influenciaram na detecção de espécies de culicídeos naturalmente infectadas por Flavivirus, sendo que estes foram detectados em espécies provenientes de pontos de coleta cuja riqueza e abundância foram altas, e a diversidade baixa. A quantidade e a variedade dos Flavivirus também foram influenciadas por esses três fatores, para ocorrer na natureza. Não foi possível correlacionar a identidade dos Flavivirus com os três fatores uma vez que as espécies detectadas com infecção natural por esses vírus não são apontadas como potenciais vetoras. Além disso, a abundância e a diversidade pareceram ter uma relação inversa entre si
Flaviviruses are transmitted by mosquitoes (Diptera: Culicidae) that take refuge in remnants of the Atlantic Forest. These green areas correspond to Conservation Units and urban parks which are spread throughout the metropolitan region of São Paulo. This study was carried out in order to identify the Culicidae fauna that circulate in Capivari-Monos Environmental Protection Area (APA), located in the South area of the city of São Paulo, and in Cantareira State Park (PEC), North area of the same municipality and to investigate natural Flaviviruses infection in this sampled Culicidae fauna. It was also proposed to relate the variety, quantity and identity of the Flaviviruses detected with patterns of richness, abundance and diversity of mosquito assemblages. Fourteen collections were carried out monthly at four collection sites in the APA and three in the PEC, all sites with different levels of anthropogenic intervention, during March 2016 to April 2017. CDC automatic traps (with attraction of CO2 and lactic acid) were installed in the canopy and on ground. The sampling effort was equivalent for all the points, and two traps were installed at each point (one in the canopy and the other on ground), with 18 hours of sampling, allowing sampling culicidae of daytime, morning and evening twilight, and nightlyl habits. The specimens were carried alive to the Public Health Laboratory of the School of Public Health of the University of São Paulo, were cryopreserved at a -70ºC temperature, identified morphologically and grouped in pools (with up to 10 individuals). The pools were submitted to the virus isolation technique in cell culture tissue (C6 / 36), followed by the indirect immunofluorescence test. The positive pools were submitted to the RT-qPCR reaction and, subsequently, sequenced. Two similarity trees were made only to confirm Flaviviruses infection. In total, 1216 specimens of culicidae were sampled (13 genera), and the richness was 42 taxa. In addition to APA recorded the highest abundance (878 specimens) and also highest richness (37 taxa). Cachoeira was the collection site in APA that showed the greatest richness and abundance as well, however, with the lowest diversity. In addition, Borracharia obtained high richness, low abundance and highest diversity. PEC sampled 338 specimens and the richness was 23 taxa. Among the collection sites of the PEC, Pinheirinho Trail showed the highest richness and also abundance. An. (Ker.) cruzii, Cx. (Cux.) sp, Cx. (Mel.) vaxus, Li. durhami, Wy. (Prl.) confusa and Wy. (Pho.) theobaldi were detected with natural Flaviviruses infection. The sequencing analyzes revealed ZIKV infection in An. (Ker.) cruzii, Li. durhami and Wy. (Prl.) confusa, and DENV-2 infection in Cx. (Cux.) sp and Cx. (Mel.) vaxus. It has concluded that the richness, abundance and also diversity are related to each other and, together, influenced the detection of species of culicidae naturally infected by Flaviviruses, which were detected in species from collection sites whose richness and abundance were high. About quantity and variety of Flaviviruses, these were also influenced by the three factors on nature. It was not possible to correlate the identity of the Flaviviruses with the three factors since the species detected with natural infection by these viruses are not indicated as potential vectors. Moreover, abundance and diversity appeared to have an inverse relation.