Description of the mitogenome and phylogeny of Aedes spp. (Diptera: Culicidae) from the Amazon region.

The genus Aedes (Diptera: Culicidae) includes species of great epidemiological relevance, particularly involved in transmission cycles of leading arboviruses in the Brazilian Amazon region, such as the Zika virus (ZIKV), Dengue virus (DENV), Yellow fever virus (YFV), and Chikungunya virus (CHIKV). We report here the first putatively complete sequencing of the mitochondrial genomes of Brazilian populations of the species Aedes albopictus, Aedes scapularis and Aedes serratus. The sequences obtained showed an average length of 14,947 bp, comprising 37 functional subunits, typical in animal mitochondria (13 PCGs, 22 tRNA, and 2 rRNA). The phylogeny reconstructed by Maximum likelihood method, based on the concatenated sequences of all 13 PCGs produced at least two non-directly related groupings, composed of representatives of the subgenus Ochlerotatus and Stegomyia of the genus Aedes. The data and information produced here may be useful for future taxonomic and evolutionary studies of the genus Aedes, as well as the Culicidae family.

Description of mitochon genome and phylogenetic considerations of Sabethes bipartipes, Sabethes cyaneus, Sabethes quasicyaneus, and Sabethes tarsopus (Diptera: Culicidae).

The genus Sabethes (Diptera: Culicidae) comprises species of great epidemiological relevance, particularly involved in transmission cycles of the Yellow fever virus in South America. Given the unavailability of information related to aspects of evolutionary biology and molecular taxonomy of species of this genus of mosquitoes, we report here the first sequencing of the mitochondrial genomes of Sabethes bipartipes, Sabethes cyaneus, Sabethes tarsopus, and Sabethes quasicyaneus. The sequences obtained showed an average length of 14,920 bp, comprising 37 functional genes (13 PCGs, 22 tRNA, and 02 rRNA). The phylogenies reconstructed by Maximum likelihood and Bayesian inference methods, based on the concatenated sequences of all 13 PCGs, produced similar topologies and strongly supported the monophyletic relationship between the Sabethes subgenera, corroborating the known taxonomic classification based on aspects of the external morphology of the taxa assessed. The data and information produced from the Sabethes species evaluated here may be useful for future taxonomic and evolutionary studies of the genus, as well as the Culicidae family.

Negeviruses isolated from mosquitoes in the Brazilian Amazon.

BACKGROUND: There are several groups of viruses including Insect Specific Viruses (ISV) such as the taxon Negevirus, a group of viruses phylogenetically related to plant viruses. Negeviruses replicate in mosquito cells, but not in vertebrate cells. METHODS: Pools of hematophagous arthropods were inoculated in Vero and C6/36 cells. The cells were observed to detect possible cytopathic effect. Then, indirect immunofluorescence, RT-PCR, and nucleotide sequencing were performed. RESULTS: Seven samples which presented negative results for flaviviruses, alphaviruses and bunyaviruses, but showed cytopathic effect in C6/36 cells were sequenced. We identified the occurrence of a variety of ISVs, most of them belonging to the taxon Negevirus: The Brejeira, Negev, Cordoba and Wallerfield viruses, including a new virus for science, tentatively named Feitosa virus. CONCLUSIONS: We detected negeviruses in the Amazon region, including two viruses that were isolated for the first time in Brazil: Cordoba virus and the Negev virus and, a new virus for science: the Feitosa virus.

Land-use effects on mosquito biodiversity and potential arbovirus emergence in the Southern Amazon, Brazil.

The Amazon rainforest is considered the largest reservoir of culicids and arboviruses in the world. It has been under intense human-driven alteration, especially in the so-called 'Arc of Deforestation', located in the eastern and southern regions. The emergence and transmission of infectious diseases are increasing, potentially due to land-use change. We used landscape-scale mosquito surveillance across a forest fragmentation gradient in the southern Amazon to evaluate the relationship between forest disturbance and the composition and structure of mosquito communities with a particular focus on the potential for arbovirus emergence in the region. Generalized linear models and logistic regression were used to associate the degree of landscape disturbance with arbovirus vectors' richness and abundance. A total of 1,960 culicids, belonging to 50 species, were collected from 2015 to 2016. Among these species, 20 have been associated with the transmission of arboviruses. Our results show an association of land use, more specifically small size of forest remnants with more irregular shape and higher edge density, with the increase of arbovirus vectors' richness and abundance. Six species of mosquito vectors exhibited a higher probability of occurrence in landscapes with medium or high degrees of disturbance. Our results indicate that land-use change influences mosquito communities with potential implications for the emergence of arboviruses.

First Description of the Mitogenome and Phylogeny of Culicinae Species from the Amazon Region.

The Culicidae family is distributed worldwide and comprises about 3587 species subdivided into the subfamilies Anophelinae and Culicinae. This is the first description of complete mitochondrial DNA sequences from Aedes fluviatilis, Aedeomyia squamipennis, Coquillettidia nigricans, Psorophora albipes, and Psorophora ferox. The mitogenomes showed an average length of 15,046 pb and 78.02% AT content, comprising 37 functional subunits (13 protein coding genes, 22 tRNAs, and two rRNAs). The most common start codons were ATT/ATG, and TAA was the stop codon for all PCGs. The tRNAs had the typical leaf clover structure, except tRNASer1. Phylogeny was inferred by analyzing the 13 PCGs concatenated nucleotide sequences of 48 mitogenomes. Maximum likelihood and Bayesian inference analysis placed Ps. albipes and Ps. ferox in the Janthinosoma group, like the accepted classification of Psorophora genus. Ae. fluviatilis was placed in the Aedini tribe, but was revealed to be more related to the Haemagogus genus, a result that may have been hampered by the poor sampling of Aedes sequences. Cq. nigricans clustered with Cq. chrysonotum, both related to Mansonia. Ae. squamipennis was placed as the most external lineage of the Culicinae subfamily. The yielded topology supports the concept of monophyly of all groups and ratifies the current taxonomic classification.

West Nile Virus in the State of Ceará, Northeast Brazil.

In June 2019, a horse with neurological disorder was diagnosed with West Nile virus (WNV) in Boa Viagem, a municipality in the state of Ceará, northeast Brazil. A multi-institutional task force coordinated by the Brazilian Ministry of Health was deployed to the area for case investigation. A total of 513 biological samples from 78 humans, 157 domestic animals and 278 free-ranging wild birds, as well as 853 adult mosquitoes of 22 species were tested for WNV by highly specific serological and/or molecular tests. No active circulation of WNV was detected in vertebrates or mosquitoes by molecular methods. Previous exposure to WNV was confirmed by seroconversion in domestic birds and by the detection of specific neutralizing antibodies in 44% (11/25) of equids, 20.9% (14/67) of domestic birds, 4.7% (13/278) of free-ranging wild birds, 2.6% (2/78) of humans, and 1.5% (1/65) of small ruminants. Results indicate that not only equines but also humans and different species of domestic animals and wild birds were locally exposed to WNV. The detection of neutralizing antibodies for WNV in free-ranging individuals of abundant passerine species suggests that birds commonly found in the region may have been involved as amplifying hosts in local transmission cycles of WNV.

Mitochondrial genome sequencing and phylogeny of Haemagogus albomaculatus, Haemagogus leucocelaenus, Haemagogus spegazzinii, and Haemagogus tropicalis (Diptera: Culicidae).

The genus Haemagogus (Diptera: Culicidae) comprises species of great epidemiological relevance, involved in transmission cycles of the Yellow fever virus and other arboviruses in South America. So far, only Haemagogus janthinomys has complete mitochondrial sequences available. Given the unavailability of information related to aspects of the evolutionary biology and molecular taxonomy of this genus, we report here, the first sequencing of the mitogenomes of Haemagogus albomaculatus, Haemagogus leucocelaenus, Haemagogus spegazzinii, and Haemagogus tropicalis. The mitogenomes showed an average length of 15,038 bp, average AT content of 79.3%, positive AT-skews, negative GC-skews, and comprised 37 functional subunits (13 PCGs, 22 tRNA, and 02 rRNA). The PCGs showed ATN as start codon, TAA as stop codon, and signs of purifying selection. The tRNAs had the typical leaf clover structure, except tRNASer1. Phylogenetic analyzes of Bayesian inference and Maximum Likelihood, based on concatenated sequences from all 13 PCGs, produced identical topologies and strongly supported the monophyletic relationship between the Haemagogus and Conopostegus subgenera, and corroborated with the known taxonomic classification of the evaluated taxa, based on external morphological aspects. The information produced on the mitogenomes of the Haemagogus species evaluated here may be useful in carrying out future taxonomic and evolutionary studies of the genus.

Chikungunya virus Detection in Aedes aegypti and Culex quinquefasciatus during an Outbreak in the Amazon Region.

Chikungunya virus (CHIKV) was first reported in Brazil in 2014 and, after it spread countrywide, an outbreak of febrile illness with reports of arthralgia happened in the municipality of Xinguara, Pará, Brazil in 2017, indicating the virus' circulation. Here, we aimed to investigate CHIKV in mosquito vectors collected during an active surveillance of virus isolation in cell culture by using molecular detection and viral genome sequencing. A total of 492 Aedes, Culex and Mansonia mosquitoes were collected and separated in 36 pools according to the species and sex, and 22.2% (8/36) were positive. CHIKV was indentified in pools of Ae. aegypti females (n = 5), an Ae. aegypti male (n = 1) and in Culex quinquefasciatus females (n = 2). However, as the mosquitoes' whole bodies were macerated and used for detection, one cannot suggest the role of the latter in the viral transmission. Despite this, vector competence studies must be carried out in the different species to investigate long-term adaptations. Viral genome sequencing has characterized the East-Central-South-African (ECSA) genotype in all positive pools analyzed, corroborating previous reports for the Amazon region.

Ecological aspects of potential arbovirus vectors (Diptera: Culicidae) in an urban landscape of Southern Amazon, Brazil.

Amazon has been under intense human pressure, especially in the so-called "Arc of Deforestation" in the Eastern and Southern regions. Changes in biodiversity due to landscape disturbance in municipalities at the Arc of Deforestation are likely to impact mosquito species leading to the potential for emergence of arboviruses. Our aim was to describe the composition and structure of culicids in Sinop, a municipality located in the Southern Amazon at the State of Mato Grosso, as well as their presence in urban habitats, analyzing climate variables and potential risk of arbovirus transmission. Mosquitoes were collected in four habitats located in urban environments for 12 months. We recovered 5,210 adult mosquitoes from mosquito traps. Among the 33 species identified, 19 are associated with arbovirus transmission. Higher abundance, richness, diversity, and equitability of mosquito species were observed in urban forest parks and in new neighborhoods located in the outskirts of the city, nearby forest fragments and agricultural areas. As the environment consolidates as urban, both abundance and richness decrease. Highly urbanized neighborhoods had higher dominance of Culex quinquefasciatus, a mosquito associated with different arboviruses, including West Nile and Saint Louis encephalitis virus. The medically important species Cx. declarator, Cx. (Melanoconion) ssp., Aedeomyia squamipennis and Aedes scapularis were found in the four habitats. It is possible that these mosquitoes are adapting to different habitats and as a consequence, they are reaching urbanized areas. Mosquito abundance was higher in the days of high temperature and low precipitation in urban forest parks and in highly urbanized neighborhoods. These results suggest that Sinop region is a key area for surveillance of arbovirus vectors.

Circulation of Chikungunya virus in Aedes aegypti in Maranhão, Northeast Brazil.

The simultaneous circulation of Dengue virus (DENV), Chikungunya virus (CHIKV) and Zika virus (ZIKV) arboviruses have placed Brazil among the main worldwide endemic areas. Brazilian Northeast region concentrates the highest incidence of infections caused by CHIKV and ZIKV. In Maranhão, the second biggest northeastern state, there are cases of human infections caused by these three arboviruses and presence of Aedes aegypti and Aedes albopictus vectors. In this context, this study aimed to investigate the circulation of CHIKV, DENV and ZIKV in Ae. aegypti and Ae. albopictus mosquitoes collected in urban areas of Barra do Corda, Caxias, Codó, São Luís and São Mateus do Maranhão municipalities in the state of Maranhão through Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) technique. 428 Ae. aegypti and 1 Ae. albopictus were collected, which formed 44 pools. Three of these showed positive results for CHIKV: AR832767 (five Ae. aegypti female collected in Caxias), AR832784 and AR832785 (both composed of 20 Ae. aegypti female collected in São Mateus do Maranhão). This study consolidates information about CHIKV circulation in state of Maranhão, as well as the role of Ae. aegypti in the transmission of CHIKV in urban area.

Not in wilderness: African vulture strongholds remain in areas with high human density.

Vultures constitute an important functional group in many ecosystems, providing crucial ecosystem services both in natural and humanized environments. These scavengers are facing massive declines worldwide, but in several African countries virtually nothing is known on populations' status and threats, hampering the development of adequate conservation strategies. In Guinea-Bissau, globally important populations of Hooded Necrosyrtes monachus and African white-backed vultures Gyps africanus were recently reported. Using the country as a study area, we aim to characterize human-vulture interactions in West Africa applying a multidisciplinary approach. We assessed the status and distribution of vulture populations using data from 1711 km of roadside transects, examined predictors of their distribution, and produced a nationwide population estimate for the Hooded Vulture, using an innovative method based on the relationship between the size of human population in settlements and vulture numbers. We conducted 47 stakeholder interviews to assess perceived roles played by vultures, and to investigate potential anthropogenic threats. Hooded vultures were strongly associated with high human population densities, whereas no relation was found between African white-backed and Rüppell's vultures and any of the tested predictors, which included cattle density, precipitation and Normalized Difference Vegetation Index, among others. We estimate a national population of 43347 Hooded vultures, the largest population reported in the species range. Respondents were generally aware of the services provided by vultures, especially waste and carcass removal, including in urban areas. Hunting for witchcraft and traditional medicine was the most frequently recognised threat, while poisoning was ranked as having the highest impact. We hypothesise that poisoning-related mortality may be affecting African white-backed and Rüppell's vultures' distribution and explain their scarcity in apparently highly suitable habitats. Our results suggest a mutualistic rather than a commensalistic relationship between vultures and humans, with important implications for designing and implementing conservation strategies.

Characterization of mitochondrial genome of Haemagogus janthinomys (Diptera: Culicidae).

Haemagogus janthinomys is a mosquito of high importance in public health due its involvement on natural wild cycles of two important arboviruses in the Brazilian Amazon region: Yellow Fever virus (Flaviviridae, Flavivirus) and Mayaro virus (Togaviridae, Alphavirus). Here, we have sequenced and described all the mitochondrial genes for the Hg. janthinomys species. The complete coding sequence is14 937 bp long and includes 37 functional genes, of which 13 codes for proteins, 22 for tRNA and 2 for ribosomal subunits. Region A + T (control region) is not presented here. The data should be helpful on further taxonomic and evolutionary studies of this important arbovirus vector.

Vector aspects in risk areas for sylvatic yellow fever in the state of Mato Grosso do Sul, Brazil

Epizootics and yellow fever epidemics in 2008 in Brazil, Paraguay and Argentina marked a significant progression of the disease further south in the continent affecting non-vaccinated human populations. The state of Mato Grosso do Sul is located between the tropical endemic region and the subtropical epidemic region and, despite being an area recommended for vaccination since the 1980s, human and animal cases of the disease are still registered. The purpose of this study was to present the results of entomological surveys of probable sites of infection (PSI) geographically describing these areas regarding human cases and disease suspicion reported in 2008. Thirteen locations in nine municipalities were investigated. A total of 305 females of the genus Haemagogus and Sabethes and five specimens of Aedes albopictus were obtained. The genus Haemagogus was more abundant but Sabethes presented twice as many species (3 x 6) and higher distribution within the collection points. The most abundant species was Hg. janthinomys, with 102 individuals in one location. The other species found were: Hg. leucocelaenus, Hg. spegazzinii, Sa. albiprivus, Sa. belisarioi, Sa. chloropterus, Sa. glaucodaemun, Sa. intermedius and Sa. soperi. It was not possible to identify predominant species in relation to environmental and geographical characteristics of the collection points. Of the nine human cases, eight corresponded to persons not resident in the State, seven of them tourists. MS features a large diversity of rural and wildlife tourist attractions where the yellow fever vectors described in this work are present. In this sense, the monitoring of vaccination coverage and flow of non-vaccinated people through the area are crucial to controlling the disease.

Emergence of new insect-restrictive viruses in the Amazon region.

The complete genome was determined for 12 viruses isolated from 8 different pools of mosquitoes (Culex sp. and Psorophora ferox) collected at Brejeira farm, Canaan dos Carajas, Para state in northern Brazil. Eight of the viruses were distantly related to Piura virus, hereafter designated as Brejeira virus; the other 4 were similar to Wallerfield virus.

Fauna of Culicidae in rural areas of Porto Velho and the first record of Mansonia (Mansonia) flaveola (Coquillet, 1906), for the state of Rondônia, Brazil.

INTRODUCTION: Knowledge concerning the fauna of Culicidae in the Brazilian Amazon States contributes to current understanding of the bionomics of the insects collected and makes it possible to observe changes in the fauna over time. METHODS: The Culicidae were captured with a BG-Sentinel® trap in extra-domiciliary area of two rural regions of Porto Velho in June and July of 2007 and 2008. RESULTS: A total of 10,695 Culicidae was collected, belonging to nine genera: Coquillettidia, Culex, Mansonia, Psorophora, Aedes, Aedeomyia, Anopheles, Uranotaenia and Wyeomyia. CONCLUSIONS: The presence of Mansonia (Mansonia) flaveola was recorded in the State of Rondônia for the first time.

Fauna of Culicidae in rural areas of Porto Velho and the first record of Mansonia (Mansonia) flaveola (Coquillet, 1906), for the state of Rondônia, Brazil / Fauna de Culicidae em áreas rurais de Porto Velho e primeiro registro de Mansonia (Mansonia) flaveola (Coquillet, 1906), para o estado de Rondônia, Brasil

INTRODUCTION: Knowledge concerning the fauna of Culicidae in the Brazilian Amazon States contributes to current understanding of the bionomics of the insects collected and makes it possible to observe changes in the fauna over time. METHODS: The Culicidae were captured with a BG-Sentinel® trap in extra-domiciliary area of two rural regions of Porto Velho in June and July of 2007 and 2008. RESULTS: A total of 10,695 Culicidae was collected, belonging to nine genera: Coquillettidia, Culex, Mansonia, Psorophora, Aedes, Aedeomyia, Anopheles, Uranotaenia and Wyeomyia. CONCLUSIONS: The presence of Mansonia (Mansonia) flaveola was recorded in the State of Rondônia for the first time.

INTRODUÇÃO: O conhecimento da fauna de culicídeos em estados da Amazônia Brasileira auxilia no conhecimento da bionomia dos insetos obtidos, além de possibilitar a observação de modificações da fauna ao decorrer do tempo. MÉTODOS: As capturas dos culicídeos foram realizadas em área extradomiciliar de duas zonas rurais de Porto Velho, durante junho e julho, nos anos de 2007 e 2008, com a armadilha BG-Sentinel®. RESULTADOs: Foram coletados 10.695 culicídeos, distribuídos em nove gêneros: Coquillettidia, Culex, Mansonia, Psorophora, Aedes, Aedeomyia, Anopheles, Uranotaenia e Wyeomyia. CONCLUSÕES: Foi registrada pela primeira vez no estado a presença de Mansonia (Mansonia) flaveola.

Infecção natural de Haemagogus janthinomys e Haemagogus leucocelaenus pelo vírus da febre amarela no Distrito Federal, Brasil, 2007-2008 / Haemagogus janthinomys and Haemagogus leucocelaenus naturally infected with yellow fever virus in Federal District, Brazil, 2007-2008

Objetivo: investigar a infectividade natural de Haemagogus janthinomys e Haemagogus leucocelaenus pelo vírus da febre amarela em áreas com notificação de casos humanos e epizootia em primatas não humanos no Distrito Federal (DF), Brasil. Métodos:realizaram-se capturas de Culicidae utilizando capturador de Castro e puçá, no período de dezembro de 2007 a fevereiro de 2008; a identificação taxonômica e o isolamento viral foram realizados no Instituto Evandro Chagas/ Ministério da Saúde; a inoculação em camundongo e o teste de imunofluorescência indireta foram realizados para identificar o agente viral. Resultados: amostras deHg. janthinomys e Hg. leucocelaenus procedentes da Região Administrativa de São Sebastião/DF apresentaram positividade para o vírus amarílico. Conclusão: a confirmação de infectividade natural por Hg. janthinomys e Hg. leucocelaenus sugere que as áreas pesquisadas são receptivas e vulneráveis ao vírus da febre amarela, tornando imprescindível aprimorar as estratégias de sua vigilância epidemiológica no Distrito Federal.

Objective: to investigate the natural infectivity of Haemagogus janthinomys and Haemagogus leucocelaenus for yellow fever virus in areas with reported human cases and epizootics in non-human primates, in the Federal District (DF), Brazil. Methods: Culicidae specimens were captured using hand net and oral aspirator, in the period since December 2007 to February 2008; taxonomic identification and virus research were performed at the Evandro Chagas Institute/ Ministry of Health; mouse inoculation and indirect immunofluorescence tests were performed to isolate the viral agent. Results: pools of Hg. janthinomys and Hg. leucocelaenus proceeding from the Administrative Region of São Sebastião/ DF were positive for yellow fever virus. Conclusion: confirmation of natural infectivity by Hg. janthinomys and Hg. leucocelaenus suggests that the area surveyed is receptive and vulnerable to yellow fever virus; therefore, it is essential to improve the strategies for epidemiological surveillance of yellow fever in the Federal District.

Yellow fever virus in Haemagogus leucocelaenus and Aedes serratus mosquitoes, southern Brazil, 2008.

Yellow fever virus (YFV) was isolated from Haemagogus leucocelaenus mosquitoes during an epizootic in 2001 in the Rio Grande do Sul State in southern Brazil. In October 2008, a yellow fever outbreak was reported there, with nonhuman primate deaths and human cases. This latter outbreak led to intensification of surveillance measures for early detection of YFV and support for vaccination programs. We report entomologic surveillance in 2 municipalities that recorded nonhuman primate deaths. Mosquitoes were collected at ground level, identified, and processed for virus isolation and molecular analyses. Eight YFV strains were isolated (7 from pools of Hg. leucocelaenus mosquitoes and another from Aedes serratus mosquitoes); 6 were sequenced, and they grouped in the YFV South American genotype I. The results confirmed the role of Hg. leucocelaenus mosquitoes as the main YFV vector in southern Brazil and suggest that Ae. serratus mosquitoes may have a potential role as a secondary vector.

Eco-epidemiologia dos arbovírus na área de influência da rodovia Cuiabá-Santarém (BR 163), Estado do Pará, Brasil / Arbovirus eco-epidemiology in the area affected by the Cuiabá-Santarém Highway (BR-163), Pará State, Brazil

O presente estudo descreve os aspectos eco-epidemiológicos sobre arbovírus nos Municípios de Novo Progresso e Trairão, Estado do Pará, Brasil, na área de influência da BR 163. Anticorpos IH foram detectados para diferentes arbovírus, com reações monotípicas para os VMAY e VORO, dois importantes arbovírus associados a epidemias na Amazônia. Anticorpos IgM para o VORO e VMAY foram detectados em soros humanos, sugerindo infecção recente por esses arbovírus. Duas cepas do VDEN-3 foram isoladas de pacientes febris residentes em Novo Progresso e identificadas como genótipo III. Em termos gerais, os dados obtidos sugerem uma área propícia para a circulação e manutenção de arbovírus e uma população pouco imunizada. Portanto, é importante um monitoramento dinâmico das populações locais e de imigrantes e de animais silvestres quanto à presença de anticorpos e isolamentos de arbovírus, o que permitirá um efetivo controle das infecções por esses agentes virais em residentes da área da rodovia dentro do território paraense.

The current study describes the eco-epidemiological aspects of arbovirus diseases in the municipalities (counties) of Novo Progresso and Trairão, Para State, Brazil, in the area affected by highway BR-163. Hemagglutination inhibition (HI) antibodies to different arboviruses were detected, with monotypic reactions to MAYV and OROV, two important arboviruses associated with epidemics in the Amazon. IgM antibodies to OROV and MAYV were found in human sera, suggesting recent infections by these viruses. Two DENV-3 strains were isolated from febrile patients in Novo Progresso and identified as genotype III strains. In general, the data suggest that the area displays ideal conditions for maintenance and circulation of arboviruses, plus a population with low immunization levels. Dynamic surveillance of local immigrants and wild animals is thus important, focusing on antibody prevalence and isolation of arboviruses, thereby allowing effective control of infections by these viral agents in the resident population along highway BR-163 in Pará State.