Evaluation of Multiple RNA Extraction Protocols for Chikungunya Virus Screening in Aedes aegypti Mosquitoes.

Chikungunya virus (Togaviridae, Alphavirus; CHIKV) is a mosquito-borne global health threat. The main urban vector of CHIKV is the Aedes aegypti mosquito, which is found throughout Brazil. Therefore, it is important to carry out laboratory tests to assist in the virus's diagnosis and surveillance. Most molecular biology methodologies use nucleic acid extraction as the first step and require quality RNA for their execution. In this context, four RNA extraction protocols were evaluated in Ae. aegypti experimentally infected with CHIKV. Six pools were tested in triplicates (n = 18), each containing 1, 5, 10, 20, 30, or 40 mosquitoes per pool (72 tests). Four commercial kits were compared: QIAamp®, Maxwell®, PureLink®, and PureLink® with TRIzol®. The QIAamp® and PureLink® with TRIzol® kits had greater sensitivity. Two negative correlations were observed: as the number of mosquitoes per pool increases, the Ct value decreases, with a higher viral load. Significant differences were found when comparing the purity and concentration of RNA. The QIAamp® protocol performed better when it came to lower Ct values and higher RNA purity and concentration. These results may provide help in CHIKV entomovirological surveillance planning.

Shotgun Metagenomic Sequencing Reveals Virome Composition of Mosquitoes from a Transition Ecosystem of North-Northeast Brazil.

A wide diversity of pathogenic mosquito-borne viruses circulate in the Brazilian Amazon, and the intense deforestation can contribute to the spread of these viruses. In this context, this study aimed to investigate the viral diversity in mosquitoes of the genera Aedes, Culex, Haemagogus, and Sabethes from a transition area between the Amazon, Cerrado, and Caatinga biomes in Brazil. Metagenomic high-throughput sequencing was used to characterize the virome of 20 mosquito pools. A total of 15 virus-like genomes were identified, comprising species genomically close to insect-specific viruses of the families Iflaviridae, Metaviridae, Lispiviridae, Rhabdoviridae, Xinmoviridae, and Parvoviridae and species of plant viruses of the families Solemoviridae, Virgaviridae, and Partitiviridae. However, sequences of viruses associated with human and animal diseases were not detected. Most of the recovered genomes were divergent from those previously described. These findings reveal that there are a large number of unknown viruses to be explored in the middle-north of Brazil.

The Importance of Entomo-Virological Investigation of Yellow Fever Virus to Strengthen Surveillance in Brazil.

The largest outbreak of sylvatic yellow fever virus (YFV) in eight decades was recorded in Brazil between 2016-2018. Besides human and NHP surveillance, the entomo-virological approach is considered as a complementary tool. For this study, a total of 2904 mosquitoes of the Aedes, Haemagogus and Sabethes genera were collected from six Brazilian states (Bahia, Goiás, Mato Grosso, Minas Gerais, Pará, and Tocantins) and grouped into 246 pools, which were tested for YFV using RT-qPCR. We detected 20 positive pools from Minas Gerais, 5 from Goiás, and 1 from Bahia, including 12 of Hg. janthinomys and 5 of Ae. albopictus. This is the first description of natural YFV infection in this species and warns of the likelihood of urban YFV re-emergence with Ae. albopictus as a potential bridge vector. Three YFV sequences from Hg. janthinomys from Goiás and one from Minas Gerais, as well as one from Ae. albopictus from Minas Gerais were clustered within the 2016-2018 outbreak clade, indicating YFV spread from Midwest and its infection in a main and likely novel bridging vector species. Entomo-virological surveillance is critical for YFV monitoring in Brazil, which could highlight the need to strengthen YFV surveillance, vaccination coverage, and vector control measures.

Vector Competence of Aedes albopictus for Yellow Fever Virus: Risk of Reemergence of Urban Yellow Fever in Brazil.

The risk of the emergence and reemergence of zoonoses is high in regions that are under the strong influence of anthropogenic actions, as they contribute to the risk of vector disease transmission. Yellow fever (YF) is among the main pathogenic arboviral diseases in the world, and the Culicidae Aedes albopictus has been proposed as having the potential to transmit the yellow fever virus (YFV). This mosquito inhabits both urban and wild environments, and under experimental conditions, it has been shown to be susceptible to infection by YFV. In this study, the vector competence of the mosquito Ae. albopictus for the YFV was investigated. Female Ae. albopictus were exposed to non-human primates (NHP) of the genus Callithrix infected with YFV via a needle inoculation. Subsequently, on the 14th and 21st days post-infection, the legs, heads, thorax/abdomen and saliva of the arthropods were collected and analyzed by viral isolation and molecular analysis techniques to verify the infection, dissemination and transmission. The presence of YFV was detected in the saliva samples through viral isolation and in the head, thorax/abdomen and legs both by viral isolation and by molecular detection. The susceptibility of Ae. albopictus to YFV confers a potential risk of reemergence of urban YF in Brazil.

Arboviruses in Free-Ranging Birds and Hematophagous Arthropods (Diptera, Nematocera) from Forest Remnants and Urbanized Areas of an Environmental Protection Area in the Amazon Biome.

The rapid and disorderly urbanization in the Amazon has resulted in the insertion of forest fragments into cities, causing the circulation of arboviruses, which can involve hematophagous arthropods and free-ranging birds in the transmission cycles in urban environments. This study aimed to evaluate the circulation of arboviruses in free-ranging birds and hematophagous arthropods captured in an Environmental Protection Area in the Belem metropolitan area, Brazil. Birds were captured using mist nets, and hematophagous arthropods were collected using a human protected attraction technique and light traps. The birds' sera were subjected to a hemagglutination inhibition test to detect antibodies against 29 arbovirus antigens. Arthropod macerates were inoculated into C6/36 and VERO cell cultures to attempt viral isolation and were tested using indirect immunofluorescence, subsequent genetic sequencing and submitted for phylogenetic analysis. Four bird sera were positive for arbovirus, and one batch of Psorophora ferox was positive for Flavivirus on viral isolation and indirect immunofluorescence. In addition, the Ilheus virus was detected in the sequencing and phylogenetic analysis. The presence of antibodies in sera from free-ranging birds and the isolation of Ilheus virus in Psorophora ferox indicate the circulation of arboviruses in forest remnants in the urban center of Belem.

Natural Infection of Aedes aegypti by Chikungunya and Dengue type 2 Virus in a Transition Area of North-Northeast Brazil.

Dengue fever, chikungunya, and Zika are diseases caused by viruses transmitted by Aedes aegypti and Aedes albopictus. In Brazil, the number of human infections is high, but few studies are performed in mosquito vectors. This study aimed to investigate the presence of Zika, Dengue and Chikungunya viruses in Ae. aegypti and Ae. albopictus from the municipalities of Alto Alegre, Caxias, Codó, and São Mateus do Maranhão, located in the state of Maranhão, Northeast Brazil. The mosquitoes were collected with a mechanical aspirator, identified, triturated, and then submitted to RNA extraction and RT-qPCR. The positive samples were confirmed by virus isolation and genome sequencing. Three hundred and forty-eight Ae. aegypti (176 males and 172 females) and 12 Ae. albopictus (eight males and four females) were collected and tested. Ae. aegypti was the only vector positive in two municipalities-Codó, with detection of Chikungunya virus (CHIKV) belonging to the East-Central-South African genotype, and in Caxias, with detection of Dengue virus (DENV)-2 belonging to the Asian/American genotype. The detection of CHIKV and DENV-2 is evidence that those viruses are maintained in arthropod vectors, and shows the epidemiological risk in the area for chikungunya cases and a possible increase of severe dengue cases, associated with the occurrence of dengue hemorrhagic fever.

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.