Tracking arboviruses, their transmission vectors and potential hosts by nanopore sequencing of mosquitoes.

The risk to human health from mosquito-borne viruses such as dengue, chikungunya and yellow fever is increasing due to increased human expansion, deforestation and climate change. To anticipate and predict the spread and transmission of mosquito-borne viruses, a better understanding of the transmission cycle in mosquito populations is needed. We present a pathogen-agnostic combined sequencing protocol for identifying vectors, viral pathogens and their hosts or reservoirs using portable Oxford Nanopore sequencing. Using mosquitoes collected in São Paulo, Brazil, we extracted RNA for virus identification and DNA for blood meal and mosquito identification. Mosquitoes and blood meals were identified by comparing cytochrome c oxidase I (COI) sequences against a curated Barcode of Life Data System (BOLD). Viruses were identified using the SMART-9N protocol, which allows amplified DNA to be prepared with native barcoding for nanopore sequencing. Kraken 2 was employed to detect viral pathogens and Minimap2 and BOLD identified the contents of the blood meal. Due to the high similarity of some species, mosquito identification was conducted using blast after generation of consensus COI sequences using RACON polishing. This protocol can simultaneously uncover viral diversity, mosquito species and mosquito feeding habits. It also has the potential to increase understanding of mosquito genetic diversity and transmission dynamics of zoonotic mosquito-borne viruses.

Possible non-sylvatic transmission of yellow fever between non-human primates in São Paulo city, Brazil, 2017-2018.

Yellow Fever (YF) is a severe disease caused by Yellow Fever Virus (YFV), endemic in some parts of Africa and America. In Brazil, YFV is maintained by a sylvatic transmission cycle involving non-human primates (NHP) and forest canopy-dwelling mosquitoes, mainly Haemagogus-spp and Sabethes-spp. Beginning in 2016, Brazil faced one of the largest Yellow Fever (YF) outbreaks in recent decades, mainly in the southeastern region. In São Paulo city, YFV was detected in October 2017 in Aloutta monkeys in an Atlantic Forest area. From 542 NHP, a total of 162 NHP were YFV positive by RT-qPCR and/or immunohistochemistry, being 22 Callithrix-spp. most from urban areas. Entomological collections executed did not detect the presence of strictly sylvatic mosquitoes. Three mosquito pools were positive for YFV, 2 Haemagogus leucocelaenus, and 1 Aedes scapularis. In summary, YFV in the São Paulo urban area was detected mainly in resident marmosets, and synanthropic mosquitoes were likely involved in viral transmission.

[Parity of Ochlerotatus scapularis in laboratory and field conditions]. / Paridade de Ochlerotatus scapularis em condições de laboratório e campo.

OBJECTIVE: To identify the parity status of Ochlerotatus scapularis females in laboratory and field conditions. METHODS: Field collections were carried out fortnightly using a hand-held aspirator in Vale do Ribeira, Southeastern Brazil, from April 2003 to March 2004. In laboratory, 100 Ochlerotatus scapularis F1 females were obtained and followed up individually, reporting the number of blood meals, the length of gonotrophic cycle, survival and number of eggs laid. The parity status and ovarian development were observed through ovarian dissection of 90 field females for each collection and all females reared in laboratory. RESULTS: The parity status diagnoses of the 100 females reared in laboratory and dissected agreed with 55% of the oviposition data, and it was underestimated in 37% and overestimated in 2%. Also in the laboratory, 106 gonotrophic cycles were completed, around 55% of females needed more than one blood meal before laying eggs. In laboratory conditions the species survival was 26 days. A total of 1,180 field females of Ochlerotatus scapularis were dissected: 418 (35.4%) nulliparous, 655 (55.5%) uniparous, 46 (3.9%) biparous and 61 (5.2%) could not be evaluated. Ninety field females were found in Christophers and Mer's phase III-V. CONCLUSIONS: The gonotrophic discordance hypothesis could be confirmed based on field and laboratory observations of Ochlerotarus scapularis females.