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Vertical transmission to progeny ensures the maintenance of arboviruses in their natural vectors. This mechanism is largely reported for dengue virus (DENV) and yellow fever virus (YFV). Few studies have addressed this mechanism for Zika virus (ZIKV), Mayaro virus (MAYV) and other arboviruses. The present study investigated the natural infection rate by arboviruses in 4490 Aedes (Stegomyia) aegypti and 296 Aedes (Stegomyia) albopictus (Diptera: Culicidae) reared from eggs collected with ovitraps in Cuiabá, Mato Grosso State, from February to July, 2017. After viral RNA extraction and reverse transcriptase‐polymerase chain reaction protocols for 10 flaviviruses and five alphaviruses, nucleotide sequencing and three passages in C6/36 cells, eight pools of Ae. aegypti positive for DENV‐4 genotype II, seven for ZIKV Asian genotype and two for MAYV genotype L were found. In addition, two Ae. albopictus pools were positive for DENV‐4 genotype II and two were positive for ZIKV Asian genotype. Infection was confirmed by viral isolation in all positive pools for DENV‐4 and for MAYV and in eight of nine for ZIKV. This mechanism may contribute to the spread of arboviruses during epidemics and also to their maintenance in natural vectors during interepidemic periods.

Zika virus (ZIKV) was first discovered in 1947 in Uganda but was not considered a public health threat until 2007 when it found to be the source of epidemic activity in Asia. Epidemic activity spread to Brazil in 2014 and continued to spread throughout the tropical and subtropical regions of the Americas. Despite ZIKV being zoonotic in origin, information about transmission, or even exposure of non-human vertebrates and mosquitoes to ZIKV in the Americas, is lacking. Accordingly, from February 2017 to March 2018, we sought evidence of sylvatic ZIKV transmission by sampling whole blood from approximately 2000 domestic and wild vertebrates of over 100 species in West-Central Brazil within the active human ZIKV transmission area. In addition, we collected over 24,300 mosquitoes of at least 17 genera and 62 species. We screened whole blood samples and mosquito pools for ZIKV RNA using pan-flavivirus primers in a real-time reverse-transcription polymerase chain reaction (RT-PCR) in a SYBR Green platform. Positives were confirmed using ZIKV-specific envelope gene real-time RT-PCR and nucleotide sequencing. Of the 2068 vertebrates tested, none were ZIKV positive. Of the 23,315 non-engorged mosquitoes consolidated into 1503 pools tested, 22 (1.5%) with full data available showed some degree of homology to insect-specific flaviviruses. To identify previous exposure to ZIKV, 1498 plasma samples representing 62 species of domestic and sylvatic Viruses 2019, 11, 11643 of 18 vertebrates were tested for ZIKV-neutralizing antibodies by plaque reduction neutralization test (PRNT 90 ). From these, 23 (1.5%) of seven species were seropositive for ZIKV and negative for dengue virus serotype 2, yellow fever virus, and West Nile virus, suggesting potential monotypic reaction for ZIKV. Results presented here suggest no active transmission of ZIKV in non-human vertebrate populations or in alternative vector candidates, but suggest that vertebrates around human populations have indeed been exposed to ZIKV in West-Central Brazil.

Viruses are ubiquitous and diverse microorganisms arising as a result of interactions within their vertebrate and invertebrate hosts. Here we report the presence of different viruses in the salivary glands of 1657 mosquitoes classified over 28 culicinae species from the North region of the Brazilian Pantanal wetland through metagenomics, viral isolation, and RT-PCR. In total, 12 viruses were found, eight putative novel viruses with relatively low similarity with pre-existing species of viruses within their families, named Pirizal iflavirus, Furrundu phlebovirus, Pixé phlebovirus, Guampa vesiculovirus, Chacororé flavivirus, Rasqueado orbivirus, Uru chuvirus, and Bororo circovirus. We also found the already described Lobeira dielmorhabdovirus, Sabethes flavivirus, Araticum partitivirus, and Murici totivirus. Therefore, these findings underscore the vast diversity of culicinae and novel viruses yet to be explored in Pantanal, the largest wetland on the planet.

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