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.

New Variants of Squash Mosaic Viruses Detected in Human Fecal Samples.

Squash mosaic virus (SqMV) is a phytovirus that infects great diversity of plants worldwide. In Brazil, the SqMV has been identified in the states of Ceará, Maranhão, Piauí, Rio Grande do Norte, and Tocantins. The presence of non-pathogenic viruses in animals, such as phytoviruses, may not be completely risk-free. Similarities in gene repertories between these viruses and viruses that affect animal species have been reported. The present study describes the fully sequenced genomes of SqMV found in human feces, collected in Tocantins, and analyzes the viral profile by metagenomics in the context of diarrhea symptomatology. The complete SqMV genome was obtained in 39 of 253 analyzed samples (15.5%); 97.4% of them belonged to children under 5 years old. There was no evidence that the observed symptoms were related to the presence of SqMV. Of the different virus species detected in these fecal samples, at least 4 (rotavirus, sapovirus, norovirus, parechovirus) are widely known to cause gastrointestinal symptoms. The presence of SqMV nucleic acid in fecal samples is likely due to recent dietary consumption and it is not evidence of viral replication in the human intestinal cells. Identifying the presence of SqMV in human feces and characterization of its genome is a relevant precursor to determining whether and how plant viruses interact with host cells or microorganisms in the human gastrointestinal tract.

High Heterogeneity of Echoviruses in Brazilian Children with Acute Gastroenteritis.

Echoviruses (E) are a diverse group of viruses responsible for various pathological conditions in humans including aseptic meningitis, myocarditis, and acute flaccid paralysis. The detection and identification of echovirus genotypes in clinical samples is challenging due to its high genetic diversity. Here, we report the complete genome sequences of nine echoviruses, obtained by next-generation sequencing of 238 fecal samples from individuals with gastroenteritis in regions of Brazil. Detected viruses were classified into six genotypes: Three E1 sequences (BRA/TO-028, BRA/TO-069 and BRA/TO-236), one E3 (BRA/TO-018), one E11 (BRA/TO-086), one E20 (BRA/TO-016), two E29 (BRA/TO-030 and BRA/TO-193), and one E30 sequence (BRA/TO-032). Phylogenetic analysis indicated that the echoviruses E1 and E29 circulating in Brazil are divergent from strains circulating worldwide. The genotype diversity identified in our study may under-represent the total echovirus diversity in Brazil because of the small sample size and the restricted geographical distribution covered by the survey.