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Abstract Classical insect-flaviviruses (cISFVs) and dual host-related insect-specific flavivirus (dISFV) are within the major group of insect-specific flavivirus. Remarkably dISFV are evolutionarily related to some of the pathogenic flavivirus, such as Zika and dengue viruses. The Evolutionary relatedness of dISFV to flavivirus allowed us to investigate the evolutionary principle of host adaptation. Additionally, dISFV can be used for the development of flavivirus vaccines and to explore underlying principles of mammalian pathogenicity. Here we describe the genetic characterization of a novel putative dISFV, termed Guapiaçu virus (GUAPV). Distinct strains of GUAPV were isolated from pools of Aedes terrens and Aedes scapularis mosquitoes. Additionally, we also detected viral GUAPV RNA in a plasma sample of an individual febrile from the Amazon region (North of Brazil). Although GUAPV did not replicate in tested mammalian cells, 3′UTR secondary structures duplication and codon usage index were similar to pathogenic flavivirus.

The simultaneous transmission of two lineages of the chikungunya virus (CHIKV) was discovered after the pathogen’s initial arrival in Brazil. In Oiapoque (Amapá state, north Brazil), the Asian lineage (CHIKV-Asian) was discovered, while in Bahia state, the East-Central-South-African lineage (CHIKV-ECSA) was discovered (northeast Brazil). Since then, the CHIKV-Asian lineage has been restricted to the Amazon region (mostly in the state of Amapá), whereas the ECSA lineage has expanded across the country. Despite the fact that the Asian lineage was already present in the Amazon region, the ECSA lineage brought from the northeast caused a large outbreak in the Amazonian state of Roraima (north Brazil) in 2017. Here, CHIKV spread in the Amazon region was studied by a Zika–Dengue–Chikungunya PCR assay in 824 serum samples collected between 2013 and 2016 from individuals with symptoms of viral infection in the Amapá state. We found 11 samples positive for CHIKV-Asian, and, from these samples, we were able to retrieve 10 full-length viral genomes. A comprehensive phylogenetic study revealed that nine CHIKV sequences came from a local transmission cluster related to Caribbean strains, whereas one sequence was related to sequences from the Philippines. These findings imply that CHIKV spread in different ways in Roraima and Amapá, despite the fact that both states had similar climatic circumstances and mosquito vector frequencies.

Plasma from patients with dengue-like symptoms was collected in 2013 to 2016 from the Brazilian states of Tocantins and Amapa. 781 samples testing negative for IgM against Dengue, Zika, and Chikungunya viruses and for flaviviruses, alphaviruses and enteroviruses RNA using RT-PCRs were analyzed using viral metagenomics. Viral particles-associated nucleic acids were enriched, randomly amplified, and deep sequenced in 102 mini-pools generating over 2 billion reads. Sequence data was analyzed for the presence of known and novel eukaryotic viral reads. Anelloviruses were detected in 80%, human pegivirus 1 in 19%, and parvovirus B19 in 17% of plasma pools. HIV and enteroviruses were detected in two pools each. Previously uncharacterized viral genomes were also identified, and their presence in single plasma samples confirmed by PCR. Chapparvovirus and ambidensovirus genomes, both in the Parvoviridae family, were partially characterized showing 33% and 34% identity in their NS1 sequences to their closest relative. Molecular surveillance using pre-existing plasma from febrile patients provides a readily scalable approach for the detection of novel, potentially emerging, viruses.

Dengue virus (DENV) is a mosquito-borne viral pathogen that plagues many tropical-climate nations around the world, including Brazil. Molecular epidemiology is a growing and increasingly invaluable tool for understanding the dispersal, persistence, and diversity of this impactful virus. In this study, plasma samples (n = 824) from individuals with symptoms consistent with an arboviral febrile illness were analyzed to identity the molecular epidemiological dynamics of DENV circulating in the Brazilian state of Amapá. Twelve DENV type 1 (DENV-1) genomes were identified, which were phylogenetically related to the BR4 lineage of genotype V. Phylodynamics analysis suggested that DENV-1 BR-4 was introduced into Amapá around early 2010, possibly from other states in northern Brazil. We also found unique amino acids substitutions in the DENV-1 envelope and NS5 protein sequences in the Amapá isolates. Characterization of the DENV-1 BR-4 sequences highlights the potential of this new lineage to drive outbreaks of dengue in the Amazon region.

Viruses are the most diverse and abundant microorganisms on earth, highly adaptive to a wide range of hosts. Viral diversity within invertebrate hosts has gained notoriety in recent years in public health as several such viruses have been of medical importance. Aedes aegypti serves as a vector for several viruses that have caused epidemics within the last year throughout Brazil; including Dengue, Zika and Chikungunya. This study aimed to identify new viral agents within Aedes aegypti mosquito in a city of the Amazonian region, where it is highly endemic. Metagenomic investigation was performed on 60 mosquito pools and viral RNA sequences present in their microbiota were characterized using genomic and phylogenetic tools. In total, we identified five putative novel virus species related to the Sobemovirus genus, Iflavirus genus and Permutatetraviridae family. These findings indicate a diverse taxonomy of viruses present in the mosquito microbiota of the Amazon, the region with the greatest invertebrate diversity in the world.

Advancements in next-generation sequencing and bioinformatics have expanded our knowledge of the diversity of viruses (pathogens and non-pathogens) harbored by mosquitoes. Hubei reo-like virus 7 (HRLV 7) was recently detected by the virome analysis of fecal samples from migratory birds in Australia. We now report the detection of RNA-dependent RNA polymerase sequences of HRLV 7 in pools of Aedes aegypti and Culex quinquefasciatus mosquitoes species from the Brazilian Amazon forest. Phylogenetic inferences indicated that all HRLV 7 strains fall within the same independent clade. In addition, HRLV 7 shared a close ancestral lineage with the Dinovernavirus genus of the Reoviridae family. Our findings indicate that HRLV 7 is present in two species of mosquitoes.

Classical insect-specific flaviviruses (cISFs) have been widely detected in different countries in the last decades. Here, we characterize the near full-length genomes of two cISFs detected in mosquitoes collected in the city of Macapá, state of Amapá, Amazon region of Brazil. A total of 105 pools of female mosquitos were analyzed by next-generation sequencing (NGS). Comparative genomics and phylogenetic analysis identified three strains of cell fusing agent virus (CFAV) and two of Culex flavivirus (CxFV). All sequences were obtained from pools of Culex sp., except for one sequence of CFAV detected in a pool of Aedes aegypti. Both CxFV strains are phylogenetically related to a strain isolated in 2012 in the Southeast region of Brazil. The CFAV strains are the first of this species to be identified in Brazil and one of them is highly divergent from other strains of CFAV that have been detected worldwide. In conclusion, CFAV and CxFV, circulate in mosquitoes in Brazil. One strain of CFAV is highly divergent from others previously described, suggesting that a novel strain of CFAV is present in this region.