Viruses were identified from male anthropophilic mosquitoes from Mato Grosso (MT) State, Midwest Brazil from February 2017 to January 2018. Mosquitoes tested included Aedes (Stegomyia) aegypti (1139 males; 84 pools), Culex quinquefasciatus (9426 males; 179 pools), Culex sp. (3 males; 3 pools) and Psorophora albigenu (1 male; 1 pool) collected from four cities of MT. Pools were subjected to viral RNA extraction followed by RT-PCRs specific for ten flaviviruses, five alphaviruses and Simbu serogroup of orthobunyaviruses. Positive pools were passaged three times in VERO cells (alphavirus and orthobunyavirus) or C6/36 cells (flavivirus), with isolates confirmed through RT-PCR and nucleotide sequencing. We detected pools positive for Ilh?us (1 pool), dengue serotype 4 (1), Mayaro (12), equine encephalitis virus (1) yellow fever (1), Oropouche (2), Zika (4) and chikungunya (12) viruses. High throughput sequencing of arbovirus positive pools identified 35 insect-specific viruses (ISVs) from the families Circoviridae (2), Parvoviridae (2), Totiviridae (1), Flaviviridae (1), Iflaviridae (2), Mesoniviridae (4), Nodaviridae (2), Luteoviridae (1), Phasmaviridae (1) Phenuiviridae (2), Rhabdoviridae (2), Orthomyxoviridae (1), Xinmoviridae (1), and unclassified Bunyavirales (1), unclassified Picornavirales (3), unclassified Riboviria (4) and taxon Negevirus (5). From these, five novel viruses were tentatively named Mojica circovirus, Kuia iflavirus, Muxirum negevirus, Lambada picorna-like virus and Tacuru picorna-like virus. Our findings underscore the diversity and wide geographical distribution of ISVs and arboviruses infecting male culicids.
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