Natural infection by Culex flavivirus in Culex quinquefasciatus mosquitoes captured in Cuiabá, Mato Grosso Mid‐Western Brazil

New species of insect‐specific viruses (ISV) have been reported worldwide. In the present study, the complete genome of Culex flavivirus (CxFV) and partial sequences of other ISVs in Culex quinquefasciatus Say 1823 females (n = 3425) sampled in 200 urban areas census tracts of Cuiaba, state of Mato Grosso, were identified via reverse transcriptase‐polymerase chain reaction for a NS5 region of flaviviruses, nucleotide and high‐throughput sequencing, and viral isolation in C6/36 cells. CxFV was detected in 16 of 403 mosquito pools; sequences found in the study presented a high similarity with isolates from São Paulo, Brazil and other countries in Latin American that belong to genotype II, supporting the geographical influence on CxFV evolution. The monthly maximum likelihood estimation for CxFV ranged from 1.81 to 9.94 per 1000 mosquitoes. In addition to the CxFV complete genome, one pool contained an ORF1 sequence (756 bp) that belongs to a novel Negevirus from the Sandewavirus supergroup most similar to the Santana virus (77.1%) and another pool presented an RNA‐dependent RNA polymerase sequence (1081 bp) of a novel Rhabdovirus most similar to Wuhan mosquito virus 9 (44%). After three passages in C6/36 cells, only CxFV was isolated from these co‐infected pools. The importance of ISVs relies on their possible ability to interfere with arbovirus replication in competent vectors. Genome sequencing: metagenomic sequences have favoured the discovery, surveillance and characterization of novel viral species and variants, allowing more accurate evolutionary studies. Virus identification: Culex quinquefasciatus females captured in Cuiabá, Mato Grosso State, were identified naturally infected by Culex flavivirus and a novel Negevirus and Rhabdovirus named Siriri and Cururu virus. Genomic analysis: phylogenetic analysis and genomic characterization contribute to the understanding of genetic variability, ecology, host interactions and evolution with respect to insect‐specific viruses, which are considered to be more ancient than their counterparts, namely the arboviruses.