This study aimed to verify the diversity of Culicidae species and their frequency of infection with flaviviruses and alphaviruses in Cuiabá, state of Mato Grosso, Brazil. Mosquitoes were captured with Nasci aspirators and hand net in 200 census tracts, identified alive at species level and pooled in one-20 (11,090 mosquitoes, 14 species). Female pools (n = 610) were subjected to multiplex seminested-reverse transcription-polymerase chain reaction (RT-PCR) for 11 flavivirus and five alphavirus. Positive pools were tested by single RT-PCR followed by nucleotide sequencing, by RT-PCR for E1 gene [Mayaro virus (MAYV)] and by inoculation in Vero cells (MAYV) or C6/36 cells (flaviviruses). One/171 Aedes aegypti was positive for dengue virus (DENV)-1, 12/403 Culex quinquefasciatus, and four/171Ae. aegypti for MAYV, which was isolated from two pools containing two nonengorged females of Ae. aegypti and two ofCx. quinquefasciatus. DENV-4 was detected in 58/171 pools of Ae. aegytpi, 105/403 Cx. quinquefasciatus, two/five Psorophora sp., two/11 Psorophora varipes/Psorophora albigenu, one/one Sabethes chloropterus, two/five Culex bidens/Culex interfor, and one/one Aedes sp. DENV-4 was isolated from two pools containing three and 16 nonengorged Cx. quinquefasciatus females. Phylogenetic analysis revealed MAYV belongs to genotype L, clustering with human samples of the virus previously identified in the city. Cuiabá has biodiversity and ecosystem favourable for vector proliferation, representing a risk for arbovirus outbreaks.

This study aimed to investigate the circulation of Orthobunyavirus species in the state of Mato Grosso (MT) Brazil. During a dengue outbreak in 2011/2012, 529 serum samples were collected from patients with acute febrile illness with symptoms for up to five days and 387 pools of female Culex quinquefasciatus captured in 2013 were subjected to nested-reverse transcription-polymerase chain reaction for segment S of the Simbu serogroup followed by nucleotide sequencing and virus isolation in Vero cells. Patients (5/529; 0.9%) from Cuiabá (n = 3), Várzea Grande (n = 1) and Nova Mutum (n = 1) municipalities were positive for the S segment of Oropouche virus (OROV). Additionally, eight/387 Cx. quinquefasciatus pools were positive for the segment, with a minimum infection rate of 2.3. Phylogenetic analysis indicated that all the samples belong to the subgenotype Ia, presenting high homology with OROV strains obtained from humans and animals in the Brazilian Amazon. The present paper reports the first detection of an Orthobunyavirus, possibly OROV, in patients and in Cx. quinquefasciatus mosquitoes in MT. This finding reinforces the notion that arboviruses frequently reported in the Amazon Region circulate sporadically in MT during dengue outbreaks.

The dengue virus (DENV), which is frequently involved in large epidemics, and the yellow fever virus (YFV), which is responsible for sporadic sylvatic outbreaks, are considered the most important flaviviruses circulating in Brazil. Because of that, laboratorial diagnosis of acute undifferentiated febrile illness during epidemic periods is frequently directed towards these viruses, which may eventually hinder the detection of other circulating flaviviruses, including the Saint Louis encephalitis virus (SLEV), which is widely dispersed across the Americas. The aim of this study was to conduct a molecular investigation of 11 flaviviruses using 604 serum samples obtained from patients during a large dengue fever outbreak in the state of Mato Grosso (MT) between 2011 and 2012. Simultaneously, 3,433 female Culex spp. collected with Nasci aspirators in the city of Cuiabá, MT, in 2013, and allocated to 409 pools containing 1-10 mosquitoes, were also tested by multiplex semi-nested reverse transcription PCR for the same flaviviruses. SLEV was detected in three patients co-infected with DENV-4 from the cities of Cuiabá and Várzea Grande. One of them was a triple co-infection with DENV-1. None of them mentioned recent travel or access to sylvatic/rural regions, indicating that transmission might have occurred within the metropolitan area. Regarding mosquito samples, one pool containing one Culex quinquefasciatus female was positive for SLEV, with a minimum infection rate (MIR) of 0.29 per 1000 specimens of this species. Phylogenetic analysis indicates both human and mosquito SLEV cluster, with isolates from genotype V-A obtained from animals in the Amazon region, in the state of Pará. This is the first report of SLEV molecular identification in MT.

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.

Dengue virus (DENV) is the most frequent arbovirus worldwide. In this study, we report a large outbreak in Mato Grosso State (MT). Serum samples from 604 patients with acute febrile illness for less than five days were inoculated in C6/36 cells, then infected cells were subjected to an indirect immunofluorescence test for DENV serotypes and yellow fever virus. Serum samples were submitted to a multiplex-semi-nested-RT-PCR for 11 flaviviruses. DENV-4 was isolated in 150/604 (24.8%) and DENV-1 in 19/604 (3.1%) specimens. By RT-PCR, 331 (54.8%) samples tested positive for DENV; 321 had single infections (DENV-4 n = 305; DENV-1 n = 15; DENV-3 n = 1), nine had co-infections of DENV-1/DENV-4, and one of DENV-2/DENV-4. DENV-4 was detected in 315/331 (95.2%) positive patients from 17 municipalities, and DENV-1 in 24/331 (7.2%) patients from five cities in north-central MT and the city of Cuiaba. The incidence of infection was higher in patients aged 20-39 (142/331; 42.9%). The NS5 partial nucleotide sequence of DENV-1 was most similar to that of genotype V, DENV-2 to Southeast Asian/American, DENV-3 to genotype III, and DENV-4 to genotype II strains, considered the most frequent strains in Brazil. This outbreak coincided with the introduction of DENV-4 in the state. Cuiaba was hyperendemic for the four DENV serotypes, highlighting the necessity for arbovirus surveillance in MT.