Ilheus virus (ILHV) was first described in 1944, when it was isolated from Aedes and Psorophora spp. mosquitoes during an epidemiological investigation of yellow fever in the city of Ilhéus, state of Bahia, northeast Brazil [1], [2]. According to the Ninth Report of the International Committee on Taxonomy of Viruses, ILHV is included in the Ntaya virus group [3]. ILHV causes mainly asymptomatic infections in humans with rare reports of encephalitis throughout northern South America [4], [5]. Human infection with ILHV has been reported in Trinidad, Panama, Colombia, French Guyana, Brazil, Ecuador and Bolivia [6], [7], [8], [9], [10], [11], [12].

ILHV is believed to be maintained in zoonotic cycles between birds and mosquitoes and has been isolated in Central and South America primarily from mosquitoes [13], [14], [15], [16]. ILHV has been previously isolated from eight mosquito genera, but also from sentinel monkeys, man and birds. In antibody surveys, neutralizing antibodies for ILHV have been found in rodents, birds, sentinel monkeys and man [17]. In the 1990s, ILHV was isolated from wild birds in southeast Brazil [18].

Little is known about ILHV infections in domestic animals. In the 1990s, a serological investigation conducted among equines from the Pantanal region of west-central Brazil found neutralizing antibodies to ILHV suggesting its circulation in the region [19]. In 2009, a flavivirus serosurvey conducted in the same area among equines from the Nhecolândia sub-region of the Pantanal detected evidence of past infection with, in increasing order of seroprevalence, Cacipacore virus (CPCV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV) and ILHV [20].

The Brazilian Pantanal is a seasonally flooded surface of approximately 140,000 km². The area is divided into 11 sub-regions based on characteristics of seasonal floods, physiography and ecology. Nhecolândia is the second largest of these sub-regions with approximately 27,000 km² and the world's largest and most diverse area of subtropical lakes encompassing approximately 10,000 lakes [21], [22], and a human population of less than 22,500 in 2010 [23].

The wetlands of the Brazilian Pantanal host large concentrations of diverse wildlife species and hematophagous arthropods, conditions that favor the circulation of zoonotic arboviruses. In spite of scarce data concerning arbovirus vectors in the area, the local movement of mosquito species previously reported as arbovirus vectors has been reported in the north and south of Pantanal [24], [25].

Serological surveys conducted in local horses have found neutralizing antibodies for at least eight arboviruses, including the bunyaviruses Tacaiuma virus and Maguari virus, the alphaviruses eastern equine encephalitis virus and western equine encephalitis virus and the flaviviruses CPCV, SLEV, WNV and ILHV [19], [20], [26], [27].

Despite the recent detection of neutralizing antibodies to various flaviviruses in horses from the Nhecolândia sub-region, circulation of these arboviruses in this area was never confirmed by virus isolation. Therefore, in an effort to detect flaviviruses currently being transmitted in this region, we collected and tested adult female mosquitoes for infectious virus and for flavivirus RNA detection.

A total of 3,234 mosquitoes of at least 16 species were collected from five out of the eight ranches where mosquito collection attempts were undertaken (Table 1). Mosquitoes of at least 11 species were identified from human landing collections, 11 from equine landing collections, 11 from CDC light traps, four from caimans and only one indoor.

Among the 1,297 specimens caught landing on horses, the most common species was Mansonia pseudotitillans (n = 177; 13,6%). Interestingly, all the specimens were only collected in October 2010, indicating a marked fluctuation of this species in the region. On the other hand, among all different methods of capture used, Uranotaenia lowii, Cx. coronator, Cx. declarator, Cx. chidesteri and Ma. humeralis were only detected in the April collections.

Among the mosquitoes identified to species, Ae. scapularis was the most prevalent with 460 (14.2%) specimens of all mosquitoes captured. Most were caught while attempting to acquire blood from humans (n = 294; 64%) and horses (n = 87; 18.7%) while only 54 (11.7%) were captured on caimans, 26 (5.7%) in CDC light traps, and none indoors, indicating anthropophilic behavior with no evidence for endophily. Ae. scapularis was the most abundant species identified in October 2009 and in April 2010, whereas Cx. quinquefasciatus was the most prevalent species among mosquitoes collected in October, 2010.

Ae. scapularis tested positive for ILHV by culture and for ILHV RNA by real-time RT-PCR, indicating a minimum infection rate of 2.5 per 1,000. Among the 331 specimens of Cx. quinquefasciatus collected in October 2010, 215 (64.9%) were removed from the walls of a local residence, 115 (34.7%) were obtained using CDC light traps and only one (0.3%) specimen was caught while blood-feeding on a horse, confirming its endophilic behavior.

A total of 292 mosquito pools were assayed by cell culture and real-time RT-PCR. One pool of 50 non-engorged female Ae. scapularis demonstrated plaques on Vero cells and CPE in C6/36 cells on the 3^rd and 6^th day post-inoculation, respectively. A 1,034-bp cDNA amplicon was obtained, and identified as closely related to ILHV. The NCBI file with the highest identity was the Ilheus virus isolate PE163615 (GenBank: EF396950.1) and had an e-value of 0.0, a maximum score of 1688, coverage of 95%, and a maximum identity of 98%.The complete genome (10,759-bp) was sequenced and the alignment with the only full-length genome available in GenBank revealed a 95% similarity to the original strain of ILHV, isolated in Brazil in 1944 (NCBI Reference Sequence: NC_009028) (Figure 2). Phylogenetic analysis grouped the NS5 and Envelope sequences of our isolate with Ilheus virus isolate FSE800 (GenBank: EF396947.1) (Figure 3) isolated from a human case in Ecuador [11]. A total of 44 nonsynonymous amino acid changes were identified between our isolate and the GenBank Ilheus 1944 isolate. Three nucleotide inserts were identified in the capsid region creating an amino acid shift between amino acid residue number 24 and 37 (nucleotides 166–204) (Figure 4; Table 2). The genome sequence of our isolate of ILHV designated as BrMS-MQ10 was deposited in the publicly accessible database GenBank under accession number KC481679. This strain was isolated from mosquitoes landing on research team members on April 18^th, 2010 from 5:35 to 7:00 pm in a natural drying lake near an artificial reservoir. Several additional viruses were detected by mosquito cell culture and/or real-time RT-PCR that did not align with known sequences on GenBank. These viruses will be described separately.

In the present work we confirm ILHV activity in the Nhecolândia sub-region of Pantanal, through the isolation of ILHV from a pool of Ae. scapularis, which was the most prevalent mosquito species identified, and one that has transmitted ILHV efficiently in laboratory vector competence studies [35]. The anthropophilic behavior, relatively high prevalence, and susceptibility to infection (minimum infection rate observed 2.5 per 1,000) implicate Ae. scapularis as a suspect vector of medical importance for ILHV in the Brazilian Pantanal. This species is known for its gonotrophic discordance, a trait that amplifies its importance as a vector [36].

The isolated strain, BrMS-MQ10 had 44 nonsynonymous mutations identified as compared to the only other ILHV full genome sequence available in GenBank. Interestingly, there were three individual nucleotide insertions identified between codon 24 and 37 of the capsid region. The capsid protein is known to form secondary structures that likely indicate the protein interacts with itself during nucleocapsid assembly [37]. Similarly, capsid has been implicating with modulating cellular pathways as well as plays a role in cis-acting interactions with other regions of the viral genome [38], [39]. Being only the isolate of ILHV with a full genome sequence record, the importance of these changes in the capsid region are as yet undefined. Future studies should strive to characterize the differences between the two known isolates and determine the significance of insertions in that region of the capsid.

Much remains undiscovered regarding mosquito populations in the Pantanal region. Four mosquito species, including Ma. pseudotitillans, Anopheles argyritarsis, Cx. coronator and Ur. lowii, are herein reported for the first time in the Nhecolândia sub-region. The abundance of several mosquito species differed greatly compared with a study conducted in the same region in 2007 [25]. This marked fluctuation of local mosquito populations was probably due to variations in annual rainfall and may influence arbovirus transmission dynamics in the region.

Little is known about human health conditions in the Brazilian Pantanal [40]. The previously reported high prevalence of neutralizing antibodies for ILHV in horses [20] combined with our isolation of ILHV in local mosquitoes confirm the activity of an arbovirus of potential medical importance in the region. The isolation of ILHV infection in mosquitoes while attempting to blood feed on a human warrants further investigation of this virus by the Brazilian arbovirus surveillance program and local physicians.