Ilheus Virus Isolation in the Pantanal, West-Central Brazil

The wetlands of the Brazilian Pantanal host large concentrations of diverse wildlife species and hematophagous arthropods, conditions that favor the circulation of zoonotic arboviruses. A recent study from the Nhecolândia sub-region of Pantanal reported serological evidence of various flaviviruses, including West Nile virus and Ilheus virus (ILHV). According to the age of seropositive horses, at least three flaviviruses, including ILHV, circulated in the Brazilian Pantanal between 2005 and 2009. To extend this study, we collected 3,234 adult mosquitoes of 16 species during 2009 and 2010 in the same sub-region. Mosquito pool homogenates were assayed for infectious virus on C6/36 and Vero cell monolayers and also tested for flaviviral RNA by a group-specific real-time RT-PCR. One pool containing 50 non-engorged female specimens of Aedes 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 1000. Full-length genomic sequence exhibited 95% identity to the only full genome sequence available for ILHV. The present data confirm the circulation of ILHV in the Brazilian Pantanal.

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 km 2 . The area is divided into 11 subregions based on characteristics of seasonal floods, physiography and ecology. Nhecolândia is the second largest of these sub-regions with approximately 27,000 km 2 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].
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.

Study area
In October 2009, April and October 2010, attempts to collect adult mosquitoes were undertaken at sites randomly selected within eight beef cattle ranches comprising a large area of approximately 1,500 km 2 in the Nhecolândia sub-region of Pantanal, west-central Brazil (18u189-19u159S, 57u059-55u249W) ( Figure 1). The collections for this study were authorized by the owners or residents of the sampled properties and also by the Animal Ethics Committee of Fundação Oswaldo Cruz (License CEUA-Fiocruz LW-1/12, protocol P-74/10-5) and the Brazilian Institute of Environment and Natural Resources (licenses IBAMA 18363-1/2009 and 18363-2/2010).

Mosquito collections
A total of 50 attempts to collect adult mosquitoes were undertaken, but only 39 were positive for mosquitoes.

Author Summary
Ilheus virus causes mainly asymptomatic infections in humans with rare reports of encephalitis in northern South America. Ilheus virus is believed to be maintained in zoonotic cycles between birds and mosquitoes in Central and South America. Aiming to detect Ilheus virus and other arboviruses in the Pantanal region of west-central Brazil, host-seeking mosquitoes were collected by aspiration as they landed on horses, research team members, and caimans. Resting mosquitoes were collected from the walls inside a local residence and additional flying mosquitoes were captured in light traps. We isolated a Brazilian strain of Ilheus virus from a pool of 50 Aedes scapularis mosquitoes that were collected while landing on a member of our research team, in April, 2010. Our finding confirms a recent report of Ilheus virus activity in the Pantanal derived from the detection of neutralizing antibodies in horses. The anthropophilic behavior of the infected mosquito(es) suggests a risk of Ilheus virus transmission from mosquitoes to humans in the region. We provide the virus' complete genomic sequence and present its phylogenetic relationship with other isolates.         conducted using CDC light traps, six collections using manual aspirators from horses, ten on research team members, three on caimans and three using manual aspirators while landing on walls inside a local residence. Mosquitoes were transported alive to a field laboratory where they were immobilized by cold anesthesia and were identified by species based on morphologic appearance according to taxonomic keys [28], [29]. The mosquitoes were grouped into pools of up to 50 individuals that were sorted with respect to collection method, site of capture, species and gender and then stored in liquid nitrogen.

Virus isolation
Mosquito samples were assayed for viral cytopathic effect (CPE) on C6/36 and plaque formation on Vero cell cultures using standard protocols [30]. Briefly, pools from one up to 50 mosquito specimens were placed in polypropylene capped 2 mL tubes with copper-clad steel beads and 1 mL of BA-1 diluent (  2 mg/mL Amphotericin B and 50 mg/mL Gentamycin) were added to the Vero cultures, respectively. Plates with C6/36 monolayers were incubated at 28uC in 5% CO 2 for 10 days, and observed daily for viral CPE. Plates with Vero monolayers were divided in two groups and then incubated at 37uC at 5% CO 2 . The first group was incubated for two days and the second group  for five days before being stained with neutral red in 3 mL of 0.5% agarose culture media. Both groups were kept at 37uC in 5% CO 2 for 10 days, and observed daily for viral plaques.

Real-time RT-PCR
A Sybr green real-time RT-PCR method for the universal detection of flaviviruses was performed to test all mosquito samples for flaviviral nucleic acid. First, RNA was extracted from triturated mosquitoes, and then tested for flavivirus genus-specific viral RNA by real-time RT-PCR using the QiaAmp viral RNA kit (Qiagen, Valencia, CA, USA). The protocol chosen is based on the amplification of a 269-272 nucleotide region at the N terminus of the NS5 gene [31]. Amplicons of samples that presented a melting curve peak above 90 RFU at a temperature above 75uC were considered positive and selected for purification and cDNA sequencing using both forward and reverse primers in separate reactions. DNA amplifications and sequencing reactions were completed on a DNA Engine PTC-200 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed on an ABI 3130 genetic analyzer (Applied Byosystems, Foster City, CA, USA). Sequences were assembled using Lasergene 9 software (DNA STAR, Madison, WI, USA) and subjected to a BLASTn analysis.

Full genome sequencing and phylogenetics
Amplification of total RNA was performed utilizing the SuperScript III one-step RT-PCR kit (Invitrogen, Carlsbad, CA, USA) and universal NS5 specific flavivirus primers (cFD3 and FU1) [32]. An initial fragment of approximately 1,000 base pairs was generated and subjected to GenBank BLAST analysis for identification. Once initial sequencing of the NS5 region indicated high identity with ILHV, primers for full-length sequencing were designed utilizing Primer 3 software in Geneious R6 based on the full-length ILHV sequence in GenBank (NC_009028). Fragments were extracted from an agarose gel with the Qiagen Qiaquick kit and subjected to nucleotide sequencing using BigDye Terminator V3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) on a DNA Engine PTC-200 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). The products were purified using BigDye Xterminator product (Applied Biosystems, Foster City, CA, USA) and then subjected to an ABI 3130 Genetic Analyzer (Applied Byosystems, Foster City, CA, USA). Sequencing files were aligned and analyzed utilizing Geneious R6 software. The full-length sequence was aligned with homologous flavivirus genomes utilizing MAFFT v7.017 [33], and trees analyzing the full genome sequence as well as the individual NS5 and E genes were generated using maximum-likelihood analyses with a PhyML algorithm in SeaView 4.0 [34] with 1,000 bootstrap replicates. Genome sequences of all flaviviruses sequences utilized are listed

Results
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 bloodfeeding 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 1,034bp 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 evalue 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.

Discussion
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. da Silva, Luis Ferreira de Arruda, Leandro Correa Ferreira and João Marcelino de Arruda. We thank Thierry Tomich, Marcia Furlan, Marcos Tadeu and all staff from Embrapa Pantanal as well as the ranch owners for granting access for mosquito collections. We thank Kristy Burkalther, Jason Velez, Janae Stovall and Karen Burroughs from CDC provided laboratory assistance. In the memory of Hermann G. Schatzmayr.