Ilheus Virus Isolation in the Pantanal, West-Central Brazil

Abstract

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.

Characterization of a novel insect-specific flavivirus from Brazil: potential for inhibition of infection of arthropod cells with medically important flaviviruses

“…Adult mosquitoes were collected between 2009 and 2010 in the Nhecolândia sub-region of the Pantanal, within the State of Mato Grosso do Sul, Brazil as previously described in Pauvolid-Correa et al (Pauvolid-Correa et al ., 2013). Pools of mosquitoes were homogenized in 300 µl Dulbecco’s modified Eagle medium (DMEM) complete with penicillin (100U/ml), streptomycin (100mg/ml), 10% fetal bovine serum (FBS), and 50µg/ml amphotericin B.…”

“…Viral RNA was extracted from 140ul of the harvested supernatant using QIAamp RNA mini kit (Qiagen, Inc., Valencia, CA). RT-PCR was performed on the extracted RNA using flavivirus-specific primers as previously described (Pauvolid-Correa et al ., 2013; Pauvolid-Correa et al ., in review). The full coding sequence was acquired with second generation sequencing (SGS) using a Mi-Seq system (Illumina Inc., San Diego, CA, USA) and the NHUV virus genome was constructed via an automated computational pipeline as previously described (Langevin et al ., 2013; Pauvolid-Correa et al ., in review).…”

Nhumirim virus, a novel flavivirus isolated from mosquitoes from the Pantanal, Brazil

“…The methods used to collect and identify mosquitoes were described previously [13]. Briefly, adult mosquitoes were caught using CDC light traps, and they were also collected from horses, research team members, caimans and the walls of a local residence using manual aspirators.…”

“…Prevalence data for specific flaviviruses in the Pantanal has relied mainly on serologic surveys rather than direct isolation. Efforts to detect flavivirus infections in mosquitoes from the Pantanal region have previously resulted in the isolation of ILHV only [13]. …”

“…As a result, the interpretive value of serological studies can be limited, resulting in an incomplete appreciation of the breadth of circulating flaviviruses. Few studies in the Brazilian Pantanal have attempted arbovirus detection directly in arthropods, and these have focused primarily on mosquitoes [11, 13, 15]. However, modern molecular techniques have facilitated virus detection and characterization, and as a result, many new flaviviruses have been discovered recently worldwide [16–19].…”