First report of Culex flavivirus infection from Culex coronator (Diptera: Culicidae), Colombia

From August 2012 to December 2014, an observational, descriptive and prospective study was conducted to identify circulating flaviviruses in different mosquito species of the department of Córdoba, Colombia. The department is located in the Caribbean region between 09° 26′16" and 07° 22′05 “N, and 74° 47’ 43” and 76° 30’01 ‘W. The department of Córdoba has areas with wetlands and tropical dry forest, the area is characterized by rainfall that does not exceed 2000 mm per year, of which, 80% is during the rainy season (May-October). The tropical climate has an average annual temperature of 28°C, with heights between 0 and 40 masl. The department is divided geographically into sub-regions according to the territorial development plan: High, Medium and Low Sinú, and the samplings were carried out in these areas. One of the geographical zones corresponded to the municipality of Montería (8 ° 43’15.80 “N -075 ° 59′27.27” W (Medium Sinú), where the circulation of WNV and SLEV have been reported [14, 15]. The two remaining areas were the municipalities of Tierralta (8° 09′19.20 “N -076° 00′ 55.40” W-High Sinu) and Purisima (9° 14’ 57.60 “N -075° 42′ 34.62” W -Low Sinú)

In each of the selected sites, a total of five samplings were set-up. Four Light-CDC traps were used to capture mosquitoes, and 2 BG Centinell ™ traps fed with CO² were used to capture gravid females of different genera. The sampling sites were close to small lagoons and areas of tropical dry forest. During each sampling, the traps were placed between 17:00 and 6:00 h. Manual aspirators were also used. The collected mosquitoes were kept at 6 °C, until being transported to the laboratory of the Biological Research Institute of the Tropic (IIBT), in less than 12 h post-capture. Once in the laboratory the mosquitoes were stored at − 80 °C for molecular analysis.

Male mosquitoes were identified first. It was necessary to clarify Terminalia [16], due to the difficulty to identify some species of the Culex genus. They were classified according to their morphology based on specialized taxonomic keys [16‐18]. To morphological confirmation and mounting of genitalia, it was verified the characters used to taxonomical identification such as: 1. Subapical lobe of the gonocoxite as a quadrate at the outer third, bearing a row of eight rods that are progressively smaller distally; gonostyle bearing a small terminal claw and two minute setae situated on inner face before tip, ventral arm of the phallosome long and curved, and lateral arm with several large teeth with angular rounded corners, using description provided by Howard (1915). Recently discussion about this species by Laurito et al. (2017), allow consider other description about genitalia of male, such as: subapical lobe of the gonocoxite more or less divided with four similar stout rod-like, setae on the proximal part of the lobe (toward the base of the gonocoxite) and several subequal filiform setae on the distal part of the lobe (toward the apex of the gonocoxite); gonocoxite with an apical cluster of fine setae that extend to mid-length of the gonostylus [19]. For females we used Forattini characters described as: 1. Feminine maxillary palps are entirely dark. 2. There are no post-spiracular scales in the pleura. 3. The wing and devoid of sets of clear scales. 4. The tarsi are clearly marked and the tarsomer 5 presents a clear basal area [20]. The identification was carried out at 14 °C. Pools of 2 to 50 individuals of engorged or non-engorged female mosquitoes of the same species, location area, date and type of trap were grouped.

RNA extraction was performed using the QIAamp Viral RNA Mini kit (QIAGEN, Inc.) following the manufacturer’s instructions. The RNA was extracted from 140 μl of the homogenized mosquitoes sample. For detection of flavivirus reverse transcription RT-PCR with universal primers previously described by Bronzoni et al. [21] were used; the primers amplify a fragment of 958 base pairs (bp) of the NS5 protein: Forward FG1-TCAAGGAACTCCACACATGAGATGTACT and Reverse FG2-GTGTCCCATCCTGCTGTGTCATCAGCATACA. The positive control used was Dengue virus 2.

The amplified fragments were sequenced in both directions in an ABI 3730 XL Automated sequencer. Thirty-six representative sequences belonging to different geographic regions were downloaded from Genbank and aligned using MUSCLE in MEGA v7 [22]. Alignments in FASTA format were also evaluated in jModelTest v2.1.4 [23], using the Akaike criterion informative to model appropriate nucleotide substitutions (Additional file 1). The XML file for bayesian analysis was built in BEAUti v1.5.4 http://​beast.​bio.​ed.​ac.​uk/​Main_​Page, to model sequence evolution, invariants, gamma distribution, size of the chain run (20 million generations), coalescent constant population and uncorrelated molecular clock were chosen for this inference [24].

Of the 334 analyzed pools, 2 pools of Culex coronator were positive for flavivirus. These mosquitoes were collected in the municipality of Montería (PM 149 and PM 212). The 958-bp amplicons from the NS5 gene were sequenced, and the sequences were edited and assembled to obtain a fragment of 758 bp for analyses. Sequences were aligned and a 99% similarity was obtained with Culex flavivirus virus sequences from position 8142 to position 8899 of the complete genome of the isolate (Strain: BR_SJRP_01_2012, Genbank accession number: KT726939). Sequences were deposited in the GenBank: CxFv COL_ PM 212 (Genbank accession number: KT307717) and CxFv COL_ PM 149 (Genbank accession number: KR014201).

Alignments of the NS5 sequences from the Culex flaviviruses, CxFv COL_ PM 212 and CxFv COL_ PM 149, with NS5 sequences from strains detected in different geographical regions allowed their grouping in a clade that includes virus strains reported in Brazil, Argentina and Mexico and belonging to the genotype II group of strains of Africa/Caribbean/Latin America (Fig. 2).