Zika Virus MB16-23 in Mosquitoes, Miami-Dade County, Florida, USA, 2016

We isolated a strain of Zika virus, MB16-23, from Aedes aegypti mosquitoes collected in Miami Beach, Florida, USA, on September 2, 2016. Phylogenetic analysis suggests that MB16-23 most likely originated from the Caribbean region.

I n 2016, outbreaks of locally transmitted Zika virus occurred in Miami (Wynwood neighborhood) and Miami Beach, in Miami-Dade County, Florida, USA (1). During these outbreaks, a Centers for Disease Control and Prevention (CDC) emergency response team was deployed to assist Miami-Dade County disease surveillance and control efforts. CDC entomologists within the CDC emergency response team worked with Miami-Dade County Mosquito Control and sampled mosquito populations using BG-Sentinel type-2 traps (Biogents AG, Regensburg, Germany) to determine basic entomological parameters. Routinely, mosquitoes were collected, identified to species on the basis of the morphologic characteristics described by Darsie and Ward (2), and shipped inactivated and preserved in RNAlater (Ambion Inc., Austin, TX, USA) to the Bronson Animal Disease Diagnostic Laboratory (Kissimmee, FL, USA) for Zika virus testing.
In addition to the routine outbreak protocol, 2 BG-Sentinel type-2 traps were placed at a construction site near the intersection of James Avenue and Lincoln Road (25°47′25.68″N, 80°07′50.24″W) in Miami Beach on September 1, 2016. This site was selected because it was adjacent to a site where Zika cases had been detected. On September 2, 2016, the mosquitoes captured were frozen and shipped on dry ice to the CDC laboratory in Fort Collins, Colorado, USA. In the laboratory, the mosquitoes were identified to species on chill tables; female Aedes aegypti mosquitoes were separated into pools of 50 mosquitoes or less. A total of 293 female Ae. aegypti mosquitoes were collected (146.5/ trap/day), grouped into 7 pools, and processed for presence of arboviral agents by cytopathic effect (CPE) assay.
We triturated pools of mosquitoes in 500 µL of Dulbecco's modified Eagle medium complete with penicillin Letters commenting on recent articles as well as letters reporting cases, outbreaks, or original research are welcome. Letters commenting on articles should contain no more than 300 words and 5 references; they are more likely to be published if submitted within 4 weeks of the original article's publication. Letters reporting cases, outbreaks, or original research should contain no more than 800 words and 10 references. They may have 1 Figure or Table and should not be divided into sections. All letters should contain material not previously published and include a word count.

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(100 U/mL), streptomycin (100 mg/mL), 20% fetal bovine serum, and 50 µg/mL amphotericin B. We used the clarified supernatants from triturated mosquito pools to inoculate Vero (mammalian) cells in 24-well plates. We observed the inoculated cells daily and harvested them upon the appearance of CPE. Of the mosquito pools processed, only 1 pool of 50 female Ae. aegypti mosquitoes caused CPE. Final titration of the Vero passage was 7.02 log 10 PFU/mL. We reinoculated the harvested supernatant onto Ae. albopictus C6/36 cells; these cell cultures were maintained at 28°C with complete Dulbecco's modified Eagle medium supplemented with 2% fetal bovine serum and penicillin/ streptomycin. We extracted viral RNA from 140 µL of the supernatant harvested from the C6/36 cell cultures using a QIAamp RNA Mini Kit (QIAGEN, Valencia, CA, USA). We performed reverse transcription PCR confirmation on the extracted RNA using flavivirus-specific primers, as described previously (3).
We performed all procedures using commercial products according to the manufacturer's protocols unless otherwise noted. We generated cDNA from extracted RNA using the NuGEN Ovation RNA-seq system V2 (NuGEN Technologies, San Carlos, CA, USA). Libraries were constructed using the Ion Xpress Plus gDNA Fragment Library preparation kit (Life Technologies, Carlsbad, CA, USA) by fragmenting cDNA for 1.5 min, generating fragments of We performed whole-genome sequencing on the Ion Torrent Personal Genome Machine system (Life Technologies) and completed preparation of template-positive ion sphere particles using the Ion OneTouch 2 system and Ion PGM Hi-Q OT2 Kit (Life Technologies). We loaded Ion spheres into an Ion 316 Chip v2 (Life Technologies) and sequenced them on the Ion Torrent Personal Genomics Machine instrument using the Ion PGM Hi-Q sequencing kit (Life Technologies). We generated full genome sequences using a templated assembly in SeqMan NGen (DNASTAR, Madison, WI, USA) and using Zika virus strain PRVABC-59 (GenBank accession no. KX377337) as a reference. We subjected consensus genomes generated by templated assemblies to BLAST analysis (http://www.ncbi. nlm.nih.gov/BLAST) and determined that they were >98% similar to the respective reference sequences.
Phylogenetic analysis showed that MB16-23 was closely related to 9 other sequences from Miami, suggesting a common origin to all these sequences ( Figure), and was also closely related to the sequence DominicanRepublic_KY014300, from Santo Domingo, Dominican Republic. This finding suggests that MB16-23 and the 9 related sequences originated from a strain or strains introduced from the Caribbean region. Our results support previous observations that genomes collected in Miami-Dade County during July 2016-November 2016 share a common ancestor with genomes localized to the Caribbean area, particularly the Dominican Republic (4,5).
In summary, we report an isolate of Zika virus, strain MB16-23, from a pool of 50 Ae. aegypti mosquitoes collected in Miami Beach, Florida. Phylogenetic analysis suggests that MB16-23 shares a common ancestor with other Florida Zika virus genomes as well as genomes localized to the Caribbean region.