Bourbon Virus in Field-collected Ticks, Missouri, USA

Harry M. Savage; Kristen L. Burkhalter; Marvin S. Godsey, Jr.; Nicholas A. Panella; David C. Ashley; William L. Nicholson; Amy J. Lambert


Emerging Infectious Diseases. 2017;23(12):2017-2022. 

In This Article

Materials and Methods

Tick Collections

We collected ticks at 6 sites in 3 counties of northwestern Missouri: Andrew, Gentry, and Nodaway (Figure 1). Five sites were properties owned by HRTV patients (sites 1, 2a, and 25–27), and 1 site (13b) was state recreational land, Honey Creek Conservation Area.[5] Ticks were collected during three 1-week-long field trips in 2013; tick collections occurred on April 22–25, June 10–13, and July 22–25. We collected ticks primarily by flagging and secondarily by use of carbon dioxide–baited tick traps as described previously.[3,5] We then froze the ticks on dry ice and shipped them to CDC (Fort Collins, CO, USA) for processing and testing.

Tick Processing, RNA Extraction, and Virus Detection

We identified ticks to species, sex, and life history stage using microscopes on refrigerated tables and standard taxonomic references[5] and grouped them into pools by site, collection date, collection type, species, sex, and stage. We homogenized tick pools in 1 mL chilled bovine albumin-1 (BA-1) in glass TenBroeck grinders (Fischer Scientific, Pittsburgh, PA, USA) as described previously.[3,5] After centrifugation, we removed a 125-μL aliquot of supernatant from each tick pool homogenate and placed the aliquot into an identically numbered tube for RNA extraction. The remaining homogenates were held at −80°C for future confirmatory testing.

We extracted RNA from a 100-μL sample removed from each aliquot tube using the QIAmp Virus BioRobot 9604 kit on a BioRobot Universal platform (both from QIAGEN, Valencia CA, USA) according to the manufacturer's protocol. Sequence and reporter information for BRBV primer/probe sets nucleoprotein (NP) 1 and polymerase basic (PB) 1, which we used for virus detection and confirmation, respectively, are as previously described.[4] We screened all samples for BRBV by using primer/probe set NP1 in a real-time reverse transcription PCR (rRT-PCR) as described for HRTV.[5] Pools positive in the screening assay were confirmed by reextracting RNA from the original tick homogenate and performing the quantitative rRT-PCR with 2 primer/probe sets, primer/probe sets NP1 and PB1. We considered pools BRBV positive if crossing threshold scores for both primer/probe sets were <37.

To estimate the detection limit associated with a crossing threshold of 37, we spiked tick pools of specimens from an A. americanum colony with serial dilutions of BRBV, original strain.[1,4] Pools comprised either 5 adult females or 25 nymphs and were ground in 1 mL BA-1. We tested 14 pools of adults and 14 pools of nymphs with each primer set.

Plaque Assays to Detect Viable Virus

We tested tick homogenates from rRT-PCR–positive samples for viable virus with 2-step cell culture assay using human hepatoma cell line 7 (HuH-7) following a published protocol.[5] Initially, we inoculated a 200-μL sample of tick homogenate into HuH-7 cells in separate T-25 flasks, monitored the flasks daily for cytopathic effect, and harvested on day 5 or 6. We then used this first viral harvest (V1) to inoculate HuH-7 cells in 6-well plates and counted plaques to estimate titer.[5]

High-throughput Sequencing and Phylogenetic Analysis

We extracted and purified BRBV RNA from HuH-7 V1 harvest from 2 pools that were rRT-PCR positive as previously described.[1,4] We then conducted high-throughput sequencing on an Ion Torrent PGM sequencer (Life Technologies, Grand Island, NY, USA) and analyzed sequence data from repeated runs using the CLC Genomics Workbench 7.5.1 (CLCbio, Cambridge, MA, USA) and NGen 4 (DNAstar, Madison, WI, USA) software program as previously described.[4] The total approximated average genome coverage across all genomic segments was 1,000×. We determined open reading frames using the EditSeq function of the Lasergene 9 package (DNAstar) and conducted phylogenetic analysis on nucleotide and amino acid sequences using MEGA5 software.[6]