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

Disclosures

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

In This Article

Results

Detection of BRBV in Ticks and Infection Rates

Based on spiked tick pools comprising 5 A. americanum adult females or 25 nymphs, ground in 1 mL BA-1, the cutoff crossing threshold of 37 corresponded to a detection limit of 102.6 PFU/mL or pool (95% CI 102.5–102.7) for primer set NP1. The crossing threshold of 37 corresponded to a detection limit of 101.4 PFU per mL or pool (95% CI 101.3–101.5) for primer set PB1. Results from adult and nymphal pools were not statistically different.

We tested 39,096 ticks representing 5 species collected from 6 sites in northwestern Missouri (Figure 1; Table 1). However, 2 species, A. americanum (L.) (97.6%) and Dermacentor variabilis (Say) (2.3%), accounted for 99.9% of ticks collected.

We tested an aliquot from all 3,073 tick pools from Missouri collections from 2013 by rRT-PCR using the screening primer/probe set NP1. Three pools were positive in the screening assay. Reextraction and testing of the original tick homogenates using both primer/probe sets NP1 and PB1 confirmed BRBV RNA in all 3 pools. All 3 tick pools yielded viable virus in cell culture. All 3 positive pools comprised A. americanum ticks (Table 2). One pool comprised 4 male adult ticks collected at site 2a on June 12; the other 2 pools each comprised 25 nymphs collected at site 27 on July 24.

The maximum-likelihood estimate[7] of the infection prevalence per 1,000 ticks, for nymphs of A. americanum from site 27 on July 24, 2013, the only day that this site was sampled, was 0.31 (95% CI 0.06–1.01), or ≈1 infected nymph per 3,226 collected nymphs. The infection prevalence for A. americanum nymphs from all sites combined during the entire 2013 season was 0.07 (95% CI 0.01–0.22), or ≈1 infected nymph per 14,286 collected nymphs.

The infection prevalence for adult male A. americanum ticks from site 2a on June 12, 2013, was 19.11 (95% CI 1.13–90.06),; for adult male A. americanum ticks from site 2a during the entire 2013 season was it .35 (95% CI 0.42–35.19); and for all adult male A. americanum ticks from all sites combined during the 2013 season it was 0.32 (95% CI 0.02–1.53). The 95% CI for difference of proportions[7] between the infection prevalence for male adults and nymphs from all sites combined during the 2013 season includes zero (95% CI −1.46 to 0.13), indicating that infection prevalence for male adults and nymphs did not significantly differ.

Phylogenetic Analyses

To confirm the molecular identification of BRBV, we selected 2 pools, MO-2013-1246 of male adults and MO-2013-2499 of nymphs, for high-throughput sequencing and phylogenetic analysis. We deposited partial genomic sequence data in GenBank (accession nos. KY825740–KY825741). Analyses revealed 6 RNA segments for strain MO-2013-1246, as previously reported for the BRBV human strain.[4] We conducted phylogenetic analysis on a 152-aa sequence of PB2 subunit of the polymerase protein (Figure 2) to assess relationships with the BRBV strain from the fatal human case and other selected members of the Orthomyxoviridae. The 3 BRBV strains form a lineage with 100% bootstrap support. The BRBV lineage is a sister group to, and mostly closely related to, Dhori virus. The BRBV-Dhori lineage appears as a sister group to a lineage of 4 tick-associated viruses and distantly related to the influenza viruses and Quaranfil virus.

Figure 2.

Phylogenetic analyses of partial polymerase basic 2 sequences of selected orthomyxoviruses. Bourbon virus sequences from 2 pools of Amblyomma americanum ticks (male adults, MO-2013-1246; nymphs, MO-2013-2499) collected in Missouri, USA, during 2013 grouped with the sequence of the original Bourbon virus isolated from a man who died in Bourbon County, Kansas, USA, during 2014. The evolutionary history was inferred using the neighbor-joining method with 2,000 replicates for bootstrap testing. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method. Scale bar indicates number of amino acid substitutions per site.

The human BRBV strain from Kansas and tick pool MO-2013-1246 comprising male adult A. ambylomma ticks were very similar for the PB2 gene segment analyzed, sharing >99.0% sequence at the amino acid level and 95.0% identity at the RNA sequence level. Furthermore, the human BRBV strain from Kansas and tick pool MO-2013-2499 (nymphs) were very similar for the PB2 gene segment analyzed, sharing 99.0% sequence identity at the amino acid and RNA sequence levels.

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