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. 2022 Jan 6;2(1):18.
doi: 10.15212/zoonoses-2022-0012. Epub 2022 Jun 14.

Eight years' advances on Bourbon virus, a tick-born Thogotovirus of the Orthomyxovirus family

Siyuan Hao  1 , Kang Ning  1 , Çağla Aksu Küz  1 , Shane McFarlin  1 , Fang Cheng  1 , Jianming Qiu  1
Affiliations

Eight years' advances on Bourbon virus, a tick-born Thogotovirus of the Orthomyxovirus family

Siyuan Hao et al. Zoonoses (Burlingt). .

Abstract

Bourbon virus (BRBV) was first isolated from a blood sample collected from a male patient living in Bourbon county, Kansas, during the spring of 2014. The patient later died due to complications associated with multiorgan failure. Currently, several BRBV infection-caused deaths have been reported in the United States, and misdiagnosed cases are often undercounted. BRBV is a member of the genus Thogotovirus of the Orthomyxoviridae family, and is transmitted through the Lone Star tick, Amblyomma Americanum, in North America. Currently, there are no specific antivirals or vaccinations available to treat or prevent BRBV infection. Several small molecular compounds have been identified to effectively inhibit BRBV infection of in vitro cell cultures at a single- or sub-micromolar level. Favipiravir, an RNA-dependent RNA polymerase inhibitor, prevented the death of Type I interferon receptor knockout mice infected with BRBV infection.

Keywords: Bourbon virus; Lone Star tick; antivirals; infection.

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Conflict of interest statement

Conflicts of Interest The authors declared no conflicts of interest exist.

Figures

Figure 1.
Figure 1.. Classification of Orthomyxoviridae.
Seven genera of the Orthomyxoviridae family are listed with species shown in parenthesis, which is based on the revised classification of Orthomyxoviridae (ICTV 2017) [44].
Figure 2.
Figure 2.. Geographical distribution of Amblyomma americanum, the Lone Star tick, in the United States.
The area highlighted in pink indicates the distribution of Lone Star ticks in North America up to the upper midwestern and northeastern regions of the United States and eastern Canada. KS, Kansas; MO, Missouri; and OK, Oklahoma of the United Sates.
Figure 3.
Figure 3.. BRBV replicon construction. (A) Schematic representation of the method for the construction of the plasmids to replicate the BRBV genome.
Viral RNA was extracted from virus particles. Reverse transcription(RT)-PCR was performed with primers containing segment-specific nucleotides and sequences for restriction enzyme BsmB I. The six viral RT-PCR fragments were digested with BsmB I and inserted into pHW2000 (linearized with BsmB I). This insertion resulted in 6 expression constructs where the viral cDNAs are precisely fused to the polymerase I promoter (Pol Ip) and the terminator (Pol It). The viral terminal sequences (AGC… and …ACT) are only shown in the PB2 segment in the black rectangles. (B) pBRBV-viral cDNA. The diagram shows the expression plasmid with a Pol Ip and a CMV pol II promoter containing viral cDNA of the PB2, PB1, PA, NP, GP, or M segment. The open reading frame for the 6 viral proteins are flanked by the segment-specific 3’ and 5’ noncoding regions (3’ NCR and 5’ NCR, respectively; pink boxes). (C) BRBV RNA replicon reporter system. pBRBV-gLuc reporter. Gaussia luciferase (gLuc) gene flanked by the 3’ and 5’ NCR of BRBV is cloned into pHW2000 that lacks the CMV pol II promoter. Thus, only replication of the gLuc gene by BRBV RNA-dependent RNA polymerase (RdRP) and NP protein produces gLuc +RNA for gLuc protein expression.
Figure 4.
Figure 4.. BRBV plaque assay.
A BRBV stock was diluted from 10 −4 to 10−9 in Dulbecco’s Modified Eagle’s medium (DMEM) media. 1 ml of each diluted virus were added in one well of a 6-well plate of Vero cells. After incubation of 1 hour at 37°C, diluted virus-containing media were removed from each well. 2 ml of DMEM with 10% fetal calf serum and 1% methylcellulose overlay were added to each well. After 3 days post-infection, the media was aspirated from each well, and the cells were stained with crystal violet [30]. Plaques, foci of infected cells, are shown as white dots in the stained cell sheet (blue background).
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