Bourbon virus is a tickborne virus that can cause human disease. Cases have been reported in Kansas, Oklahoma, and Missouri, USA. We identified Bourbon virus-specific neutralizing antibodies in patients from North Carolina. Bourbon virus infections are likely more common than previously thought, highlighting the need for improved diagnostics and surveillance

Bamunuarachchi, Gayan

Boon, Adrianus C M

Boyce, Ross M

Commins, Scott P

Zychowski, Diana L

English

Digital Commons@Becker

Washington University School of Medicine Digital Commons@Becker 2020-Current year OA Pubs Open Access Publications 11-1-2024 Evidence of human Bourbon virus infections, North Carolina, USA Diana L Zychowski University of North Carolina at Chapel Hill Gayan Bamunuarachchi Washington University School of Medicine in St. Louis Scott P Commins University of North Carolina at Chapel Hill Ross M Boyce University of North Carolina at Chapel Hill Adrianus C M Boon Washington University School of Medicine in St. Louis Follow this and additional works at: https://digitalcommons.wustl.edu/oa_4  Part of the Medicine and Health Sciences Commons Please let us know how this document benefits you. Recommended Citation Zychowski, Diana L; Bamunuarachchi, Gayan; Commins, Scott P; Boyce, Ross M; and Boon, Adrianus C M, "Evidence of human Bourbon virus infections, North Carolina, USA." Emerging infectious diseases. 30, 11. 2396 - 2399. (2024). https://digitalcommons.wustl.edu/oa_4/4749 This Open Access Publication is brought to you for free and open access by the Open Access Publications at Digital Commons@Becker. It has been accepted for inclusion in 2020-Current year OA Pubs by an authorized administrator of Digital Commons@Becker. For more information, please contact vanam@wustl.edu. 2396 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 30, No. 11, November 2024DISPATCHESVectorborne diseases are a growing public health concern in the United States. Whereas bacterial pathogens are responsible for most infections, tick-borne viruses represent an emerging and poorly un-derstood threat (1).Bourbon virus (BRBV), a tickborne virus be-longing to the family Orthomyxoviridae, was first isolated from a patient living in Bourbon County, Kansas, USA, in 2014 (2). To date, human cases have been reported only in the United States, with 5 cases reported in 3 states: Kansas, Oklahoma, and Missouri (2–5). However, serosurveillance of St. Louis, Missouri, residents identified BRBV-specific serum-neutralizing antibodies in 0.7% (3/440) of persons, suggesting that BRBV infections are likely underrecognized (6).Because of the scarcity of confirmed cases, de-scriptions of the clinical disease spectrum are limited. Fever, arthralgia, diarrhea, headache, and rash are complaints recorded early in the course of infection, followed by progression to critical illness in some cases. Laboratory abnormalities include leukopenia, thrombocytopenia, and elevated levels of aspartate and alanine aminotransferase (2,3,7).The primary vector of BRBV is the lone star tick (Amblyomma americanum), which is widely distributed throughout the central, eastern, southeastern, and south-central United States (4,8). Although no cases of human BRBV disease have been confirmed in North Carolina, BRBV was isolated from ticks (North Caro-lina Division of Public Health, pers. comm., email, 2022 Jul 13), and neutralizing antibodies were detect-ed in white-tailed deer across the state (Figure 1) (9). BRBV may be circulating in North Carolina and be-ing transmitted to humans, possibly causing disease, but is undiagnosed or interpreted as other tickborne diseases. We used previously collected human serum samples to screen for the presence of BRBV-neutraliz-ing antibodies.The StudySerum from 518 residents of North Carolina, with variable known tick exposure, were screened for the presence of BRBV-neutralizing antibodies. Anoth-er 162 samples came from a cohort of patients with confirmed alpha-gal syndrome (AGS), a delayed-onset reaction following ingestion of mammal meat products that is associated with the bite of a lone star tick (10). An additional 156 samples were from a re-pository of recent heart valve recipients undergoing surveillance for the development of immunoglobu-lin E to galactose-α-1,3-galactose. The remaining 200 samples were from antenatal women. Those sample sources were selected because of availability, with the AGS group having the highest risk for tick exposure. Samples were collected during 2021–2023 and tested for BRBV antibodies in 2023.We conducted testing by using previously vali-dated methods (6). We diluted the serum samples 1:60 and screened in a rapid neutralization assay with a chimeric vesicular stomatitis virus (VSV) expressing the BRBV envelope protein to assess for the presence of BRBV-specific neutralizing antibodies. We con-ducted confirmatory testing on samples with >90% inhibition of VSV-BRBV by using a focus reduction neutralization test (FRNT) that used a BRBV St. Louis Evidence of Human Bourbon Virus Infections, North Carolina, USADiana L. Zychowski,1 Gayan Bamunuarachchi,1 Scott P. Commins, Ross M. Boyce, Adrianus C.M. BoonAuthor affiliations: University of North Carolina at Chapel Hill,  Chapel Hill, North Carolina, USA (D.L. Zychowski,  S.P. Commins, R.M. Boyce); Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA (G. Bamunuarachchi, A.C.M. Boon).DOI: https://doi.org/10.3201/eid3011.240499 1These first authors contributed equally to this article.Bourbon virus is a tickborne virus that can cause hu-man disease. Cases have been reported in Kansas, Oklahoma, and Missouri, USA. We identified Bourbon virus–specific neutralizing antibodies in patients from North Carolina. Bourbon virus infections are likely more common than previously thought, highlighting the need for improved diagnostics and surveillance. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 30, No. 11, November 2024 2397strain. We serially diluted serum samples 3-fold and incubated them with BRBV to reach a final serum di-lution of 1:40–1:29,160 in media. We tested samples in duplicate. Each assay included convalescent se-rum from BRBV-infected mice as positive controls and samples without serum as negative controls. We calculated inhibitory values at a concentration of 90% (IC90) by using log (agonist) versus response. We de-fined samples with BRBV-neutralizing antibodies of IC90 >1:40 as positive and undertook a 10-year chart review of outpatient and inpatient records. The col-lection of the data used in the study was approved by the institutional review boards of Duke University (Durham, NC, USA) and the University of North Car-olina, Chapel Hill (Chapel Hill, NC, USA).Of the 518 samples, 6 (1.15%) demonstrated >90% inhibition of infection (Figure 2) in the VSV-BRBV rapid assay and underwent further testing by using BRBV FRNT. From the AGS cohort samples, 1 sample was positive for BRBV neutralizing antibodies, and from the heart valve group, 3 samples were positive for BRBV neutralizing antibodies. The IC90 for those 4 serum samples ranged from 1:100 to 1:200.Among the 4 positive samples, all were residents of North Carolina (Table). Of those residents, 3 sought care for nonspecific viral respiratory symptoms with-in the previous 10 years, but there was no suspicion for a febrile tickborne illness. Only 1 person, with known AGS, had a documented history of tick bites.ConclusionsThe presence of BRBV-specific neutralizing antibod-ies in 4/518 (0.77%) persons provides evidence of hu-man BRBV infection in North Carolina. Whereas some of the patients did seek care for respiratory infections, there was no documented history of illness compat-ible with previous descriptions of BRBV infection. This finding suggests that there are likely to be asymptom-atic or more subtle manifestations of BRBV infection, particularly in immunocompetent hosts, which would be consistent with other arboviral infections. Overall, our findings suggest that BRBV may be an underap-preciated cause of vectorborne disease in NC.Our study likely underestimates the number of persons infected with BRBV in North Carolina. It is possible we missed exposed persons who have wan-ing immunity from more remote infections, or the in-fection did not induce sufficiently high levels of neu-tralizing antibodies to be detected in our assays.The first limitation of our study is that the pres-ence of neutralizing antibodies does not equate to clinical disease. This study aimed to demonstrate the presence of human infection in North Carolina but was not designed to capture a history of typical symptoms or risk factors for disease severity. Sec-ond, we cannot exclude travel of persons positive for BRBV neutralizing antibody to states with known cases. However, given the evidence of BRBV in North Carolina wildlife and ticks (9), it is not surprising that Figure 1. Distribution of the lone star tick (Amblyomma americanum) and evidence of Bourbon virus detection throughout North Carolina, USA. The map is adapted from a previous publication (8) and updated with locations of known bourbon virus carrying ticks (North Carolina Division of Public Health, pers. comm., email, 2022 Jul 13), white-tailed deer with positive bourbon virus–specific neutralizing antibodies (9), and persons with bourbon virus–specific neutralizing antibodies. Created by using http://www.mapchart.net.Human Bourbon Virus Infections, USADISPATCHES2398 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 30, No. 11, November 2024local BRBV transmission to humans has occurred. Finally, whereas we found a higher proportion of persons with BRBV-specific neutralizing antibodies within the heart valve group compared with those with known AGS, this study was not designed to look for statistical differences between groups and is sub-ject to small frequencies.The laboratory diagnosis of BRBV infection is challenging because there are no commercially avail-able tests within the United States. Samples can be sent for serologic and nucleic acid amplification testing at public health laboratories or FRNT at the Centers for Disease Control and Prevention. Unfor-tunately, those tests have limitations. The viremic window for nucleic acid detection may be short or limited to the asymptomatic phase, and antibodies may not be detectable until 1 week after symptom onset. New diagnostic approaches are needed to im-prove accessibility and time to diagnosis, which can-not only prevent further invasive testing and unnec-essary antimicrobial exposure but can also provide anticipatory guidance.In addition to improved diagnostics, clinicians must remain vigilant to identify patients in need of viral testing. A single, acute serologic titer result can-not be used for the diagnosis of tickborne Rickettsia or Ehrlichia infections. For example, a North Caro-lina seroprevalence study revealed high population point prevalence rates for Ehrlichia infection of 8.6% (95% CI 5.9%–11.3%) and Rickettsia infection of 17.1% (95% CI 12.6–21.5) (11). Therefore, a single positive bacterial antibody titer should not preclude further tickborne diagnostic workup, particularly in cases of severe disease or where the patient fails to respond to antimicrobials.Because of the clinical manifestation of nonspe-cific viral symptoms, challenging laboratory diagnos-tics, and the lack of commercially available tests, the true incidence and clinical symptomatology of BRBV remain unknown and active surveillance for acute cases is needed. Our findings substantially expand the known geographic area at risk for this emerging virus and demonstrate the need for further investi-gation and more widespread testing in patients with suspected BRBV infection. Table. Documented characteristics of patients with bourbon virus–specific neutralizing antibodies, North Carolina, USA* Case Group Age, y County Recent travel? Tickborne illness? Tick bite history? Outdoor exposures Comorbidities Medical history 1 Heart valve 77 Cumberland No No No Outdoor walks AS, essential thrombocytopenia, prior nephrectomy, OSA, HTN Received care for allergies, possible viral upper respiratory infection, spring 2022. 2 Heart valve 78 Durham No No No None AS, DMII, CKD, HTN, HLD, MGUS, ILD Received antibiotics for possible lower respiratory infection superimposed on ILD, spring 2023. 3 Heart valve 79 Wake No No No None AS, CAD, childhood rheumatic fever, HTN, HLD, hypothyroidism Received care for respiratory viral infection or possible conjunctivitis, 2014. 4 AGS 63 Duplin Arkansas, Montana No Yes, many None HTN, nephrolithiasis Received multiple empiric doxycycline courses. *AGS, alpha-gal syndrome; AS, aortic stenosis; CAD, coronary artery disease; CKD, chronic kidney disease; DMII, type two diabetes mellitus; HLD, hyperlipidemia; HTN, hypertension; ILD, interstitial lung disease; MGUS, monoclonal gammopathy of undetermined significance; OSA, obstructive sleep apnea.  Figure 2. Results from a vesicular stomatitis virus and Bourbon virus rapid neutralization assay using serum samples from residents of North Carolina, USA. Normalized vesicular stomatitis virus-Bourbon virus neutralization (percentage foci compared with the control without serum) results are shown from 3 groups: antenatal women, persons with alpha-gal syndrome, and recent heart valve recipients. Colored dots represent singular serum sample. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 30, No. 11, November 2024 2399Human Bourbon Virus Infections, USAAcknowledgmentsWe thank Joseph Turek, Joseph Nellis, Berk Aykut, and Sean Whelan. A.C.M.B. was supported by the National Institute of  Allergy and Infectious Disease (grant nos. R01-AI173327 and U01-AI151810), R.M.B. was supported by a Creativity Hub Award from the University of North Carolina Office of the Vice Chancellor for Research, D.L.Z. was supported by the National Institute of Allergy and Infectious Disease (grant no. AI070114). S.P.C. was supported by the National Institute of Allergy and Infectious Disease (grant no. R01-AI135049).Author contributions: study conception and design, A.C.M.B., R.M.B., and S.P.C.; funding procurement, A.C.M.B., R.M.B., and S.P.C.; laboratory testing, G.B.;  first draft of manuscript, D.L.Z. and G.B.About the AuthorDr. Zychowski is an Infectious Disease Fellow at the University of North Carolina at Chapel Hill. Her primary research focus is on the intersection of tickborne infections, public health surveillance, and population health sciences.References  1. Rosenberg R, Lindsey NP, Fischer M, Gregory CJ,  Hinckley AF, Mead PS, et al. Vital signs: trends in reported vectorborne disease cases—United States and territories, 2004–2016. MMWR Morb Mortal Wkly Rep. 2018;67:496–501. https://doi.org/10.15585/mmwr.mm6717e1  2. Kosoy OI, Lambert AJ, Hawkinson DJ, Pastula DM,  Goldsmith CS, Hunt DC, et al. Novel thogotovirus  associated with febrile illness and death, United States, 2014. Emerg Infect Dis. 2015;21:760–4. https://doi.org/10.3201/eid2105.150150  3. Roe MK, Huffman ER, Batista YS, Papadeas GG, Kastelitz SR,  Restivo AM, et al. Comprehensive review of emergence and virology of tickborne bourbon virus in the United States. Emerg Infect Dis. 2023;29:1–7. https://doi.org/10.3201/eid2901.212295  4. Savage HM, Burkhalter KL, Godsey MS Jr, Panella NA,  Ashley DC, Nicholson WL, et al. Bourbon virus in field- collected ticks, Missouri, USA. Emerg Infect Dis. 2017;23:2017–22. https://doi.org/10.3201/eid2312.170532  5. Bricker TL, Shafiuddin M, Gounder AP, Janowski AB,  Zhao G, Williams GD, et al. Therapeutic efficacy of  favipiravir against bourbon virus in mice. PLoS  Pathog. 2019;15:e1007790. https://doi.org/10.1371/ journal.ppat.1007790  6. Bamunuarachchi G, Harastani H, Rothlauf PW, Dai YN,  Ellebedy A, Fremont D, et al. Detection of bourbon  virus-specific serum neutralizing antibodies in human  serum in Missouri, USA. MSphere. 2022;7:e0016422.  https://doi.org/10.1128/msphere.00164-22  7. Centers for Disease Control and Prevention. Bourbon virus. [cited 2024 Jan 3]. https://www.cdc.gov/bourbon-virus/hcp/clinical-diagnosis-treatment   8. Dupuis AP II, Lange RE, Ciota AT. Emerging tickborne viruses vectored by Amblyomma americanum (Ixodida: Ixodidae): heartland and bourbon viruses. J Med Entomol. 2023;60:1183–96. https://doi.org/10.1093/jme/tjad060  9. Komar N, Hamby N, Palamar MB, Staples JE, Williams C. Indirect evidence of bourbon virus (Thogotovirus,  Orthomyxoviridae) infection in North Carolina. N C Med J. 2020;81:214–5. https://doi.org/10.18043/ncm.81.3.21410. Kersh GJ, Salzer J, Jones ES, Binder AM, Armstrong PA, Choudhary SK, et al. Tick bite as a risk factor for alpha- gal-specific immunoglobulin E antibodies and development of alpha-gal syndrome. Ann Allergy Asthma Immunol. 2023;130:472–8. https://doi.org/10.1016/j.anai.2022.11.02111. Zychowski DL, Alvarez C, Abernathy H, Giandomenico D, Choudhary SK, Vorobiov JM, et al. Tick-borne disease  infections and chronic musculoskeletal pain. JAMA Netw Open. 2024;7:e2351418–2351418. https://doi.org/10.1001/jamanetworkopen.2023.51418Address for correspondence: Diana Zychowski, University of North Carolina at Chapel Hill, 111 Mason Farm Rd, Chapel Hill, NC 27514, USA; email: diana.zychowski@unchealth.unc.edu

Evidence of human Bourbon virus infections, North Carolina, USA

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Evidence of human Bourbon virus infections, North Carolina, USA

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