Serum from 518 residents of North Carolina, with variable known tick exposure, were screened for the presence of BRBV-neutralizing antibodies. Another 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 repository of recent heart valve recipients undergoing surveillance for the development of immunoglobulin 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 validated 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 conducted confirmatory testing on samples with >90% inhibition of VSV-BRBV by using a focus reduction neutralization test (FRNT) that used a BRBV St. Louis strain. We serially diluted serum samples 3-fold and incubated them with BRBV to reach a final serum dilution of 1:40–1:29,160 in media. We tested samples in duplicate. Each assay included convalescent serum 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 defined samples with BRBV-neutralizing antibodies of IC₉₀ >1:40 as positive and undertook a 10-year chart review of outpatient and inpatient records. The collection 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 Carolina, Chapel Hill (Chapel Hill, NC, USA).

Figure 2

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...

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 within 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.

The presence of BRBV-specific neutralizing antibodies in 4/518 (0.77%) persons provides evidence of human BRBV infection in North Carolina. Whereas some of the patients did seek care for respiratory infections, there was no documented history of illness compatible with previous descriptions of BRBV infection. This finding suggests that there are likely to be asymptomatic 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 underappreciated 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 waning immunity from more remote infections, or the infection did not induce sufficiently high levels of neutralizing antibodies to be detected in our assays.

The first limitation of our study is that the presence 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. Second, 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 local 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 subject to small frequencies.

The laboratory diagnosis of BRBV infection is challenging because there are no commercially available 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. Unfortunately, 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 improve accessibility and time to diagnosis, which cannot only prevent further invasive testing and unnecessary 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 cannot be used for the diagnosis of tickborne Rickettsia or Ehrlichia infections. For example, a North Carolina 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 nonspecific viral symptoms, challenging laboratory diagnostics, 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 investigation and more widespread testing in patients with suspected BRBV infection.