Detection of Bourbon Virus-Specific Serum Neutralizing Antibodies in Human Serum in Missouri, USA

Abstract

Bourbon virus (BRBV) was first discovered in 2014 in a fatal human case. Since then it has been detected in the tick Amblyomma americanum in the states of Missouri and Kansas in the United States. Despite the high prevalence of BRBV in ticks in these states, very few human cases have been reported, and the true infection burden of BRBV in the community is unknown. Here, we developed two virus neutralization assays, a vesicular stomatitis virus (VSV)-BRBV pseudotyped rapid assay and a BRBV focus reduction neutralization assay, to assess the seroprevalence of BRBV neutralizing antibodies in human sera collected in 2020 in St. Louis, MO. Of 440 human serum samples tested, three (0.7%) were able to potently neutralize both VSV-BRBV and wild-type BRBV. These findings suggest that human infections with BRBV are more common than previously recognized. IMPORTANCE Since the discovery of the Bourbon virus (BRBV) in 2014, a total of five human cases have been identified, including two fatal cases. BRBV is thought to be transmitted by the lone star tick, which is prevalent in the eastern, southeastern, and midwestern United States. BRBV has been detected in ticks in Missouri and Kansas, and serological evidence suggests that it is also present in North Carolina. However, the true infection burden of BRBV in humans is not known. In the present study, we developed two virus neutralization assays to assess the seroprevalence of BRBV-specific antibodies in human sera collected in 2020 in St. Louis, MO. We found that a small subset of individuals are seropositive for neutralizing antibodies against BRBV. Our data suggest that BRBV infection in humans is more common than previously thought.

Keywords: Bourbon virus; glycoprotein; human serum; monoclonal antibody; seroprevalence; virus neutralization.

FIG 1

Generation of a replication-competent VSV expressing eGFP and BRBV GP. (A) Genome schematic. Genes in the viruses are to scale (except the leader [Le] and trailer [Tr]). N, nucleoprotein; P, phosphoprotein; M, matrix protein; G or GP, glycoprotein; L, large protein or polymerase. Glycoprotein gene length excludes the stop codon. (B) Vero-CCL81 cells were infected with VSV or VSV-BRBV at an MOI of 1, and images were acquired 6 hpi. (C) Representative plaque assay of VSV or VSV-BRBV on Vero-CCL81 cells. Images were acquired 24 hpi.

FIG 2

Generation and characterization of humanized monoclonal antibodies against BRBV GP. (A) Immunization regimen. Eleven- to twelve-week-old C57BL/6 mice were immunized intramuscularly with 500 ng BPL-inactivated BRBV, followed by boosters with 5 μg of rGP of BRBV 33 days later. Sera were collected at days 21 and 38. Spleens were harvested 5 days after the booster (day 38) (B) IgG serum Ab ELISA for rGP of BRBV for naive and immunized mice at days 21 and 38. Results are averages from two independent experiments. (C) Representative plots of BRBV GP ASC (CD4⁻ CD19⁺ IgD^lo CD95⁺ GL7⁻ CD138⁺ BRBV GP⁺ live singlet lymphocytes) in naive mice (top) and BRBV-immunized mice (bottom). (D) ELISA measuring binding of nine MAbs to rGP of BRBV. Each line represents a different antibody, and the results are averages from two independent experiments. (E) Binding of MAb B02 and E02 to BRBV-infected cells. Vero cells were infected with ~100 infectious units of BRBV for 24 h and fixed with 5% formalin, and virus-infected cells were visualized by incubating the cells with 2 to 3 μg/mL of MAbs B02 and E02 or a control. Images are representative of 2 experiments.

FIG 3

Development of a rapid eGFP-based BRBV neutralizing assay. (A) Effective neutralization of VSV-BRBV by BRBV-neutralizing antibodies. Monolayers of Vero cells incubated for 8 h with VSV-BRBV, expressing eGFP, in the presence of mouse serum-containing BRBV neutralizing antibodies (positive control), control mouse serum, or no serum were visualized with DAPI (blue). VSV-BRBV-infected cells were visualized by the expression of GFP from the VSV genome. Representative images of the no-serum (left), negative-serum (middle), and positive-serum (right) controls are shown at a magnification of ×4. (B) Normalized VSV-BRBV neutralization (percent foci compared to the no-serum control) of the positive- and negative-control sera. Each symbol represents an individual mouse serum sample (15 negative and 5 positive controls). (C) Normalized VSV-BRBV infection (percent foci compared to the no-serum control) of 440 human serum samples at a 1:20 (left) and 1:60 (right) dilution of the sera. Each symbol represents a serum sample. The dotted and dashed lines represent the cutoffs for 60% and 80% inhibition.

FIG 4

Detection of serum neutralizing antibodies against BRBV in human serum samples. A FRNT was developed for BRBV. Serially diluted heat-inactivated human or mouse serum (starting at a 1:40 dilution) was incubated for 1 h with 100 FFU of BRBV prior to being added to Vero cells. One hour later, the inoculum was removed, and the cells were overlaid with 1% methylcellulose and incubated for 24 h. Clusters of infected cells (foci) were visualized using the E02 MAb as described in Materials and Methods. The number of foci was normalized to the value for no-serum controls. (A) (Left) FRNT with serum from mock-infected control animals. No BRBV-neutralizing activity was detected in the negative-control sera. (Right) FRNT demonstrating virus-neutralizing activity in convalescent-phase serum collected from C57BL/6 mice 22 days after inoculation with 10⁴ PFU of BRBV. Neutralization of BRBV was detected in all five mouse serum samples. (B) FRNT on human serum samples with distinct neutralizing activities in the rapid assay. Human serum samples showing complete neutralization (0 to 20%) (left), partial neutralization (20 to 40%) (middle), or no neutralization (100%) (right) in the VSV-BRBV rapid assay were tested by FRNT. Three human sera were able to neutralize BRBV by FRNT.