key: cord-0269523-72rkyjk1 authors: Bamunuarachchi, G.; Rothlauf, P. W.; Harastani, H. H.; Dai, Y.-N.; Ellebedy, A.; Fremont, D. H.; Whelan, S. P. J.; Wang, D.; Boon, A. C. title: Detection of Bourbon virus specific serum neutralizing antibodies in human serum in Missouri, USA date: 2022-03-18 journal: nan DOI: 10.1101/2022.03.17.22272570 sha: 47f78a0071178b86102938297d481f98bc778448 doc_id: 269523 cord_uid: 72rkyjk1 Bourbon virus (BRBV) was first discovered in 2014 in a fatal human case. Since then it has been detected in the Amblyomma americanum tick 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 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, Missouri. Out 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. Emerging and reemerging infectious diseases cause substantial global health and next-generation sequencing of the blood of this patient identified BRBV [5] . In 2017, a 64 state park official from Missouri was diagnosed positive for BRBV. This patient later died BRBV is isolated from lone star ticks (Amblyomma americanum) [7] [8] [9] [10] . These ticks 68 are abundant and aggressive human-biting ticks that are widely distributed across the 69 East, Southeast, and Midwest US [8, 11] . Rabbits fed on by BRBV-infected ticks 70 developed high antibody titers to the virus suggesting the lone star tick is a competent 71 vector of BRBV [10] . BRBV neutralizing antibodies have been identified in different wild 72 animal species. Using plaque-reduction neutralization test (PRNT), 50% and 86% 73 seroprevalence of BRBV neutralizing antibodies was found in sera from raccoons and 74 white-tailed deer in Missouri respectively [12] . Moreover, 56% seroprevalence was 75 observed in white-tailed deer sera from North Carolina [13] . These observations 76 demonstrate that the rate of infection of wild animals is significant, raising questions as to 77 the true infection rate and clinical burden of BRBV in humans. 78 Serology can provide an important benchmark on population immunity against 79 pathogens. However, to date there have been no serosurveillance studies assessing the 80 seroprevalence of BRBV specific neutralizing antibodies in humans in the US and world. 81 Thus the objectives of our study were to develop BRBV neutralization assays and 82 measure the human seroprevalence of BRBV infection. We found that nearly 1% of the 83 human sera, obtained in 2020, contained BRBV neutralizing antibodies. These data 84 suggest that BRBV infection in people is more common than previously thought. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 RT-PCR Kit with primers targeting VSV M-G (forward) and G-L (reverse) intergenic 106 regions. Stock sequences were confirmed by Sanger sequencing. BRBV (strain BRBV-107 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 STL) [2] was grown on Vero E6 cells. Virus stocks were sequenced by next generation, 108 aliquoted, stored at -80°C, and used for all subsequent studies. Louis. Eleven-to-twelve weeks-old female C57BL/6 mice were obtained from Jackson CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Sigma-Aldrich)) was added to all wells and incubated for 5 min before the reaction was 198 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Since 263 authentic BRBV is a biosafety level 3 (BSL3) pathogen, we sought to develop a tool to 264 study aspects of the BRBV lifecycle at reduced biocontainment. To do this, we replaced 265 the glycoprotein gene in a molecular clone of eGFP-expressing VSV with the GP gene of 266 BRBV (VSV-BRBV) (Fig 1A) . VSV-BRBV was capable of robust eGFP expression in 267 Vero-CCL81 and grew to high titers (>10 8 FFU/mL) with similar plaque size to VSV 268 containing its native glycoprotein (Fig 1B, C) . to-twelve weeks-old C57BL/6 mice were immunized intramuscularly with inactivated 271 BRBV followed by a single intramuscular boost with rGP of BRBV 33 days later. Sera 272 were collected 21 and 5 days after the first and second immunization respectively ( Fig 273 2A ) and used to measure GP specific antibody responses by ELISA. Compared to the 274 primary immunization, mice boosted with rGP of BRBV showed ~6-fold increase in serum 275 antibody titer for binding to BRBV rGP (Fig 2B) . Five days after the boost, the spleens 276 from one immunized and a control animal were isolated and processed for detection and 277 isolation of BRBV GP-specific B cells. Nearly 2% of the ASC B-cells (CD4 -, CD19 + , IgD lo , 278 CD95 + , GL7 -, CD138 + ) were specific for BRBV-GP (Fig 2C) . No BRBV-GP specific ASC CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 VH and VL gene from each clonal group were cloned into human IgG1 expression 285 vectors, transfected in HEK293 cells, and purified. These mAbs are further referred to as 286 BRBV-GP-A05, -A08, -A10, -B02, -B03, -B04, -E02, -E04, and -F07. Purified mAbs were 287 tested for specificity and affinity to rGP by ELISA (Fig 2D) . All nine antibodies were 288 specific for BRBV-GP, three (BRBV-GP-A05, -A08, and -B02) of which demonstrated 289 binding affinity to BRBV rGP with an EC50 < 500 ng/mL. The mAbs were further evaluated in a FFA with BRBV for their ability to detect surface 291 expressed GP on infected cells. In cells infected with BRBV, only two of the nine tested 292 mAbs (B02, and E02) were able to detect the GP protein on the surface of infected cells 293 and produce distinct foci in this assay (Fig 2E) . The remaining seven mAbs were unable 294 to detect GP expression in BRBV-infected Vero E6 cells. Of the two mAbs, E02 produced 295 the most distinct and intense foci in the BRBV FFA (Fig 2E) . This data combined with the 296 ELISA data (Fig 2D) , demonstrate that we produced the first BRBV specific monoclonal 297 antibodies that can be used for diagnostic assays. A rapid, eGFP-based neutralization assay for BRBV. To facilitate throughput and 299 reduce the costs associated with BSL3 work, we developed a rapid and high-throughput 300 eGFP-based neutralization assay using a replication competent BRBV-GP pseudotyped 301 VSV that also expressed eGFP (VSV-BRBV). To demonstrate the utility of this virus, cells 302 were inoculated with 200 FFU of VSV-BRBV with or without sera containing neutralizing 303 antibodies against BRBV. After 8 h without serum, eGFP expression is clearly visible in 304 a subset of the cells (Fig 3A, left panel) . The addition of mouse sera obtained from naïve 305 C57BL/6 animals did not inhibit the infection and replication of VSV-BRBV and similar 306 number of eGFP positive cells were visible (Fig 3A, middle panel) . In contrast, the 307 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 addition of serum collected 22 days after inoculation of C57BL/6 mice with 10 4 infectious 308 units of BRBV, completely neutralized VSV-BRBV and no eGFP-positive cells were 309 detected (Fig 3A, right panel) . Analysis of the positive and negative control sera show a 310 significant difference (P < 0.0001) in neutralization of VSV-BRBV (Fig 3B) . Combined, 311 these data show that VSV-BRBV can be used as a high-throughput alternative for live 312 BRBV to detect BRBV neutralizing antibodies. assay. The vast majority of the sera did not neutralize VSV-BRBV at either the 1:20 or 317 1:60 dilution of the sera (Fig 3C) . Interestingly, three human serum samples 318 demonstrated > 80% reduction in VSV-BRBV infection at both serum dilutions (Fig 3C) . 319 In addition, 54 and 14 sera were identified as potentially positive (> 60%, but < 80% 320 inhibition) at the 1:20 and 1:60 dilution of the sera respectively (Fig 3C) . The 14 samples, FRNT for BRBV using the BRBV-GP specific mAb (E02) described above. Sera collected 327 from naïve mice was unable to neutralize BRBV and the number of foci was comparable 328 to the no serum controls (Fig 4A, left panel) . In contrast, convalescent sera from mice 329 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 infected with BRBV was able to potently neutralize live BRBV (Fig 4A, right panel) . The 330 IC50 for these sera ranged between 1:500 and 1:1350. Next, the BRBV FRNT assay was used to test the 17 human serum samples that 332 previously demonstrated complete or partial neutralization of VSV-BRBV (Fig 3C) . The 333 three serum samples that strongly inhibited (> 80% inhibition) VSV-BRBV also inhibited 334 BRBV in the FRNT assay (Fig 4B, left panel) . The IC50 values for these three serum 335 samples were 1:2631, 1:1259 and 1:938. The remaining 14 human serum samples that 336 partially inhibited VSV-BRBV infection (> 60%, but <80% inhibition) did not neutralize 337 BRBV in the FRNT assay (Fig 4B, middle panel) . As a control we tested 19 human 338 serum samples that did not neutralize VSV-BRBV. None of the 19 control sera were able 339 to inhibit BRBV infection and their IC50 values were at the limit of detection (1:40) (Fig 340 4B, right panel) . Collectively, these data demonstrate that BRBV infections occur in the 341 St Louis community and that the seroprevalence of BRBV neutralizing antibodies is 342 ~0.7%. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Here we defined the first seroprevalence of BRBV serum neutralizing antibodies in 345 human sera collected from the greater St. Louis Metropolitan area. We developed two 346 virus neutralization assays, a rapid VSV-BRBV eGFP based neutralization assay, and a 347 BRBV focus reduction neutralization assay, to systematically evaluate the presence of 348 BRBV neutralizing antibodies in human serum. These assays identified 3 (0.7%) human 349 sera that contained BRBV specific neutralizing antibodies. These finding indicate that the One of the major limitation of studying emerging pathogens is the lack of reagents 363 such as mAbs, recombinant proteins, reporter viruses etc. This is the first report 364 describing mAbs specific for GP of BRBV. Two of these mAbs were able to detect GP 365 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 BRBV to be much higher than what was previously expected based on the number of 389 confirmed BRBV infections per year. Three human sera showed the highest neutralization activity (> 80% inhibition) in the 391 VSV-BRBV eGFP-based assay. These same samples were confirmed positive for BRBV-392 specific neutralizing antibody by FRNT. These findings suggest that the rapid assay and 393 the BRBV FRNT are consistent in detecting the true seropositive samples and that our 394 cut-off of 80% is rigorous. These two BRBV neutralization assays will expand our 395 knowledge of BRBV seroprevalence in humans and in wild animals. Previous studies 396 have shown that serum from white-tailed deer, raccoons, domestic dogs, horses, and 397 eastern cottontails also contains BRBV neutralizing antibodies [12, 13] . The assays 398 developed in this study will facilitate additional testing of animal species in BRBV endemic 399 and non-endemic areas and help identify the breadth of host species affected by this 400 virus. In conclusion, our study established two neutralization assays for BRBV and for the 402 first time evaluated BRBV seroprevalence in a cohort of human sera. We found 3 people 403 that were positive for BRBV neutralizing antibodies. 404 405 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 18, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Emerging Pandemic Diseases: How We Got to COVID-19 Therapeutic efficacy of favipiravir against Bourbon virus in mice Postfusion structure of human-infecting Bourbon virus envelope glycoprotein Immune protection conferred by the baculovirus-related glycoprotein of 426 Thogoto virus (Orthomyxoviridae). Virology Novel thogotovirus associated with febrile illness and death, United States Bourbon Virus Now Listed as Emerging Illness -New Case in St. Louis County Molecular, serological and in vitro culture-based characterization of 434 Bourbon virus, a newly described human pathogen of the genus Thogotovirus Emerg Infect Dis Surveillance for Tick-Borne Viruses Near the Location of a Fatal Human Case 439 of Bourbon Virus (Family Orthomyxoviridae: Genus Thogotovirus) in Eastern Kansas Orthomyxoviridae: Thogotovirus) Surveillance for Heartland and Bourbon Viruses in Eastern Kansas Bourbon Virus in Wild and Domestic Animals Indirect Evidence of Bourbon Virus (Thogotovirus, Orthomyxoviridae) Infection 448 in North Carolina Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent 450 and a Clinical Isolate of SARS-CoV-2. Cell host & microbe Structure and receptor specificity of the hemagglutinin from an H5N1 influenza 453 virus A herpesvirus encoded Qa-1 mimic inhibits natural killer cell cytotoxicity through 455 CD94/NKG2A receptor engagement. Elife IMGT/V-QUEST: the highly customized and 459 integrated system for IG and TR standardized V-J and V-D-J sequence analysis IMGT/V-QUEST: IMGT standardized analysis of the 462 immunoglobulin (IG) and T cell receptor (TR) nucleotide sequences Refined protocol for generating monoclonal antibodies from single human and 465 murine B cells Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection Seroprevalence of SARS-CoV-2 Antibodies in Children and Adults in Seroprevalence of Heartland Virus Antibodies in Blood Donors, Northwestern 471 Missouri, USA. Emerg Infect Dis 406 We thank Maxene Ilagan at Washington University School of Medicine High 407 Throughput Center for supporting with imaging. This study was supported by NIH/NIAID