RESEARCH Essential Role of Interferon Response in Containing Human Pathogenic Bourbon Virus Jonas Fuchs, Tobias Straub, Maximilian Seidl, Georg Kochs Bourbon virus (BRBV) is a recently discovered tick-trans- spleen, leading to a fatal acute hepatitis. This severe dis- mitted viral pathogen that is prevalent in the Midwest and ease progression is accompanied by a massive induction southern United States. Since 2014, zoonotic BRBV infec- of interferon (IFN) α without an apparent protective effect tions have been verified in several human cases of severe (11,12). febrile illness, occasionally with fatal outcomes, indicating a We conducted our study with the aim to evaluate the possible public health threat. We analyzed the pathology of virulence and pathogenesis of BRBV in vivo. Furthermore, BRBV infection in mice and found a high sensitivity of the virus to the host interferon system. Infected standard labo- we assessed the antiviral effect of the host IFN system on ratory mice did not show clinical signs or virus replication. BRBV replication. However, in mice carrying defects in the type I and type II interferon system, the virus grew to high titers and caused Materials and Methods severe pathology. In cell culture, BRBV was blocked by an- tiviral agents like ribavirin and favipiravir (T705). Our data Biosafety and Animal Ethics suggest that persons having severe BRBV infection might Because of the unknown health risk associated with the hu- have a deficiency in their innate immunity and could benefit man BRBV isolate, all work with infectious virus was per- from an already approved antiviral treatment. formed under Biosafety Level 3 conditions. The animals were handled in accordance with guidelines of the Federa- n the past 4 years, several reports from the Midwest and tion for Laboratory Animal Science Association and the Isouthern United States have described the detection of national animal welfare body of Germany. Animal experi- a new viral pathogen, called Bourbon virus (BRBV), as- ments were performed in compliance with animal protec- sociated with severe febrile illness (1–4). The isolation of tion laws in Germany and approved by the local animal BRBV from patients with a history of tick bites was unex- welfare committee (Regierungspraesidium Freiburg, per- pected, because BRBV belongs to tick-transmitted viruses mit no. G-15/127). of the genus Thogotoviruses, which are largely unknown in the United States. However, recent tick surveillance Cells and Viruses campaigns confirmed the prevalence of BRBV in the af- We cultivated human lung epithelial A549 cells (ATCC fected region (5,6). CCL-185), human hepatoma Huh7 cells (13), human cer- Thogotoviruses are influenza virus–like arboviruses vix carcinoma HeLa cells (ATCC CCL-2), African green with a segmented RNA genome; they are frequently iso- monkey kidney Vero cells (ATCC CCL-81), and trans- lated in Africa, the Middle East, Asia, and southern Europe formed human dermal fibroblast cultures 14( ) in Dulbec- and are usually associated with diseases in livestock (7). co modified eagle medium supplemented with 10% fetal BRBV is genetically most similar to Dhori virus (DHOV) calf serum at 37°C and 5% CO2. We treated Huh7 cells from India (8). Although serologic surveys suggest the oc- with recombinant human IFN-α2a (PBL Assay Science, currence of zoonotic transmission of Thogotoviruses, few https://www.pblassaysci.com) or recombinant human human cases have been well-documented (9,10). Howev- IFN-γ (R&D Systems, https://www.rndsystems.com) 16 er, in laboratory mice, Thogotoviruses show an aggressive h before and 2 h after infection. To test antivirals, we systemic infection affecting mainly the liver, lungs, and treated Huh7 cells with ribavirin (Sigma-Aldrich, https:// www.sigmaaldrich.com) or favipiravir/T-705 (BioVi- Author affiliation: University of Freiburg Medical Center, Faculty of sion, https://www.biovision.com) 2 h after infection. To Medicine, University of Freiburg, Freiburg, Germany evaluate the cytotoxicity of these substances, we treated uninfected cells for 48 h with the maximum doses as DOI: https://doi.org/10.3201/eid2507.181062 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 7, July 2019 1297 RESEARCH described. Afterward, we assessed the viability of the infection and production of specific antiserum directed cells by measuring the activity of lactate dehydrogenase against DHOV, as described previously (18). in the cell culture supernatant (Pierce LDH Cytotoxic- We performed antibody treatment of the animals by ity Assay Kit; ThermoFisher, https://www.thermofisher. intraperitoneal injection. To deplete IFN-γ, we injected 0.5 com). To establish a positive control, we treated cells with mg of IFN-γ monoclonal antibody (mAb) (XMG1.2; Bio- the lysis buffer provided by the manufacturer. legend, https://www.biolegend.com) at 1 day preinfection and 2 days postinfection. We achieved blockage of the type Viruses and Infection I IFN receptor (IFNAR) by treating the mice with 1 mg of BRBV (strain NR-50132/ATCC VR-1842) (1) was kindly anti-IFNAR-1 mAb (MAR1–5A3; BioXCell, https://bx- provided by Amy J. Lambert of the Centers for Disease cell.com) at 1 day preinfection and 1 day postinfection. Control and Prevention (Fort Collins, CO, USA). We pro- To deplete natural killer (NK) cells, we treated IFNAR–/– duced virus stocks of BRBV, DHOV (strain India/1313/61) mice infected with 100 PFU of BRBV with 0.25 mg of (15), and Thogoto virus (THOV) (strain SiAr126) (16) on NK1.1 mAb (PK136, BioXcell) at 3 days preinfection and Vero cells. To perform growth kinetics, we infected the 1 day postinfection. At 4 days postinfection, we harvested cells with BRBV at a multiplicity of infection (MOI) of organs and used fluorescence-activated cell sorting analy- 0.001 in Dulbecco modified eagle medium with 1% fetal sis to determine virus titers and confirm the depletion of calf serum and 20 mmol/LM HEPES. We harvested su- NK1.1+ cells. pernatants at the indicated time points. We stored virus- We administered 20 mg or 40 mg of ribavirin (5 mg/ containing supernatants at –80°C, and we determined viral mL in 0.9% NaCl; Sigma-Aldrich, https://www.sigmaal- titers by using plaque assay on Vero cells. drich.com) per kilogram bodyweight each day intraperito- neally, starting immediately postinfection. We mock-treat- Animal Infections ed control animals with 0.9% NaCl only. We purchased wild-type C57BL/6 mice from Janvier For histologic analysis, we harvested the organs, which Labs (https://www.janvier-labs.com). We bred mice with we washed once in PBS and then fixed in 4% formaldehyde defects in the IFN pathway (17) in house. We conducted in PBS overnight. For cryoprotection, we incubated organs all experiments by using sex- and age-matched animals once in 15% sucrose (in H2O) at 4°C for 4 h and afterward (7–9 week-old mice). We infected animals intraperitone- in 30% sucrose at 4°C overnight. After embedding in OCT ally with BRBV or DHOV diluted in 100 µL PBS with medium (Tissue-Tek; Sakura, https://www.sakuraus.com), 0.3% bovine serum albumin. Depending on the experimen- we performed 5-µm cryosections and stained them with he- tal setup, we measured weight, survival, and clinical score matoxylin and eosin. daily for each animal (Appendix, https://wwwnc.cdc.gov/ EID/article/25/7/18-1062-App1.pdf). We euthanized the Western Blot Analysis and Antibodies animals by using cervical dislocation at the indicated time We infected Vero cells with the indicated viruses points. To determine survival of the animals after infec- (MOI 0.25) for 24 h and then lysed them in Passive Lysis tion, we euthanized animals if the weight loss was >25% Buffer (Promega, https://www.promega.com). We denatur- or the mice showed severe clinical signs. We harvested or- ated proteins in Lämmli buffer and incubated them at 95°C gans (liver, lung, spleen, and kidney) at day 4 postinfection for 5 min. We separated the protein lysates by using 12% and homogenized them by using FastPrep Homogenizer SDS-polyacrylamid gel electrophoresis and transferred (MP Biomedicals, https://www.mpbio.com) in PBS. Af- them onto a PVDF membrane (Millipore Sigma, http:// ter centrifugation of the supernatants at 5,000 × g for 10 www.emdmillipore.com). We detected viral proteins by min at 4°C, we analyzed them by using plaque assay on using polyclonal mouse antisera. We used β-actin–specific Vero cells. We collected whole blood from animals anes- rabbit antiserum (Sigma-Aldrich) as an internal control. thetized with ketamine/xylazine by using heart puncture We detected primary antibodies by using fluorescent-la- before cervical dislocation. We prepared serum samples by beled anti-mouse secondary antibodies (LI-COR, https:// using incubation at 37°C for 10 min and centrifugation at www.licor.com). 5,000 × g for 10 min. We used serum samples directly to To test the antiserum for virus neutralization, we pre- determine alanine transaminases by using an alanine color pared serial dilutions of the polyclonal mouse serum in endpoint assay (MaxDiscovery; Bio Scientific, http://www. PBS and incubated them with a fixed amount of 100 PFU biooscientific.com), or we stored the samples at –20°C. We of BRBV for 1 h at room temperature. To establish a con- generated postinfectious serum directed against BRBV by trol, we incubated virus with PBS or an unspecific mouse challenging C57BL/6 mice with 105 PFU/animal. Four- serum. We transferred the virus–serum mixture onto Vero teen days after infection, we harvested the serum. Because cells and performed a plaque assay. We normalized the of the lethality of DHOV, we used Mx1+/+ mice for the PFU of the antibody-treated viruses to the control virus. 1298 Emerging Infectious Diseases • www.cdc.gov/eid • Vol.