TIM-1 Serves as a Nonredundant Receptor for Ebola Virus, Enhancing Viremia and Pathogenesis

Bethany Brunton; Kai Rogers; Elisabeth K. Phillips; Rachel B. Brouillette; Ruayda Bouls; Noah S. Butler; Wendy Maury

have demonstrated viral antigen in many different organs including: liver, spleen, lymph nodes, 73 kidney, adrenal glands, lungs, gastrointestinal tract, skin, brain and heart [3] [4] [5] [6] [7] . -/genotype (referred to as TIM-1 -/throughout this study). All expected genotypes 164 were produced in normal Mendelian ratios. Genomic DNA from mouse tail-clips was assessed 165 by PCR for genotypes. The primers and protocol for Ifnar -/screening has been previously 166 Organs were harvested from control and TIM-1 -/mice at 1, 3 or 5 days following infection 204 from with EBOV GP∆O/rVSV. Prior to euthanasia, mice were anesthetized with isoflurane to 205 perform retro-orbital bleeds for serum. Mice were euthanized and perfused with 10 mL of PBS 206 through the heart and organs harvested, weighed and frozen at -80ºC. To determine virus titers, 207 organs or sera were thawed and organs were homogenized in PBS and filtered through a 0.45 µ 208 syringe filter. Viral titers were determined by end point dilution on Vero cell as previously 209 Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to detect 216 proinflammatory cytokine and chemokines levels from organs of mice challenged with EBOV 217 GP∆O/rVSV. At time of harvest, organs were placed in Trizol and frozen at -80ºC until further 218 use. Total RNA was isolated using TRIzol LS reagent (Life Technologies) according 219 to manufacturer's tissue RNA isolation procedure. RNA was quantified by Nanodrop (Thermo 220 Scientific). Total RNA (2 µg) was reverse transcribed into cDNA using random primers and the 221 High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). SYBR Green based 222 quantitative PCR reactions (Applied Biosystems) were performed using 1.5µL of a 1:100 223 dilution of cDNA from each reaction and specific primers for murine cytokines and 224 chemokines. Primer sequences are found in Supplemental Table I The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/466102 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. has not been examined. Further, WT VSV serves as an excellent control for in vivo studies with 287 EBOV GP/rVSV. We challenged TIM-1 -/and control mice with 10 5 iu of VSV by i.v. injection. 288