Human, Nonhuman Primate, and Bat Cells Are Broadly Susceptible to Tibrovirus Particle Cell Entry

Yingyun Cai; Shuiqing Yu; Rohit Jangra; Elena Postnikova; Jiro Wada; Robert Tesh; Sean Whelan; Michael Lauck; Michael Wiley; Courtney Finch; Sheli Radoshitzky; David O'Connor; Gustavo Palacios; Kartik Chandran; Charles Chiu; Jens Kuhn

Recently, the coding-complete BAV genome sequence (13,296 nt) was determined (Lauck et To further understand which cell types tibrovirus particles may enter and in which cell 133 types authentic tibroviruses may replicate, we exposed 53 human, 11 bat, 7 nonhuman primate, 1 134 hispid cotton rat, 1 boa constrictor, and 1 Asian tiger mosquito cell lines to newly established 135 infectious recombinant vesicular stomatitis Indiana viruses (rVSIVs) encoding the eight diverse 136 tibrovirus Gs and to four authentic tibroviruses (BAV, CPV, SWBV, and TIBV). The obtained 137 rVSIV infections rates, which reflect tibrovirus particle cell entry (host cell susceptibility), 138 indicate that particles of all tibroviruses, not only those of BASV, easily enter a broad range of 139 human and non-human cells. We confirm pH dependency of BASV G-mediated entry, show that 140 tibrovirus particle cell entry is likely dynamin-dependent and cholesterol-independent, and 141 extrapolate these findings to all tibroviruses. Using the four authentic tibroviruses and cross-142 reacting anti-tibrovirus N antibodies in western blots, we demonstrate, however, that many cell 143 lines that are susceptible to tibrovirus G-mediated particle entry are not likely to be permissive to 144 replication of most tibroviruses after particle entry. Kleinfelter et al., 2015) to yield 161 rVSIV-BAV G, rVSIV-BHV G, rVSIV-CPV G, and rVSIV-TIBV G, respectively ( Figure 1A) . 162 The sequences of the G-encoding regions of all created rVISV genomes were confirmed to be 163 identical to those of the respective GenBank accession numbers by Sanger sequencing. Primers 164 were designed to amplify rVSIV-tibrovirus G-encoding regions by RT-PCR, and these fragments 165 were sequenced by Macrogen, Rockville, MD, USA. All rVSIVs were rescued, grown, and were designed using Antigen Profiler software (ThermoFisher Scientific). Two 19-mers were 268 designed, produced, and injected into rabbits by the company. Rabbit sera containing antibodies 269 were harvested and purified by the company 72 days after peptide injection. To detect authentic 270 tibroviruses, virus-exposed cells were harvested and lysed using Cell Lysis Buffer (Cell Tris Gels (ThermoFisher Scientific), and gels were run with MOPS SDS Running Buffer 277 (ThermoFisher Scientific). Gels were dry-transferred using the iBlot 2 Gel Transfer Device 278 (ThermoFisher Scientific). Primary antibodies were diluted to 1:1,000; goat anti-rabbit IgG 279 coupled to horseradish peroxidase (HRP) (Sigma Aldrich) was diluted to 1:3,000. Protein 280 loading was controlled by detecting cellular β-actin using anti-beta actin antibody ab8227 281 This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.