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

In 2012, the genome of a novel rhabdovirus, Bas-Congo virus (BASV), was discovered in the acute-phase serum of a Congolese patient with presumed viral hemorrhagic fever. In the absence of a replicating virus isolate, fulfilling Koch’s postulates to determine whether BASV is indeed a human virus and/or pathogen has been impossible. However, experiments with vesiculoviral particles pseudotyped with Bas-Congo glycoprotein suggested that BASV particles can enter cells from multiple animals, including humans. In 2015, genomes of two related viruses, Ekpoma virus 1 (EKV-1) and Ekpoma virus 2 (EKV-2), were detected in human sera in Nigeria. Isolates could not be obtained. Phylogenetic analyses led to the classification of BASV, EKV-1, and EKV-2 in the same genus, Tibrovirus, together with five biting midge-borne rhabdoviruses [i.e., Beatrice Hill virus (BHV), Bivens Arm virus (BAV), Coastal Plains virus (CPV), Sweetwater Branch virus (SWBV), and Tibrogargan virus (TIBV)] not known to infect humans. Using individual recombinant vesiculoviruses expressing the glycoproteins of all eight known tibroviruses and more than 75 cell lines representing different animal species, we demonstrate that the glycoproteins of all tibroviruses can mediate vesiculovirus particle entry into human, bat, nonhuman primate, cotton rat, boa constrictor, and Asian tiger mosquito cells. Using four of five isolated authentic tibroviruses (i.e., BAV, CPV, SWBV, and TIBV), our experiments indicate that many cell types may be partially resistant to tibrovirus replication after virion cell entry. Consequently, experimental data solely obtained from experiments using tibrovirus surrogate systems (e.g., vesiculoviral pseudotypes, recombinant vesiculoviruses) cannot be used to predict whether BASV, or any other tibrovirus, infects humans

FDA-FRNA50-07MAR17_plos

Operetta and chemiluminescent raw dat

Origin of tested bat cell lines.

<p>Origin of tested bat cell lines.</p

Simian Hemorrhagic Fever Virus Cell Entry Is Dependent on CD163 and Uses a Clathrin-Mediated Endocytosis-Like Pathway

Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques but is thought to be nonpathogenic for humans. To date, the SHFV life cycle is almost completely uncharacterized on the molecular level. Here, we describe the first steps of the SHFV life cycle. Our experiments indicate that SHFV enters target cells by low-pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-β-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knockout study and electron microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, α-chymotrypsin, and trypsin) abrogated entry, indicating that the SHFV cell surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component

Expression of human CD26/DPP4 confers MERS-CoV susceptibility to otherwise resistant bat cells.

<p>(A) Viral yields from MERS-CoV-resistant PESU-B5L, R05T, R06E, and Tb1Lu bat cells. Cells were transfected with a plasmid expressing human CD26/DPP4 or empty control plasmid and exposed 48 h later to MERS-CoV/EMC at an MOI of 3. Supernatants were harvested at 24 h after virus exposure for quantification of virus yields by plaque assay. (B) Same experiment: representative immunofluorescence assay (IFA) images of cells stained with anti-MERS-CoV spike protein antibody (green, top) or anti-human CD26/DPP4 antibody (red, bottom). (C) Merged IFA images demonstrate colocalization of MERS-CoV spike protein and CD26/DPP4. (D). Viral yields from MERS-CoV-susceptible bat cells transfected with a plasmid expressing human CD26/DPP4 or empty control plasmid using procedures identical to resistant cells in (A) except that cells were exposed to virus 24 h after transfection. Error bars indicate the standard deviation of duplicate samples.</p

Cell-surface expression of CD26/DPP4 on bat cells.

<p>Cells of different bat cell lines were analyzed by flow cytometry after staining with goat anti-human DPP4/CD26 antibody (red line) or control antibody (black lines).</p

Six of ten tested bat cell lines are susceptible to MERS-CoV infection.

<p>(A and B) Ten different bat cell lines were exposed to MERS-CoV/EMC (A) or MERS-CoV/Jor (B) at an MOI of 1. Supernatants were harvested at days 0, 1, 3, and 5 after virus exposure, and virus yields were determined by plaque assay on Vero cells. Error bars indicate the standard deviation of triplicate samples. (C and D) Same experiment: immunofluorescence assay (IFA) images of bat cell lines exposed to MERS-CoV/EMC (C) or MERS-CoV/Jor. (D) 1 (D1) or 3 (D3) days after virus exposure and stained against MERS-CoV spike protein (green). (E) Same experiment: TEM images of bat cells infected with MERS-CoV/EMC at day 1 after virus exposure. Red arrows point at double-membrane vesicles (DMVs) typical of coronavirus infections.</p

Persistent MERS-CoV infection of bat cells induces downregulation of bat cell CD26/DPP4 expression.

<p>Bat cell lines susceptible to infection were infected with MERS-CoV/EMC (A) or MERS-CoV/Jor (B) at an MOI of 1. After 7 days, supernatants were harvested for virus yield analysis by plaque assay, and the cells were subcultured at a 1∶10 dilution in new flasks. Subsequently, the persistently infected cells were passaged at a 1∶10 dilution weekly. Error bars indicate the standard deviation of duplicate samples. (C and D) Same experiment: immunofluorescence assay (IFA) images of bat cells persistently infected with MERS-CoV/EMC (C) or MERS-CoV/Jor (D) at day +33 stained with anti-MERS-CoV spike protein antibody (green). (E) Same experiment: TEM images of bat cells persistently infected with MERS-CoV/EMC at day 56. (F) Flow cytometry data of CD26/DPP4 surface expression (red line: anti-human CD26-/DPP4 antibody; black line: control antibody) in persistently infected cells. (G) CD26/DPP4 expression in persistently infected EidNi/41.3 cells (day 63) as detected by western blot.</p

<i>In vitro</i> detection of KRCV-1 RNA in infected cells using RNAscope^® <i>in situ</i> hybridization.

<p>(A) SHFV-infected MA-104 cells labeled with SHFV- (left) or KRCV-1-specific (right) probes. (B) KRCV-1-infected MARC-145 cells labeled with SHFV- (left) or KRCV-1-specific (right) probes. Positive results manifest as brown staining after amplification. All images were originally taken at 400X magnification.</p

Anti-human CD26/DPP4 antibody inhibits MERS-CoV infection of bat cells.

<p>RoNi/7.1 or Huh-7 cells (control) were treated with increasing concentrations (0, 1.25, 2.5, 5, 10, and 20 µg/ml) of anti-human CD26/DPP4 antibody or control antibody and then exposed to MERS-CoV/EMC at an MOI of 1. (A) After 24 h, viral yields in supernatants were determined by plaque assay. (B) Cellular infection was determined by immunofluorescence assay (IFA) with an anti-MERS-CoV spike protein antibody (green). (B left) The percentage of infected cells was analyzed by high content imaging. (B right) Representative IFA images. Error bars indicate the standard deviation of triplicate samples.</p