Primate hemorrhagic fever-causing arteriviruses are poised for spillover to humans

Abstract

Simian arteriviruses are endemic in some African primates and can cause fatal hemorrhagic fevers when they cross into primate hosts of new species. We find that CD163 acts as an intracellular receptor for simian hemorrhagic fever virus (SHFV; a simian arterivirus), a rare mode of virus entry that is shared with other hemorrhagic fever-causing viruses (e.g., Ebola and Lassa viruses). Further, SHFV enters and replicates in human monocytes, indicating full functionality of all of the human cellular proteins required for viral replication. Thus, simian arteriviruses in nature may not require major adaptations to the human host. Given that at least three distinct simian arteriviruses have caused fatal infections in captive macaques after host-switching, and that humans are immunologically naive to this family of viruses, development of serology tests for human surveillance should be a priority.

Keywords: CD163 receptor; arms race; arteriviruses; disease emergence; hemorrhagic fever; positive selection; primates; virus entry; virus evolution; zoonosis.

Figure 1.. Historical outbreaks and natural reservoirs for simian arteriviruses.

(A) Documented outbreaks of simian arteriviruses in primate facilities. Locations of affected facilities are shown by colored circles on the map and timeline. Several facilities experienced multiple outbreaks. (B) Phylogeny of representative viruses within the order Nidovirales, including all published simian arteriviruses, based on an alignment of concatenated RNA-directed RNA polymerase (RdRp) and helicase genes. Arteriviruses known to have caused outbreaks in primate facilities are written in red. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site and bootstrap values shown for major nodes. Asterisks indicate nodes with 100% support. (C) Geographical ranges of primate species known to be infected with simian arteriviruses. Sampling sites are indicated by black stars. *Only detected in captivity; ^†Kafue kinda chacma baboon virus (KKCBV) was discovered in kinda x chacma hybrid baboons, within Kafue National Park, as shown as an inset in red (Chiou et al., 2021).

Figure 2.. CD163 is necessary and sufficient as the receptor for SHFV infection.

(A) SHFV (at a multiplicity of infection [MOI] of 3) was incubated with cells from four different grivet cell lines (MA-104, MARC-145, COS-7, and Vero). Infectious virion production over a time course was quantified from cell supernatants by plaque assay (PFU/mL) on permissive MA-104 cells. (B) Single-step (MOI = 3) and multi-step (MOI = 0.03) growth curves of SHFV on wild-type MA-104 cells and its derivatives: ΔCD163, ΔCD163 complemented with empty vector (ΔCD163+vector), and ΔCD163 with grivet CD163 (ΔCD163+gCD163). (C) Untransformed primary skin fibroblasts derived from a 6-mo-old (#1) and a 20-yr-old patas monkey (#2) were complemented with empty vector or patas monkey CD163 (pCD163) and exposed to SHFV as described in panel A. (A–C) Lysates from cells were probed for CD163 expression using western blotting. Actin beta served as a loading control. Data show the mean +/− standard error of the mean (SEM) from three independent experiments, with one replicate per experiment. Dotted lines represent the limit of detection for the assay. All plots include error bars; no error bars are shown when the SEM was smaller than the size of the symbols. Statistical tests used: (A) Two-way analysis of variance (ANOVA) with Dunnett’s post-test when compared to MA-104 cells (***P < 0.001, ****P < 0.0001, NS, not significant). (B) Two-way ANOVA with Sidak’s post-test for wild-type compared to ΔCD163 (****P < 0.0001), or for ΔCD163+vector compared to ΔCD163+gCD163 (++++P < 0.0001), and (C) for +pCD163 compared to +vector cells (***P < 0.001, ****P < 0.0001). See also Figures S1 and S2. SHFV, simian hemorrhagic fever virus.

Figure 3.. CD163 is an intracellular receptor.

(A) Wild-type MA-104 or (B) ΔCD163 cells were cultured on glass coverslips and mock-exposed or exposed to simian hemorrhagic fever virus (SHFV) at a multiplicity of infection (MOI) of 10 for 1, 4, 6, or 8 h prior to fixation. Cell membranes were immunolabeled (anti-occludin antibody; red) and cells were probed for DNA (4′,6-diamidino-2-phenylindole [DAPI]; blue) and viral RNA (detected by single-molecule RNA fluorescence in situ hybridization [smFISH]; gray). Arrows indicate regions of viral RNA signal. (C) Rhesus macaque monocytes were isolated from whole blood and differentiated into macrophages using macrophage colony-stimulating factor (M-CSF). Exposure to SHFV was performed as described above but for 45 min, representing the earliest timepoint that intracellular viral RNA could reliably be detected inside cells. In addition to Hoechst (DNA, nucleus) and occludin staining, cells were immunolabeled to detect CD163 (green). (A–C) Cells were imaged by laser scanning confocal microscopy (LSCM); a representative maximum intensity projection from each condition is shown. Arrows indicate regions of viral RNA signal. (D) Pearson’s correlation analysis of the LSCM images generated in the experiment in panel C. A Pearson’s correlation coefficient was calculated between viral RNA signal and either Hoechst (blue), occludin (red), or CD163 (green) signal. Average and standard deviations of each correlation are indicated; each data point represents a single cell. (E) Single z-planes are shown for two representative images in which viral RNA (white) and CD163 (green) co-localized and were analyzed in three-dimensional space. The YZ and XZ images are shown for the regions of XY, denoted by the yellow lines and spanning from the top of the imaged cell to the bottom. Actual and simplified illustrations of each orthogonal view are shown, illustrating the relative positions of the viral RNA (white), CD163 (green), nucleus (blue), and cellular boundaries (red dots). In the simplified view, the red profile is the outline of the cell compiled from Z-stack images. See also Figures S3 and S4.

Figure 4.. CD163 is a dynamic barrier to host-switching of SHFV, but the human ortholog is fully functional for virus entry.

(A) Illustration of the CD163 receptor, showing the extracellular scavenger receptor cysteine-rich (SRCR domains 1–9) and proline domains rich with serine threonine (PST) I and II, and the short cytoplasmic tail. Residues evolving under positive selection are shown with red circles, and the putative arterivirus interaction domain is denoted with a black line. The N-terminus (amine terminus; NH₂) and C-terminus (carboxyl terminus; COOH) denote the beginning and end of the protein polypeptide chain, respectively. (B) MA-104 ΔCD163 cells stably expressing the indicated primate CD163 ortholog (X-axis) were exposed to SHFV at a multiplicity of infection (MOI) of 3. Viral titers in cell supernatants 12 h post-exposure were assessed by plaque assay. Bars are color-coded by log-fold differences in SHFV titers compared to cells expressing the receptor of the presumed natural host (patas monkey; #7). The data show the mean +/− standard error of the mean (SEM) from three independent experiments, with one replicate per experiment. One-way analysis of variance (ANOVA) with Dunnett’s post-test compared to control cells (*P < 0.05, **P < 0.01, ****P < 0.0001, NS not significant). The dotted line represents the limit of detection for the assay. CD163 receptor expression was confirmed by western blotting, and actin beta served as a protein loading control. See also Figure S5. SHFV, simian hemorrhagic fever virus.

Figure 5.. Human blood monocyte-derived macrophages do not support SHFV infection.

(A) Monocytes were isolated from human peripheral blood mononuclear cells (PBMCs; n = 3 donors) and differentiated into M0 macrophages. M0 macrophages were further polarized into M1 or M2 subsets using IFN-γ or IL-4. (B) Monocyte-derived macrophages were exposed to SHFV at a multiplicity of infection (MOI) of 3 or Ebola virus (MOI = 3), and viral titers were enumerated by plaque assay over time. Exposures were performed in technical triplicates, and the mean viral titers from three donors were plotted. (Error bars represent standard error of the mean [SEM].) (C) In parallel, virus stocks were verified as infectious by exposing them to positive control cell lines (MA-104 for SHFV, Vero E6 for Ebola virus). Data are the mean +/− the standard deviation from technical triplicates. See also Figure S6. SHFV, simian hemorrhagic fever virus; EBOV, Ebola virus.

Figure 6.. SHFV is fully competent for replication in human cells.

(A) Three human monocytic cell lines were exposed to SHFV at a multiplicity of infection (MOI) of 0.3. Infectious virus production over a time course was quantified in cell supernatant by plaque assay (PFU/mL) on SHFV-permissive MA-104 cells. (B) A human cell line was transduced with human CD163 (hCD163) or empty retroviral vectors, and antibiotics were used to select for stable integration. Cells were exposed to recombinant SHFV expressing enhanced green fluorescent protein (rSHFV-eGFP), MOI = 1. Shown are high-content images of mock-exposed or virus-exposed cells counterstained with Hoechst (blue) at 72 h post-exposure. Green fluorescence is indicative of productive viral infection. MA-104 monkey cells served as a positive control. (C) Wild-type MA-104, along with ACHN kidney cells stably transduced with human CD163 (hCD163) or empty vector, were exposed to wild-type SHFV (MOI = 0.3). (A, C) Data shown are representative of two independent experiments, each with three technical replicates. Error bars represent the mean +/− standard deviation. See also Figure S7.