Human cell tropism and innate immune system interactions of human respiratory coronavirus EMC compared to those of severe acute respiratory syndrome coronavirus

Abstract

Infections with human coronavirus EMC (HCoV-EMC) are associated with severe pneumonia. We demonstrate that HCoV-EMC resembles severe acute respiratory syndrome coronavirus (SARS-CoV) in productively infecting primary and continuous cells of the human airways and in preventing the induction of interferon regulatory factor 3 (IRF-3)-mediated antiviral alpha/beta interferon (IFN-α/β) responses. However, HCoV-EMC was markedly more sensitive to the antiviral state established by ectopic IFN. Thus, HCoV-EMC can utilize a broad range of human cell substrates and suppress IFN induction, but it does not reach the IFN resistance of SARS-CoV.

Fig 1

Virus multiplication in differentiated cultures of human tracheobronchial epithelial cells. Differentiated HTBE cultures grown on Transwell permeable membrane supports under air-liquid interface conditions were apically inoculated with SARS-CoV strain FFM-1 (34) or the HCoV-EMC isolate (3) at an MOI of 0.1. Virus yields from the apical and basolateral sides were determined at 24, 48, and 72 h p.i. by a TCID₅₀ assay. Mean values plus standard deviations (error bars) of 3 replicate experiments are shown.

Fig 2

Cell tropism and IFN sensitivity. Multiplication and type I IFN sensitivity of HCoV-EMC in comparison to SARS-CoV were studied by applying high or low doses of IFN. (A to C) Cultures of primary nondifferentiated HTBE cells (A), the human bronchial epithelial cell line Calu-3 (B), and the primate kidney cell line Vero (C) were pretreated with 0, 100, 500, or 1,000 units per ml of recombinant human IFN-β (Betaferon; Schering). After 18 h of incubation, cells were infected with SARS-CoV (top panel) or HCoV-EMC (bottom panel) at an MOI of 0.01. Viral titers in the supernatants were determined at 24 h and 48 h p.i., using the TCID₅₀ assay in Vero cells. (D and E) Application of low doses of IFN. IFN sensitivity of the viruses was tested in Calu-3 (D) and Vero (E) cells, using 5, 10, and 50 units per ml of recombinant human IFN-β. Mean values plus standard deviations (error bars) of 3 replicate experiments are shown.

Fig 3

Cytokine responses and IRF-3 activation. (A to C) Real-time RT-PCR analyses for cytokine induction (26) and viral RNA production (–37). The human bronchial epithelium cell line Calu-3 was infected with SARS-CoV, HCoV-EMC, or the recombinant Rift Valley fever virus (RVFV) mutant RVFVΔNSs::Ren (control [CTRL]) at an MOI of 1. Total cell RNA was assayed at the indicated time points for changes in the levels of RNAs for IFN-β, ISG56, and IP-10 (A and B) or viral RNAs (C). rel.u., relative units. (D to F) A parallel experiment measuring cytokine induction and viral RNA detection in the human lung adenocarcinoma cell line A549 at the indicated time points p.i. Mean values plus standard deviations (error bars) of 3 replicate experiments are shown. (G) Activation of IRF-3. Calu-3 cells (left panels) or A549 cells (right panels) were infected with the indicated viruses at an MOI of 1, fixed, and stained for endogenous IRF-3 (24), viral dsRNA (29), and RVFV N protein as described previously (38). Note that for reasons of antibody compatibility, the RVFV N signals shown in the small insets are from different coverslips which were infected in parallel. In all IRF-3 images, the contrast was enhanced using the autocontrast feature of Adobe Photoshop.