key: cord-255709-tm3we5sd
authors: Mossel, Eric C.; Sainz, Bruno; Garry, Robert F.; Peters, C. J.
title: Synergistic Inhibition of Sars-Coronavirus Replication by Type I and Type II IFN
date: 2006
journal: The Nidoviruses
DOI: 10.1007/978-0-387-33012-9_89
sha: 
doc_id: 255709
cord_uid: tm3we5sd

nan

It has been previously shown that treatment of cells with both type I and type II IFN produces an antiviral state greater in magnitude than can be explained by additive effects alone. [8] [9] [10] [11] [12] We sought to determine the effect of such an enhanced antiviral state on the replication of SARS-CoV.

has been extensively examined in culture, animals, and the clinic. IFN-α, relatively ineffective in cell culture, showed suggestive but inconclusive efficacy in monkeys and SARS patients. 1,2 IFN-β has most potent antiviral activity, though concentrations of greater than 1000 U/ml result in only marginal reduction of virus titer. 3-7 IFN-γ is ineffective against SARS-CoV in cell culture. 3, 5, 6 To characterize the inhibitory effect of IFN-β and IFN-γ treatment on SARS-CoV replication, three-day viral growth assays were performed. IFN pretreated Vero E6 cells were infected with SARS-CoV at a MOI of 0.01. Cultures treated with 100 U/ml of IFNor IFN-γ were significantly refractory for SARS-CoV Urbani and HK replication (P < 0.001) at 24 and 48 hpi (Figures 1a and 1b) . By 72 hpi, however, viral titers in IFN-β-or IFN-γ-treated cultures approached levels detected in vehicle-treated groups. A potent inhibitory effect was observed when Vero E6 cultures were treated with both IFN-β and IFN-γ. The inhibitory effect achieved with combination IFN-β and IFN-γ treatment was consistently greater than 3000-fold at all time points tested and reached levels of greater than 1×10 5 -fold at 72 hpi relative to vehicle treated Vero E6 cells.

Cytopathic effect (CPE) was extensive in vehicle-treated groups infected with either SARS-CoV strain at 120 hpi ( Fig. 2A and 2E) , as evident by the reduced monolayer staining with crystal violet. Relative to vehicle-treated and individual IFN-treated cultures, CPE is less evident in cells treated with both IFN-β and IFN-γ at 120 hpi; monolayers appeared evenly stained with little to no visible CPE ( Fig. 2D and 2H ). To determine whether this phenomenon is limited to Vero E6 cells, additional cell lines were examined. Calu-3 cells showed the same gradated CPE as Vero E6 cells with little or no CPE present in cells treated with both IFN-β and IFN-γ (Fig. 3) . Contrary to the observations of others, gross CPE does not occur in SARS-CoV-infected Caco-2 cells in our hands. 3, 13 As such, SARS-CoV-infected Caco-2 cell monolayers remained confluent regardless of treatment. However, the CPE profile observed in Calu-3 cells suggests that the synergistic inhibitory effect on SARS-CoV replication by IFN-β and IFN-γ is not Vero E6 cell specific.

It has been known for more than 25 years that treatment of cells with type I and type II IFN potentiates the antiviral response to levels greater than can be explained by simple additive effects. 10 Since then, the effect has been shown for a wide variety of viruses, including human cytomegalovirus, HSV-1, vesicular stomatitis virus (VSV), Lassa virus, and others. 8, 9, 11, 12, 14 The mechanism of synergistic inhibition of virus replication by type I and type II IFN has not been determined for any virus. However, it was recently shown that NO, induced by a combination of IFN-γ and IL-1 , inhibits SARS-CoV replication. 15 Further, it was shown in an avian system that type I and type II IFN potentiate the antiviral response as well as the secretion of NO. 16 Based on this evidence, a role for NO and iNOS in the potentiated anti-SARS-CoV response induced by type I and type II IFN cotreatment deserves consideration.

The authors wish to thank Dr. Dr. Li-Kuang Chen from Tzu-Chi University at Hualien, Taiwan for kindly supplying the SARS-CoV strain HK. This work is supported by National Institutes of Health grants AI007536 (E.C.M.), AI0543818 (B.S.), and AI054626, AI054238, RR018229, and CA08921 (R.F.G.) and contract NO1 AI 25489 (C.J.P.).

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