MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment

Adriaan H De Wilde,V Stalin Raj,Ronald W A L Limpens,Diede Oudshoorn,Stefan Van Nieuwkoop,Clara C Posthuma,Montserrat Bárcena,Eric J Snijder,Yvonne Van Der Meer,Theo M Bestebroer,Bart L Haagmans,Bernadette G Van Den Hoogen

doi:10.1099/vir.0.052910-0

Abstract

Coronavirus (CoV) infections are commonly associated with respiratory and enteric disease in humans and animals. The 2003 outbreak of severe acute respiratory syndrome (SARS) highlighted the potentially lethal consequences of CoV-induced disease in humans. In 2012, a novel CoV (Middle East Respiratory Syndrome coronavirus; MERS-CoV) emerged, causing 49 human cases thus far, of which 23 had a fatal outcome. In this study, we characterized MERS-CoV replication and cytotoxicity in human and monkey cell lines. Electron microscopy of infected Vero cells revealed extensive membrane rearrangements, including the formation of double-membrane vesicles and convoluted membranes, which have been implicated previously in the RNA synthesis of SARS-CoV and other CoVs. Following infection, we observed rapidly increasing viral RNA synthesis and release of high titres of infectious progeny, followed by a pronounced cytopathology. These characteristics were used to develop an assay for antiviral compound screening in 96-well format, which was used to identify cyclosporin A as an inhibitor of MERS-CoV replication in cell culture. Furthermore, MERS-CoV was found to be 50–100 times more sensitive to alpha interferon (IFN-α) treatment than SARS-CoV, an observation that may have important implications for the treatment of MERS-CoV-infected patients. MERS-CoV infection did not prevent the IFN-induced nuclear translocation of phosphorylated STAT1, in contrast to infection with SARS-CoV where this block inhibits the expression of antiviral genes. These findings highlight relevant differences between these distantly related zoonotic CoVs in terms of their interaction with and evasion of the cellular innate immune response.

Highlights

In June 2012, a previously unknown coronavirus was isolated from a 60-year-old Saudi Arabian patient who died from acute respiratory distress syndrome and multiple organ failure (Zaki et al, 2012)
The CoV replicative cycle starts with translation of the positive-sense RNA genome into replicase polyproteins that are cleaved into 16 nsps (Gorbalenya et al, 2006; van Boheemen et al, 2012)
Hybridization analysis of the accumulation of viral RNA revealed the presence of genome RNA and seven sg transcripts, with sizes closely matching those predicted previously from the positions of conserved transcription regulatory sequences (TRSs) in the viral genome (Fig. 1a)

Summary

Introduction

In June 2012, a previously unknown coronavirus was isolated from a 60-year-old Saudi Arabian patient who died from acute respiratory distress syndrome and multiple organ failure (Zaki et al, 2012). A cluster of respiratory infections in Jordan (April 2012) was linked to the same agent, no convincing evidence for human-to-human transmission was obtained. This was clearly different for a cluster of three UK cases in early 2013, consisting of a patient who had travelled to Saudi Arabia and two family members without recent travel history outside the UK. Up to May 2013, 49 confirmed MERS cases, including 23 fatalities, have been recorded (http://www.who.int/csr/don/archive/disease/coronavirus_ infections/en/)

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Conclusion