gms | German Medical Science

International Conference on SARS - one year after the (first) outbreak

08. - 11.05.2004, Lübeck

Early events of SARS-coronavirus replication in Vero E6 cells


  • Marcel Müller - Robert Koch-Institut, ZBS-1, Berlin, Germany
  • Nadine Litzba - Philipps-Universität Marburg, Marburg, Germany
  • Andreas Nitsche - Robert Koch-Institut, ZBS-1, Berlin, Germany
  • Kim Hattermann - Robert Koch-Institut, ZBS-1, Berlin, Germany
  • corresponding author presenting/speaker Matthias Niedrig - Robert Koch-Institut, ZBS-1, Berlin, Germany

International Conference on SARS - one year after the (first) outbreak. Lübeck, 08.-11.05.2004. Düsseldorf, Köln: German Medical Science; 2004. Doc04sarsP6.02

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Veröffentlicht: 26. Mai 2004

© 2004 Müller et al.
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The severe acute respiratory syndrome associated coronavirus (SARS-CoV) is the cause for a life-threatening form of pneumonia. Viral replication is characterised by discontinuous transcription and is apparently controlled by transcription-regulating sequences. Moreover it was discovered that SARS-CoV attaches, enters and uncoats the nucleocapsids all within a 30 minute period.

In this study we investigated the abundance of SARS-CoV mRNA with emphasis on the open-reading-frames (ORF) 3a and 7a in the first seven hours post infection (p.i.). The aim was to determine the efficiency of viral replication in newly infected cells. Vero E6 cells were infected with SARS- CoV Hong Kong and total RNA was isolated 15, 30, 45, 60, 90, 120, 180, 240, 300, 360 and 420 minutes p.i. and analysed by conventional RT-PCR and real-time TaqMan-PCR. For RT-PCR analysis a 5´-primer from the 5´-leader part and a 3´-primer specific for different ORFs enabled us to amplify particular subgenomic transcripts. RT-PCR revealed the existence of SARS-CoV mRNAs already 15 min after inoculation. A clear increase of viral mRNA transcripts could be detected with different primer combinations in RT-PCR and real-time PCR 300 minutes post infection.

To investigate functional influences of nonstructural proteins on early-phase viral replication the ORF 3a and 7a were cloned in a mammalian expression vector. In future experiments stably and transiently transfected Vero E6 cells will be co-infected with SARS-CoV to find out if an overexpression of one of the ORFs will have an influence on viral replication efficiency.