Abstract
Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity—but also aided in the control—of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6,7,8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples—in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.
Data availability
Sequence data are available in Gisaid under accession number EPI_ISL_406862. All other data are available from C.D. upon reasonable request.
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Acknowledgements
This work was funded by grants from the German Ministry of Research (01KI1723A) and the European Union (602525) to C.D. as well as by the German Bundeswehr Medical Service Biodefense Research Program. The funders had no role in study design, data collection and analysis or decision to publish. We thank P. Mackeldanz, E. Möncke-Buchner, A. Richter, M. Schmidt and J. Beheim-Schwarzbach for technical assistance.
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Contributions
R.W. and V.M.C. planned and supervised laboratory testing, and evaluated data. W.G. and M.S. managed patients and evaluated clinical data. S.Z., T.B., S.B., J.S., R.E. and K.Z. performed laboratory testing. M.A.M. managed serological laboratory testing. D.N. managed and performed virus isolation studies. T.C.J. analysed sequences and population-specific polymorphisms. P.V. managed laboratory testing. C.R. managed initial patient contacts. M.H. managed initial patient contacts and evaluated clinical data. C.D. designed and supervised laboratory studies, and wrote the manuscript. C.W. designed and supervised clinical management and clinical data.
Corresponding authors
Correspondence to Christian Drosten or Clemens Wendtner.
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Peer review information Nature thanks Peter Openshaw and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 Sequence analysis of E gene subgenomic mRNA.
The leader sequence (purple), putative transcription regulatory sequences (TRS) (grey) and nucleotides coding for the 5′-proximal part of the E gene (yellow box) are shown. PCR primer binding sites used for amplification and RT–PCR detection are shown as green arrows, and the 5′-nuclease PCR probe is shown as a red arrow.
Extended Data Fig. 2 Recombinant SARS-CoV-2-spike-based immunofluorescence test shows seroconversion of patient no.
4. Representative outcome of a recombinant immunofluorescence test using serum dilutions 1:10, 1:100, 1:1,000 and 1:10,000 of patient no. 4 at 5 and 17 days after the onset of symptoms. Secondary detection was done by using a goat-anti human immunoglobulin labelled with Alexa Fluor 488 (shown in green). The experiment was performed in duplicate.
Supplementary information
Supplementary Information
This file contains Supplementary Methods, which include a description of RNA extraction and RT-PCR methods, a description of cell culture and antibody detection methods and Supplementary References.
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Wölfel, R., Corman, V.M., Guggemos, W. et al. Virological assessment of hospitalized patients with COVID-2019. Nature (2020). https://doi.org/10.1038/s41586-020-2196-x
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