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Virological assessment of hospitalized patients with COVID-2019

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Virological assessment of hospitalized patients with COVID-2019

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Coronavirus disease 2019 (COVID-19) is an acute respiratory tract infection that emerged in late 20191,2. Initial outbreaks in China involved 13.8% cases with severe, and 6.1% with critical courses³. This severe presentation corresponds to the usage of a virus receptor that is expressed predominantly in the lung2,4. By causing an early onset of severe symptoms, this same receptor tropism is thought to have determined pathogenicity, but also aided the control, of severe acute respiratory syndrome (SARS) in 2003⁵. However, there are reports of COVID-19 cases with mild upper respiratory tract symptoms, suggesting the potential for pre- or oligosymptomatic transmission6–8. There is an urgent need for information on body site-specific virus replication, immunity, and infectivity. Here we provide a detailed virological analysis of nine cases, providing proof of active virus replication in upper respiratory tract tissues. Pharyngeal virus shedding was very high during the first week of symptoms (peak at 7.11 × 10⁸ RNA copies per throat swab, day 4). Infectious virus was readily isolated from throat- and lung-derived samples, but not from stool samples, in spite of high virus RNA concentration. Blood and urine never yielded virus. Active replication in the throat was confirmed by viral replicative RNA intermediates in throat samples. Sequence-distinct virus populations were consistently detected in throat and lung samples from the same patient, proving independent replication. Shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (14 days in all), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild upper respiratory tract illness. Active virus replication in the upper respiratory tract puts the prospects of COVID-19 containment in perspective.
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Nature | Vol 581 | 28 May 2020 | 465
Article
Virological assessment of hospitalized
patients with COVID-2019
Roman Wölfel1,6, Victor M. Corman2,6, Wolfgang Guggemos3,6, Michael Seilmaier3,
Sabine Zange1, Marcel A. Müller2, Daniela Niemeyer2, Terry C. Jones2,4, Patrick Vollmar1,
Camilla Rothe5, Michael Hoelscher5, Tobias Bleicker2, Sebastian Brünink2, Julia Schneider2,
Rosina Ehmann1, Katrin Zwirglmaier1, Christian Drosten2,7 ✉ & Clemens Wendtner3,7 ✉
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
transmission6–8. There is an urgent need for information on virus replication,
immunity and infectivity in specic 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 rst 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
conrmed 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 conrmation of active virus replication in the upper respiratory
tract has implications for the containment of COVID-19.
There is a close genetic relationship between SARS coronavirus
(SARS-CoV) and the causative agent of COVID-19, SARS-CoV-2. The pre-
dominant expression of ACE2 in the lower respiratory tract is believed
to have determined the natural history of SARS as an infection of the
lower respiratory tract
5
. Although the positive detection of SARS-CoV-2
in clinical specimens from the upper respiratory tract has previously
been described9,10, these observations do not address the principal dif-
ferences between SARS and COVID-19 in terms of clinical pathology. The
patients who were studied here were enrolled because they acquired their
infections upon known close contact to an index case, thereby avoiding
representational biases owing to symptom-based case definitions. All
patients were treated in a single hospital in Munich, Germany. Virological
testing was done by two closely collaborating laboratories that used the
same standards of technology for PCR with reverse transcription (RT
PCR) and virus isolation; these two laboratories confirmed each other’s
results in almost all of the individual samples. Owing to the extremely
high congruence of results, all data—except for the serological data
(which are based on results from one laboratory only)—are presented
together. The patients are part of a larger cluster of epidemiologically
linked cases that occurred after 23 January 2020 in Munich, as discovered
on 27 January (ref. 11). The present study uses samples taken during the
clinical course in the hospital, as well as from initial diagnostic testing
before admission. In cases in which this initial diagnostic testing was done
by other laboratories, the original samples were retrieved and retested
under the rigorous quality standards of the present study.
RT–PCR, replication sites and infectivity
To first understand whether the described clinical presentations are
solely caused by infection with SARS-CoV-2, samples from all patients
https://doi.org/10.1038/s41586-020-2196-x
Received: 1 March 2020
Accepted: 24 March 2020
Published online: 1 April 2020
Check for updates
1Bundeswehr Institute of Microbiology, Munich, Germany. 2Charité Universitätsmedizin Berlin, Berlin, Germany. 3Klinikum München-Schwabing, Munich, Germany. 4Center for Pathogen
Evolution, Department of Zoology, University of Cambridge, Cambridge, UK. 5University Hospital LMU Munich, Munich, Germany. 6These authors contributed equally: Roman Wölfel,
Victor M. Corman, Wolfgang Guggemos. 7These authors jointly supervised this work: Christian Drosten, Clemens Wendtner. e-mail: christian.drosten@charite.de;
clemens.wendtner@muenchen-klinik.de
Content courtesy of Springer Nature, terms of use apply. Rights reserved
... Right from the beginning of COVID-19, careful clinical studies have been conducted. It was found that COVID-19 patients shed larger quantities of viral particles than those infected with SARS-CoV-1 [92]. The lifecycle of SARS-CoV-2 is also longer, with some patients shedding particles even after 6 months [92]. ...
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Background In December, 2019, the newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, causing COVID-19, a respiratory disease presenting with fever, cough, and often pneumonia. WHO has set the strategic objective to interrupt spread of SARS-CoV-2 worldwide. An outbreak in Bavaria, Germany, starting at the end of January, 2020, provided the opportunity to study transmission events, incubation period, and secondary attack rates. Methods A case was defined as a person with SARS-CoV-2 infection confirmed by RT-PCR. Case interviews were done to describe timing of onset and nature of symptoms and to identify and classify contacts as high risk (had cumulative face-to-face contact with a confirmed case for ≥15 min, direct contact with secretions or body fluids of a patient with confirmed COVID-19, or, in the case of health-care workers, had worked within 2 m of a patient with confirmed COVID-19 without personal protective equipment) or low risk (all other contacts). High-risk contacts were ordered to stay at home in quarantine for 14 days and were actively followed up and monitored for symptoms, and low-risk contacts were tested upon self-reporting of symptoms. We defined fever and cough as specific symptoms, and defined a prodromal phase as the presence of non-specific symptoms for at least 1 day before the onset of specific symptoms. Whole genome sequencing was used to confirm epidemiological links and clarify transmission events where contact histories were ambiguous; integration with epidemiological data enabled precise reconstruction of exposure events and incubation periods. Secondary attack rates were calculated as the number of cases divided by the number of contacts, using Fisher's exact test for the 95% CIs. Findings Patient 0 was a Chinese resident who visited Germany for professional reasons. 16 subsequent cases, often with mild and non-specific symptoms, emerged in four transmission generations. Signature mutations in the viral genome occurred upon foundation of generation 2, as well as in one case pertaining to generation 4. The median incubation period was 4·0 days (IQR 2·3–4·3) and the median serial interval was 4·0 days (3·0–5·0). Transmission events were likely to have occurred presymptomatically for one case (possibly five more), at the day of symptom onset for four cases (possibly five more), and the remainder after the day of symptom onset or unknown. One or two cases resulted from contact with a case during the prodromal phase. Secondary attack rates were 75·0% (95% CI 19·0–99·0; three of four people) among members of a household cluster in common isolation, 10·0% (1·2–32·0; two of 20) among household contacts only together until isolation of the patient, and 5·1% (2·6–8·9; 11 of 217) among non-household, high-risk contacts. Interpretation Although patients in our study presented with predominately mild, non-specific symptoms, infectiousness before or on the day of symptom onset was substantial. Additionally, the incubation period was often very short and false-negative tests occurred. These results suggest that although the outbreak was controlled, successful long-term and global containment of COVID-19 could be difficult to achieve. Funding All authors are employed and all expenses covered by governmental, federal state, or other publicly funded institutions.
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The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
Preprint
The emergence of a novel, highly pathogenic coronavirus, 2019-nCoV, in China, and its rapid national and international spread pose a global health emergency. Coronaviruses use their spike proteins to select and enter target cells and insights into nCoV-2019 spike (S)-driven entry might facilitate assessment of pandemic potential and reveal therapeutic targets. Here, we demonstrate that 2019-nCoV-S uses the SARS-coronavirus receptor, ACE2, for entry and the cellular protease TMPRSS2 for 2019-nCoV-S priming. A TMPRSS2 inhibitor blocked entry and might constitute a treatment option. Finally, we show that the serum form a convalescent SARS patient neutralized 2019-nCoV-S-driven entry. Our results reveal important commonalities between 2019-nCoV and SARS-coronavirus infection, which might translate into similar transmissibility and disease pathogenesis. Moreover, they identify a target for antiviral intervention. One sentence summary The novel 2019 coronavirus and the SARS-coronavirus share central biological properties which can guide risk assessment and intervention.