Virological assessment of hospitalized cases of coronavirus disease 2019
Roman Wölfel*, Victor M. Corman*, Wolfgang Guggemos*, Michael Seilmaier, Sabine
Zange, Marcel A. Müller, Daniela Niemeyer, Terence C. Jones Kelly, Patrick Vollmar, Camilla
Rothe, Michael Hoelscher, Tobias Bleicker, Sebastian Brünink, Julia Schneider, Rosina
Ehmann, Katrin Zwirglmaier, Christian Drosten**, Clemens Wendtner**
*equal contribution
**senior authors with equal contribution
Author affiliations:
Bundeswehr Institute of Microbiology, Munich, Germany (Roman Wölfel, M.D.; Sabine
Zange, M.D.; Patrick Vollmar, M.D.; Rosina Ehmann DVM; Katrin Zwirglmaier, Ph.D.)
Klinikum München-Schwabing, Munich, Germany (Clemens Wendtner, M.D.; Wolfgang
Guggemos, M.D.; Michael Seilmaier, M.D.)
Charité Universitätsmedizin Berlin, Berlin, Germany (Victor M. Corman, M.D.; Marcel A.
Müller, Ph.D.; Daniela Niemeyer, Ph.D.; Terence C Jones Kelly, Ph.D., Tobias Bleicker,
Sebastian Brünink, Julia Schneider, MSc; Christian Drosten, M.D.)
University Hospital LMU Munich, Munich, Germany (Camilla Rothe, M.D; Michael Hoelscher
(M.D., Ph.D.)
Coronavirus disease 2019 (COVID-19) is an acute respiratory tract infection that
emerged in late 2019
1,2
. Initial outbreaks in China involved 13.8% cases with severe-,
and 6.1% with critical courses
3
. This severe presentation corresponds to the usage of
a virus receptor that is expressed predominantly in the lung
2,4
. 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 2003
5
. However, there are reports of COVID-19 cases with mild upper respiratory
tract symptoms, suggesting a potential for pre- or oligosymptomatic transmission
6-8
.
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
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NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
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 X 10
8
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 of one same patient. Shedding of viral RNA from sputum outlasted the end of
symptoms. Seroconversion occurred after 6-12 days, but was not followed by a rapid
decline of viral loads. COVID-19 can present as a mild upper respiratory tract illness.
Active virus replication in the upper respiratory tract puts prospects of COVID-19
containment in perspective.
There is a close genetic relatedness between severe acute respiratory syndrome coronavirus
(SARS-CoV) and the causative agent of COVID-19, SARS-CoV-2. The predominant
expression of ACE2 in the lower respiratory tract is believed to have determined the natural
history of SARS as a lower respiratory tract infection. Whereas positive SARS-CoV-2
detection in clinical specimens from the upper respiratory tract has been described
9
, these
observations do not address principal differences between SARS and COVID-19 in terms of
clinical pathology. The here-studied patients were enrolled because they acquired their
infections upon known close contact to an index case, thereby avoiding representational
biases due 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
using the same standards of technology for RT-PCR and virus isolation, confirming each
other’s results based on almost all individual samples. Due to extremely high congruence of
results, all data are presented together. Only the serological data are based on results from
one laboratory. The patients are part of a larger cluster of epidemiologically-linked cases that
occurred after January 23
rd
, 2020 in Munich, Germany, as discovered on January 27
th
(Böhmer et al., accompanying manuscript). The present study uses samples taken during the
clinical course in the hospital, as well as from initial diagnostic testing before admission. In
cases when this initial diagnostic testing was done by other laboratories, the original samples
were retrieved and re-tested under the rigorous quality standards of the present study.
RT-PCR sensitivity, sites of replication, and correlates of infectivity based on aggregated
data
To first understand whether the described clinical presentations are solely caused by SARS-
CoV-2 infection, samples from all patients were tested against a panel of typical agents of
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respiratory viral infection, including HCoV-HKU1, -OC43, -NL63, -229E; Influenza virus A and
B, Rhinovirus, Enterovirus, Respiratory syncytial virus, Human Parainfluenza virus 1-4,
Human metapneumovirus, Adenovirus, and Human bocavirus. Interestingly, no co-infection
was detected in any patient.
All patients were initially diagnosed by RT-PCR
10
from oro- or nasopharyngeal swab
specimens. Both specimen types were collected over the whole clinical course in all patients.
There were no discernible differences in viral loads or detection rates when comparing naso-
vs. oropharyngeal swabs (Figure 1B). The earliest swabs were taken on day 1 of symptoms,
with symptoms often being very mild or prodromal. All swabs from all patients taken between
days 1 and 5 tested positive. The average virus RNA load was 6.76x10
5
copies per whole
swab until day 5 (maximum, 7.11X10
8
copies/swab). Swab samples taken after day 5 had an
average viral load of 5.13x10
3
copies per swab and a detection rate of 45.95%. The last
swab sample was taken on day 22 post-onset. Average viral load in sputum was 1.18 x 10
6
copies per mL (maximum, 6.65x10
8
copies per mL).
Because swab samples had limited sensitivity for initial diagnosis of cases of SARS
13,14
, we
analyzed the first paired swab and sputum samples taken on the same occasion from seven
patients (Table 1). All samples were taken between 2 and 4 days post-onset. In two cases,
swab samples had clearly higher virus concentrations than sputum samples, as indicated by
a difference greater than 3 in threshold cycle (Ct) value. The opposite was true in two others
cases, while the other 5 cases had similar concentrations in both sample types.
None of 27 urine samples and none of 31 serum samples were tested positive for SARS-
CoV2 RNA.
To understand infectivity, live virus isolation was attempted on multiple occasions from
clinical samples (Figure 1 D). Whereas virus was readily isolated during the first week of
symptoms from a significant fraction of samples (16.66% in swabs, 83.33% in sputum
samples), no isolates were obtained from samples taken after day 8 in spite of ongoing high
viral loads.
Virus isolation from stool samples was never successful, irrespective of viral RNA
concentration, based on a total of 13 samples taken between days six to twelve from four
patients. Virus isolation success also depended on viral load: samples containing <10
6
copies/mL (or copies per sample) never yielded an isolate. For swab and sputum,
interpolation based on a probit model was done to obtain laboratory-based infectivity criteria
for discharge of patients (Figures 1 E, F).
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High viral loads and successful isolation from early throat swabs suggested potential virus
replication in upper respiratory tract tissues. To obtain proof of active virus replication in
absence of histopathology, we conducted RT-PCR tests to identify viral subgenomic
messenger RNAs (sgRNA) directly in clinical samples. Viral sgRNA is only transcribed in
infected cells and is not packaged into virions, therefore indicating the presence of actively-
infected cells in samples. Viral sgRNA was compared against viral genomic RNA in the same
sample. In sputum samples taken on days 4/5, 6/7, and 8/9, a time in which active replication
in sputum was obvious in all patients as per longitudinal viral load courses (see below), mean
normalized sgRNA per genome ratios were ~0.4% (Figure 1G). Swabs taken up to day 5
were in the same range, while no sgRNA was detectable in swabs thereafter. Together,
these data indicate active replication of SARS-CoV-2 in the throat during the first 5 days after
symptoms onset. No, or only minimal, indication of replication in stool was obtained by the
same method (Figure 1G).
During our study we sequenced full virus genomes from all patients. A G6446A exchange
was first detected in one patient and later transmitted to other patients in the cluster
(Böhmer, accompanying manuscript). In the first patient, this mutation was found in a throat
swab while a sputum sample from the same day still showed the original allele, 6446G. The
SNP was analyzed by RT-PCR and Sanger sequencing in all sequential samples available
from that patient (Table 1). The presence of separate genotypes in throat swabs and sputum
strongly supported our suspicion of independent virus replication in the throat, rather than
passive shedding there from the lung.
Virus shedding, antibody response, and clinical correlation in individual courses
Daily measurements of viral load in sputum, pharyngeal swabs, and stool are summarized in
Figure 2. In general, viral RNA concentrations were very high in initial samples. In all
patients except one, throat swab RNA concentrations seemed to be already on the decline at
the time of first presentation. Sputum RNA concentrations declined more slowly, with a peak
during the first week of symptoms in three of eight patients. Stool RNA concentrations were
also high. Courses of viral RNA concentration in stool seemed to reflect courses in sputum in
many cases (e.g., Figure 2 A, B, C). In only one case, independent replication in the
intestinal tract seemed obvious from the course of stool RNA excretion (Figure 2 D).
Whereas symptoms mostly waned until the end of the first week (Table 2), viral RNA
remained detectable in throat swabs well into the second week. Stool and sputum samples
remained RNA-positive over even longer periods, in spite of full resolution of symptoms.
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All cases had comparatively mild courses (Table 2). The two patients who showed some
signs of pneumonia were the only cases where sputum viral loads showed a late and high
peak around day 10/11, whereas sputum viral loads were on the decline by this time in all
other patients (Figure 2 F,G).
Seroconversion was detected by IgG and IgM immunofluorescence using cells expressing
the spike protein of SARS-CoV-2 and a virus neutralization assay using SARS-CoV-2 (Table
3). In early sera, taken between day 3 and 6, none of the patients showed detectable
antibody. The patients monitored long enough to yield a serum sample after two weeks all
showed neutralizing antibodies, the titer levels of which did not suggest any correlation with
clinical courses. Of note, case #4, with the lowest virus neutralization titer at end of week 2,
seemed to shed virus from stool over prolonged time (Figure 2 D). Results on differential
recombinant immunofluorescence assay indicated no significant rise in titer against the four
endemic human Coronaviruses (Table S1).
Conclusions
The clinical courses in subjects under study were mild, all being young- to middle-aged
professionals without significant underlying disease. Apart from one patient, all cases were
first tested when symptoms were still mild or in the prodromal stage, a period in which most
patients would present once there is general awareness of a circulating pandemic disease
5
.
Diagnostic testing suggests that simple throat swabs will provide sufficient sensitivity at this
stage of infection. This is in stark contrast to SARS. For instance, only 38 of 98 nasal or
nasopharyngeal swab samples tested positive by RT-PCR in SARS patients in Hong Kong
15
.
Also, viral load differed considerably. In SARS, it took 7 to 10 days after onset until peak
RNA concentrations (of up to 5x10
5
copies per swab) were reached
13,14
. In the present study,
peak concentrations were reached before day 5, and were more than 1000 times higher.
Successful live virus isolation from throat swabs is another striking difference from SARS, for
which such isolation was rarely successful
16-18
. Altogether, this suggests active virus
replication in upper respiratory tract tissues, where only minimal ACE-2 expression is found
and SARS-CoV is therefore not thought to replicate
19
. At the same time, the concurrent use
of ACE-2 as a receptor by SARS-CoV and SARS-CoV-2 corresponds to a highly similar
excretion kinetic in sputum, with active replication in the lung. SARS-CoV was found in
sputum at mean concentrations of 1.2-2.8x10
6
copies per mL, which corresponds to
observations made here
13
.
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