The clinical courses in the patients under study—all of whom were young- to middle-aged professionals without notable underlying disease—were mild. 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 disease5). 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 patients with SARS in Hong Kong15. Viral load also differs considerably between SARS and COVID-19. For SARS, it took 7 to 10 days after the onset of symptoms until peak RNA concentrations (of up to 5 × 105 copies per swab) were reached13,14. In the present study, peak concentrations were reached before day 5, and were more than 1,000 times higher. Successful isolation of live virus from throat swabs is another notable difference between COVID-19 and SARS, for which such isolation was rarely successful16,17,18. This suggests active virus replication in tissues of the upper respiratory tract, where SARS-CoV is not thought to replicate in spite of detectable ACE2 expression19,20. At the same time, the concurrent use of ACE2 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 previously found13 in sputum at mean concentrations of 1.2–2.8 × 106 copies per ml, which corresponds to observations made here.

Whereas proof of replication by histopathology is awaited, extended tissue tropism of SARS-CoV-2 with replication in the throat is strongly supported by our studies of cells that transcribe subgenomic mRNA in throat swab samples, particularly during the first 5 days of symptoms. Notable additional evidence for independent replication in the throat is provided by sequence findings in one patient, who consistently showed a distinct virus in the throat as opposed to the lung. In addition, the disturbance of gustatory and olfactory senses points at an infection of the tissues of the upper respiratory tract.

Critically, the majority of patients in the present study seemed to be beyond their shedding peak in samples from the upper respiratory tract when they were first tested, whereas the shedding of infectious virus in sputum continued throughout the first week of symptoms. Together, these findings suggest a more efficient transmission of SARS-CoV-2 than SARS-CoV, through active pharyngeal viral shedding at a time at which symptoms are still mild and typical of infections of the upper respiratory tract. Later in the disease, COVID-19 resembles SARS in terms of replication in the lower respiratory tract. Of note, the two patients who showed some symptoms of the lungs being affected showed a prolonged viral load in sputum. Our study is limited, in that no severe cases were observed. Future studies that include severe cases should look at the prognostic value of an increase of viral load beyond the end of week 1, potentially indicating an aggravation of symptoms.

One of the most interesting hypotheses to explain the potential extension of tropism to the throat is the presence of a polybasic furin-type cleavage site at the S1–S2 junction in the SARS-CoV-2 spike protein that is not present in SARS-CoV17. The insertion of a polybasic cleavage site in the S1–S2 region in SARS-CoV has previously been shown to lead to a moderate, but discernible, gain-of-fusion activity that might result in increased viral entry in tissues with a low density of ACE2 expression21.

The combination of very high concentrations of virus RNA and the occasional detection of cells in stools that contain subgenomic mRNA indicate active replication in the gastrointestinal tract. Active replication is also suggested by a much higher detection rate compared to the Middle East respiratory system coronavirus (MERS-CoV), for which stool-associated RNA was found in only 14.6% of samples from 37 patients hospitalized in Riyadh (Saudi Arabia)22,23. If SARS-CoV-2 was only passively present in the stool (such as after swallowing respiratory secretions), similar detection rates as for MERS-CoV would be expected. Replication in the gastrointestinal tract is also supported by analogy with SARS-CoV, which was regularly excreted in stool (from which it could be isolated in cell culture24). Our failure to isolate live SARS-CoV-2 from stools may be due to the mild courses of cases, with only one case showing intermittent diarrhoea. In China, diarrhoea was seen in only 2 of 99 cases25. Further studies should therefore address whether SARS-CoV-2 shed in stools is rendered noninfectious though contact with the gut environment. Our initial results suggest that measures to contain viral spread should aim at droplet-, rather than fomite-, based transmission.

The prolonged viral shedding in sputum is relevant not only for the control of infections in hospitals, but also for discharge management. In a situation characterized by a limited capacity of hospital beds in infectious disease wards, there is pressure for early discharge after treatment. On the basis of the present findings, early discharge with ensuing home isolation could be chosen for patients who are beyond day 10 of symptoms and have less than 100,000 viral RNA copies per ml of sputum. Both criteria predict that there is little residual risk of infectivity, on the basis of cell culture.

The serological courses of all patients suggest a timing of seroconversion similar to, or slightly earlier than, in SARS-CoV infection18. Seroconversion in most cases of SARS occurred during the second week of symptoms. As in SARS and MERS, IgM was not detected considerably earlier than IgG in immunofluorescence; this might in part be due to technical reasons, as the higher avidity of IgG antibodies outcompetes IgM for viral epitopes in the assay. IgG depletion can only partially alleviate this effect. Because immunofluorescence assay is a labour-intensive method, enzyme-linked immunosorbent assay tests should be developed as a screening test. Neutralization testing is necessary to rule out cross-reactive antibodies directed against endemic human coronaviruses. On the basis of the frequently low neutralizing antibody titres observed in coronavirus infection26,27, we have here developed a particularly sensitive plaque-reduction neutralization assay. Considering the titres we observed, a simpler microneutralization test format is likely to provide sufficient sensitivity in routine application and population studies.

When aligned to viral load courses, it seems there is no abrupt virus elimination at the time of seroconversion. Rather, seroconversion early in week 2 coincides with a slow but steady decline of viral load in sputum. Whether properties such as the glycosylation pattern at critical sites of the glycoprotein have a role in the attenuation of the neutralizing antibody response needs further clarification. In any case, vaccine approaches targeting mainly the induction of antibody responses should aim to induce particularly strong antibody responses to be effective.