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To determine whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can establish a faecal–oral transmission route, it is essential to confirm that infectious virus particles

are shed in faeces from patients with COVID-19. We would like to thank Pedersen et al. for their Correspondence on our Perspective (Guo, M. et al. Potential intestinal infection and

faecal–oral transmission of SARS-CoV-2. _Nat. Rev. Gastroenterol. Hepatol._ 18, 269–283 (2021))1, which raises some important issues (Pedersen et al. Rectally shed SARS-CoV-2 lacks

infectivity: time to rethink faecal–oral transmission? _Nat. Rev. Gastroenterol. Hepatol._ https://doi.org/10.1038/s41575-021-00501-w (2021))2. Our Perspective refers to four studies that

discuss isolation of infectious virus particles from patient faeces3,4,5,6. Two of these studies5,6 only reported data obtained by electron microscopy, which are indeed insufficient to

determine infectivity, as argued by Pedersen et al.2. However, Zhou and colleagues reported the extraction of infectious virus particles from a patient with COVID-19 based on increases in

viral load in human intestinal organoids4. Also, Xiao et al. reported isolation of SARS-CoV-2 particles from two of three stool samples3; the viral isolates, first observed by electron

microscopy, could successfully infect Vero cells (confirmed by immunofluorescence staining)3. More recently, when three viral isolates were extracted from stool samples of patients with

COVID-19 and added to Vero cells7, the culture supernatants tested positive for SARS-CoV-2 by quantitative reverse transcription PCR. The infectious viral particles were collected from

culture supernatant viral stock, then frozen and subsequently thawed stock were able to infect Vero and Calu-3 (human lung adenocarcinoma) cells. Two of the three viral faecal isolates were

more infectious in cells than viral isolates extracted from nasopharyngeal swabs and sputum in vitro7. These results strongly suggested that infectious virus could be shed in faecal samples

from patients with COVID-19. Notably, three of five studies mentioned here also included patient information3,6,7, and all of the viral isolates in these studies were extracted from patients

with severe disease. Without more information, we cannot exclude the possibility that one study mentioned by Pederson et al. that failed to isolate infectious SARS-CoV-2 from stool could be

attributable to a mild disease course in those patients8. Moreover, Xiao et al. failed to isolate virus from stool at later time points, indicating the absence of infectious virus, with

only viral fragments apparently shed in faeces during late stages of the disease3. Thus, disease severity and time course for sampling could be determining factors in the isolation of

infectious viral particles. Research in animals might also provide valuable insights. One study investigating SARS-CoV-2 infection in rhesus macaques reported the successful isolation of

infectious viruses from faeces, and confirmed this finding by electron microscopy and TCID50 (median tissue culture infective dose) assays (105/ml)9. Similarly, a study of SARS-CoV-2

infection in ferrets showed that oral inoculation with faecal supernatants of infected specimens could establish infection in naive ferrets10. These animal studies therefore demonstrated the

presence of infectious virus in the faeces of COVID-19 animal models. Although research has suggested that infectious viral particles can be isolated from both humans and animals, based on

the available evidence (Table 1), a clear faecal–oral transmission route has not been established for SARS-CoV-2. REFERENCES * Guo, M., Tao, W., Flavell, R. A. & Zhu, S. Potential

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Google Scholar  Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Digestive Disease, The First Affiliated Hospital of USTC, Division of Life Sciences and

Medicine, University of Science and Technology of China, Hefei, China Meng Guo, Wanyin Tao & Shu Zhu * Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key

Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China Meng

Guo, Wanyin Tao & Shu Zhu * Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA Richard A. Flavell * Howard Hughes Medical Institute, Chevy Chase, MD, USA

Richard A. Flavell * School of Data Science, University of Science and Technology of China, Hefei, China Shu Zhu * CAS Centre for Excellence in Cell and Molecular Biology, University of

Science and Technology of China, Hefei, China Shu Zhu Authors * Meng Guo View author publications You can also search for this author inPubMed Google Scholar * Wanyin Tao View author

publications You can also search for this author inPubMed Google Scholar * Richard A. Flavell View author publications You can also search for this author inPubMed Google Scholar * Shu Zhu

View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHORS Correspondence to Richard A. Flavell or Shu Zhu. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare no competing interests. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Guo, M., Tao, W., Flavell, R.A. _et al._ Reply to:

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