• diarrhoea
• intestinal gene regulation
• intestinal epithelium

A series of pneumonia cases caused by 2019 novel coronavirus (2019-nCoV, also named COVID-19) are being reported globally. Based on recent publications,1–3 the most common symptoms in patients infected by 2019-nCoV were fever and cough. However, the incidence of other clinical features differs in different reports. To address this issue, we collected the data from three reports1–3 and compared the incidence accordingly. We found that the incidence of leucopenia, fever and diarrhoea in the three studies showed a statistically significant difference (table 1). Among these symptoms, diarrhoea displayed the smallest p-value (p=0.016), suggesting that the criteria for diagnosing diarrhoea may differ in different hospitals. Due to the different criteria, clinicians may underestimate the value of this symptom in clinical practice, and it may affect the preliminary diagnostic accuracy.

Recent studies showed that the spike protein of 2019-nCoV shared the same cell entry receptor ACE2 as SARS-CoV.4 5 In terms of the pathological importance of ACE2 in modulating intestinal inflammation and diarrhoea,6 we examined the expression profiles of ACE2 in various human tissues and found that ACE2 was highly expressed in the human small intestine (online supplementary file 1). Intriguingly, the RNA level of ACE2 was quite low in lung tissues from healthy donors.

Given that the distribution of ACE2 may determine the route of 2019-nCoV infection, we next evaluated the expression of ACE2 in different cell populations of the small intestine by analysing the single-cell RNA sequencing (scRNA-Seq) data. Based on the scRNA-Seq data (GSE92332),7 we analysed 7216 individual cells derived from the small intestine of normal C57BL/6 mice. Using the unsupervised graph-based clustering, we found that the small intestine tissues contained at least eight distinct cell clusters according to their corresponding marker gene expression profiles (figure 1A,B). For instance, the LGR5 gene was highly expressed in the stem cell cluster of the small intestine, and it was significantly reduced in other cell clusters (figure 1B).

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Figure 1

High expression of 2019-nCoV viral receptor ACE2 in enterocytes of small intestine. (A) Analysis of single-cell sequencing data identified eight cell subpopulations within mouse small intestine. (B) Cluster identity was identified by the expression of marker genes and the annotations provided in the UMI-barcode matrix. (C&E&G) Expression profile of three virus receptors ACE2 (2019-nCoV and SARS-CoV), ANPEP (HCoV-229E) and DPP4 (MERS-CoV) in eight subset cell clusters. (D&F&H) Violin plots displayed that ACE2, ANPEP and DPP4 are highly expressed in proximal and distal enterocytes. nCoV, novel coronavirus.

We then found that ACE2 was highly expressed in proximal and distal enterocytes (figure 1C,D). Enterocytes are simple columnar epithelial cells located in the inner surface of the small and large intestines. Thus, enterocytes are directly exposed to food and foreign pathogens. Interestingly, when we examined expression profiles of another two virus receptors (ANPEP receptor for HCoV-229E virus and DPP4 receptor for MERS-CoV virus), we found that the RNA levels of these two virus entry receptors were also highly expressed in proximal or distal enterocytes (figure 1E–H), consistent with the expression profile of ACE2.

Currently, the infection routes of 2019-nCoV remain elusive. The distribution of 2019-nCoV entry receptor may determine the path of infection, and the route of infection is essential for understanding the pathogenesis, both of which are vital for infection control in hospitals and society. Based on the current findings, we proposed that (1) the incidence of diarrhoea may be underestimated in previous investigations; (2) ACE2-expressing small intestinal epithelium cells might be more vulnerable to attack by 2019-nCoV.

In this study, we displayed that ACE2 was highly expressed in the small intestine, especially in proximal and distal enterocytes. Consistently, another group has recently reported a similar expression pattern in the human digestive system.8 Interestingly, other virus receptors like DPP4 displayed similar expression patterns as ACE2 in the small intestine. DPP4 is a known receptor for MERS-CoV through interacting with MERS-CoV spike protein. According to the recent publication, Zhou et al reported the DPP4-expressing human intestine cells were highly susceptible to MERS-CoV and supported robust viral replication,9 suggesting that the human intestinal tract may serve as an alternative infection route for MERS-CoV. In terms of the fact that most of the patients in the outbreak reported a link to a wild animal market, this observation raises an important question about whether this virus is transmitted via contaminated food when the food arrives at the small intestine.

It is known that ACE2 controls intestinal inflammation and diarrhoea. Therefore, mutual interaction between 2019-nCoV and ACE2 might disrupt the function of ACE2 and results in diarrhoea. Here, we found that the incidence of diarrhoea significantly differed in different reports. As 2019-nCoV is highly homologous to SARS-CoV and around 20%–25% of SARS patients have diarrhoea,10 it is confusing to observe the relatively low incidence (2%–3%) of diarrhoea in two cohorts from hospitals in Wuhan. The underestimation may result from that we still do not have a precise criterion for diarrhoea. The definition of diarrhoea by the WHO is having three or more loose or liquid stools per day or having more stools than a person’s health condition. To a certain extent, this criterion is subjective. Besides, we still cannot exclude the effect of the small sample size (n=6) of the Hong Kong cohort, which affects the result of statistical analysis. Emerging evidence shows that 2019-nCoV RNA can be detected in stool samples as SARS. Based on the postulation from the epidemiological features of SARS, which is transmitted through fecal-oral, 2019-nCoV might use the same path for transmission. Thus, future efforts at prevention and control must take into consideration the potential for fecal-mediated spread of this virus.

Taken together, the symptoms of diarrhoea could be underestimated. The information on discharge frequencies and the Bristol stool scale should be carefully collected. When infected patients with diarrhoea visit the gastroenterology department, it may increase the risk of infection of healthcare workers. To reduce healthcare-associated infection, clinicians should be careful when their patients complain of diarrhoea.