key: cord-0965622-9rg9xe57
authors: Ding, Siyuan; Liang, T. Jake
title: Is SARS-CoV-2 Also an Enteric Pathogen with Potential Fecal-Oral Transmission: A COVID-19 Virological and Clinical Review
date: 2020-04-27
journal: Gastroenterology
DOI: 10.1053/j.gastro.2020.04.052
sha: 33c44fcceb71359a929f20fc66885e92a09c8a92
doc_id: 965622
cord_uid: 9rg9xe57

Abstract In as short as 3 months, COVID-19 has spread and ravaged the world in an unprecedented speed in modern history rivaling the 1918 flu pandemic. SARS-CoV-2, the culprit virus, is highly contagious and stable in the environment and predominantly transmits among humans via the respiratory route. Accumulating evidence suggest that this virus, like many of its related viruses, may also be an enteric virus that can spread via the fecal-oral route. Such a hypothesis would also contribute to the rapidity and proliferation of this pandemic. Here we briefly summarize what is known about this family of viruses and literature basis of the hypothesis that SARS-CoV-2 is capable of infecting the gastrointestinal tract and shedding in the environment for potential human-to-human transmission.

Coronaviruses (CoVs) are ubiquitous in nature and infect a wide range of animals, causing diseases involving the respiratory, gastrointestinal (GI), and neurological systems 1 has emerged as a world pandemic. Globally SARS-CoV-2 has infected 2,667,532 people, of which 850,116 cases are identified in the United States alone. Currently, no clinically approved specific antivirals, other therapeutic remedies, or vaccines are available for this disease. While spread of the virus among humans is predominantly through respiratory droplets, questions remain regarding other potential modes of transmission that may contribute to the initial crossspecies infection, a large number asymptomatic cases, and the rapid and unusual pattern of dissemination across the globe. In this review, we provide a summary of the molecular biology of the virus, evidence for its infection of cells within the gastrointestinal and hepatotropic/biliary tracts, and implications for potential fecal-oral transmission of the virus.

For more extensive review of the virus and its associated diseases, we refer to other review articles and brief summaries [5] [6] [7] [8] [9] .

CoVs belong to the Coronaviridae family within the Nidovirales order. They are enveloped, nonsegmented, positive-sense RNA viruses with a large genome of approximately 30 kb. Fig. 1 illustrates the schematic replication cycle of the virus. The initial attachment of the CoV to the host cell is mediated by interactions between the spike glycoprotein (S) and its cognate receptor. This molecular interaction is a major determinant of species, tissue, and cell tropism of a CoV. Many CoVs utilize cell-surface peptidases as their receptors, but the peptidase activity seems to be dispensable for viral entry 10 . Many alphacoronaviruses use aminopeptidase N (ANPEP) 11, 12 . In the case of SARS-CoV and SARS-CoV-2, angiotensin I converting enzyme 2 (ACE2) mediates entry into host cells [13] [14] [15] , whereas dipeptidyl-peptidase 4 (DPP4) is the receptor for MERS-CoV 16 . Of note, ACE2 is a X-linked gene and have sex-specific expression profiles 17 that may contribute to the observed more severe clinical manifestations in males over females with COVID-19 18 . Smokers and individuals suffering from chronic obstructive pulmonary disease have higher ACE2 expression level 19 . Innate immune signaling such as interferon (IFN) also seems to regulate ACE2 levels and thus susceptibility to SARS-CoV-2 infection 20 . In the context   of the GI tract, patients with enteric virus infections and other inflammatory conditions may have a different cytokine profile and thus distinct ACE2 levels in the gut. In addition, genetic polymorphisms in the ACE2 gene have been associated with diabetes and hypertension 21, 22 .

Whether they are linked to clinical outcomes in COVID-19 patients remains to be tested and may shed light on the role of genetic predisposition to more severe diseases.

Interestingly, these viral receptors have been the targets of drug development for cardiac disease, hypertension and diabetes, with ACE inhibitors blocking the renin-angiotensinaldosterone system and gliptins inhibiting the DPP4 action to improve glucose control. While the enzymatic actions of these peptidases are dispensable for viral infection, these inhibitors can result in the upregulation of the protein, offering an intriguing hypothesis that patients with hypertension and diabetes are often on these drugs and thus may be more susceptible to more severe COVID-19 disease 4 .

Multiple Cryo-EM structures of the recombinant S receptor binding domain and ACE2 complex have already been solved with an unprecedented speed [23] [24] [25] [26] . These data indicate that the receptor-binding domain of S binds tightly to human and bat ACE2, suggestive of a zoonotic origin. A recent study demonstrated that the virus can indeed be transmitted to cats (including a recent news of transmission to tigers in the Bronx Zoo) and ferrets, but not dogs, chickens or pigs 24 , although porcine ACE2 mediates viral entry in cell culture 14 . This broad species-tropism raises concerns of potential transmission from domestic pets to humans and vice versa. From a structural and immunogen design perspective, more information is needed regarding the native S protein trimeric state on virions, how the trimer interacts with ACE2, and how such interaction is disrupted by neutralizing antibodies. A recent publication on the crystal structure of an antibody in complex with the receptor-binding domain of the SARS-CoV-2 S protein provides important molecular insight into antibody recognition of the virus and a potential strategy for vaccine development 27 . Besides ACE2, other potential entry factors such as CD147 28 and integrins 29 are currently under investigation.

Following receptor engagement, SARS-CoV-2 gains access into the host cell. Like other human CoVs, this process is generally accomplished by acid-dependent proteolytic cleavage of S protein proteases such as cathepsins that exposes the fusion domain of the S protein in the endosome 30 or by transmembrane serine protease 2 (TMPRSS2) at the plasma membrane 31, 32 .

For MERS-CoV, furin-mediated cleavage and fusion also occurs during virus entry 33 , and may be relevant for SARS-CoV-2 34 . This step takes place prior to fusion of the viral and cellular membranes and is also a key determinant of tissue and species tropism of the virus 35 . This aciddependent process may explain the proposed efficacy of chloroquine or hydroxychloroquine, as lysosomotropic agent, in the treatment of COVID-19 36 (Fig. 1 ). Recent publications suggest that similar to SARS-CoV, trypsin and TMPRSS2 also prime SARS-CoV-2 S protein for efficient infection 15, 37 . Overexpression of TMPRSS2 in African Green Monkey Vero-E6 cells significantly enhanced SARS-CoV-2 infectivity 38 . Serine protease inhibitor camostat blocked SARS-CoV-2 entry into host cells in a dose-dependent manner 15 , making it and other similar inhibitors such as nafamostat 39 and Pharos compounds (CHEMBL1229259) candidate small-molecule inhibitors in the treatment of COVID-19 patients (Fig. 1 ).

Besides the respiratory tract including oral mucosa 40 , the GI tract, in particular the small intestine, has high expression levels of ACE2 and TMPRSS2 in both humans 41 and mice 42 .

Multiple datasets of single-cell RNA-seq analysis indicated that mature absorptive enterocytes from ileum and colon have high ACE2 mRNA expression levels [43] [44] [45] . ACE2 protein levels have been validated by immunostaining in human small intestinal epithelium 46 . On the luminal surface of intestinal epithelial cells, ACE2 associates with the neutral amino acid transporter B0AT1 and regulates intestinal microflora 47, 48 . Thus SARS-CoV-2 infection of GI tract, by altering the levels of ACE2 at the brush border, may cause microbial dysbiosis and inflammation. In addition, high ACE2 expression is also evident in cholangiocytes and to a less extent, hepatocytes, and suggests possible hepatobiliary infection by SARS-CoV-2 49 .

As major human pathogens of medical significance, CoVs cause a variety of diseases in animals 50 . For instance, the prototypic mouse hepatitis virus (MHV) infects the lung and spreads systemically to the GI tract, liver and brain, causing gastroenteritis, hepatitis and encephalitis.

Transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) cause severe gastroenteritis in young piglets, leading to significant morbidity and mortality. Avian infectious bronchitis virus (IBV) is a major cause of economic loss within the poultry industry.

Bat CoV infects the GI and respiratory tracts of the bats without apparent diseases. Feline enteric CoV causes a mild or asymptomatic infection in domestic cats. It is clear from the names of these animal CoVs that they are entero-pathogens and the primary symptoms point to an intestinal tropism. Figure 2 illustrates the species and tissue tropisms of the common human and animal CoVs and diseases they are known to cause.

The receptors of the human pathogens, HCoV-229E, SARS-CoV, and MERS-CoV, are ANPEP (also known as CD13), ACE2, and DPP4 (also known as CD26), respectively, all brush-border enzymes highly expressed on the apical surface of mature enterocytes 51 

Similar to SARS-CoV and MERS-CoV, there is mounting evidence that SARS-CoV-2 infection also involves the GI tract. In early reports from the city of Wuhan, 2-10% of patients with COVID-19

had GI symptoms such as abdominal pain, diarrhea, nausea, or vomiting [66] [67] [68] [69] [70] . A recent metaanalysis of >4,000 East Asian patients with COVID-19 describes up to 20% had GI symptoms and viral RNA was detected in the stool of almost 50% of patients 71 . Notable GI symptoms had also been reported in a several other studies and meta-analysis, and can either precede or follow respiratory symptoms 72, 73 . In another cohort study that included more than 200 individuals, digestive symptoms were observed in 50.5% of patients and those with GI symptoms took longer to be discharged from the hospital 74 .

In addition to clinical symptoms, SARS-CoV-2 RNA was detected in the endoscopic specimens of the esophagus, stomach, duodenum and rectum from several patients 72 . Substantial amounts of SARS-CoV-2 RNA have been consistently detected in the stool specimens from COVID-19 patients 71 . The first reported case of COVID-19 in the United States experienced diarrhea and tested positive for viral RNA in his feces but not serum 75 . These results were subsequently confirmed by other studies. In some cases, by day 5 of admission, more anal swabs tested positive for viral RNA than oral swabs 76 . Stool samples from patients tested positive somewhere between 36% and 53% of all confirmed cases 8, 71 . Prolonged presence of SARS-CoV-2 viral RNA was noted in fecal samples 77, 78 . The stool specimens of many patients remained positive after a negative nasopharyngeal swab test 79 . Persistent fecal viral shedding was also observed in SARS-CoV-2 infected children 80 . Although detection of high copy numbers of viral RNA in the stool does not equate to shedding of infectious viruses or transmission of the disease, these findings raise the possibility that SARS-CoV-2 may also be an enteric virus and can be transmitted via the fecal-oral route.

Vis-à-vis direct evidence of viral infection of gut tissues, SARS-CoV-2 antigen was positively stained in the intestinal epithelium of one COVID-19 patient 78 and high viral loads were observed in the intestines of infected macaques 81 . In one study, no infectious virus was successfully isolated from the feces of COVID-19 patients 82 , but the cohort size is small and it is unclear how sensitive the cell assay system is. In a recently published ferret model, fecal shedding was seen in naïve animals in direct or indirect contact with the infected host 83 .

However, respiratory transmission was not specifically blocked, making it difficult to attribute the transmission to fecal-oral route.

On the other hand, evidence for direct liver involvement by SARS-CoV-2 is less clear. Recent studies on COVID-19 patients from Asia showed the presence of liver injury, indicated by elevated aminotransferases, ranged from 15-50% of the patients 66, 67, 70 . But the elevations were mostly mild except for patients with more severe COVID-19 84 , who might be experiencing drug-induced or sepsis/shock-related liver injury. Limited post-mortem pathology of the liver of COVID-19 patients showed moderate microvesicular steatosis and mild lobular and portal activity, which are nonspecific changes 85 . In a US-based study, serum aminotransferase levels were also not significantly altered in COVID-19 patients 86 . Further studies are necessary to define whether SARS-CoV-2 is indeed a hepato-or cholangiotropic virus that can cause direct liver injury or be secreted into the bile.

Many key questions remain before we can definitively address whether SARS-CoV-2 is an enteric virus that can be transmitted via fecal-oral route. Figure 3 In basic research, much work is needed to examine the full extent of GI and liver aspects of COVID-19. Primary human intestinal epithelial cells, hepatocytes or cholangiocytes as well as donor-derived human intestinal enteroids 88 and liver organoids will be valuable models to study SARS-CoV-2 infectivity and replication. So far, there is one report of SARS-CoV-2 infection of cholangiocytes 49 Murine models would also help investigate the disease in vivo, such as potential intestinal and/or hepatic injury, effects on various digestive functions and interactions with gut and systemic immunity. Other larger animal models, such as ferrets 82, 94 , cats 93 , and macaques 81, 83, 95 , all recently shown to be infectible by SARS-CoV-2 with similar disease, will also be valuable models to study the biology of infection and testing of vaccine and drug therapies. Finally, host innate immunity to SARS-CoV-2, including IFN signaling 96 and the inflammasome related cytokines 97 , in particular those pathways at the mucosal surfaces 98, 99 , the short-and long-term adaptive immune responses against this virus, and how these antiviral activities vary in children, adults and the elderly, are largely unknown and fertile for future research.

As COVID-19 continues to have a devastating impact on countless people's lives, we do not know for certainty what the future holds. Whether the virus will persist in human populations with recurrent bouts of outbreaks, like influenza or other emerging infections, attenuate after accumulating mutations to the likes of HCoV-OC43 and HCoV-229E common cold CoVs, or disappear after the primary outbreak, like SARS-CoV, remains a vital question for the coming year. Regardless of the answer, it is indisputable that future pandemic like this one, will likely occur again. We are at the crossroads of science, medicine and societal policies; our actions and commitments will profoundly shape the future of our world. For now, lessons learned from studying this virus, its infection modes, pathogenesis and disease manifestations will not only be invaluable for developing effective vaccines and therapies against this emerging disease but also prepare our world better for future pandemics. Other potential organs of involvement include intestine, hepatobiliary system, heart, kidney or central nervous system, many of which express high levels of ACE2, the main receptor for viral entry. Whether the virus can directly infect the intestine bypassing the respirstory system is unknown. Either way, the virus may infect, replicate and shed from the enterocytes and possibly hepatocytes/cholangiocytes and be excreted as fecal materials into the environment contaminating water and food supplies. The hypothesis that virus may either be directly transmitted to other humans via fecal-oral route, or infect household pets, like cats, or wildlife first before passing to humans, remains a key question to answer.

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