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SARS-CoV-2 infection of the intestinal cells at the apical side damages the integrity of the intestinal epithelial barrier

The interaction between the gastrointestinal (GI) tract and pathogens that disrupt the complex mucosal barrier is critically important in the pathogenesis of viral infections. The GI symptoms, like abdominal pain, nausea, vomiting, and diarrhea are among the most commonly reported extrapulmonary clinical features of COVID-19. In this study, the authors from France investigated the possibility that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the intestinal epithelium as an alternative route of infection. They also examined the susceptibility of intestinal cells to SARS-CoV-2 infection via the apical and/or basal side.

The intestinal epithelium is composed of a monolayer of self-renewing epithelial cells linked together. The structural tissue integrity is preserved through intercellular junctions involving tight junctions, adherent junctions, desmosomes, and gap junctions.

The epithelial cells layer is polarized and organized into an apical domain that faces the lumen of the intestine and a basolateral domain that is divided into a basal domain facing the basal membrane and a lateral domain facing the neighboring cells. The polarity of the epithelial cells allows a balanced communication between the intestinal lumen and the tissues and protects against GI pathogens.

Two host-cell factors are important for SARS-CoV-2 viral entry into many cell types: angiotensin-converting enzyme 2 (ACE2), which is bound by spike (S) protein, and transmembrane serine protease 2 (TMPRSS2), which cleaves S protein, allowing this binding to take place. The SARS-CoV-2 enters enterocytes by binding to ACE2 and TMPRSS2.

Patients who developed a long COVID syndrome often test negative for SARS-CoV-2, but numerous studies reported the persistence of viral RNA and/or antigen(s) in specimens from the GI tract. The SARS-CoV-2 RNA and specific viral antigens were found for up to 180 days in the GI of COVID-19 convalescents who did not develop long COVID syndrome. But, SARS-CoV-2 was detected in the stools of 12.7% of individuals diagnosed with long COVID 120 days after the infection whereas 3.8% of them still excreted the virus 210 days after the infection. A recent study reported that SARS-CoV-2 specific antigens, spike (S), and nucleocapsid (N) proteins were detected in the appendix sample 426 days after the infection. Accordingly, the GI tract is considered a potential viral reservoir for SARS-CoV-2.

Previous data suggest that SARS-CoV-2 initiates GI pathology from the luminal side. The infection of the enterocytes rapidly induces T-cell infiltration in the intestinal epithelium and lamina propria, whereas, infection-induced destruction of the epithelium leads to the transfer of the intestinal microbiota to the lamina propria and then systemically (Frontiers in Immunology 2022; 13: 899559)

Original illustration from the Frontiers in Immunology 2022; 13: 899559.

About the study

To investigate the possibility that the intestinal epithelium serves as an alternative infection route, the researchers employed a model of polarized intestinal cell monolayers inoculated with SARS-CoV-2. They used the human colon adenocarcinoma Caco-2 cell line, susceptible to SARS-CoV-2 infection and intense viral replication. After 21 days of culture, Caco-2 cells adopted an adherent polarized cell architecture and formed an epithelial monolayer resembling an epithelial permeability barrier. However, the authors stated that Caco-2 cell culture monolayer systems are oversimplified and fail to replicate numerous cellular components and complex intestinal structures. Particularly, they lack the mucin microenvironment, a typical characteristic of the intestinal barrier that plays a major role in the dynamics of viral infections, including SARS-CoV-2. Therefore, they used the HT29 human tumor epithelial cell line of intestinal origin, which produces mucin, to mimic intestinal goblet cells.

The permeability barrier’s integrity was assessed by measuring the transepithelial electronic resistance (TEER). A stable TEER of ~500–700 Ω.cm2 was considered a strong indicator of the integrity of the epithelial permeability barrier.

The Caco-2 and HT29 cells were co-cultured to form a single monolayer barrier that closely resembles the physiological conditions encountered in the mucosal epithelial layer. The mucin produced by the HT29 cells is expected to protect the Caco-2 cells, similar to the protection of enterocytes in the intestinal mucosa. The Caco-2 and HT29 cells were infected with an inoculum of SARS-CoV-2 either on the apical (upper chamber) or basolateral (lower chamber) sides. The TEER was measured 4, 8, 16, 24, and 48 hours after the infection. SARS-CoV-2 RNA was detected by reverse transcription polymerase chain reaction (rt-PCR). Tissue culture infectious dose 50 (TCID50) was used to evaluate viral release.


24 hours after SARS-CoV-2 inoculation, TEER values decreased, with the mean TEER value of 443 Ω.cm2 after apical and 556 Ω.cm2 after basolateral inoculation. 

Confocal immunofluorescence, used to examine the expression of intercellular adhesion proteins in Caco-2 cell monolayers, demonstrated that high levels of E-cadherin (E-cad) expression decreased significantly 24 hours after SARS-CoV-2 inoculation via the apical side. In contrast, after SARS-CoV-2 inoculation via the basolateral side, the expression of E-cad protein was almost similar to that observed in virus-free Caco-2 cell monolayers, indicating that integrity of the intercellular junctions was preserved. Decreased E-cad expression after SARS-CoV-2 inoculation via the apical side correlated with significantly reduced TEER. These findings were almost undetectable when SARS-CoV-2 was inoculated at the basolateral side.

After infection at the apical side of the monolayer, the Ct values (results of qRT-PCR were expressed as Ct) indicated the presence of the virus in samples from the upper chamber. The Ct values from the lower chamber were negative during the first 16 hours after the infection, but after 24 hours, they indicated the presence of the virus and damage to intercellular junctions. 

After infection at the basolateral side of the monolayer, the Ct values showed the presence of the virus in the lower chamber, but not in the upper chamber, suggesting that the virus was kept in the lower chamber due to the maintained integrity of the epithelial barrier. 

48 hours after infection at the apical side, the assay that evaluates viral release by TCID50 revealed viral particles in the upper and lower chambers. Higher TCID50 titer in the samples collected from the apical side after apical inoculation indicated de novo synthesis of virions. After the infection at the basolateral side, no viral particles were detected in the upper chamber, showing that apical inoculation was more effective in establishing a productive viral infection than basolateral inoculation.


This study has shown that SARS-CoV-2 infection of the intestinal cells at the apical side resulted in severe damage to the integrity of the intestinal epithelial barrier. According to the authors, although they cannot rule out transcytosis events, these findings indicate a preferential disruption of tight junctions, damage to monolayer barrier integrity, and paracellular trafficking of the virus.

This suggests that the virus present in the intestinal lumen in individuals infected with SARS-CoV-2 could damage the intestinal epithelial barrier and enter the blood vessels to spread to various organs.


This study has been published on a preprint server and is currently being peer-reviewed.


Journal Reference

Garrec G, Arrindell J, Andrieu J et al. Preferential apical infection of intestinal cell monolayers by SARS-CoV-2 is associated with damage to cellular barrier integrity: Implications for the physiopathology of COVID-19. bioRxiv preprint.



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