Across all age groups, diarrhea is one of the top five causes of death worldwide; while in children less than 5 years old, diarrhea is the second leading cause of death. Diarrhea etiology includes bacteria, viruses, parasites, toxins, or drugs. Acute viral gastroenteritis remains a major public health concern and a common cause of morbidity and mortality worldwide. Rotaviruses, caliciviruses (particularly norovirus), astroviruses, and enteric adenoviruses are the four predominant causes of viral gastroenteritis, inducing symptoms ranging from self-limiting watery diarrhea that resolves within 7 days to severe dehydration (with complications including cerebral edema, hypovolemic shock, renal failure) and death. Two recently developed rotavirus vaccines (RotaTeq® RV5 and Rotarix® RV1) have reduced rotavirus disease in developed countries but are much less effective in low income settings (Babji and Kang, 2012; Tate et al., 2013). The recent Global Enteric Multicenter Study (GEMS) confirmed that rotavirus is the leading cause of infant diarrhea among the more than 20,000 children studied in seven sites across Asia and Africa (Kotloff et al., 2013). Although Oral Rehydration Solution (ORS) remains the only safe, effective, and low cost therapeutic option to prevent life-threatening dehydration due to acute severe diarrheal diseases (Binder et al., 2014), the GEMS study reported that each episode of severe diarrhea in children increased the risk of delayed physical and intellectual development as well as increased mortality by 8.5-fold (Kotloff et al., 2013). Therefore, there remains a great need to develop additional therapies to treat severe diarrheal diseases.

Diarrhea often involves the dysregulation of absorptive processes and/or loss of epithelial barrier integrity, resulting in nutrient malabsorption and/or activated secretion of fluid into the intestinal lumen. In addition to the contribution of the enteric nervous system to diarrhea, roles for intestinal microbiota and epithelial immunity have been described (Moens and Veldhoen, 2012). While numerous studies of bacterial pathogens have improved our understanding of the pathogenesis of gastroenteritis, other studies suggest that viral diarrhea occurs by distinctly different mechanisms (Lorrot and Vasseur, 2007). This chapter will describe the current understanding of normal human intestinal physiology and compare the molecular mechanisms that are dysregulated by enteric bacterial and viral pathogens in the various model systems studied to date.

The intestinal epithelium serves as a barrier separating luminal dietary and microbial content from the bloodstream. A single monolayer of polarized columnar epithelial cells is connected by tight junctions that segregate the epithelial apical and basolateral membranes. Tight junctions are formed and maintained by dynamic multiprotein complexes that regulate the paracellular movement of water, ions, and small molecules. Occludin and the claudin family of proteins regulate paracellular movement of ions and small molecules through selectivity based on electrical conductance, size, and charge (Anderson and Van Itallie, 2006). Of these, electrical conductance is the most variable (>fivefold difference) between the “leaky” and “tight” epithelium of the duodenum and distal colon, respectively. The expression of different claudin isoforms varies along the horizontal and vertical axes conferring intestinal segment-specific regulation of paracellular transport (Holmes et al., 2006). Movement through tight junctions is a passive process generated by chemical and osmotic gradients that result from active transcellular transport during the postprandial state. Tight junctions regulate the rate of paracellular fluid flow through the opening and closing of the claudin pores (Anderson and Van Itallie, 2006).

The intestinal epithelium is comprised of the major epithelial cell types: enterocytes, entero-endocrine, goblet, tuft and Paneth cells. The leucine-rich G-protein coupled receptor 5, Lgr5, has been established as the molecular marker for the actively dividing intestinal stem cells that give rise to all epithelial cell types (Sato et al., 2011a). In the small intestine, Lgr5+ stem cells reside at the base of the crypt, interspersed among Paneth cells, and undergo asymmetric division producing immature enterocytes which comprise the crypt transit amplifying zone. These enterocytes terminally differentiate into the other epithelial cell types, including mature enterocytes and secretory cell lineages, as they migrate towards the villus tips (Barker et al., 2008). The life cycle of the small intestinal epithelium is about 5–7 days; while Paneth and stem cells turn over approximately every 30 days. This rapid renewal is necessary to handle the harsh luminal environment, and thus the intestinal epithelium is considered the most regenerative region in the body.

In addition to promoting nutrient digestion and absorption, the intestinal epithelial barrier protects the body from luminal pathogens and their secreted products. The intestinal epithelium is exposed to the largest population of microbial species (ranging from <10⁴/mL in the small intestine to >10¹⁵/mL in the colon) and serves as the first line of defense against harmful microbial pathogens. Goblet cells constantly secrete highly glycosylated proteins, called mucins (MUC), to produce a mucus layer that serves as a physical barrier, preventing direct interaction of commensal microbes and enteric pathogens with the host epithelium. The mucus layer is a complex organization of transmembrane and gel-forming (ie, secreted) MUC molecules that develop a tightly adhered inner layer and loose outer layer, respectively (Johansson et al., 2013). The small intestine has a single, loose unattached mucus layer comprised exclusively of the gel forming MUC2 while the colon has a two-layered system in which MUC2 serves as the foundation for both layers. The mucus of the small intestine serves to provide a nearly sterile environment through hourly turnover of the mucus layer and antimicrobial peptide secretion by Paneth cells (Bevins et al., 1999). The constant turnover occurs through regulated anion secretion during digestion and between meals. The inner mucus layer of the colon is constantly regenerated and maintained by surface goblet cells. The outer layer of the colonic mucus originates from the inner layer which becomes loose and unattached allowing commensal bacteria to reside there (Johansson et al., 2013). While the intestinal mucus is the first line of defense against enteric pathogens, enteric viruses evade this protective barrier by mechanisms requiring further study. A single report suggested that small intestinal and colonic mucins (colonic mucus had stronger effect) can prevent binding and entry of rotavirus and thus viral replication (Chen et al., 1993). Further studies are required to understand the mechanisms responsible for this effect.

Comparison of the four major viral causes of gastroenteritis (ie, rotaviruses, noroviruses, astroviruses, and enteric adenoviruses) in humans reveals similar pathologic characteristics such as site of infection, mechanisms of action, and type of diarrhea. These enteric viruses invade the intestinal epithelium of the small intestine, elevate intracellular Ca²⁺ concentrations, decrease nutrient absorption, stimulate Cl⁻ secretion, and induce epithelial cell loss, which all contribute to the moderate/severe diarrhea phenotype observed in human disease (Karst et al., 2015; Moser and Schultz-Cherry, 2005; Ramig, 2004). Each type of virus infection is associated with various extraintestinal phenotypes (eg, vomiting, nausea, viremia), and some of these are reviewed in Chapters 2.6, 2.9, and 3.4. The ontogeny of viral gastroenteritis in humans has been difficult to characterize due to limited availability of patient tissue samples during the early stages of infection. Thus, much of our understanding of viral pathogenesis has come from animal models, cancer derived intestinal epithelial cell lines, and the infection of the African green monkey kidney cell line, MA104, and other cell lines. The use of multiple model systems has confounded some observations which identified altered cellular and/or molecular events as mechanisms for diarrhea (Lorrot and Vasseur, 2007). For example, some data have indicated that viral infections induce intestinal epithelial cell loss resulting in epithelial lesions and thus a decreased capacity to absorb water, ions, and nutrients due to decreased intestinal surface area (Davidson et al., 1977). However, more recent animal studies of rotavirus, norovirus and astrovirus diarrhea have suggested that diarrhea occurs in the presence of an intact intestinal epithelial barrier and prior to any evidence of epithelial loss (Karst et al., 2015; Moser and Schultz-Cherry, 2005; Ramig, 2004; Lundgren and Svensson, 2001). Furthermore, decreased activity and amount of BB digestive enzymes were also observed in the absence of cell death (Jourdan et al., 1998). Taken together, these data suggest that viral pathogenesis disrupts normal intestinal epithelial physiology that may lead to the subsequent loss of epithelial cells observed in the later stages of viral infection. For further details see Chapter 2.4.

The lack of consistent evidence that epithelial damage is responsible for malabsorption and water loss suggests that viral-induced diarrhea may also result from alterations in intestinal absorption and secretion. Transport physiology studies designed to elucidate the exchangers, symporters, and/or channels responsible for the decreased ion and nutrient absorption and increase Cl⁻ secretion associated with viral gastroenteritis are incomplete. In rotavirus diarrhea, nutrient malabsorption is correlated with decreased expression of SGLT1 while increased Cl⁻ secretion has been suggested to involve CaCCs since RV-mediated Cl⁻ secretion can still occur in the absence of functional CFTR (Halaihel et al., 2000). A recent study reported that rotavirus can inhibit NHE3 activity as early as 30 min post infection in intestinal epithelial cells, suggesting that inhibition of electroneutral Na⁺ absorption may contribute to RV diarrhea (Foulke-Abel et al., 2014). Astrovirus, enteric adenovirus, and norovirus diarrhea also present with primary malabsorptive and secretory phenotypes (Karst et al., 2015; Moser and Schultz-Cherry, 2005) and thus, additional transport studies are needed to identify the transporters involved.

Over the last two decades, considerable progress has been made to understand the molecular mechanisms responsible for normal ion and water absorption as well as for each of the previously described contributors to diarrhea. The identification and characterization of bacterial and viral enterotoxins (NSP4 for rotavirus; see Chapter 2.4) have provided valuable insights into the cellular and molecular mechanisms of diarrhea diseases. Mechanisms invoked include pathogen entry/invasion, further characterization of signal transduction pathways, identification of multiprotein regulatory complexes, predicting structure/function relationships, and protein trafficking (Hodges and Gill, 2010; Viswanathan et al., 2009). For example, several intestinal ion transporters and channels, including NHE3 and CFTR, contain PDZ [post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 (ZO-1) protein] protein–protein interacting domains at their carboxy-terminal ends. These PDZ domains directly bind regulatory proteins that increase/decrease transporter/channel activity based on the binding partner. The NHERF (Na⁺/H⁺ exchanger regulatory factor) family of multi-PDZ domain containing proteins has been shown to regulate the activity of NHE3, CFTR, and DRA (among nearly 70 other targets) by affecting their turnover number and/or trafficking to and from the enterocyte BB (Donowitz et al., 2005). Other PDZ proteins have been shown to regulate tight junction function by affecting tight and adherence junctional complex formation, thereby modulating ion and small solute permeability through the paracellular network (Van Itallie and Anderson, 2014). Studies of bacterial infection, including enterotoxigenic, enteroaggregative, enterohemorrhagic, and enteropathogenic E. coli, have demonstrated decreased tight junction function due to mislocalization or extracellular damage of tight junction proteins (Nassour and Dubreuil, 2014; Simonovic et al., 2000; Strauman et al., 2010; Tomson et al., 2004). The effects of enteric viruses on tight junction function are limited as these studies are difficult to assess in vivo. Studies in MDCK and Caco-2 cell lines have reported decreased TER and increase permeability to large cargo (20 kDa) after RV infection (Dickman et al., 2000; Tafazoli et al., 2001). Whether these effects occur in other enteric viral infections in the human intestine remains to be determined.

Since the use of multiple models to study viral pathogens has limited our understanding of human disease, the recent development of the methodologies to culture primary human differentiated epithelial cells from the recently identified Lgr5+ stem cell has reinvigorated the study of gastrointestinal physiology and pathophysiology in general (Jung et al., 2011; Sato et al., 2011b). Primary human intestinal epithelial cultures may be derived from either human crypt based stem cells (termed enteroids/colonoids) or from a single inducible pluripotent stem cell (iPSC) line (termed intestinal organoid) (Jung et al., 2011; Sato et al., 2011b; McCracken et al., 2011). These cultures are comprised of the complete complement of intestinal epithelial cells including immature enterocytes (that may be differentiated into more mature absorptive enterocytes), enteroendocrine, goblet, Paneth, tuft, and Lgr5+ stem cells. Enteroid/colonoid cultures represent the intestinal segment from which they are derived, and recent modifications generating intestinal organoid cultures have developed organoids with proximal and distal intestinal phenotypes (Watson et al., 2014). Furthermore, the human enteroid model has been shown to perform Na⁺ absorptive and Cl⁻ secretory functions and is being used to understand the pathogenesis of host–pathogen interactions (Foulke-Abel et al., 2014; Kovbasnjuk et al., 2013). Recent work by the laboratory of Mary Estes has demonstrated that intestinal organoids and enteroids may be infected by RV and that each model has the capacity to facilitate viral replication resulting in the production of infectious viral progeny (Finkbeiner et al., 2012; Saxena et al., 2015). The development of enteroids/colonoids and intestinal organoids as models to study the human intestine offer the opportunity to study the pathogenesis of enteric viral infections.