Activation of the enteric nervous system

In addition to epithelial and immune cells, the enteric nervous system is involved in enteric virus-associated diarrhea development. Rotavirus infection primarily activates the secretion of serotonin from enterochromaffin cells, a subset of enteroendocrine cells [11]. This process is largely mediated by the viral nonstructural protein 4 (NSP4), which has been identified as a viroporin, which we will discuss below, and viral infection induced extracellular purinergic signaling [11]. The released serotonin acts on the serotonin receptors, which are key players in the gastrointestinal system, leading to increased intestinal motility and enhanced fluid secretion [11]. Studies have shown that blocking the serotonin receptors with small-molecule antagonists such as Ondansetron significantly reduces the severity and duration of diarrhea in infected mice, particularly in mouse pups more susceptible to rotavirus [11]. Moreover, serotonin receptors blockade also appears to reduce overall viral shedding and improves weight gain during infection, suggesting a direct link between serotonin signaling and viral replication [11]. The role of the enteric nervous system extends beyond just motility and fluid secretion; it also influences the viral impacts on electrolyte balance and intestinal inflammatory responses, further highlighting its critical role in the pathophysiology of rotavirus-induced diarrhea and presenting potential new avenues for therapeutic intervention. In a similar manner, enteric adenovirus, through its short fiber knob and hexon proteins, engages with enterochromaffin cells and stimulates serotonin release [12].

Activities of viroporins

Viroporins are virally encoded proteins that oligomerize in the host membranes and form hydrophilic pores [13]. The mechanisms of diarrhea induced by the viroporin activities of rotavirus NSP4 and rhesus calicivirus Tulane virus NS1-2 highlight yet another shared viral strategy involving the manipulation of cellular calcium signaling to facilitate viral replication and induce gastrointestinal symptoms. As a viroporin localized to the endoplasmic reticulum (ER), NSP4 is the first viral enterotoxin discovered. It interacts with the stromal interaction molecule 1, depletes ER calcium stores, and triggers the activation of plasma membrane calcium influx channels [13]. Furthermore, rotavirus NSP4-induced calcium dysregulation is necessary for activation of purinergic intercellular calcium waves [14]. The resultant elevation in cytoplasmic calcium levels is crucial for viral replication and contributes to diarrhea by disturbing cellular functions and promoting secretory processes in intestinal cells. Similar to rotavirus NSP4, Tulane virus NS1-2 protein also functions as a viroporin within the ER, enhancing intracellular calcium signaling by facilitating the release of calcium from ER stores [15]. The NS1-2 protein specifically activates this process through its viroporin domain, which is essential for the effective replication of the viruses [15]. Increased cytoplasmic calcium, a direct outcome from NS1-2 activity, supports viral replication dynamics and is linked to the pathogenesis of diarrhea through disruption of cellular and systemic calcium balance. Three other examples of viroporins are Ebola virus delta peptide [16], astrovirus XP protein [17], and poliovirus 2B protein [18]. This highly convergent mechanism of diarrhea induction underscores the critical role of viroporins in viral life cycles and their impact on host gastrointestinal physiology. By inducing calcium imbalance, both NSP4 and NS1-2 not only ensure an environment conducive to viral replication but also contribute to the diarrheal output seen in infections, making these viroporin functions potential targets for therapeutic interventions aimed at mitigating disease symptoms and curtailing viral spread.

Importantly, SARS-CoV-2, a noncanonical enteric virus, also causes gastrointestinal symptoms. One potential mechanism is through an enterotoxin-like spike protein [19]. In a loop injection model, direct administration of SARS-CoV-2 spike protein induces intestinal lumen fluid accumulation, inflammation, mucosal histoarchitecture disruption, and altered innate immunity activation [19]. However, this has not been interrogated in the context of live virus infection nor the new variants and the underlying mechanisms are unclear.

Induction of diarrhea by nonreplicating viral particles

There is one intriguing report that after psoralen UV treatment, transcription- and replication-defective rotaviruses can still cause diarrhea in suckling mouse pups [20]. This finding challenges the paradigm that diarrhea results solely from rotavirus replication-induced cell destruction and/or NSP4, which is only produced from cells with actively replicating viruses, highlighting a potentially alternative mechanism of viral attachment and entry into intestinal cells in diarrheal pathogenesis. Rotavirus-specific IgA response in the gut was not observed in mice receiving the oral inoculation of inactivated rotavirus [20], suggesting that if immunopathology is driving the diarrheal disease, it is more likely on the innate immune arm of the host response. These findings open new avenues for understanding the pathogenesis of rotavirus-induced diarrhea and suggest potential targets for therapeutic intervention.