Intestinal mucus: the unsung hero in the battle against viral gastroenteritis

Rotavirus (RV) is a genus of multilayered non-enveloped viruses of about 100 nm diameter in the family Reoviridae [42, 43]. The triple-layered icosahedral capsid encloses a genome of 11 segments of double-stranded RNA (dsRNA), each coding for one or two viral proteins [44]. The species are based on serogroups A-J and putative species RVK and RVL are already reported [45]. The outer layer of the capsid contains VP7 and VP4 proteins that are important for viral attachment, entry, and antigenicity while also determining rotavirus serotype and strain [46]. These proteins are also called G (for glycoprotein) and P (for protease-sensitive) types, respectively [47]. Sialic acids, integrins and hsc70 have been reported as functional receptors for many RVs [48, 49]. Additionally, our lab’s work on primary porcine enterocytes co-cultured with porcine myofibroblasts infected with porcine rotavirus hinted towards the usage of a basolateral intercellular receptor [50]. There are at least 27 G types, and 37 P types of rotavirus identified so far, though only a few of them are common in humans and animals [51]. They also go under genetic reassortment and carry a zoonotic potential [52]. However, genome characterization of ancient and recent Belgian pig RVAs from our lab has indicated a different evolutionary path with human Wa-like RVAs [53], suggesting a better adaptation of pig RVAs to porcine enterocytes and less chances of spread to human populations. In humans, the diseases severity of rotavirus infections is higher than other enteric pathogens [54]. Inflammation of the stomach and intestines leading to diarrhea, vomiting, fever, and dehydration are the main clinical signs [55]. Neonates are generally less affected probably due to protection from maternal antibodies, yet infections are more severe in young children and animals [56]. Work on porcine rotavirus from our lab in Belgian pig farms has identified RVA with heterogenous VP7/VP4 genotype combinations [48], subclinical RVA [57], along with RVA and RVC [58]. A detail of common rotavirus species along with common genotypes with respect to their host is provided in Fig. 1 [59‐61].

Coronaviruses (CoV) are large, enveloped viruses of the family Coronaviridae containing positive-sense single-stranded RNA (ssRNA) enclosed within the nucleocapsid protein of around 30 kbps size [62]. They have four main structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N), arranged in a specific pattern that gives the virus its characteristic crown-shaped (corona) appearance [63]. Certain members of the Betacoronavirus genus, particularly those in lineage A, possess an additional hemagglutinin-esterase (HE) structural protein, exhibiting lectin-like activity by binding to sialic acid [64], making it crucial for studying its interactions with mucus. The S protein has two subunits: a receptor-binding domain (RBD, S1) and the membrane-fusion domain (S2) [65], making it a target of many vaccines and therapeutics against coronaviruses [66]. Receptor usage depends on virus type. For example, SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) [67] while porcine coronaviruses like transmissible gastroenteritis virus (TGEV) mainly use aminopeptidase N (APN) [68]. Another study from our lab reported a two-to-seven-fold higher infectivity of TGEV Miller in APN positive cells than in APN negative cells, but TGEV Purdue replicated better in APN negative cells [69]. The same study reported that terminal sialic acids are not determinants of TGEV infection. This shows that receptor usage of coronaviruses is highly variable. Although coronaviruses are mainly associated with respiratory infections in humans, they are known causative agents of neonatal diarrhea in many animals [70], as shown in Fig. 1. In humans, studies on severe acute respiratory syndrome-CoV-2 have also identified the presence of the virus in fecal samples, suggesting that it may be capable of causing gastrointestinal infections as well [71]. In addition, there are several other coronaviruses that have been detected during gastrointestinal infections in children, including 229E, OC43, HKU1, NL63 [72]. Moreover, research on the replication of swine acute diarrhea syndrome coronavirus in primary human cells indicates that humans may be susceptible to this virus, highlighting a potential zoonotic threat [73]. Similarly, the zoonotic nature of porcine delta coronavirus (PDCoV) has also recently demonstrated with detection in plasma samples of hospitalized children in Haiti [74]. Animal coronaviruses cause extensive necrosis of mature jejunal and ileal enterocytes within 24 h of infection, reducing enzymatic activity (mainly alkaline phosphatase and lactase) and disrupting digestion and electrolyte imbalance, leading to fluid deposition in the intestinal lumen, causing acute malabsorptive diarrhea [75]. The resulting dehydration and loss of extravascular proteins is fatal and could lead to metabolic acidosis, increased K⁺ levels, and eventually cardiac arrest [76]. Main symptoms include severe diarrhea, anorexia, emaciation, and dehydration [77]. Infection is more severe in newborn animals with mortality rates reaching 100% in the absence of lactogenic immunity [78].

Adenoviruses are non-enveloped, icosahedral viruses of the family Adenoviridae, of 70–90 nm diameter, and have a linear dsDNA genome of approximately 26–48 kbps [79]. The genome is packaged into a protein capsid that consists of three main structural components: (1) penton base, a five-fold symmetric protein structure that forms the vertex and is responsible for virus attachment to the host cell; (2) hexon, the capsid protein forming the bulk of the virus particle; (3) fiber, a trimeric protein binds to specific receptors on the host cell surface [80]. The genome is bound to histone-like proteins known as protamines [81]. They are highly regarded for their use in vector-based vaccines due to their ease of genetic manipulation, inherent stability, and wide tissue tropism [82]. Enteric adenoviruses cause gastrointestinal infections in both humans and animals. Among 111 types of human adenovirus (HAdV), enteric types like F40/41 are significant acute gastroenteritis agents in children [83]. Enterocolitis-like symptoms have been associated with different adenovirus species isolated from various domestic and wild animals as well, as shown in Fig. 1 [84, 85].

Astroviruses are small, non-enveloped viruses of the family Astroviridae containing a single-stranded RNA genome of approximately 6.8 to 7.9 kbps [86]. Common to most RNA viruses, their genomes are prone to a high mutation and recombination rate, resulting in species that can infect a wide variety of hosts [87, 88]. They are a common cause of gastroenteritis in human children and young animals all over the world [89, 90]. The symptoms include diarrhea, nausea, vomiting, abdominal cramps, low-grade fever and malaise [91]. They mainly infect the intestinal goblet cells, leading to increased mucus production and electrolyte imbalance [92]. Some common astroviruses infecting human and different animal species are described in Fig. 1 [84, 90].

There are several other viruses that can cause gastrointestinal infections in humans and animals, including:

The number of mucus-producing cells along the intestinal length can give a good indication of mucus production in a particular species, age-group or individual [141]. We demonstrated that the number of mucus-producing cells per total epithelial cells increased with pig’s age along the whole intestinal length, suggesting more mucus production per unit of area in older pigs [135]. This could provide an explanation why younger piglets are more prone to severe infections and high mortality. As the fixation process during mucus staining strongly impacts the preservation of mucus in histological sections [142], traditional PAS staining on paraffin sections and a novel method of fixing cryosections on the positively charged Blotting-Nylon 66 membranes could be used [135]. The results corresponded to each other. This shows that relatively simple staining techniques can still be efficient in providing valuable data related to virology. Furthermore, region-specific changes in the intestines can also provide significant data related to enteric viral infections. The Brunner’s glands found in the duodenum produce a large amount of mucus as observed in our study [135]. Mucus produced from these merocrine glands is more alkaline to counteract the acidic content coming from the stomach [143]. This could explain why the duodenum is relatively less infected by enteric viruses as compared to jejunum and ileum. A study using in-situ hybridization for the detection and localization of PEDV found strong signals in villus enterocytes of jejunum and ileum of all ten pigs, while duodenum was positive in only one pig [144].

Thus, quantifying intestinal mucus or mucus-producing cells with respect to the intestinal region, host species or age holds significant translational value across various medical fields. Measuring mucus levels can enhance the diagnosis and monitoring of conditions like inflammatory bowel disease (IBD) and ulcerative colitis, as variations in mucus production may indicate disease progression or treatment response [145]. Understanding individual differences in mucus production can help tailor treatments for gastrointestinal diseases, offering specific therapeutic approaches based on mucus levels [146]. Insights into mucus dynamics can guide the creation of new drugs, allowing researchers to design medications that modulate mucus production to improve patient outcomes [32, 146]. Additionally, studying mucus interactions with gut microbiome can lead to the development of probiotics or other interventions that support overall health [147]. As a protective barrier for the gut lining, mucus quantification can help researchers understand and address barrier dysfunctions in diseases, potentially preventing infections and inflammation.

Mucus composition is mostly explored by performing a proteomic profile consisting of mucins, enzymes, immunoglobulins, and non-mucin proteins including the immunomodulatory elements like antimicrobial peptides (AMPs) [148]. However, apart from proteins, intestinal mucus is a blend of lipids, electrolytes, and water [9]. In a study from our lab, a label-free proteomic analysis of the ex vivo intestinal mucus from 3-day-old and 3-week-old pigs revealed that around 2.75% proteomic profile was unique between the age groups [109]. There is a variety of non-mucin proteins that are in play during different infectious diseases. Secretory IgA can bind to certain bacteria forming immune complexes that promote phagocytosis [149]. ZG16 is known to combine with the peptidoglycans found in the gram-positive bacterial cell wall, forming large aggregates that cannot cross the mucus layer [150]. Receptor proteins like NLRP6 are activated during certain viral and gram-positive bacterial infections [151]. AMPs like defensins and cathelicidins possess antibacterial and antiviral properties that are mainly released into the intestinal lumen by the Paneth cells (PCs) [152‐154]. Thus, characterization of these molecules in mucus samples from different ages of host species can provide a better overview of the onset, progression, and severity of enteric diseases, especially in newborn humans and suckling and naïve animals.

Non-proteinaceous parameters of intestinal mucus like the yield, pH, water content, sugars, lipids, and metabolites also considerably affect the mucus composition. Mucus pH affects virus stability as evidenced by the high antiviral activity of cervicovaginal mucus against human immunodeficiency virus 1 (HIV-1) at acidic pH [155]. Lipid profile variation in different sputum samples affects influenza and rhinovirus infection [156]. Mucus electrolytes alter mucus properties as divalent cations (Mg²⁺, Ca²⁺) are known to collapse the mucus gel structure while monovalent cations (Na⁺, K⁺) are known to reduce mucus viscosity [32], which in turn can affect virus tropism and infection. Thus, these non-proteinaceous intestinal mucosal components offer much room for future investigation in relation to viral gastroenteritis. The absolute mucus yield can be measured as the weight of mucus in grams per meter of intestinal length, whereas the relative yield (%) can be calculated by dividing the mucus weight by total tissue weight before mucus collection for a specific length [157]. The pH measurements can be done on fresh mucus samples within 1 h of collection using a micro-electrode pH meter, while the water content can be analyzed by freeze-drying the mucus samples and comparing its weight with fresh samples for similar quantities [158]. Glycomic, lipidomic and metabolomic analyses can be performed like proteomic analysis by using mass spectrometry techniques like LC-MS or GC-MS [158]. Hence, it can be concluded that the mucus compositional changes occurring with respect to age and disease status of the host is a key research area during infection diseases and host-pathogen interaction studies.

Intestinal mucosa serves as the frontline in between invading pathogens and the intestinal immune system. Intestinal mucosal immunity involves a complex interplay of cells like PCs, enterocytes, Goblet cells (GCs), innate lymphoid cells, intraepithelial lymphocytes, and lymphoid systems such as the ileal Peyer’s patches, which together form the gut’s innate immune system and regulate adaptive responses upon interaction with microbes [159]. Immune cells like dendritic cells (DCs) and neutrophils are in direct contact with the intestinal lumen, while specialized cells like γδ T receptor-expressing intraepithelial lymphocytes do not have access to the intestinal lumen but maintain homeostasis by producing AMPs and limiting pathogen invasion after barrier breaches [160]. CXCR1 + chemokine receptor 1 (CXC3CR1) expressing DCs can intercalate between epithelial cells to uptake antigens from luminal mucus, while chemosensory tuft cells, crucial for helminth infections, and M cells, which also uptake luminal antigens, may also be present in the intestinal mucus [161, 162]. Intestinal epithelial cells and local innate immunity form a barrier to pathogens via Toll-like receptors (TLRs), while adaptive immunity is established through antigen recognition by antigen-presenting cells (APCs) in Peyer’s patches and mesenteric lymph nodes [163]. TLR1, TLR2, TLR4, TLR6, and TLR10 are specifically related to the recognition of viral proteins [164]. The T and B lymphocytes of the gut-associated lymphoid tissues (GALTs) are involved in IgA responses, further strengthening the adaptive immune response [165]. IgA is known to bind bacteria or viruses and slow down their diffusion and bacterial motility in the mucus [29]. Antimicrobial components such as lysozymes, defensins, and DMBT1 are also present in small intestinal mucus produced by PCs located in the intestinal crypts [30]. In humans, intestinal epithelial cells produce REG3A, which has bactericidal properties, typically against Gram-negative bacteria [31]. Additionally, GCs secrete immune modulators such as CLCA1, FCGBP, AGR2, ZG16, KLK1, and TFF3, though their specific functions remain largely unknown [9]. Hence, significant gaps remain in our understanding of how these immunological factors of the intestinal mucosa interact with enteric viruses, offering abundant opportunities for future research.

Among the various constituents of the mucosal system, mucins stand out as the primary functional elements of intestinal mucus, as reported in various studies [166‐169]. Within the realm of mucins, it is the mucin glycans that present the most compelling and relatively unexplored area of study in relation to enteric viral infections. TGEV has an affinity for different sialic acids, such as Neu5Gc, Neu5Ac, and Neu5,9Ac2, which also act as receptors or receptor binding co-factors for other coronaviruses [26, 27]. MUC2 is rich in these sialic acids and may play a role in determining virus pathogenicity [170, 171]. The sialic acids are known to bind and trap respiratory viruses aiding in their clearance via mucociliary transport of the respiratory system [172]. Other viruses like rotavirus, influenza B and C viruses, respirovirus and certain parvoviruses are also known to use sialic acids as receptors through their sialolectins [173], where mucin glycans might be of interest as they may provide decoy receptors for these viruses. The glycan-dependent inhibitory effect of mucins against coronavirus infection of live cells is also reported [138]. Thus, performing a glycan analysis and subsequently using them in virology assays can provide a good idea about their potential protective role against enteric viruses. Commercially available mucins like porcine gastric mucin are available to carry out these studies [174]. However, the ex vivo mucus extracted directly from the host species can also be purified to extract mucins. Addition of guanidine hydrochloride or urea in the extracted mucus can weaken the hydrophobic bonds between mucins and other mucosal components easing subsequent purification [175, 176]. Using density gradients like cesium chloride or rate zonal centrifugation, further impurities like cellular and fecal debris can be removed [177‐179]. Chromatographic separation like size exclusion chromatography or gel filtration can be employed to isolate relatively larger sized mucins from other mucosal components [180]. Mucin fractions isolated under this fashion can be monitored by UV absorbance at 215 nm and visualized by mucin-specific staining like PAS [181]. These isolated mucins can then be desalted, concentrated, and lyophilized for storage, while mass spectrometry can be used to assess their composition [182]. From these purified mucins, O-glycans can be extracted using non-reductive alkaline β-elimination ammonolysis, which conserves the structure of glycans [183]. From these extracted glycans, core glycan structures can be isolated using partial acid hydrolysis and analyzed by mass spectrometry [184]. These glycans can be incorporated into virology experiments like virus plaque-blocking assays. Concludingly, mucin glycans isolated from different host species can give an idea of the glycomic changes that might play a key role in protecting against enteric viral infections.

Although the idea of using purified mucins extracted from intestinal mucus as a therapeutic tool to combat enteric infections, particularly in newborn humans and animals, may seem far-fetched, there is promising research in this area. However, the use of synthetic mucins as therapeutics is a viable possibility. For instance, Nason et al. have developed special HEK293 cells that are ‘glyco-engineered’ to express representative short tandem repeats (∼ 200 amino acids) of human O-glycodomains [185]. This approach could potentially be adapted to enhance the therapeutic application of mucins. With tunable structures and patterns of O-glycans, that are representative of actual mucin repertoire of the body, large quantities of specific mucin glycodomains can be sustainably manufactured from natural mucin polymers and their therapeutic potential as dietary pre-biotic material can be assessed. These mucins can either offer their protective ability by directly blocking the invading viruses or bacteria, or by stimulating the natural commensals of the body in maintaining gut health.

Recombinant N-glycoproteins such as IgG and erythropoietin have already been investigated and characterized for their use as biopharmaceutical products [186‐188]. Wohlschlager et al.. also described multiple N-glycans and up to 26 O-glycans attached to the chimeric TNF-α receptor fusion protein with a therapeutic potential [187]. Thus, similar quality control studies can be utilized for mucin O-glycan standardization. However, an important consideration here is that the therapeutic effect of mucus can be specific to host species, and the pathogens adapted to that species. Bovine mucins inhibited the infection of bovine-originated human coronavirus OC43 in susceptible cells in a concentration- and glycan-dependent manner but could not inhibit Mouse Hepatitis Virus (MHV), a mouse coronavirus [138]. This suggests that mucin biophysics and biochemistry is dependent on small conformational and glycoform changes that may be related to species genetics, metabolism, or the environment [12, 189, 190]. Hence, isolation of species-specific mucins and mucin glycans should be considered. Regardless, it is ambitious but an achievable idea that offers huge research potential. Apart from mucins, other antimicrobial components of mucus like AMPs or lipids also hold potential for synthetical engineering and be used as therapeutics. Moreover, with the progress in fecal microbiota transplantation and its success in treating gastrointestinal disorders [191], there is a potential for intestinal mucus transplants from healthy individuals to those with viral gastroenteritis, which should be explored.

Mucus composition varies across different physiological systems with a species [192]. This is another potentially important area to study viral pathogenesis and tropism along with changes or shifts occurring in them. For example, in 1995, key deletions in TGEV at nucleotides 621–681 gave rise to porcine respiratory coronavirus which mainly causes respiratory symptoms [193]. The variations in mucus structure and mucosal components between the two systems may have driven TGEV to mutate, enabling it to shift its virus tropism to PRCV. Previously in our lab, we analyzed the interaction of pseudorabies virus (PRV, diameter = ∼ 250 nm) with porcine respiratory mucus using SPT [194]. Only PEGylated particles displayed chaotic Brownian movement in respiratory mucus, while negatively and positively charged particles, and negatively charged PRV were restricted. However, our study in the interaction of TGEV and similar-sized control particles showed that both the carboxylated (-) and PEGylated (=) particles showed chaotic Brownian movement in the intestinal mucus of 3-day and 3-week-old pigs [109]. This is likely due to the different composition of respiratory and intestinal mucus. Respiratory mucus contains mainly the MUC1/MUC4/MUC5AB-C complex, while MUC2 is the primary gel-forming mucin of small intestinal mucus [195]. Furthermore, the diversity of the host’s immune components and microflora in mucus from both systems is critical in combating the invading pathogens [196]. Another study from our lab showed that PRV showed more penetration through porcine respiratory mucus at 4 °C compared to 37 °C after 30 min [197]. However, our study on TGEV in the case of 3-week intestinal mucus showed that its movement in both short- and long-duration diffusion assays showed was hindered at lower temperatures [109]. This indicates that intestinal and respiratory mucus behave differently when temperature is decreased, further highlighting the compositional differences between the two. Thus, similar studies can be designed for PRCV in terms of respiratory mucus and cross examining of both viruses in both types of mucus systems. The interaction of PRCV with the respiratory mucus along with availability of a different repertoire of available mucin glycans can provide a new angle on the shift in tissue tropism. Combining the analysis of physiochemical properties of porcine respiratory mucus, particle-like behavior of PRCV in respiratory mucus and assessing its blocking activity offers a lot of opportunities for further research. In Fig. 3, a schematic outline of the key compositional differences between mucus from the respiratory and gastrointestinal systems is presented to understand and aid in designing cross-system mucus studies toward unravelling the TGEV/PRCV tissue tropism.