Noroviruses as a Cause of Diarrhea in Immunocompromised Pediatric Hematopoietic Stem Cell and Solid Organ Transplant Recipients.

Case reports describe significant norovirus gastroenteritis morbidity in immunocompromised patients. We evaluated norovirus pathogenesis in prospectively enrolled solid organ (SOT) and hematopoietic stem cell transplant (HSCT) patients with diarrhea who presented to Texas Children's Hospital and submitted stool for enteric testing. Noroviruses were detected by real-time reverse transcription polymerase chain reaction. Clinical outcomes of norovirus diarrhea and non-norovirus diarrhea patients, matched by transplanted organ type, were compared. Norovirus infection was identified in 25 (22%) of 116 patients, more frequently than other enteropathogens. Fifty percent of norovirus patients experienced diarrhea lasting ≥14 days, with median duration of 12.5 days (range 1-324 days); 29% developed diarrhea recurrence. Fifty-five percent of norovirus patients were hospitalized for diarrhea, with 27% requiring intensive care unit (ICU) admission. One HSCT recipient developed pneumatosis intestinalis. Three HSCT patients expired ≤6 months of norovirus diarrhea onset. Compared to non-norovirus diarrhea patients, norovirus patients experienced significantly more frequent ICU admission (27% vs. 0%, p = 0.02), greater serum creatinine rise (median 0.3 vs. 0.2 mg/dL, p = 0.01), and more weight loss (median 1.6 vs. 0.6 kg, p < 0.01). Noroviruses are an important cause of diarrhea in pediatric transplant patients and are associated with significant clinical complications.

Intestinal epithelia activate anti-viral signaling via intracellular sensing of rotavirus structural components.

Rotavirus (RV), a leading cause of severe diarrhea, primarily infects intestinal epithelial cells (IECs) causing self-limiting illness. To better understand innate immunity to RV, we sought to define the extent to which IEC activation of anti-viral responses required viral replication or could be recapitulated by inactivated RV or its components. Using model human intestinal epithelia, we observed that RV-induced activation of signaling events and gene expression typically associated with viral infection was largely mimicked by administration of ultraviolet (UV)-inactivated RV. Use of anti-interferon (IFN) neutralizing antibodies revealed that such replication-independent anti-viral gene expression required type I IFN signaling. In contrast, RV-induction of nuclear factor-κB-mediated interleukin-8 expression was dependent on viral replication. The anti-viral gene expression induced by UV-RV was not significantly recapitulated by RV RNA or RV virus-like particles although the latter could enter IEC. Together, these results suggest that RV proteins mediate viral entry into epithelial cells leading to intracellular detection of RV RNA that generates an anti-viral response.

Anti-VP6 IgG antibodies against group A and group C rotaviruses in South India.

In an epidemiological survey from South India, 936 serum samples were tested for IgG against recombinant baculovirus-expressed VP6 proteins from human group A and group C rotaviruses. The overall seroprevalence for group A was 100% and for group C was 25.32% (95% CI 22.64-28.21). The lowest seroprevalence for group C was in children aged <10 years (16.79%). An age-related rise in seroprevalence in group C, but not group A, suggests different patterns of exposure. Seroprevalence was similar in rural and urban subjects, unlike the higher prevalence in rural subjects in studies elsewhere.

Infant morbidity in an Indian slum birth cohort.

OBJECTIVE: To establish incidence rates, clinic referrals, hospitalisations, mortality rates and baseline determinants of morbidity among infants in an Indian slum. DESIGN: A community-based birth cohort with twice-weekly surveillance. SETTING: Vellore, South India. SUBJECTS: 452 newborns recruited over 18 months, followed through infancy. MAIN OUTCOME MEASURES: Incidence rates of gastrointestinal illness, respiratory illness, undifferentiated fever, other infections and non-infectious morbidity; rates of community-based diagnoses, clinic visits and hospitalisation; and rate ratios of baseline factors for morbidity. RESULTS: Infants experienced 12 episodes (95% confidence interval (CI) 11 to 13) of illness, spending about one fifth of their infancy with an illness. Respiratory and gastrointestinal symptoms were most common with incidence rates (95% CI) of 7.4 (6.9 to 7.9) and 3.6 (3.3 to 3.9) episodes per child-year. Factors independently associated with a higher incidence of respiratory and gastrointestinal illness were age (3-5 months), male sex, cold/wet season and household involved in beedi work. The rate (95% CI) of hospitalisation, mainly for respiratory and gastrointestinal illness, was 0.28 (0.22 to 0.35) per child-year. CONCLUSIONS: The morbidity burden due to respiratory and gastrointestinal illness is high in a South Indian urban slum, with children ill for approximately one fifth of infancy, mainly with respiratory and gastrointestinal illnesses. The risk factors identified were younger age, male sex, cold/wet season and household involvement in beedi work.

Rotavirus proteins: structure and assembly.

Rotavirus is a major pathogen of infantile gastroenteritis. It is a large and complex virus with a multilayered capsid organization that integrates the determinants of host specificity, cell entry, and the enzymatic functions necessary for endogenous transcription of the genome that consists of 11 dsRNA segments. These segments encode six structural and six nonstructural proteins. In the last few years, there has been substantial progress in our understanding of both the structural and functional aspects of a variety of molecular processes involved in the replication of this virus. Studies leading to this progress using of a variety of structural and biochemical techniques including the recent application of RNA interference technology have uncovered several unique and intriguing features related to viral morphogenesis. This review focuses on our current understanding of the structural basis of the molecular processes that govern the replication of rotavirus.

Rotavirus NSP4 induces a novel vesicular compartment regulated by calcium and associated with viroplasms.

Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.

Emerging themes in rotavirus cell entry, genome organization, transcription and replication.

Rotaviruses, causative agents of gastroenteritis in young animals and humans, are large icosahedral viruses with a complex architecture. The double-stranded RNA (dsRNA) genome composed of 11 segments, which codes for 6 structural and 6 non-structural proteins, is enclosed within three concentric capsid layers. In addition to facilitating host-specific interactions, the design of the capsid architecture in rotaviruses as in other dsRNA viruses should also be conducive to the requirement of transcribing the enclosed genome segments repeatedly and simultaneously within the capsid interior. Several non-structural proteins facilitate the subsequent processes of genome replication and packaging. Electron cryomicroscopy studies of intact virions, recombinant virus-like particles, functional complexes, together with recent X-ray crystallographic studies on rotavirus proteins have provided structural insights into the capsid architecture, genome organization, antibody interaction, cell entry, trypsin-enhanced infectivity, endogenous transcription and replication. These studies underscore contrasting features and unifying themes between rotavirus and other dsRNA viruses.

Intussusception in southern Indian children: lack of association with diarrheal disease and oral polio vaccine immunization.

BACKGROUND: Intussusception is the most common cause of intestinal obstruction in young children and has been reported as a complication of a recently withdrawn tetravalent reassortant rotavirus vaccine. METHODS: We studied the history, clinical presentation, management and outcome of intussusception presenting to a tertiary care hospital in southern India over a 10-year period, in order to assess potential association with diarrheal disease and immunization. RESULTS: Data from 137 index cases and 280 control subjects indicated that the risk of diarrheal disease or oral polio vaccine administration in the month prior to presentation was similar in the index cases and controls. Mean time to presentation to hospital after developing symptoms was 1.8 days, and 77.3% of patients required surgery, with 47.4% undergoing intestinal resection. Mortality was 0.006%. CONCLUSIONS: No association could be demonstrated between gastroenteritis or oral poliovirus vaccine immunization and intussusception in southern Indian children. These children presented later and required operative intervention more frequently than has been reported in other studies, but had a good outcome with low mortality.

Cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (NSP4-EGFP) expressing cells loaded with Fura-2.

The green fluorescent protein (GFP) and its analogs are standard markers of protein expression and intracellular localization of proteins. The fluorescent properties of GFP complicate accurate measurement of intracellular calcium using calcium sensitive fluorophores, which show a great degree of spectral overlap with GFP, or their K(d) values are too high for accurate measurement of subtle changes in cytoplasmic calcium concentrations. Here we describe a simple modification of the standard microscope-based Fura-2 calcium-imaging technique which permits the quantitative measurement of intracellular calcium levels in cells expressing enhanced green fluorescent protein (EGFP) fusion proteins. Longpass emission filtering of the Fura-2 signal in cells expressing an EGFP fusion protein is sufficient to eliminate the EGFP-Fura-2 emission spectra overlap and allows quantitative calibration of intracellular calcium. To validate this technique, we investigated the ability of rotavirus enterotoxin NSP4-EGFP to elevate intracellular calcium levels in mammalian HEK 293 cells. We show here that inducible intracellular expression of NSP4-EGFP fusion protein elevates basal intracellular calcium more than two-fold by a phospholipase C (PLC) independent mechanism.

Norwalk virus gastroenteritis among Israeli soldiers: lack of evidence for flyborne transmission.

BACKGROUND: Paired sera collected from subjects before and after a fly-control intervention trial conducted in the Israel Defense Force (IDF) were tested for seroconversion to Norwalk virus (NV) to examine the role of NV as a cause of diarrhea in this population and to ascertain whether flies might also be implicated in transmission. MATERIALS AND METHODS: An enzyme-linked immunosorbent assay (ELISA), using recombinant NV capsid proteins (rNV) as antigen was employed to determine the seroconversion rate in a sample of 444 subjects. RESULTS: During 11-week field training cycles, 18% of IDF soldiers who were tested had an NV infection defined as a > or = 4-fold rise in antibody, yielding a cumulative incidence of nearly one infection (0.95) per soldier per year. The rate of seroconversion was nearly twice as high among soldiers who recalled having diarrhea as among those who did not, but the rates did not differ significantly between soldiers in the fly intervention areas and those in the control areas. CONCLUSION: NV is a common cause of enteric infections and diarrhea among Israeli soldiers who serve under field conditions, but unlike infections with Shigella and enterotoxigenic Escherichia coli, transmission of NV cannot be interrupted with an aggressive program of fly-control.

Differential infection of polarized epithelial cell lines by sialic acid-dependent and sialic acid-independent rotavirus strains.

Infection of epithelial cells by some animal rotaviruses, but not human or most animal rotaviruses, requires the presence of N-acetylneuraminic (sialic) acid (SA) on the cell surface for efficient infectivity. To further understand how rotaviruses enter susceptible cells, six different polarized epithelial cell lines, grown on permeable filter membrane supports containing 0.4-microm pores, were infected apically or basolaterally with SA-independent or SA-dependent rotaviruses. SA-independent rotaviruses applied apically or basolaterally were capable of efficiently infecting both sides of the epithelium of all six polarized cell lines tested, while SA-dependent rotaviruses only infected efficiently through the apical surface of five of the polarized cell lines tested. Regardless of the route of virus entry, SA-dependent and SA-independent rotaviruses were released almost exclusively from the apical domain of the plasma membrane of polarized cells before monolayer disruption or cell lysis. The transepithelial electrical resistance (TER) of cells decreased at the same time, irrespective of whether infection with SA-independent rotaviruses occurred apically or basolaterally. The TER of cells infected apically with SA-dependent rotaviruses decreased earlier than that of cells infected basolaterally. Rotavirus infection decreased TER before the appearance of cytopathic effect and cell death and resulted in an increase in the paracellular permeability to [(3)H]inulin as a function of loss of TER. The presence of SA residues on either the apical or basolateral side was determined using a Texas Red-conjugated lectin, wheat germ agglutinin (WGA), which binds SA residues. WGA bound exclusively to SA residues on the apical surface of the cells, confirming the requirement for SA residues on the apical cell membrane for efficient infectivity of SA-dependent rotaviruses. These results indicate that the rotavirus SA-independent cellular receptor is present on both sides of the epithelium, but SA-dependent and SA-independent rotavirus strains infect polarized epithelial cells by different mechanisms, which may be relevant for pathogenesis and selection of vaccine strains. Finally, rotavirus-induced alterations of the epithelial barrier and paracellular permeability suggest that common mechanisms of pathogenesis may exist between viral and bacterial pathogens of the intestinal tract.

Recombinant Norwalk virus-like particles administered intranasally to mice induce systemic and mucosal (fecal and vaginal) immune responses.

Recombinant Norwalk virus-like particles (rNV VLPs) were administered to BALB/c mice by the intranasal (i.n.) route to evaluate the induction of mucosal antibody responses. The results were compared to systemic and mucosal responses observed in new and previous studies (J. M. Ball, M. E. Hardy, R. L. Atmar, M. E. Connor, and M. K. Estes, J. Virol. 72:1345-1353, 1998) after oral administration of rNV VLPs. Immunizations were given in the presence or absence of a mucosal adjuvant, mutant Escherichia coli heat-labile toxin LT(R192G). rNV-specific immunoglobulin G (IgG) and fecal IgA were evaluated by enzyme-linked immunosorbent assay. The i.n. delivery of rNV VLPs was more effective than the oral route at inducing serum IgG and fecal IgA responses to low doses of rNV particles. Vaginal responses of female mice given VLPs by the i.n. and oral routes were also examined. All mice that received two immunizations with low doses i.n. (10 or 25 microg) of rNV VLPs and the majority of mice that received two high doses orally (200 microg) in the absence of adjuvant had rNV-specific serum IgG, fecal, and vaginal responses. Additional experiments evaluated whether rNV VLPs can function as a mucosal adjuvant by evaluating the immune responses to two soluble proteins, keyhole limpet hemocyanin and chicken egg albumin. Under the conditions tested, rNV VLPs did not enhance the serum IgG or fecal IgA response to these soluble proteins when coadministered by the i.n. or oral route. Low doses of nonreplicating rNV VLPs are immunogenic when administered i.n. in the absence of adjuvant, and addition of adjuvant enhanced the magnitude and duration of these responses. Recombinant NV VLPs represent a candidate mucosal vaccine for NV infections in humans.

Interactions between rotavirus and gastrointestinal cells.

Rotaviruses are the leading cause of life-threatening diarrheal disease in infants and in young animals worldwide. The outcome of rotavirus infection of intestinal epithelial cells is more complex and involves induction of more diverse cellular responses than initially appreciated. Similar to bacteria, the pathogenesis of rotavirus-induced disease involves an enterotoxin, activation of the enteric nervous system and malabsorption, suggesting that common mechanisms of pathogenesis may exist between viral and bacterial pathogens.

Structural studies on gastroenteritis viruses.

There are many recent advances in our understanding of the structure-function relationships in rotavirus, a major pathogen of infantile gastroenteritis, and Norwalk virus, a causative agent of epidemic gastroenteritis in humans. Rotavirus is a large (1000 A) and complex icosahedral assembly formed by three concentric capsid layers that enclose the viral genome of 11 dsRNA segments. Because of its medical relevance, intriguing structural complexity, and several unique strategies in the morphogenesis and replication, this virus has been the subject of extensive biochemical, genetic and structural studies. Using a combination of electron cryomicroscopy and computer image processing together with atomic resolution X-ray structural information, we have been able to provide not only a better description of the rotavirus architecture, but also a better understanding of the structural basis of various biological functions such as trypsin-enhanced infectivity, virus assembly and the dynamic process of endogenous transcription. In contrast to rotavirus, Norwalk virus has a simple architecture with an icosahedral capsid made of 180 copies of a single protein. We have determined the structure of the Norwalk virus capsid to a resolution of 3.4 A using X-ray crystallographic techniques. These studies have provided valuable information on domain organization in the capsid protein, and residues that may be critical for dimerization, assembly, strain-specificity and antigenicity.

Pathogenesis of rotavirus gastroenteritis.

The outcome of intestinal infection with rotaviruses is more complex than initially appreciated, and it is affected by a complex interplay of host and viral factors. Rotaviruses infect intestinal enterocytes, and the early events in infection are mediated by virus-epithelial cell interactions. Diarrhoea may be caused by several mechanisms including (i) malabsorption that occurs secondary to the destruction of enterocytes, (ii) villus ischaemia and activation of the enteric nervous system that may be evoked by release of a vasoactive agent from infected epithelial cells in the absence of significant pathologic lesions or enterocyte damage, and (iii) intestinal secretion stimulated by the intracellular or extracellular action of the rotavirus non-structural protein, NSP4, a novel enterotoxin and secretory agonist with pleiotropic properties. New studies of rotavirus infection of polarized intestinal epithelial cells show that rotaviruses infect cells differently depending on whether or not they require sialic acid for initial binding, and infection alters epithelial cell functions. NSP4 also affects epithelial cell function and interactions. NSP4 (i) induces an age- and dose-dependent diarrhoeal response in young rodents that is similar to virus-induced disease, (ii) stimulates a Ca(2+)-dependent cell permeability where the secretory response is age-dependent, and (iii) alters epithelial cell integrity. Antibody to NSP4 protects mouse pups from diarrhoea induced by homotypic and heterotypic viruses. These data support a new mechanism of rotavirus-induced diarrhoea whereby a viral enterotoxin triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. This new information about how a gastrointestinal virus causes disease demonstrates common pathogenic mechanisms for viral and bacterial pathogens not previously appreciated. These results also suggest new approaches to prevent or treat rotavirus-induced diarrhoea.

Microbes and microbial toxins: paradigms for microbial-mucosal interactions. VIII. Pathological consequences of rotavirus infection and its enterotoxin.

Rotaviral infection in neonatal animals and young children leads to acute self-limiting diarrhea, but infected adults are mainly asymptomatic. Recently, significant in-roads have been made into our understanding of this disease: both viral infection and virally manufactured nonstructural protein (NSP)4 evoke intracellular Ca(2+) ([Ca(2+)]i) mobilization in native and transformed gastrointestinal epithelial cells. In neonatal mouse pup mucosa models, [Ca(2+)]i elevation leads to age-dependent halide ion movement across the plasma membrane, transepithelial Cl(-) secretion, and, unlike many microbial enterotoxins, initial cyclic nucleotide independence to secretory diarrhea. Similarities between rotavirus infection and NSP4 function suggest that NSP4 is responsible for these enterotoxigenic effects. NSP4-mediated [Ca(2+)]i mobilization may further facilitate diarrhea by signaling through other Ca(2+)-sensitive cellular processes (cation channels, ion and solute transporters) to potentiate fluid secretion while curtailing fluid absorption. Apart from these direct actions in the mucosa at the onset of diarrhea, innate host-mediated defense mechanisms, triggered by either or both viral replication and NSP4-induced [Ca (2+)]i mobilization, sustain the diarrheal response. This secondary component appears to involve the enteric nervous system and may be cyclic nucleotide dependent. Both phases of diarrhea occur in the absence of significant inflammation. Thus age-dependent rotaviral disease represents an excellent experimental paradigm for understanding a noninflammatory diarrhea.

Trypsin cleavage stabilizes the rotavirus VP4 spike.

Trypsin enhances rotavirus infectivity by an unknown mechanism. To examine the structural basis of trypsin-enhanced infectivity in rotaviruses, SA11 4F triple-layered particles (TLPs) grown in the absence (nontrypsinized rotavirus [NTR]) or presence (trypsinized rotavirus [TR]) of trypsin were characterized to determine the structure, the protein composition, and the infectivity of the particles before and after trypsin treatment. As expected, VP4 was not cleaved in NTR particles and was cleaved into VP5(*) and VP8(*) in TR particles. However, surprisingly, while the VP4 spikes were clearly visible and well ordered in the electron cryomicroscopy reconstructions of TR TLPs, they were totally absent in the reconstructions of NTR TLPs. Biochemical analysis with radiolabeled particles indicated that the stoichiometry of the VP4 in NTR particles was the same as that in TR particles and that the VP8(*) portion of NTR, but not TR, particles is susceptible to further proteolysis by trypsin. Taken together, these structural and biochemical data show that the VP4 spikes in the NTR TLPs are icosahedrally disordered and that they are conformationally different. Structural studies on the NTR TLPs after trypsin treatment showed that spike structure could be partially recovered. Following additional trypsin treatment, infectivity was enhanced for both NTR and TR particles, but the infectivity of NTR remained 2 logs lower than that of TR particles. Increased infectivity in these particles corresponded to additional cleavages in VP5(*), at amino acids 259, 583, and putatively 467, which are conserved in all P serotypes of human and animal group A rotaviruses and also corresponded with a structural change in VP7. These biochemical and structural results show that trypsin cleavage imparts order to VP4 spikes on de novo synthesized virus particles, and these ordered spikes make virus entry into cells more efficient.

Pathogen filtration, heterogeneity, and the potable reuse of wastewater.

Filtration is commonly employed in water and wastewater treatment to remove particles and reduce the concentration of microbial pathogens. All physical models of packed-bed filtration are based on a proportional relationship between particle removal per unit depth of bed and the local particle concentration, dC/dz = -C/l, where l is the filtration length scale. Although l is known to vary with time and filter depth for heterogeneous suspensions or "dirty" beds, this paper demonstrates that the filtration rates of even seemingly monodisperse particle suspensions under clean-bed filtration conditions cannot be described with a single filtration length scale. A new model is derived for particle filtration that accounts for heterogeneity at two different spatial scales. Heterogeneity at the scale of the pathogen and/or collector (microscale heterogeneity) leads to a slow power-law decay of contaminant concentration with distance, instead of the fast exponential decay predicted by the standard model. Heterogeneity at the filter scale (macroscale heterogeneity) provides another level of complexity that can be evaluated once microscale heterogeneity effects are characterized. This model for microscale and macroscale heterogeneous particle filtration is verified by filtration experiments on a recombinant analogue of the waterborne pathogen Norwalk virus.

Membrane interactions of a novel viral enterotoxin: rotavirus nonstructural glycoprotein NSP4.

The rotavirus enterotoxin, NSP4, is a novel secretory agonist that also plays a role in the unique rotavirus morphogenesis that involves a transient budding of newly made immature viral particles into the endoplasmic reticulum. NSP4 and an active peptide corresponding to NSP4 residues 114 to 135 (NSP4(114-135)) mobilize intracellular calcium and induce secretory chloride currents when added exogenously to intestinal cells or mucosa. Membrane-NSP4 interactions may contribute to these alterations; however, details of a lipid-binding domain are unresolved. Therefore, circular dichroism was used to determine (i) the interaction(s) of NSP4 and NSP4(114-135) with model membranes, (ii) the conformational changes elicited in NSP4 upon interacting with membranes, (iii) if NSP4(114-135) is a membrane interacting domain, and (iv) the molar dissociation constant (K(d)) of NSP4(114-135) with defined lipid vesicles. Circular dichroism revealed for the first time that NSP4 and NSP4(114-135) undergo secondary structural changes upon interaction with membrane vesicles. This interaction was highly dependent on both the membrane surface curvature and the lipid composition. NSP4 and NSP4(114-135) preferentially interacted with highly curved, small unilamellar vesicle membranes (SUV), but significantly less with low-curvature, large unilamellar vesicle membranes (LUV). Binding to SUV, but not LUV, was greatly enhanced by negatively charged phospholipids. Increasing the SUV cholesterol content, concomitant with the presence of negatively charged phospholipids, further potentiated the interaction of NSP4(114-135) with the SUV membrane. The K(d) of NSP4(114-135) was determined as well as partitioning of NSP4(114-135) with SUVs in a filtration-binding assay. These data confirmed NSP4 and its active peptide interact with model membranes that mimic caveolae.