Judith M. Ball

Age-Dependent Diarrhea Induced by a Rotaviral Nonstructural Glycoprotein

The rotavirus nonstructural glycoprotein NSP4 is an intracellular receptor that mediates the acquisition of a transient membrane envelope as subviral particles bud into the endoplasmic reticulum. NSP4 also causes an increase in intracellular calcium in insect cells. Purified NSP4 or a peptide corresponding to NSP4 residues 114 to 135 induced diarrhea in young (6 to 10 days old) CD1 mice. This disease response was age-dependent, dose-dependent, and specific. Electrophysiologic data from intestinal mucosa showed that the NSP4 114–135 peptide potentiates chloride secretion by a calcium-dependent signaling pathway. Diarrhea is induced when NSP4, acting as a viral enterotoxin, triggers a signal transduction pathway.

Norwalk Virus Open Reading Frame 3 Encodes a Minor Structural Protein

ABSTRACT Norwalk virus (NV) is a causative agent of acute epidemic nonbacterial gastroenteritis in humans. The inability to cultivate NV has required the use of molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characterization of the NV ORF 3 protein expressed in a cell-free translation system and in insect cells and show its association with recombinant virus-like particles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbit reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3′ end of the genome beginning with the ORF 2 coding sequences (ORFs 2+3), and the second recombinant contained ORF 3 alone. Expression from the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with ORF 3-specific peptide antisera. However, expression from a construct containing only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect-immunofluorescence studies using an ORF 3 peptide antiserum showed that the ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purified from the media of insect cells infected with a baculovirus recombinant containing the entire 3′ end of the NV genome. Western blot analysis of NV purified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple higher-molecular-weight bands specifically recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.

Recombinant Norwalk virus–like particles given orally to volunteers: Phase I study

BACKGROUND & AIMS Norwalk virus (NV) is a major cause of epidemic gastroenteritis. The NV capsid is composed of a single protein that forms recombinant (rNV) virus-like particles (VLPs). In mice, these VLPs are immunogenic when administered orally without adjuvant, and they elicit serum immunoglobulin (Ig) G and intestinal IgA responses. The aim of this study was to evaluate the safety and immunogenicity of rNV VLPs in healthy volunteers. METHODS Twenty antibody-positive adults were orally administered rNV VLPs in sterile Milli-Q water on days 1 and 21. Vaccine safety and serum rNV-specific total and subclass IgG and IgA antibody responses were monitored. The immune response induced by the VLPs was compared with the response elicited by replicating virus. RESULTS No side effects were observed or reported by the volunteers. Serum IgG responses to rNV VLPs were dose-dependent, and all vaccinees given 250 microgram of rNV VLPs responded with >/=4-fold increases in serum IgG titers. Most of the volunteers (83%; 15 of 18) responded after the first rNV VLP dose and showed no increase in serum IgG titer after the second dose. CONCLUSIONS Orally administered rNV VLPs are safe and immunogenic in healthy adults when administered without adjuvant and are useful to test the mucosal delivery of immunogens.

NSP4 elicits age-dependent diarrhea and Ca2+mediated I− influx into intestinal crypts of CF mice

Homologous disruption of the murine gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to the loss of cAMP-mediated ion transport. Mice carrying this gene defect exhibit meconium ileus at birth and gastrointestinal plugging during the neonatal period, both contributing to high rates of mortality. We investigated whether infectious mammalian rotavirus, the recently characterized rotaviral enterotoxin protein NSP4, or its active NSP4114-135 peptide, can overcome these gastrointestinal complications in CF (CFTRm3Bay null mutation) mice. All three agents elicited diarrhea when administered to wild-type (CFTR+/+), heterozygous (CFTR+/-), or homozygous (CFTR-/-) 7- to 14-day-old mouse pups but were ineffective when given to older mice. The diarrheal response was accompanied by non-age-dependent intracellular Ca2+ mobilization within both small and large intestinal crypt epithelia. Significantly, NSP4 elicited cellular I-influx into intestinal epithelial cells from all three genotypes, whereas both carbachol and the cAMP-mobilizing agonist forskolin failed to evoke influx in the CFTR-/- background. This unique plasma membrane halide permeability pathway was age dependent, being observed only in mouse pup crypts, and was abolished by either the removal of bath Ca2+or the transport inhibitor DIDS. These findings indicate that NSP4 or its active peptide may induce diarrhea in neonatal mice through the activation of an age- and Ca2+-dependent plasma membrane anion permeability distinct from CFTR. Furthermore, these results highlight the potential for developing synthetic analogs of NSP4114-135 to counteract chronic constipation/obstructive bowel syndrome in CF patients.Homologous disruption of the murine gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to the loss of cAMP-mediated ion transport. Mice carrying this gene defect exhibit meconium ileus at birth and gastrointestinal plugging during the neonatal period, both contributing to high rates of mortality. We investigated whether infectious mammalian rotavirus, the recently characterized rotaviral enterotoxin protein NSP4, or its active NSP4(114-135) peptide, can overcome these gastrointestinal complications in CF (CFTR(m3Bay) null mutation) mice. All three agents elicited diarrhea when administered to wild-type (CFTR(+/+)), heterozygous (CFTR(+/-)), or homozygous (CFTR(-/-)) 7- to 14-day-old mouse pups but were ineffective when given to older mice. The diarrheal response was accompanied by non-age-dependent intracellular Ca(2+) mobilization within both small and large intestinal crypt epithelia. Significantly, NSP4 elicited cellular I(-) influx into intestinal epithelial cells from all three genotypes, whereas both carbachol and the cAMP-mobilizing agonist forskolin failed to evoke influx in the CFTR(-/-) background. This unique plasma membrane halide permeability pathway was age dependent, being observed only in mouse pup crypts, and was abolished by either the removal of bath Ca(2+) or the transport inhibitor DIDS. These findings indicate that NSP4 or its active peptide may induce diarrhea in neonatal mice through the activation of an age- and Ca(2+)-dependent plasma membrane anion permeability distinct from CFTR. Furthermore, these results highlight the potential for developing synthetic analogs of NSP4(114-135) to counteract chronic constipation/obstructive bowel syndrome in CF patients.

Recombinant Norwalk Virus-Like Particles Administered Intranasally to Mice Induce Systemic and Mucosal (Fecal and Vaginal) Immune Responses

ABSTRACT 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 coliheat-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 μg) of rNV VLPs and the majority of mice that received two high doses orally (200 μg) 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.

Norwalk Virus Vaccines: Challenges and Progress

Human caliciviruses (HuCVs) are the major cause of outbreaks of acute nonbacterial gastroenteritis throughout the world. An increasing recognition of the clinical significance of these viruses as human pathogens causing foodborne and waterborne disease indicates that an effective vaccine would be useful. This article reviews the current challenges that exist for the development of a vaccine for the HuCVs as well as the status of development of a candidate vaccine. HuCVs are viruses that exhibit a restricted tropism for infection of the gastrointestinal tract of humans, and a volunteer model of infection and disease is available. As pathogens with a restricted host range, the HuCVs are excellent models for understanding the mechanisms that mediate and regulate viral infection of the gastrointestinal tract and mucosal immunity in humans.

Direct Inhibitory Effect of Rotavirus NSP4(114-135) Peptide on the Na+-d-Glucose Symporter of Rabbit Intestinal Brush Border Membrane

ABSTRACT The direct effect of a rotavirus nonstructural glycoprotein, NSP4, and certain related peptides on the sodium-coupled transport ofd-glucose and of l-leucine was studied by using intestinal brush border membrane vesicles isolated from young rabbits. Kinetic analyses revealed that the NSP4(114-135) peptide, which causes diarrhea in young rodents, is a specific, fully noncompetitive inhibitor of the Na+-d-glucose symporter (SGLT1). This interaction involves three peptide-binding sites per carrier unit. In contrast, the Norwalk virus NV(464-483) and mNSP4(131K) peptides, neither of which causes diarrhea, both behave inertly. The NSP4(114-135) and NV(464-483) peptides inhibited Na+-l-leucine symport about equally and partially via a different transport mechanism, in that Na+behaves as a nonobligatory activator. The selective and strong inhibition caused by the NSP4(114-135) peptide on SGLT1 in vitro suggests that during rotavirus infection in vivo, NSP4 can be one effector directly causing SGLT1 inhibition. This effect, implying a concomitant inhibition of water reabsorption, is postulated to play a mechanistic role in the pathogenesis of rotavirus diarrhea.

Temporal changes in cytokine expression of foals during the first month of life

Foals are uniquely susceptible to a wide variety of opportunistic infections normally associated with immunodeficiencies. Little is understood about the immune system of foals during the neonatal period. An apparent age-related susceptibility predisposes neonatal foals to infectious diseases and hinders therapeutic and preventative interventions for these diseases. Cytokine expression is correlated with the type of immune response as well as the severity of a disease. In this study, we measured foal peripheral blood mononuclear cell (PBMC)-specific mRNA cytokine expression from 72 foals from three different farms during the first 4 weeks of life. Interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p35, IL-12p40, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1) were cloned and transcribed in vitro to generate antisense probes for ribonuclease protection assays. Using linear mixed-effect models, we determined that IFN-gamma, TGF-beta1, and IL-1alpha increased significantly (P<0.05) with age.

Feline Immunodeficiency Virus Infection Is Characterized by B7+CTLA4+ T Cell Apoptosis

The B7.1 and B7.2 costimulatory molecules on antigen-presenting cells provide second signals for regulating T cell immune responses via CD28 and cytotoxic T lymphocyte antigen 4 (CTLA4) on T cells. CD28 signals cell proliferation, whereas CTLA4 signals for anergy or apoptosis, terminating the immune response. Because T cell apoptosis and immunodeficiency is a characteristic of feline immunodeficiency virus (FIV)-infected cats, it is possible that negative T cell signaling via B7 and CTLA4 may be favored in these cats. Flow cytometry revealed high percentages of CD8+ and CD4+ cells expressing B7.1, B7.2, and CTLA4 in lymph nodes of FIV-positive cats and a large fraction of CTLA4+ T cells coexpressing B7.1 and B7.2. Three-color analysis with anti-B7.1, anti-B7.2, or anti-CTLA4 and TUNEL (terminal deoxynucleotidyl transferase nick-end-labeling) analysis revealed that apoptosis was a characteristic of B7.1+ B7.2+ CTLA4+ T cells. These data support the hypothesis that lymph node apoptosis and immune deterioration in FIV-infected cats results from chronic B7.1- and/or B7.2-CTLA4-mediated T-T interactions.

The Rotavirus Enterotoxin NSP4 Directly Interacts with the Caveolar Structural Protein Caveolin-1

ABSTRACT Rotavirus nonstructural protein 4 (NSP4) is known to function as an intracellular receptor at the endoplasmic reticulum (ER) critical to viral morphogenesis and is the first characterized viral enterotoxin. Exogenously added NSP4 induces diarrhea in rodent pups and stimulates secretory chloride currents across intestinal segments as measured in Ussing chambers. Circular dichroism studies further reveal that intact NSP4 and the enterotoxic peptide (NSP4114-135) that is located within the extended, C-terminal amphipathic helix preferentially interact with caveola-like model membranes. We now show colocalization of NSP4 and caveolin-1 in NSP4-transfected and rotavirus-infected mammalian cells in reticular structures surrounding the nucleus (likely ER), in the cytosol, and at the cell periphery by laser scanning confocal microscopy. A direct interaction between NSP4 residues 112 to 140 and caveolin-1 was determined by the Pro-Quest yeast two-hybrid system with full-length NSP4 and seven overlapping deletion mutants as bait, caveolin-1 as prey, and vice versa. Coimmunoprecipitation of NSP4-caveolin-1 complexes from rotavirus-infected mammalian cells demonstrated that the interaction occurs during viral infection. Finally, binding of caveolin-1 from mammalian cell lysates to Sepharose-bound, NSP4-specific synthetic peptides confirmed the yeast two-hybrid data and further delineated the binding domain to amino acids 114 to 135. We propose that the association of NSP4 and caveolin-1 contributes to NSP4 intracellular trafficking from the ER to the cell surface and speculate that exogenously added NSP4 stimulates signaling molecules located in caveola microdomains.