The magic of MAGI-1: A scaffolding protein with multi signalosomes and functional plasticity.

MAGI-1 is a critical cellular scaffolding protein with over 110 different cellular and microbial protein interactors. Since the discovery of MAGI-1 in 1997, MAGI-1 has been implicated in diverse cellular functions such as polarity, cell-cell communication, neurological processes, kidney function, and a host of diseases including cancer and microbial infection. Additionally, MAGI-1 has undergone nomenclature changes in response to the discovery of an additional PDZ domain, leading to lack of continuity in the literature. We address the nomenclature of MAGI-1 as well as summarize many of the critical functions of the known interactions. Given the importance of many of the interactors, such as human papillomavirus E6, the Coxsackievirus and adenovirus receptor (CAR), and PTEN, the enhancement or disruption of MAGI-based interactions has the potential to affect cellular functions that can potentially be harnessed as a therapeutic strategy for a variety of diseases.

Human Norovirus Triggers Primary B Cell Immune Activation In Vitro.

Human norovirus (HNoV) is a global health and socioeconomic burden, estimated to infect every individual at least five times during their lifetime. The underlying mechanism for the potential lack of long-term immune protection from HNoV infections is not understood and prompted us to investigate HNoV susceptibility of primary human B cells and its functional impact. Primary B cells isolated from whole blood were infected with HNoV-positive stool samples and harvested at 3 days postinfection (dpi) to assess the viral RNA yield by reverse transcriptase quantitative PCR (RT-qPCR). A 3- to 18-fold increase in the HNoV RNA yield was observed in 50 to 60% of donors. Infection was further confirmed in B cells derived from splenic and lymph node biopsy specimens. Next, we characterized infection of whole-blood-derived B cells by flow cytometry in specific functional B cell subsets (naive CD27- IgD+, memory-switched CD27+ IgD-, memory-unswitched CD27+ IgD+, and double-negative CD27- IgD- cells). While the susceptibilities of the subsets were similar, changes in the B cell subset distribution upon infection were observed, which were also noted after treatment with HNoV virus-like particles and the predicted recombinant NS1 protein. Importantly, primary B cell stimulation with the predicted recombinant NS1 protein triggered B cell activation and induced metabolic changes. These data demonstrate that primary B cells are susceptible to HNoV infection and suggest that the NS1 protein can alter B cell activation and metabolism in vitro, which could have implications for viral pathogenesis and immune responses in vivo. IMPORTANCE Human norovirus (HNoV) is the most prevalent causative agent of gastroenteritis worldwide. Infection results in a self-limiting disease that can become chronic and severe in the immunocompromised, the elderly, and infants. There are currently no approved therapeutic and preventative strategies to limit the health and socioeconomic burdens associated with HNoV infections. Moreover, HNoV does not elicit lifelong immunity as repeat infections are common, presenting a challenge for vaccine development. Given the importance of B cells for humoral immunity, we investigated the susceptibility and impact of HNoV infection on human B cells. We found that HNoV replicates in human primary B cells derived from blood, spleen, and lymph node specimens, while the nonstructural protein NS1 can activate B cells. Because of the secreted nature of NS1, we put forward the hypothesis that HNoV infection can modulate bystander B cell function with potential impacts on systemic immune responses.

The Coxsackievirus and Adenovirus Receptor Has a Short Half-Life in Epithelial Cells.

The coxsackievirus and adenovirus receptor (CAR) is an essential cellular protein that is involved in cell adhesion, cell signaling, and viral infection. The 8-exon encoded isoform (CAREx8) resides at the apical surface of polarized epithelia, where it is accessible as a receptor for adenovirus entering the airway lumen. Given its pivotal role in viral infection, it is a target for antiviral strategies. To understand the regulation of CAREx8 and determine the feasibility of receptor downregulation, the half-life of total and apical localized CAREx8 was determined and correlated with adenovirus transduction. Total and apical CAREx8 has a relatively short half-life of approximately 2 h. The half-life of apical CAREx8 correlates well with adenovirus transduction. These results suggest that antiviral strategies that aim to degrade the primary receptor for apical adenovirus infection will be effective within a relatively short time frame after application.

MAGI-1 PDZ2 Domain Blockade Averts Adenovirus Infection via Enhanced Proteolysis of the Apical Coxsackievirus and Adenovirus Receptor.

Adenoviruses (AdVs) are etiological agents of gastrointestinal, heart, eye, and respiratory tract infections that can be lethal for immunosuppressed people. Many AdVs use the coxsackievirus and adenovirus receptor (CAR) as a primary receptor. The CAR isoform resulting from alternative splicing that includes the eighth exon, CAREx8, localizes to the apical surface of polarized epithelial cells and is responsible for the initiation of AdV infection. We have shown that the membrane level of CAREx8 is tightly regulated by two MAGI-1 PDZ domains, PDZ2 and PDZ4, resulting in increased or decreased AdV transduction, respectively. We hypothesized that targeting the interactions between the MAGI-1 PDZ2 domain and CAREx8 would decrease the apical CAREx8 expression level and prevent AdV infection. Decoy peptides that target MAGI-1 PDZ2 were synthesized (TAT-E6 and TAT-NET1). PDZ2 binding peptides decreased CAREx8 expression and reduced AdV transduction. CAREx8 degradation was triggered by the activation of the regulated intramembrane proteolysis (RIP) pathway through a disintegrin and metalloproteinase (ADAM17) and γ-secretase. Further analysis revealed that ADAM17 interacts directly with the MAGI-1 PDZ3 domain, and blocking the PDZ2 domain enhanced the accessibility of ADAM17 to the substrate (CAREx8). Finally, we validated the efficacy of TAT-PDZ2 peptides in protecting the epithelia from AdV transduction in vivo using a novel transgenic animal model. Our data suggest that TAT-PDZ2 binding peptides are novel anti-AdV molecules that act by enhanced RIP of CAREx8 and decreased AdV entry. This strategy has additional translational potential for targeting other viral receptors that have PDZ binding domains, such as the angiotensin-converting enzyme 2 receptor. IMPORTANCE Adenovirus is a common threat in immunosuppressed populations and military recruits. There are no currently approved treatments/prophylactic agents that protect from most AdV infections. Here, we developed peptide-based small molecules that can suppress AdV infection of polarized epithelia by targeting the AdV receptor, coxsackievirus and adenovirus receptor (CAREx8). The newly discovered peptides target a specific PDZ domain of the CAREx8-interacting protein MAGI-1 and decrease AdV transduction in multiple polarized epithelial models. Peptide-induced CAREx8 degradation is triggered by extracellular domain (ECD) shedding through ADAM17 followed by γ-secretase-mediated nuclear translocation of the C-terminal domain. The enhanced shedding of the CAREx8 ECD further protected the epithelium from AdV infection. Taken together, these novel molecules protect the epithelium from AdV infection. This approach may be applicable to the development of novel antiviral molecules against other viruses that use a receptor with a PDZ binding domain.

Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells.

Human adenoviruses (HAdV) cause a variety of infections in human hosts, from self-limited upper respiratory tract infections in otherwise healthy people to fulminant pneumonia and death in immunocompromised patients. Many HAdV enter polarized epithelial cells by using the primary receptor, the Coxsackievirus and adenovirus receptor (CAR). Recently published data demonstrate that a potent neutrophil (PMN) chemoattractant, interleukin-8 (IL-8), stimulates airway epithelial cells to increase expression of the apical isoform of CAR (CAREx8), which results in increased epithelial HAdV type 5 (HAdV5) infection. However, the mechanism for PMN-enhanced epithelial HAdV5 transduction remains unclear. In this manuscript, the molecular mechanisms behind PMN mediated enhancement of epithelial HAdV5 transduction are characterized using an MDCK cell line that stably expresses human CAREx8 under a doxycycline inducible promoter (MDCK-CAREx8 cells). Contrary to our hypothesis, PMN exposure does not enhance HAdV5 entry by increasing CAREx8 expression nor through activation of non-specific epithelial endocytic pathways. Instead, PMN serine proteases are responsible for PMN-mediated enhancement of HAdV5 transduction in MDCK-CAREx8 cells. This is evidenced by reduced transduction upon inhibition of PMN serine proteases and increased transduction upon exposure to exogenous human neutrophil elastase (HNE). Furthermore, HNE exposure activates epithelial autophagic flux, which, even when triggered through other mechanisms, results in a similar enhancement of epithelial HAdV5 transduction. Inhibition of F-actin with cytochalasin D partially attenuates PMN mediated enhancement of HAdV transduction. Taken together, these findings suggest that HAdV5 can leverage innate immune responses to establish infections.

Antiviral effects of bovine lactoferrin on human norovirus.

Human noroviruses cause significant morbidity and mortality worldwide, but lack approved antivirals or vaccines to treat or prevent infections. The recent development of two cell culture systems in human transformed B cells (BJABs) and non-transformed human intestinal enteroid cultures overcomes a main limitation in identifying molecules with anti-norovirus activities. Lactoferrin is an iron-binding glycoprotein found in the milk of most mammals, with broad spectrum antimicrobial activities, including against the related murine norovirus in cell culture. In a Japanese clinical trial, ingestion of lactoferrin reduced the incidence of infectious gastroenteritis in the participants. Because human noroviruses were the most common cause of gastroenteritis in Japan during the clinical trial period, we sought to determine whether lactoferrin could inhibit infection with human norovirus. Our study, using a B cell culture model, demonstrates that lactoferrin reduces human norovirus infection. The mechanism of antiviral action is likely indirect and may involve the induction of innate interferon responses. Therefore, future studies are warranted to test the antiviral efficacy of lactoferrin against human norovirus infection in patients.

Production of isoform-specific knockdown/knockout Madin-Darby canine kidney epithelial cells using CRISPR/Cas9.

CRISPR-Cas9 gene editing has made it possible to specifically edit genes in a myriad of target cells. Here, a method for isoform-specific editing and clonal selection in Madin-Darby canine kidney (MDCK) epithelial cells is described in detail. This approach was used to address a long-standing question in virology of how adenovirus enters polarized epithelia from the apical surface. Our method relies on selecting two sgRNA sequences, cloning them into a suitable fluorescently labeled Cas9 vector system, and subsequently transfecting our MDCK epithelium and selecting isoform-specific Coxsackievirus and adenovirus receptor knockout clones. Utilization of this method is readily applicable to many other genetic targets in epithelial cells.•Simultaneous utilization of an sgRNA upstream and an sgRNA downstream of a target sequence allows for deletion of the intervening sequence, including whole exons.•Sorting of cells positive for fluorescent marker gene expression enhances the identification of partial and biallelic gene knockout.•PCR screening allows relatively fast and efficient determination of isoform-specific deletion.

Infectious Norovirus Is Chronically Shed by Immunocompromised Pediatric Hosts.

Noroviruses are a leading cause of gastroenteritis worldwide. Although infections in healthy individuals are self-resolving, immunocompromised individuals are at risk for chronic disease and severe complications. Chronic norovirus infections in immunocompromised hosts are often characterized by long-term virus shedding, but it is unclear whether this shed virus remains infectious. We investigated the prevalence, genetic heterogeneity, and temporal aspects of norovirus infections in 1140 patients treated during a 6-year period at a pediatric research hospital. Additionally, we identified 20 patients with chronic infections lasting 37 to >418 days. Using a new human norovirus in vitro assay, we confirmed the continuous shedding of infectious virus for the first time. Shedding lasted longer in male patients and those with diarrheal symptoms. Prolonged shedding of infectious norovirus in immunocompromised hosts can potentially increase the likelihood of transmission, highlighting the importance of isolation precautions to prevent nosocomial infections.

CD300lf is the primary physiologic receptor of murine norovirus but not human norovirus.

Murine norovirus (MNoV) is an important model of human norovirus (HNoV) and mucosal virus infection more broadly. Viral receptor utilization is a major determinant of cell tropism, host range, and pathogenesis. The bona fide receptor for HNoV is unknown. Recently, we identified CD300lf as a proteinaceous receptor for MNoV. Interestingly, its paralogue CD300ld was also sufficient for MNoV infection in vitro. Here we explored whether CD300lf is the sole physiologic receptor in vivo and whether HNoV can use a CD300 ortholog as an entry receptor. We report that both CD300ld and CD300lf are sufficient for infection by diverse MNoV strains in vitro. We further demonstrate that CD300lf is essential for both oral and parenteral MNoV infection and to elicit anti-MNoV humoral responses in vivo. In mice deficient in STAT1 signaling, CD300lf is required for MNoV-induced lethality. Finally, we demonstrate that human CD300lf (huCD300lf) is not essential for HNoV infection, nor does huCD300lf inhibit binding of HNoV virus-like particles to glycans. Thus, we report huCD300lf is not a receptor for HNoV.

Intestinal Enteroid Culture for Human Astroviruses.

Human astroviruses (HAstV) are non-enveloped, positive-sense single stranded RNA viruses that typically cause gastroenteritis in children, the elderly and among immunocompromised individuals. Some HAstV species have also been implicated in neurological diseases. It is important to study these viruses to understand the pathogenesis and develop therapeutics. Here we describe HAstV infection in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. Although different HAstV clades have been propagated in transformed immortalized cell lines such as A549, Caco-2, HEK293T and Huh7.5, we chose HIE because they better mimic the human intestine and thus are more physiologically relevant. Additionally, HIE support the replication of all HAstV clades including clinical samples, thus making HIE a valuable potential universal model to study HAstV biology.

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape.

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.

Glycolysis Is an Intrinsic Factor for Optimal Replication of a Norovirus.

The metabolic pathways of central carbon metabolism, glycolysis and oxidative phosphorylation (OXPHOS), are important host factors that determine the outcome of viral infections and can be manipulated by some viruses to favor infection. However, mechanisms of metabolic modulation and their effects on viral replication vary widely. Herein, we present the first metabolomics and energetic profiling of norovirus-infected cells, which revealed increases in glycolysis, OXPHOS, and the pentose phosphate pathway (PPP) during murine norovirus (MNV) infection. Inhibiting glycolysis with 2-deoxyglucose (2DG) in macrophages revealed that glycolysis is an important factor for optimal MNV infection, while inhibiting the PPP and OXPHOS showed a relatively minor impact of these pathways on MNV infection. 2DG affected an early stage in the viral life cycle after viral uptake and capsid uncoating, leading to decreased viral protein production and viral RNA. The requirement of glycolysis was specific for MNV (but not astrovirus) infection, independent of the type I interferon antiviral response, and unlikely to be due to a lack of host cell nucleotide synthesis. MNV infection increased activation of the protein kinase Akt, but not AMP-activated protein kinase (AMPK), two master regulators of cellular metabolism, implicating Akt signaling in upregulating host metabolism during norovirus infection. In conclusion, our findings suggest that the metabolic state of target cells is an intrinsic host factor that determines the extent of norovirus replication and implicates glycolysis as a virulence determinant. They further point to cellular metabolism as a novel therapeutic target for norovirus infections and improvements in current human norovirus culture systems.IMPORTANCE Viruses depend on the host cells they infect to provide the machinery and substrates for replication. Host cells are highly dynamic systems that can alter their intracellular environment and metabolic behavior, which may be helpful or inhibitory for an infecting virus. In this study, we show that macrophages, a target cell of murine norovirus (MNV), increase glycolysis upon viral infection, which is important for early steps in MNV infection. Human noroviruses (hNoV) are a major cause of gastroenteritis globally, causing enormous morbidity and economic burden. Currently, no effective antivirals or vaccines exist for hNoV, mainly due to the lack of high-efficiency in vitro culture models for their study. Thus, insights gained from the MNV model may reveal aspects of host cell metabolism that can be targeted for improving hNoV cell culture systems and for developing effective antiviral therapies.

Evolution on the Biophysical Fitness Landscape of an RNA Virus.

Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.

Epidemiology of ovarian cancers in Zaria, Northern Nigeria: a 10-year study.

BACKGROUND: Globally, the absence of a premalignant stage of ovarian cancer and a reliable screening tool make early diagnosis difficult. Locally, poverty, ignorance, and lack of organized cancer services make prognosis poor. We describe the epidemiological features of ovarian cancer seen at Ahmadu Bello University Teaching Hospital Zaria, Northern Nigeria, a tertiary referral center, over a 10-year period in this challenging setting. METHODS: All cases of histologically diagnosed ovarian cancer between January 1, 2004 and December 31, 2013 were included in the study. Case notes were retrieved to collect clinical data including age, parity, clinical stage of disease at presentation, and known associated factors. Results were analyzed using Epi info™. RESULTS: A total of 78 patients were included in the study. About 4-13 cases were seen every year with a tendency to increasing incidence. The patients were aged 8-80 years with mean of 37 years. Sixty-two (79.5%) patients were premenopausal while postmenopausal women accounted for only seven cases or 9.0%. There were 17 cases (22.3%) of aggressive cancers in patients aged ≤20 years. A majority of the patients, 65 (83.3%), were parous with only nine (11.5%) patients being nulliparous. Serous cyst adenocarcinoma accounted for 32 (41%) cases. Granulosa cell tumor was the second commonest with 18 cases (23.1%). The mean age of occurrence of serous cyst adenocarcinoma was 31 years and for epithelial ovarian cancers in general it was 33.5 years. Endometrioid adenocarcinoma was rare with only one case in 10 years. Factors like age, parity, and premenopausal status did not appear to be protective to the occurrence of malignant ovarian tumor in this group. CONCLUSION: Increasing numbers of patients with ovarian cancer were seen over the 10-year period. Young, premenopausal, parous women made up the majority of cases. Serous cystadenocarcinoma was the most common histological variant.

Norovirus Escape from Broadly Neutralizing Antibodies Is Limited to Allostery-Like Mechanisms.

Ideal antiviral vaccines elicit antibodies (Abs) with broad strain recognition that bind to regions that are difficult to mutate for escape. Using 10 murine norovirus (MNV) strains and 5 human norovirus (HuNoV) virus-like particles (VLPs), we identified monoclonal antibody (MAb) 2D3, which broadly neutralized all MNV strains tested. Importantly, escape mutants corresponding to this antibody were very slow to develop and were distal to those raised against our previously studied antibody, A6.2. To understand the atomic details of 2D3 neutralization, we determined the cryo-electron microscopy (cryo-EM) structure of the 2D3/MNV1 complex. Interestingly, 2D3 binds to the top of the P domain, very close to where A6.2 binds, but the only escape mutations identified to date fall well outside the contact regions of both 2D3 and A6.2. To determine how mutations in distal residues could block antibody binding, we used molecular dynamics flexible fitting simulations of the atomic structures placed into the density map to examine the 2D3/MNV1 complex and these mutations. Our findings suggest that the escape mutant, V339I, may stabilize a salt bridge network at the P-domain dimer interface that, in an allostery-like manner, affects the conformational relaxation of the P domain and the efficiency of binding. They further highlight the unusual antigenic surface bound by MAb 2D3, one which elicits cross-reactive antibodies but which the virus is unable to alter to escape neutralization. These results may be leveraged to generate norovirus (NoV) vaccines containing broadly neutralizing antibodies. IMPORTANCE The simplest and most common way for viruses to escape antibody neutralization is by mutating residues that are essential for antibody binding. Escape mutations are strongly selected for by their effect on viral fitness, which is most often related to issues of protein folding, particle assembly, and capsid function. The studies presented here demonstrated that a broadly neutralizing antibody to mouse norovirus binds to an exposed surface but that the only escape mutants that arose were distal to the antibody binding surface. To understand this finding, we performed an in silico analysis that suggested that those escape mutations blocked antibody binding by affecting structural plasticity. This kind of antigenic region-one that gives rise to broadly neutralizing antibodies but that the virus finds difficult to escape from-is therefore ideal for vaccine development.

Recent advances in understanding noroviruses.

Noroviruses are the leading cause of acute gastroenteritis around the world. An individual living in the United States is estimated to develop norovirus infection five times in his or her lifetime. Despite this, there is currently no antiviral or vaccine to combat the infection, in large part because of the historical lack of cell culture and small animal models. However, the last few years of norovirus research were marked by a number of ground-breaking advances that have overcome technical barriers and uncovered novel aspects of norovirus biology. Foremost among them was the development of two different in vitro culture systems for human noroviruses. Underappreciated was the notion that noroviruses infect cells of the immune system as well as epithelial cells within the gastrointestinal tract and that human norovirus infection of enterocytes requires or is promoted by the presence of bile acids. Furthermore, two proteinaceous receptors are now recognized for murine norovirus, marking the first discovery of a functional receptor for any norovirus. Recent work further points to a role for certain bacteria, including those found in the gut microbiome, as potential modulators of norovirus infection in the host, emphasizing the importance of interactions with organisms from other kingdoms of life for viral pathogenesis. Lastly, we will highlight the adaptation of drop-based microfluidics to norovirus research, as this technology has the potential to reveal novel insights into virus evolution. This review aims to summarize these new findings while also including possible future directions.

Post-exposure antiviral treatment of norovirus infections effectively protects against diarrhea and reduces virus shedding in the stool in a mortality mouse model.

Noroviruses are a leading cause of gastroenteritis across the world in all age groups and are linked to increased hospitalization and mortality in children, the elderly and immunocompromised. The development of specific antiviral treatment for norovirus gastroenteritis is urgently needed. We explored in a mouse model whether an inhibitor of norovirus replication could be used therapeutically post murine norovirus (MNV)-infection of mice. Using the MNV, we previously discovered that the viral polymerase inhibitor 2'-C-methylcytidine (2CMC) is able to protect against diarrhea and mortality in mice when used prophylactically and to block the transmission of MNV between mice. Here, we investigated whether 2CMC could be used therapeutically, starting treatment between 12 h and 3 days post-infection with 2CMC. Post-exposure treatment of MNV-infected mice with 2CMC was efficient up to 2 days after infection, preventing norovirus-induced diarrhea, delaying and reducing MNV shedding in stool of treated mice. Rehydration of 2CMC-treated animals did not result in a further improvement of the disease evolution compared to antiviral treatment only. The presence of MNV antigens and inflammation in the small intestine of infected mice inversely correlated with the effectiveness of delayed antiviral treatment. Anti-MNV IgGs were detected in re-challenged mice 10 weeks after the first contact, these protected the mice from re-infection. We here demonstrate the benefit of antiviral treatment in ongoing norovirus infections.

Inhibition of human norovirus by a viral polymerase inhibitor in the B cell culture system and in the mouse model.

The recently developed human norovirus (HuNoV) B cell culture and mouse models hold promise for drug discovery and development but their suitability for antiviral studies has not been assessed. We demonstrate the inhibitory effect of the nucleoside analogue 2'-C-methylcytidine (2CMC) on HuNoV replication in the human B cell BJAB cell line and in Balb/c Rag/gamma chain-deficient (Rag-γc(-/-)) mice. These data suggest the applicability of both models for future study and development of antiviral drugs for the treatment of HuNoV infections.