Dasabuvir Inhibits Human Norovirus Infection in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are acute viral gastroenteritis pathogens that affect all age groups, yet no approved vaccines and drugs to treat HuNoV infection are available. In this study, we screened an antiviral compound library to identify compound(s) showing anti-HuNoV activity using a human intestinal enteroid (HIE) culture system in which HuNoVs are able to replicate reproducibly. Dasabuvir (DSB), which has been developed as an anti-hepatitis C virus agent, was found to inhibit HuNoV infection in HIEs at micromolar concentrations. Dasabuvir also inhibited severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human rotavirus A (RVA) infection in HIEs. To our knowledge, this is the first study to screen an antiviral compound library for HuNoV using HIEs, and we successfully identified dasabuvir as a novel anti-HuNoV inhibitor that warrants further investigation. IMPORTANCE Although there is an urgent need to develop effective antiviral therapy directed against HuNoV infection, compound screening to identify anti-HuNoV drug candidates has not been reported so far. Using a human HIE culture system, our compound screening successfully identified dasabuvir as a novel anti-HuNoV inhibitor. Dasabuvir's inhibitory effect was also demonstrated in the cases of SARS-CoV-2 and RVA infection, highlighting the usefulness of the HIE platform for screening antiviral agents against various viruses that target the intestines.

Protective effect of pogostone on murine norovirus infected-RAW264.7 macrophages through inhibition of NF-κB/NLRP3-dependent pyroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Pogostemon cablin, the dry overground parts of Pogostemon cablin (Blanco) Benth, has been widely used in the treatment of gastrointestinal dysfunction, such as nausea, diarrhea, headaches and fever. Pogostone (PO) is a major component of Pogostemon cablin which has a variety of pharmacological properties, including antiinflammatory, and immunosuppressive activities, and antioxidant. However, the effect of PO on norovirus gastroenteritis and the underlying molecular mechanism remain unclear. AIM OF THE STUDY: The purpose of our study is to investigate the effects of PO against MNV infection using RAW264.7 cells and to elucidate its active mechanisms. MATERIALS AND METHODS: The cell viability was assessed using Cell Counting Kit-8 (CCK-8) assay and Fluorescein diacetate (FDA) staining. The activation of nuclear factor kappa B (NF-κB) signaling and NOD-like receptor 3 (NLRP3) inflammasome was evaluated by assessing the level of phospho-NF-κB p65, interleukin (IL)-6, TNF-α, NLRP3, cleaved caspase-1, IL-18, IL-1ß using Western blot and quantitative real-time PCR (qPCR), respectively. The number of infected cells were determined by immunofluorescence microscopic assay. RESULTS: PO did not possess a cytotoxic effect toward RAW264.7 cells. The cytotoxic damage caused by MNV infection in RAW264.7 cells decreased significantly in the presence of PO. Cell viability assays showed that pyroptosis is the major mechanism of death in MNV-infected RAW264.7 cells. PO could decreased the expression levels of p-p65, IL-6, TNF-α, NLRP3, cleaved caspase-1, IL-1ß, and IL-18. CONCLUSIONS: These results demonstrate that PO decreases MNV-induced RAW264.7 macrophages death and MNV replication through repressing NF-κB/NLRP3-dependent pyroptosis. Therefore PO may be considered as a potential therapeutic agent for preventing and treating norovirus gastroenteritis.

Antiviral efficacy of orally delivered neoagarohexaose, a nonconventional TLR4 agonist, against norovirus infection in mice.

The neoagarohexaose (NA6) is an oligosaccharide that is derived from agarose, the major component of red algae cell walls, by enzymatic hydrolysis. Here we show that NA6 is a noncanonical Toll-like receptor 4 (TLR4) agonist with antiviral activity against norovirus. Its TLR4 activation was dependent on myeloid differentiation factor 2 (MD2) and cluster of differentiation 14 (CD14), leading to interferon-ß (IFN-ß) and tumor necrosis factor-α (TNF-α) production. This effect was abolished by TLR4 knockdown or knockout in murine macrophages. NA6 inhibited murine norovirus (MNV) replication with an EC50 of 1.5 µM in RAW264.7 cells. It also lowered viral RNA titer in a human hepatocellular carcinoma Huh7-derived cell line harboring a human norovirus subgenomic replicon. The antiviral activity of NA6 was mainly attributed to IFN-ß produced through the TLR4-TRIF signaling pathway. NA6-induced TNF-α, which had little effect on norovirus replication per se, primed macrophages to mount greater antiviral innate immune responses when IFN signaling was activated. NA6 boosted the induction of IFN-ß in MNV-infected RAW264.7 cells and upregulated IFN-regulatory factor-1, an IFN-stimulated gene. NA6 induced IFN-ß expression in the distal ileum with Peyer's patches and oral administration of NA6 reduced MNV loads through activation of TLR4 signaling, highlighting its potential contribution to protective antiviral innate immunity against norovirus.

Effects of famciclovir in cats with spontaneous acute upper respiratory tract disease.

OBJECTIVES: The aim of this study was to assess the effects of famciclovir administration in cats with spontaneously acquired acute upper respiratory tract disease. METHODS: Twenty-four kittens with clinical signs of acute upper respiratory tract disease were randomly allocated to receive doxycycline (5 mg/kg PO q12h) alone (group D; n = 12) or with famciclovir (90 mg/kg PO q12h; group DF; n = 12) for up to 3 weeks. Clinical disease severity was scored at study entry and daily thereafter. Oculo-oropharyngeal swabs collected at study entry and exit were assessed using quantitative PCR for nucleic acids of feline herpesvirus type 1 (FHV-1), feline calicivirus (FCV), Chlamydia felis, Bordetella bronchiseptica and Mycoplasma felis. RESULTS: The median (range) age of cats was 1.5 (1-6) months in group D vs 1.6 (1-5) months in group DF (P = 0.54). Pathogens detected in oculo-oropharyngeal swabs at study entry included FCV (n = 13/24; 54%), M felis (n = 8/24; 33%), FHV-1 (n = 7/24; 29%), C felis (n = 7/24; 29%) and B bronchiseptica (n = 3/24; 12%). Median (range) duration of clinical signs was 11.5 (3-21) days in group DF and 11 (3-21) days in group D (P = 0.75). Median (range) total disease score at the end of the study did not differ between groups (group D 1 [1-1] vs group DF 1 [1-3]; P = 0.08). CONCLUSIONS AND RELEVANCE: This study revealed no significant difference in response to therapy between cats treated with doxycycline alone or with famciclovir; cats improved rapidly in both groups. However, identification of FHV-1 DNA was relatively uncommon in this study and clinical trials focused on FHV-1-infected cats are warranted to better evaluate famciclovir efficacy.

Human Norovirus Cultivation in Nontransformed Stem Cell-Derived Human Intestinal Enteroid Cultures: Success and Challenges.

Noroviruses, in the genus Norovirus, are a significant cause of viral gastroenteritis in humans and animals. For almost 50 years, the lack of a cultivation system for human noroviruses (HuNoVs) was a major barrier to understanding virus biology and the development of effective antiviral strategies. This review presents a historical perspective of the development of a cultivation system for HuNoVs in human intestinal epithelial cell cultures. Successful cultivation was based on the discovery of genetically-encoded host factors required for infection, knowledge of the site of infection in humans, and advances in the cultivation of human intestinal epithelial cells achieved by developmental and stem cell biologists. The human stem cell-derived enteroid cultivation system recapitulates the multicellular, physiologically active human intestinal epithelium, and allows studies of virus-specific replication requirements, evaluation of human host-pathogen interactions, and supports the pre-clinical assessment of methods to prevent and treat HuNoV infections.

The Adenosine Analogue NITD008 has Potent Antiviral Activity against Human and Animal Caliciviruses.

The widespread nature of calicivirus infections globally has a substantial impact on the health and well-being of humans and animals alike. Currently, the only vaccines approved against caliciviruses are for feline and rabbit-specific members of this group, and thus there is a growing effort towards the development of broad-spectrum antivirals for calicivirus infections. In this study, we evaluated the antiviral activity of the adenosine analogue NITD008 in vitro using three calicivirus model systems namely; feline calicivirus (FCV), murine norovirus (MNV), and the human norovirus replicon. We show that the nucleoside analogue (NA), NITD008, has limited toxicity and inhibits calicivirus replication in all three model systems with EC50 values of 0.94 µM, 0.91 µM, and 0.21 µM for MNV, FCV, and the Norwalk replicon, respectively. NITD008 has a similar level of potency to the most well-studied NA 2'-C-methylcytidine in vitro. Significantly, we also show that continual NITD008 treatment effectively cleared the Norwalk replicon from cells and treatment with 5 µM NITD008 was sufficient to completely prevent rebound. Given the potency displayed by NITD008 against several caliciviruses, we propose that this compound should be interrogated further to assess its effectiveness in vivo. In summary, we have added a potent NA to the current suite of antiviral compounds and provide a NA scaffold that could be further modified for therapeutic use against calicivirus infections.

Putative structural rearrangements associated with the interaction of macrocyclic inhibitors with norovirus 3CL protease.

Human noroviruses are the primary cause of outbreaks of acute gastroenteritis worldwide. The problem is further compounded by the current lack of norovirus-specific antivirals or vaccines. Noroviruses have a single-stranded, positive sense 7 to 8 kb RNA genome which encodes a polyprotein precursor that is processed by a virus-encoded 3C-like cysteine protease (NV 3CLpro) to generate at least six mature nonstructural proteins. Processing of the polyprotein is essential for virus replication, consequently, NV 3CLpro has emerged as an attractive target for the discovery of norovirus therapeutics and prophylactics. We have recently described the structure-based design of macrocyclic transition state inhibitors of NV 3CLpro. In order to gain insight and understanding into the interaction of macrocyclic inhibitors with the enzyme, as well as probe the effect of ring size on pharmacological activity and cellular permeability, additional macrocyclic inhibitors were synthesized and high resolution cocrystal structures determined. The results of our studies tentatively suggest that the macrocyclic scaffold may hamper optimal binding to the active site by impeding concerted cross-talk between the S2 and S4 subsites.

Antiviral Drug Discovery: Norovirus Proteases and Development of Inhibitors.

Proteases are a major enzyme group playing important roles in a wide variety of biological processes in life forms ranging from viruses to mammalians. The aberrant activity of proteases can lead to various diseases; consequently, host proteases have been the focus of intense investigation as potential therapeutic targets. A wide range of viruses encode proteases which play an essential role in viral replication and, therefore, constitute attractive targets for the development of antiviral therapeutics. There are numerous examples of successful drug development targeting cellular and viral proteases, including antivirals against human immunodeficiency virus and hepatitis C virus. Most FDA-approved antiviral agents are peptidomimetics and macrocyclic compounds that interact with the active site of a targeted protease. Norovirus proteases are cysteine proteases that contain a chymotrypsin-like fold in their 3D structures. This review focuses on our group's efforts related to the development of norovirus protease inhibitors as potential anti-norovirus therapeutics. These protease inhibitors are rationally designed transition-state inhibitors encompassing dipeptidyl, tripeptidyl and macrocyclic compounds. Highly effective inhibitors validated in X-ray co-crystallization, enzyme and cell-based assays, as well as an animal model, were generated by launching an optimization campaign utilizing the initial hit compounds. A prodrug approach was also explored to improve the pharmacokinetics (PK) of the identified inhibitors.

Potential Therapeutic Agents for Feline Calicivirus Infection.

Feline calicivirus (FCV) is a major cause of upper respiratory tract disease in cats, with widespread distribution in the feline population. Recently, virulent systemic diseases caused by FCV infection has been associated with mortality rates up to 50%. Currently, there are no direct-acting antivirals approved for the treatment of FCV infection. Here, we tested 15 compounds from different antiviral classes against FCV using in vitro protein and cell culture assays. After the expression of FCV protease-polymerase protein, we established two in vitro assays to assess the inhibitory activity of compounds directly against the FCV protease or polymerase. Using this recombinant enzyme, we identified quercetagetin and PPNDS as inhibitors of FCV polymerase activity (IC50 values of 2.8 µM and 2.7 µM, respectively). We also demonstrate the inhibition of FCV protease activity by GC376 (IC50 of 18 µM). Using cell culture assays, PPNDS, quercetagetin and GC376 did not display antivirals effects, however, we identified nitazoxanide and 2'-C-methylcytidine (2CMC) as potent inhibitors of FCV replication, with EC50 values in the low micromolar range (0.6 µM and 2.5 µM, respectively). In conclusion, we established two in vitro assays that will accelerate the research for FCV antivirals and can be used for the high-throughput screening of direct-acting antivirals.

Nitazoxanide Inhibits Human Norovirus Replication and Synergizes with Ribavirin by Activation of Cellular Antiviral Response.

Norovirus is the main cause of viral gastroenteritis worldwide. Although norovirus gastroenteritis is self-limiting in immunocompetent individuals, chronic infections with debilitating and life-threatening complications occur in immunocompromised patients. Nitazoxanide (NTZ) has been used empirically in the clinic and has demonstrated effectiveness against norovirus gastroenteritis. In this study, we aimed at uncovering the antiviral potential and mechanisms of action of NTZ and its active metabolite, tizoxanide (TIZ), using a human norovirus (HuNV) replicon. NTZ and TIZ, collectively referred to as thiazolides (TZD), potently inhibited replication of HuNV and a norovirus surrogate, feline calicivirus. Mechanistic studies revealed that TZD activated cellular antiviral response and stimulated the expression of a subset of interferon-stimulated genes (ISGs), particularly interferon regulatory factor 1 (IRF-1), not only in a Huh7 cell-based HuNV replicon, but also in naive Huh7 and Caco-2 cells and novel human intestinal organoids. Overexpression of exogenous IRF-1 inhibited HuNV replication, whereas knockdown of IRF-1 largely attenuated the antiviral activity of TZD, suggesting that IRF-1 mediated TZD inhibition of HuNV. By using a Janus kinase (JAK) inhibitor, CP-690550, and a STAT1 knockout approach, we found that TZD induced antiviral response independently of the classical JAK-signal transducers and activators of transcription (JAK-STAT) pathway. Furthermore, TZD and ribavirin synergized to inhibit HuNV replication and completely depleted the replicons from host cells after long-term treatment. In summary, our results demonstrated that TZD combated HuNV replication through activation of cellular antiviral response, in particular by inducing a prominent antiviral effector, IRF-1. NTZ monotherapy or combination with ribavirin represent promising options for treating norovirus gastroenteritis, especially in immunocompromised patients.

Clinical presentation and outcomes of norovirus infection in intestinal allograft compared to native intestine.

BACKGROUND: No data are available on clinical manifestations and course of norovirus gastroenteritis (NVE) in intestinal allograft (from intestinal and multivisceral transplant recipients, ITR) compared to native intestine (from other allograft recipients, nITR). METHODS: This was a retrospective study of solid organ transplant recipients with NVE at two centers from January 1, 2010 to April 1, 2014. Chi-square, t-test, linear and logistic regression analyses were done to compare NVE in ITR vs nITR patients. RESULTS: The ITR (45 patients) were compared to nITR (107 patients). ITR were younger (odds ratio [OR]=0.90; P<.0001), less likely to receive anti-lymphocyte induction therapy (OR=0.15; P<.0001), and had shorter time from transplant to NVE (OR=0.99; P=.008). On presentation ITR had less frequent nausea (OR=0.11; P<.0001) or vomiting (OR=0.36; P=.01), higher white blood cell count (OR=1.09; P=.001), and higher glomerular filtration rate (OR=1.02; P<.0001). ITR were less likely to receive anti-motility agents (OR=9.6; P<.0001). ITR were more likely to stay longer on intravenous (IV) fluids (OR=1.18; P<.0001); have recurrent NVE (OR=4.25; P<.0001); have longer hospital stay (OR=1.07; P<.0001); develop acute rejection (OR=5.1; P=.006); and have lower overall survival (OR=0.28; P=.006). CONCLUSIONS: Compared to nITR, the ITR with NVE were significantly younger, had less nausea and vomiting at presentation, received less anti-motility agents, required more IV fluids, and had longer hospital stay. A trend was seen for lower survival with NVE in ITR.

Antiviral effect of vitamin A on norovirus infection via modulation of the gut microbiome.

The effect and underlying mechanism of vitamin A on norovirus infection are largely unknown. This study aimed to investigate how vitamin A administration affects the gut microbiome after norovirus infection. Here, we demonstrate that treatment with either retinol or retinoic acid (RA) inhibits murine norovirus (MNV) replication using both in vitro and in vivo models. Compositional changes in the gut microbiome associated with RA administration and/or norovirus infection were also investigated. Oral administration of RA and/or MNV significantly altered intestinal microbiome profiles. Particularly, bacterial species belonging to the Lactobacillaceae families were remarkably increased by MNV inoculation and RA administration, suggesting that the antiviral effects of RA occur via the modulation of specific microbiota. The antiviral causal effect of Lactobacillus was identified and demonstrated using in vitro models in RAW264.7 cells. The antiviral immune response to MNV was mediated by IFN-ß upregulation. This study represents the first comprehensive profiling of gut microbiota in response to RA treatment against norovirus infection. Moreover, we conclude that the abundance of Lactobacillus through gut microbiota modulation by RA is at least partially responsible for norovirus inhibition.

Melatonin inhibits the sphingosine kinase 1/sphingosine-1-phosphate signaling pathway in rabbits with fulminant hepatitis of viral origin.

The sphingosine kinase (SphK)1/sphingosine-1-phosphate (S1P) pathway is involved in multiple biological processes, including liver diseases. This study investigate whether modulation of the SphK1/S1P system associates to the beneficial effects of melatonin in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10(4) hemagglutination units of a RHDV isolate and received 20 mg/kg of melatonin at 0, 12, and 24 hr postinfection. Liver mRNA levels, protein concentration, and immunohistochemical labeling for SphK1 increased in RHDV-infected rabbits. S1P production and protein expression of the S1PR1 receptor were significantly elevated following RHDV infection. These effects were significantly reduced by melatonin. Rabbits also exhibited increased expression of toll-like receptor (TLR)4, tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, nuclear factor-kappa B (NF-κB) p50 and p65 subunits, and phosphorylated inhibitor of kappa B (IκB)α. Melatonin administration significantly inhibited those changes and induced a decreased immunoreactivity for RHDV viral VP60 antigen in the liver. Results obtained indicate that the SphK1/S1P system activates in parallel to viral replication and the inflammatory process induced by the virus. Inhibition of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in this animal model of ALF, and supports the potential of melatonin as an antiviral agent.

Evaluation of intranasal vaccine administration and high-dose interferon- α2b therapy for treatment of chronic upper respiratory tract infections in shelter cats.

Clinical signs of upper respiratory tract infection can be hard to manage in cats, particularly those in shelters. In this study, clinical data were collected from chronically ill (3-4 weeks' duration) cats with suspected feline herpesvirus-1 (FHV-1) or feline calicivirus (FCV) infections after administration of one of two novel therapies. Group A cats were administered a commercially available formulation of human interferon-α2b at 10,000 U/kg subcutaneously for 14 days, and group B cats were administered one dose of a FHV-1 and FCV intranasal vaccine. Molecular assays for FHV-1 and FCV were performed on pharyngeal samples, and a number of cytokines were measured in the blood of some cats. A clinical score was determined daily for 14 days, with cats that developed an acceptable response by day 14 returning to the shelter for adoption. Those failing the first treatment protocol were entered into the alternate treatment group. During the first treatment period, 8/13 cats in group A (61.5%) and all 12 cats in group B (100%) had apparent responses. The seven cats positive for nucleic acids of FHV-1 or FCV responded favorably, independent of the treatment group. There were no differences in cytokine levels between cats that responded to therapy or failed therapy. Either protocol assessed here may be beneficial in alleviating chronic clinical signs of suspected feline viral upper respiratory tract disease in some cats that have failed other, more conventional, therapies. The results of this study warrant additional research involving these protocols.

Recent Advances in the Discovery of Norovirus Therapeutics.

Noroviruses are members of the family Caliciviridae. Norovirus infections are a global health burden that impacts >20 million individuals annually in the U.S. alone. Noroviruses are associated with high morbidity among vulnerable populations, particularly immunocompromised patients. This perspective highlights recent developments related to the discovery and development of norovirus-specific small-molecule therapeutics as well as recent advances in our understanding of norovirus biology and pathogenesis. Most of the work in this area is at the early discovery stage and has been primarily focused on inhibitors of norovirus 3C-like protease and RNA dependent RNA polymerase. However, recent discoveries emanating from basic studies in norovirus research have resulted in the identification of new host-related drug targets that can be exploited. A repurposed compound has been advanced to human clinical studies.

Atividade in vitro de plantas condimentares (Rosmarinus officinalis L., Lippia graveolens HBK e Thymus vulgaris L. ) contra o calicivírus felino / In vitro activity of plants used as condiments (Rosmarinus officinalis L., Lippia graveolens HBK e Thymus vulgaris L. ) against the feline calicivirus

O calicivírus felino (FCV) é um importante patógeno de gatos que causa lesões ulcerativas orais e infecções respiratórias. O vírus tem sido utilizado como modelo experimental para avaliação de agente antivirais contra norovírus (NoVs). Nesse estudo, investigou-se a ação dos óleos essenciais de alecrim (Rosmarinus officinalis L.), orégano mexicano (Lippia graveolens HBK.) e tomilho (Thymus vulgaris L.) frente ao FCV, in vitro. A toxicidade celular foi testada pelo método de MTT e os ensaios antivirais pelo teste de redução de placas. Três protocolos foram aplicados: a) diferentes concentrações não tóxicas dos óleos essenciais (CNTOE) foram incubadas com o vírus por 1 hora antes da inoculação (ensaio virucida); b) CNTOE foram adicionadas às células CRFK e incubadas por 1 hora antes da adsorção viral (ensaio de pré-tratamento); c) CNTOE foram adicionadas às células após a inoculação do FCV e mantidas por 18 horas (ensaio de pós-tratamento). A CC 50 para os óleos de alecrim, orégano mexicano e tomilho foram: 1300,21 ?g mL -1 ; 435,92 ?g mL -1 e 675,34 ?g mL -1 ; respectivamente. O óleo essencial de tomilho apresentou índice de seletividade [IS=CC 50 /CI 50 ] de 8,57 para o ensaio de pré-tratamento e 6,2 no ensaio virucida. O óleo de alecrim mostrou atividade antiviral no ensaio virucida (IS=6,54) e de pós-tratamento (IS=6,86). O orégano mexicano apresentou IS de 5,75 no ensaio virucida e 5,59 no de pós-tratamento. Conclui-se que os óleos essenciais de tomilho e alecrim apresentaram atividade frente ao FCV em diferentes momentos da infecção viral.(AU)

The feline calicivirus (FCV) is an important pathogen of feline causing oral ulcerative lesions and respiratory disease. This virus has been used as a model to evaluate antiviral compounds against Norovirus (NoVs). In this study, the essential oils of rosemary (Rosmarinus officinalis L.), mexican oregano (Lippia graveolens HBK) and thyme (Thymus vulgaris L.) were examined for their activity towards FCV, in vitro. The cytotoxicity was determined by the MTT test and the antiviral assays were performed by the plaque reduction test. Three protocols were applied: a) different non-toxic concentrations of the essential oils (NTCEO) were incubated with the virus for 1 hour before viral inoculation (virucidal assay); b) NTCEO were added to CRFK cells and incubated for 1 hour before viral adsorption (pre-treatment assay); c) NTCEO were added to cells after virus inoculation and maintained for 18 hours (post-treatment assay). The cytotoxic concentration at 50% (CC 50 ) for the essential oils of rosemary, mexican oregano, and thyme were: 1300.21 ?g mL -1 ; 435.92 ?g mL -1 and 675.34 ?g mL -1 ; respectively. The essential oil of thyme showed a selectivity index (IS=CC 50 /CI 50 ) of 8.57 at the cell pre-treatment assay and 6.2 at the virucidal assay. The essential oil of rosemary showed antiviral activity at the virucidal assay (IS=6.54) and, also, at the post- treatment assay (IS=6.86). The mexican oregano showed an IS of 5.75 at the virucidal assay and 5.59 at the post-treatment. Therefore, it can be concluded that the essential oils of thyme and rosemary show antiviral activity against FCV in different times of the infection.(AU)

The role of chronic norovirus infection in the enteropathy associated with common variable immunodeficiency.

OBJECTIVES: A severe enteropathy of unknown etiology can be associated with common variable immunodeficiency (CVID). METHODS: S tool and archived small intestinal mucosal biopsies from patients with CVID enteropathy were analyzed by PCR for the presence of Norovirus RNA. The PCR products were sequenced to determine the relationship of viral isolates. Stool samples from 10 patients with CVID but no enteropathy served as controls. RESULTS: All eight patients in our CVID cohort with enteropathy showed persistent fecal excretion of Norovirus. Analysis of archived duodenal biopsies revealed a strong association between the presence of Norovirus and villous atrophy over a period of up to 8 years. Analysis of the viral isolates from each patient revealed distinct strains of genogroup II.4. Sequence analysis from consecutive biopsy specimens of one patient demonstrated persistence of the same viral strain over a 6-year period. CVID patients without enteropathy showed no evidence of Norovirus carriage. Viral clearance occurred spontaneously in one patient and followed oral Ribavirin therapy in two further patients, and resulted in complete symptomatic and histological recovery. However, Ribavirin treatment in two further patients was unsuccessful. CONCLUSIONS: Norovirus is an important pathogen for patients with CVID and a cause of CVID enteropathy, as viral clearance, symptom resolution, and histological recovery coincide. Ribavirin requires further evaluation as a potential therapy.

Development of Lactobacillus paracasei harboring nucleic acid-hydrolyzing 3D8 scFv as a preventive probiotic against murine norovirus infection.

The protein 3D8 single-chain variable fragment (3D8 scFv) has potential anti-viral activity due to its ability to penetrate into cells and hydrolyze nucleic acids. Probiotic Lactobacillus paracasei engineered to secrete 3D8 scFv for oral administration was used to test the anti-viral effects of 3D8 scFv against gastrointestinal virus infections. We found that injection of 3D8 scFv into the intestinal lumen resulted in the penetration of 3D8 scFv into the intestinal villi and lamina propria. 3D8 scFv secreted from engineered L. paracasei retained its cell-penetrating and nucleic acid-hydrolyzing activities, which were previously shown with 3D8 scFv expressed in Escherichia coli. Pretreatment of RAW264.7 cells with 3D8 scFv purified from L. paracasei prevented apoptosis induction by murine norovirus infection and decreased messenger RNA (mRNA) expression of the viral capsid protein VP1. In a mouse model, oral administration of the engineered L. paracasei prior to murine norovirus infection reduced the expression level of mRNA encoding viral polymerase. Taken together, these results suggest that L. paracasei secreting 3D8 scFv provides a basis for the development of ingestible anti-viral probiotics active against gastrointestinal viral infection.

Nonnucleoside inhibitors of norovirus RNA polymerase: scaffolds for rational drug design.

Norovirus (NoV) is the leading cause of acute gastroenteritis worldwide, causing over 200,000 deaths a year. NoV is nonenveloped, with a single-stranded RNA genome, and is primarily transmitted person to person. The viral RNA-dependent RNA polymerase (RdRp) is critical for the production of genomic and subgenomic RNA and is therefore a prime target for antiviral therapies. Using high-throughput screening, nearly 20,000 "lead-like" compounds were tested for inhibitory activity against the NoV genogroup II, genotype 4 (GII.4) RdRp. The four most potent hits demonstrated half-maximal inhibitory concentrations (IC50s) between 5.0 µM and 9.8 µM against the target RdRp. Compounds NIC02 and NIC04 revealed a mixed mode of inhibition, while NIC10 and NIC12 were uncompetitive RdRp inhibitors. When examined using enzymes from related viruses, NIC02 demonstrated broad inhibitory activity while NIC04 was the most specific GII.4 RdRp inhibitor. The antiviral activity was examined using available NoV cell culture models; the GI.1 replicon and the infectious GV.1 murine norovirus (MNV). NIC02 and NIC04 inhibited the replication of the GI.1 replicon, with 50% effective concentrations (EC50s) of 30.1 µM and 71.1 µM, respectively, while NIC10 and NIC12 had no observable effect on the NoV GI.1 replicon. In the MNV model, NIC02 reduced plaque numbers, size, and viral RNA levels in a dose-dependent manner (EC50s between 2.3 µM and 4.8 µM). The remaining three compounds also reduced MNV replication, although with higher EC50s, ranging from 32 µM to 38 µM. In summary, we have identified novel nonnucleoside inhibitor scaffolds that will provide a starting framework for the development and future optimization of targeted antivirals against NoV.