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Norovirus (NV) is a major cause of gastroenteritis worldwide. Antivirals against such important pathogens are on demand. Among the viral proteins that orchestrate viral replication, RNA-dependent-RNA-polymerase (RdRp) is a promising drug development target. From an in silico-docking search focused on the RdRp active site, we selected the compound PPNDS, which showed low micromolar IC50 vs. murine NV-RdRp in vitro. We report the crystal structure of the murine NV-RdRp/PPNDS complex showing that two molecules of the inhibitor bind in antiparallel stacking interaction, properly oriented to block exit of the newly synthesized RNA. Such inhibitor-binding mode mimics two stacked nucleotide-bases of the RdRp/ssRNA complex. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Noroviruses are members of the Caliciviridae family of positive sense RNA viruses. In humans Noroviruses cause rapid onset diarrhea and vomiting. Currently Norovirus infection is responsible for 21 million gastroenteritis yearly cases in the USA. Nevertheless, despite the obvious public health and socio-economic relevance, no effective vaccines/antivirals are yet available to treat Norovirus infection. Since the activity of RNA-dependent RNA polymerase (RdRp) plays a key role in genome replication and in the synthesis/amplification of subgenomic RNA, the enzyme is considered a promising target for antiviral drug development. In this context, following the identification of suramin and NF023 as Norovirus RdRp inhibitors, we analyzed the potential inhibitory role of naphthalene di-sulfonate (NAF2), a fragment derived from these two molecules. Although NAF2, tested in enzymatic polymerase inhibition assays, displayed low activity against RdRp (IC 50 = 14 μM), the crystal structure of human Norovirus RdRp revealed a thumb domain NAF2 binding site that differs from that characterized for NF023/suramin. To further map the new potential inhibitory site, we focused on the structurally related molecule pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-nitro-4′,8′-disulfonate) tetrasodium salt (PPNDS). PPNDS displayed below-micromolar inhibitory activity versus human Norovirus RdRp (IC 50 = 0.45 μM), similarly to suramin and NF023. Inspection of the crystal structure of the RdRp/PPNDS complex showed that the inhibitor bound to the NAF2 thumb domain site, highlighting the relevance of such new binding site for exploiting Norovirus RdRp inhibitors.

Caliciviridae are human or non-human pathogenic viruses with a high diversity. Some members of the Caliciviridae, i.e. human pathogenic norovirus or rabbit hemorrhagic disease virus (RHDV), are worldwide emerging pathogens. The norovirus is the major cause of viral gastroenteritis worldwide, accounting for about 85% of the outbreaks in Europe between 1995 and 2000. In the United States, 25 million cases of infection are reported each year. Since its emergence in 1984 as an agent of fatal hemorrhagic diseases in rabbits, RHDV has killed millions of rabbits and has been dispersed to all of the inhabitable continents. In view of their successful and apparently increasing emergence, the development of antiviral strategies to control infections due to these viral pathogens has now become an important issue in medicine and veterinary medicine. Antiviral strategies have to be based on an understanding of the epidemiology, transmission, clinical symptoms, viral replication and immunity to infection resulting from infection by these viruses. Here, we provide an overview of the mechanisms underlying calicivirus infection, focusing on the molecular aspects of replication in the host cell. Recent experimental data generated through an international collaboration on structural biology, virology and drug design within the European consortium VIZIER is also presented. Based on this analysis, we propose antiviral strategies that may significantly impact on the epidemiological characteristics of these highly successful viral pathogens.

In immunocompromised patients, the diagnosis of infections with herpesviruses and adenoviruses relies mainly on PCR amplification of viral genomic DNA from clinical samples. In the case of co-infections with two or more viruses, single amplification of viral DNA from clinical samples has proven to be time-consuming and expensive, hampering the efficient diagnosis and therapy of viral co-infections. In this study, a diagnostic DNA-microarray allowing simultaneous detection of herpes simplex virus types 1 and 2 (HSV 1/2), varicella zoster virus (VZV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus-6 types A and B (HHV-6 A/B), and adenovirus in clinical samples was developed and validated. The assay displays a high analytical sensitivity (10 genome equivalents (GE)/reaction) and specificity, being cost-effective and time-saving. Because the DNA-microarray uses the same analytical conditions as real-time quantitative PCR, it can be used as a screening device for multiple viral infections, followed by selective viral load measurement depending on the clinical context. Those features make the DNA-microarray an attractive device for the management of viral infections in immunocompromised patients.

Sapoviruses are one of the major agents of acute gastroenteritis in childhood. They form a tight genetic cluster (genus) in the Caliciviridae family that regroups both animal and human pathogenic strains. No permissive tissue culture has been developed for human sapovirus, limiting its characterization to surrogate systems. We report here on the first extensive characterization of the key enzyme of replication, the RNA-dependent RNA polymerase (RdRp) associated with the 3D pol -like protein. Enzymatically active sapovirus 3D pol and its defective mutant were expressed in Escherichia coli and purified. The overall structure of the sapovirus 3D pol was determined by X-ray crystallography to 2.32-Å resolution. It revealed a right hand fold typical for template-dependent polynucleotide polymerases. The carboxyl terminus is located within the active site cleft, as observed in the RdRp of some (norovirus) but not other (lagovirus) caliciviruses. Sapovirus 3D pol prefers Mn 2+ over Mg 2+ but may utilize either as a cofactor in vitro. In a synthetic RNA template-dependent reaction, sapovirus 3D pol synthesizes a double-stranded RNA or labels the template 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurs de novo on heteropolymeric templates or in a primer-dependent manner on polyadenylated templates. Strikingly, this mode of initiation of RNA synthesis was also described for norovirus, but not for lagovirus, suggesting structural and functional homologies in the RNA-dependent RNA polymerase of human pathogenic caliciviruses. This first experimental evidence makes sapovirus 3D pol an attractive target for developing drugs to control calicivirus infection in humans.

The aim of this study was to investigate the clinical impact of reactivation of human herpes virus-6 (HHV-6) and HHV-7 infections in stem cell transplantation recipients, and to examine a possible increase in virulence of the two roseoloviruses when a reactivation of CMV (HHV-5) simultaneously occurs. For this purpose, quantitative real-time PCR systems were developed to assess the viral load of CMV, HHV-6, or HHV-7 in the plasma of haematopoetic stem cell recipients. One hundred and ninety-eight plasma samples from 37 patients who underwent allogeneic stem cell transplantation were tested for CMV, HHV-6, and HHV-7 by a 5'-exonuclease (TaqMan) quantitative real-time PCR. The CMV load obtained by the real-time PCR assay was compared retrospectively with results generated previously with a commercially available test (COBAS AMPLICOR CMV MONITOR Test, Roche). The results suggest that CMV and HHV-6 may be associated with post-transplantation end-organ disease, while HHV-7 reactivation had no impact on the patients included in this study. No evidence for a potential interaction of the roseoloviruses and CMV infections was found.

Noroviruses ( Caliciviridae ) are RNA viruses with a single-stranded, positive-oriented polyadenylated genome. To date, little is known about the replication strategy of norovirus, a so-far noncultivable virus. We have examined the initiation of replication of the norovirus genome in vitro, using the active norovirus RNA-dependent RNA polymerase (3D pol ), homopolymeric templates, and synthetic subgenomic or antisubgenomic RNA. Initiation of RNA synthesis on homopolymeric templates as well as replication of subgenomic polyadenylated RNA was strictly primer dependent. In this context and as observed for other enteric RNA viruses, i.e., poliovirus, a protein-primed initiation of RNA synthesis after elongation of the VPg by norovirus 3D pol was postulated. To address this question, norovirus VPg was expressed in Escherichia coli and purified. Incubation of VPg with norovirus 3D pol generated VPg-poly(U), which primed the replication of subgenomic polyadenylated RNA. In contrast, replication of antisubgenomic RNA was not primer dependent, nor did it depend on a leader sequence, as evidenced by deletion analysis of the 3′ termini of subgenomic and antisubgenomic RNA. On nonpolyadenylated RNA, i.e., antisubgenomic RNA, norovirus 3D pol initiated RNA synthesis de novo and terminated RNA synthesis by a poly(C) stretch. Interestingly, on poly(C) RNA templates, norovirus 3D pol initiated RNA synthesis de novo in the presence of high concentrations of GTP. We propose a novel model for initiation of replication of the norovirus genome by 3D pol , with a VPg-protein-primed initiation of replication of polyadenylated genomic RNA and a de novo initiation of replication of antigenomic RNA.

Noroviruses are single-stranded RNA viruses with high genomic variability. They have emerged in the last decade as a major cause of acute gastroenteritis. It remains so far unclear whether norovirus evolution is driven by sequence mutation and/or recombination. In this study, we have assessed the occurrence of recombination in the norovirus capsid gene. For this purpose, 69 complete capsid sequences of norovirus strains accessible in GenBank as well as 25 complete capsid sequences generated from norovirus-positive clinical samples were examined. Unreported recombination was detected in about 8% of norovirus strains belonging to genetic clusters I/1 ( n = 1), II/1 ( n = 1), II/3 ( n = 1), II/4 ( n = 3), and II/5 ( n = 1). Recombination breakpoints were mainly located at the interface of the putative P1-1 and P2 domains of the capsid protein and/or within the P2 domain. The recombination region displayed features such as length, sequence composition (upstream and downstream GC- and AU-rich sequences, respectively), and predicted RNA secondary structure that are characteristic of homologous recombination activators. Our results suggest that recombination in the norovirus capsid gene may naturally occur, involving capsid domains presumably exposed to immunological pressure.

Norovirus RNA-dependent RNA polymerase (RdRp) is a promising target enzyme for the development of new antiviral drugs. Starting from the crystal structure of norovirus RdRp, we had previously performed an in silico docking search using a library of low-molecular-weight compounds that enabled us to select molecules with predicted enzyme inhibitory activity. Among these, the polysulfonated naphthylurea suramin proved to inhibit in vitro both murine and human norovirus polymerases, with IC50 values in the low micromolar range. The negatively charged inhibitor, however, displayed poor cell permeability in cell-based experiments. Therefore, we produced different suramin-loaded liposome formulations and evaluated their activities in cell-based assays using murine norovirus cultivated in RAW 264.7 macrophages, as a model for norovirus genus. The results obtained show that suramin, when delivered through liposomes, can effectively inhibit murine norovirus replication.

Noroviruses (NV) are +ssRNA viruses responsible for severe gastroenteritis; no effective vaccines/antivirals are currently available. We previously identified Suramin (9) as a potent inhibitor of NV-RNA dependent RNA polymerase (NV-RdRp). Despite significant in vitro activities versus several pharmacological targets, Suramin clinical use is hampered by pharmacokinetics/toxicity problems. To improve Suramin access to NV-RdRp in vivo, a Suramin-derivative, 8, devoid of two sulphonate groups, was synthesized, achieving significant anti-human-NV-RdRp activity (IC50 = 28 nM); the compound inhibits also murine NV (mNV) RdRp. The synthesis process led to the isolation/characterization of lower molecular weight intermediates (3–7) hosting only one sulphonate head. The crystal structures of both hNV/mNV-RdRps in complex with 6, were analyzed, providing new knowledge on the interactions that a small fragment can establish with NV-RdRps, and establishing a platform for structure-guided optimization of potency, selectivity and drugability.

Caliciviridae are RNA viruses with a single-stranded, positively oriented polyadenylated genome, responsible for a broad spectrum of diseases such as acute gastroenteritis in humans. Recently, analyses on the structures and functionalities of the RNA-dependent RNA polymerase (RdRp) from several Caliciviruses have been reported. The RdRp is predicted to play a key role in genome replication, as well as in synthesis and amplification of additional subgenomic RNA. Starting from the crystal structures of human Norovirus (hNV) RdRp, we performed an in silico docking search to identify synthetic compounds with predicted high affinity for the enzyme active site. The best-ranked candidates were tested in vitro on murine Norovirus (MNV) and hNV RdRps to assay their inhibition of RNA polymerization. The results of such combined computational and experimental screening approach led to the identification of two high-potency inhibitors: Suramin and NF023, both symmetric divalent molecules hosting two naphthalene-trisulfonic acid heads. We report here the crystal structure of MNV RdRp alone and in the presence of the two identified inhibitors. Both inhibitory molecules occupy the same RdRp site, between the fingers and thumb domains, with one inhibitor head close to residue 42 and to the protein active site. To further validate the structural results, we mutated Trp42 to Ala in MNV RdRp and the corresponding residue (i.e., Tyr41 to Ala) in hNV RdRp. Both NF023 and Suramin displayed reduced inhibitory potency versus the mutated hNV RdRp, thus hinting at a conserved inhibitor binding mode in the two polymerases.

We report on the case of a healthy young boy who developed a fulminant myocarditis due to acute coinfection with erythrovirus (parvovirus B19) and human herpesvirus 6 (HHV-6) in the absence of an antiviral immune response. We suggest that the HHV-6-induced immunosuppression enhanced dissemination of parvovirus B19, which led to fatal myocarditis.

Norovirus (NV) is a leading cause of gastroenteritis worldwide and a major public health concern. So far, the replication strategy of NV remains poorly understood, mainly because of the lack of a cell system to cultivate the virus. In this study, the function and the structure of a key viral enzyme of replication, the RNA-dependent RNA polymerase (RdRp, NS7), was examined. The overall structure of the NV NS7 RdRp was determined by X-ray crystallography to a 2.3 Å (0.23 nm) resolution (PDB ID 2B43), displaying a right-hand fold typical of the template-dependent polynucleotide polymerases. Biochemical analysis evidenced that NV NS7 RdRp is active as a homodimer, with an apparent K d of 0.649 μM and a positive cooperativity (Hill coefficient nH=1.86). Crystals of the NV NS7 homodimer displayed lattices containing dimeric arrangements with high shape complementarity statistics. This experimental data on the structure and function of the NV RdRp may set the cornerstone for the development of polymerase inhibitors to control the infection with NV, a medically relevant pathogen.

Astroviruses are single-stranded RNA viruses with a replication strategy based on the proteolytic processing of a polyprotein precursor and subsequent release of the viral enzymes of replication. So far, the catalytic properties of the astrovirus protease as well as its structure have remained uncharacterized. In this study, the three-dimensional crystal structure of the predicted protease of human pathogenic astrovirus has been solved to 2.0 A resolution. The protein displays the typical properties of trypsin-like enzymes but also several characteristic features: (i) a catalytic Asp-His-Ser triad in which the aspartate side chain is oriented away from the histidine, being replaced by a water molecule; (ii) a non-common conformation and composition of the S1 pocket; and (iii) the lack of the typical surface beta-ribbons together with a "featureless" shape of the substrate-binding site. Hydrolytic activity assays indicate that the S1 pocket recognises Glu and Asp side chains specifically, which, therefore, are predicted to occupy the P1 position on the substrate cleavage site. The positive electrostatic potential featured by the S1 region underlies this specificity. The comparative structural analysis highlights the peculiarity of the astrovirus protease, and differentiates it from the human and viral serine proteases.

Sapovirus is a positive-stranded RNA virus with a translational strategy based on processing of a polyprotein precursor by a chymotrypsin-like protease. So far, the molecular mechanisms regulating cleavage specificity of the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the predicted forms of the viral protease, the 3C-like protease (NS6) and the 3CD-like protease-polymerase (NS6-7), were examined in vitro. The purified NS6 and NS6-7 were able to cleave synthetic peptides (15 to 17 residues) displaying the cleavage sites of the sapovirus polyprotein, both NS6 and NS6-7 proteins being active forms of the viral protease. High-performance liquid chromatography and subsequent mass spectrometry analysis of digested products showed a specific trans cleavage of peptides bearing Gln-Gly, Gln-Ala, Glu-Gly, Glu-Pro, or Glu-Lys at the scissile bond. In contrast, peptides bearing Glu-Ala or Gln-Asp at the scissile bond (NS4-NS5 and NS5-NS6, or NS6-NS7 junctions, respectively) were resistant to trans cleavage by NS6 or NS6-7 proteins, whereas cis cleavage of the Glu-Ala scissile bond of the NS5-NS6 junction was evidenced. Interestingly, the presence of a Phe at position P4 overruled the resistance to trans cleavage of the Glu-Ala junction (NS5-NS6), whereas substitutions at the P1 and P2′ positions altered the cleavage efficiency. The differential cleavage observed is supported by a model of the substrate-binding site of the sapovirus protease, indicating that the P4, P1, and P2′ positions in the substrate modulate the cleavage specificity and efficiency of the sapovirus chymotrypsin-like protease.

Coutard, B. et al., Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules. © 2007 Elsevier B.V. All rights reserved., This work was supported by the VIZIER integrated project (LSHG-CT-2004-511960) of the European Union 6th Framework Programme (FP6)

TBZE-029 {1-(2,6-difluorophenyl)-6-trifluoromethyl-1 H ,3 H -thiazolo[3,4-a]benzimidazole} is a novel selective inhibitor of the replication of several enteroviruses. We show that TBZE-029 exerts its antiviral activity through inhibition of viral RNA replication, without affecting polyprotein processing. To identify the viral target of TBZE-029, drug-resistant coxsackievirus B3 (CVB3) was selected. Genotyping of resistant clones led to the identification of three amino acid mutations in nonstructural protein 2C, clustered at amino acid positions 224, 227, and 229, immediately downstream of NTPase/helicase motif C. The mutations were reintroduced, either alone or combined, into an infectious full-length CVB3 clone. In particular the mutations at positions 227 and 229 proved essential for the altered sensitivity of CVB3 to TBZE-029. Resistant virus exhibited cross-resistance to the earlier-reported antienterovirus agents targeting 2C, namely, guanidine hydrochloride, HBB [2-(alpha-hydroxybenzyl)-benzimidazole], and MRL-1237 {1-(4-fluorophenyl)-2-[(4-imino-1,4-dihydropyridin-1-yl)methyl]benzimidazole hydrochloride}. The ATPase activity of 2C, however, remained unaltered in the presence of TBZE-029.

In order to assess the risk of transmission of viral diseases during floods, the viral burden in flooded areas of the city of Dresden (Germany) in August 2002 was investigated. Water samples were collected from 9 sampling sites and tested for the presence of 11 enteric viral pathogens. As a control, water samples from the same sites were analyzed in seasonal intervals over the following year. A total of 36 samples were collected, 92% (33/36) being positive for at least one virus. Adenovirus type 40/41 was the most frequently detected (53%), followed by astrovirus (50%) and enterovirus (50%). In all samples, low levels of bacteriophages were detected with no specificity as to sampling site and season, indicating a moderate river contamination with wastewater. A striking association between water temperature and viral genome detection was observed, as illustrated in August 2002 (mean water temperature of 17.8°, 8 sites positive for 17 viruses), in comparison to November 2002 (mean water temperature of 7.6°, 9 sites positive for 45 viruses). Importantly, hepatitis A viral RNA was not detected in the flooded area. In conclusion, our results indicate no increased risk for transmission of viral diseases through water contact in flooded areas.

A single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay that detects and identifies Norovirus, Astrovirus and Adenovirus in clinical stool samples is described. Four hundred sixty stool samples were tested from patients with non-rotavirus acute gastroenteritis, that were either stored at -80 degrees C and tested retrospectively, or tested immediately after viral nucleic acid extraction in a prospective manner, including outbreaks of gastroenteritis that occurred in Germany during the winter of 2003. The multiplex RT-PCR was validated against simplex RT-PCR with published primers for Norovirus (JV12/JV13 and p289/p290) and Astrovirus (Mon340/348), and against simplex PCR for Adenovirus. In both retrospective and prospective settings, the multiplex RT-PCR was equally sensitive and specific in detecting non-rotavirus infections compared with simplex RT-PCR/PCR. The specificity of the multiplex RT-PCR was assessed by sequencing of the amplicons that showed high nucleotide identities to Norovirus genogroup I/1, I/4, II/2, or II/4 clades, as well as to Astrovirus serotypes 1, 2, 4, or 8. The multiplex RT-PCR was also more sensitive than Astrovirus and Norovirus antigen enzyme immunoassays (IDEIA, Dako), as well as Astrovirus isolation in cell culture. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of non-rotavirus acute gastroenteritis.

Infections with herpesvirus and adenovirus is a major cause of morbidity and mortality in patients undergoing stem cell transplantation (SCT). In those patients, the frequency of herpesvirus and adenovirus co-infections as well as their contribution to the infectious burden remain so far unclear. In this study, we have postulated that co-infections with herpesviruses and adenoviruses are frequent in the 100 days following SCT, contributing significantly to the infectious burden. To address this issue, 615 blood samples collected from patients undergoing SCT (n=35) were retrospectively tested by quantitative real-time PCR for the presence of HSV 1/2, VZV, EBV, CMV, HHV6 A/B, and adenovirus. Infections with a single virus were detected in 46.1% (283/615) of the samples, whereas co-infections with two or three viruses were detected in 9.9% (61/615) and 2.1% (13/615) of the samples, respectively. Interestingly, about 50% of the patients (17/35) displayed a co-infection with two or more viruses, with [CMV+HHV6 A/B], and [EBV + HHV6 A/B +adenovirus] being most frequently detected, respectively. Comparison of viral loads indicated that the infectious burden increases proportionally to the number of viruses detected. Moreover, a DNA-microarray allowing simultaneous detection of HSV 1/2, VZV, EBV, CMV, HHV6 A/B, and adenovirus was developed. The assay displayed a high diagnostic accuracy, being an attractive device for routine assessment of the infectious burden in immunocompromised patients after SCT.

Herpesviruses and adenovirus are an important cause of end-organ disease (EOD) and are associated with graft versus host disease (GVHD) in patients undergoing allogeneic stem cell transplantation (SCT). Detection and monitoring of those viruses is routinely performed by simplex quantitative real-time PCR. Since more than one herpes- or adenovirus are reported to be present shortly after transplantation, detection of all possible (sub-)clinical viral infections is still time-consuming and cost-intense. We therefore designed a DNA-microarray for the simultaneous detection of herpes-simplex-virus-1 and -2 (HSV-1/2), cytomegalovirus (CMV), varicella-zoster-virus (VZV), Epstein-Barr-virus (EBV), human herpesvirus-6 (HHV-6) and adenovirus. The DNA-microarray was validated in comparison to simplex quantitative PCR routinely used in our diagnostic laboratory. We retrospectively screened 608 samples of 35 patients undergoing allogeneic SCT on a regular basis until day 100 post-transplantation. The detection limit of the DNA-microarray is 10 genome equivalents (GE)/ml. We detected one or more viruses in 367 samples (60,2%). Of the positive samples, CMV was detected in 272 (74,1%), EBV in 103 (28,1%), HHV-6 in 81 (22,1%), adenovirus in 35 (9,5%), HSV-1/2 in 21 (5,7%) and VZV in 14 samples (5,2%), respectively. Of the samples containing two viruses (n=62), we detected CMV/EBV in 19 (30,6%), CMV/HHV-6 in 27 (43,6%) and HHV-6/EBV in 7 (11,3%) samples, respectively. CMV/EBV/HHV-6 was detected in 4 (36,4%) of the samples containing 3 viruses (n=11). Results of the DNA-microarray showed good overall agreement in comparison to the simplex quantitative PCR. We conclude that the DNA-microarray is a highly sensitive and specific method for the simultaneous detection of up to six viruses in a single sample. By screening and monitoring SCT-patients for viruses known to be associated with EOD or GVHD we herewith introduce an innovative molecular technique that can simultaneously detect multiple viral infections on a large scale. The assay is currently used to determine the impact of multi-virus detection on clinical end-points like GVHD and infectious complications in patients undergoing allogeneic stem cell transplantation.

Caliciviridae are RNA viruses with single stranded positive-oriented genome causing a broad spectrum of diseases such as acute gastroenteritis in humans. The structures of the RNA-dependent RNA-polymerase (RdRp) of several Caliciviruses have been reported. The RdRp is predicted to play a key role in genome replication, as well as in the synthesis and amplification of subgenomic RNA.Starting from crystal structures of human (hNV) and Murine Norwalk virus (MNV) RdRp, we performed an in silico docking search to identify commercially available compounds with predicted high affinity for the enzyme active site. The best candidates were tested in vitro to assay their effective inhibition of MNV and hNV RdRp. The results of such combined computational and experimental screening approach led to the identification of two high-potency inhibitors: EM01 and EM02. The crystal structure of MNV in the presence of the two inhibitors showed a common binding site close to the protein active site where the addition of new nucleotides to the nascent RNA occurs. From such structures we could identify the main residues involved in inhibitor binding. We inserted a point mutation in one of these key residues showing the reduction of inhibitory potency in both MNV and hNV RdRps. Finally, we identified a common moiety present in the two inhibitors likely carrying most of the inhibitory power. Such fragment can be elongated/modified to enhance its druglikeness in order to develop novel anti-viral drugs.