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There are currently no antivirals available to treat infection with enterovirus A71 (EV-A71) or any other enterovirus. The extensively studied capsid binders rapidly select for drug-resistant variants. We here explore whether the combination of two direct-acting enterovirus inhibitors with a different mechanism of action may delay or prevent resistance development to the capsid binders. To that end, the in vitro dynamics of resistance development to the capsid binder pirodavir was studied either alone or in combination with a viral 2C-targeting compound (SMSK_0213), a viral 3C-protease inhibitor (rupintrivir) or a viral RNA-dependent RNA polymerase inhibitor (7DMA). We demonstrate that combining pirodavir with either rupintrivir or 7DMA delays the development of resistance to pirodavir and that no resistance to the protease or polymerase inhibitor develops. The combination of pirodavir with the 2C inhibitor results in a double-resistant virus population, where only the minority carries the resistant mutation. ispartof: ACS INFECTIOUS DISEASES vol:7 issue:10 pages:2801-2806 ispartof: location:United States status: published

Akt (Protein kinase B) is a key signaling protein in eukaryotic cells that controls many cellular processes such as glucose metabolism and cell proliferation for survival. As obligate intracellular pathogens, viruses modulate host cellular processes, including Akt signaling, for optimal replication. The mechanisms by which viruses modulate Akt and the resulting effects on the infectious cycle differ widely depending on the virus. In this study, we explored the effect of Akt serine 473 phosphorylation (p-Akt) during murine norovirus (MNV) infection. p-Akt increased during infection of murine macrophages with acute MNV-1 and persistent CR3 and CR6 strains. Inhibition of Akt with MK2206, an inhibitor of all three isoforms of Akt (Akt1/2/3), reduced infectious virus progeny of all three virus strains. This reduction was due to decreased viral genome replication (CR3), defective virus assembly (MNV-1), or diminished cellular egress (CR3 and CR6) in a virus strain-dependent manner. Collectively, our data demonstrate that Akt activation increases in macrophages during the later stages of the MNV infectious cycle, which may enhance viral infection in unique ways for different virus strains. The data, for the first time, indicate a role for Akt signaling in viral assembly and highlight additional phenotypic differences between closely related MNV strains. Importance Human noroviruses (HNoV) are a leading cause of viral gastroenteritis, resulting in high annual economic burden and morbidity; yet there are no small animal models supporting productive HNoV infection, or robust culture systems producing cell culture-derived virus stocks. As a result, research on drug discovery and vaccine development against norovirus infection has been challenging, and no targeted antivirals or vaccines against HNoV are approved. On the other hand, murine norovirus (MNV) replicates to high titers in cell culture and is a convenient and widespread model in norovirus research. Our data demonstrate the importance of Akt signaling during the late stage of the MNV life cycle. Notably, the effect of Akt signaling on genome replication, virus assembly and cellular egress is virus strain specific, highlighting the diversity of biological phenotypes despite small genetic variability among norovirus strains. This study is the first to demonstrate a role for Akt in viral assembly.

Background Previous studies demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be detected for weeks after infection. The significance of this finding is unclear and, in most patients, does not represent active infection. Detection of subgenomic RNA has been proposed to represent productive infection and may be a useful marker for monitoring infectivity. Methods We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR) to quantify total and subgenomic nucleocapsid (sgN) and envelope (sgE) transcripts in 185 SARS-CoV-2–positive nasopharyngeal swab samples collected on hospital admission and to relate to symptom duration. Results We find that all transcripts decline at the same rate; however, sgE becomes undetectable before other transcripts. The median duration of symptoms to a negative test is 14 days for sgE and 25 days for sgN. There is a linear decline in subgenomic compared to total RNA, suggesting that subgenomic transcript copy number is dependent on copy number of total transcripts. The mean difference between total and sgN is 16-fold and the mean difference between total and sgE is 137-fold. This relationship is constant over duration of symptoms, allowing prediction of subgenomic copy number from total copy number. Conclusions Subgenomic RNA may be no more useful in determining infectivity than a copy number threshold determined for total RNA.

Vapendavir is a rhino/enterovirus inhibitor that targets a hydrophobic pocket in the viral capsid preventing the virus from entering the cell. We set out to study and compare the molecular mechanisms of resistance to vapendavir among clinically relevant Picornavirus species. To this end in vitro resistance selection of drug-resistant isolates was applied in rhinovirus 2 and 14, enterovirus-D68 and Poliovirus 1 Sabin. Mutations in the drug-binding pocket in VP1 (C199R/Y in hRV14; I194F in PV1; M252L and A156T in EV-D68), typical for this class of compounds, were identified. Interestingly, we also observed mutations located outside the pocket (K167E in EV-D68 and G149C in hRV2) that contribute to the resistant phenotype. Remarkably, the G149C substitution rendered the replication of human rhinovirus 2 dependent on the presence of vapendavir. Our data suggest that the binding of vapendavir to the capsid of the G149C isolate may be required to stabilize the viral particle and to allow efficient dissemination of the virus. We observed the dependency of the G149C isolate on other compounds of this class, suggesting that this phenotype is common for capsid binders. In addition the VP1 region containing the G149C substitution has not been associated with antiviral resistance before. Our results demonstrate that the phenotype and genotype of clinically relevant vapendavir-resistant picornavirus species is more complex than generally believed. ispartof: ANTIVIRAL RESEARCH vol:195 ispartof: location:Netherlands status: published

Human noroviruses are the leading cause of severe childhood diarrhea worldwide, yet we know little about their pathogenic mechanisms. Murine noroviruses cause diarrhea in interferon-deficient adult mice but these hosts also develop systemic pathology and lethality, reducing confidence in the translatability of findings to human norovirus disease. Herein we report that a murine norovirus causes self-resolving diarrhea in the absence of systemic disease in wild-type neonatal mice, thus mirroring the key features of human norovirus disease and representing a norovirus small animal disease model in wild-type mice. Intriguingly, lymphocytes are critical for controlling acute norovirus replication while simultaneously contributing to disease severity, likely reflecting their dual role as targets of viral infection and key components of the host response., Here the authors show that a murine norovirus causes self-resolving diarrhea in the absence of systemic disease in wild-type neonatal mice and show that lymphocytes affect disease in a dual manner. The data suggests that neonatal mice are a promising model for further studies.

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.

Human noroviruses are the leading cause of severe childhood diarrhea worldwide yet we know very little about their pathogenic mechanisms. Murine noroviruses cause diarrhea in interferon-deficient adult mice but these hosts also develop systemic pathology and lethality, reducing confidence in the translatability of findings to human norovirus disease. Herein we report that a murine norovirus causes self-resolving diarrhea in the absence of systemic disease in wild-type neonatal mice, thus mirroring the key features of human norovirus disease and representing a robust norovirus small animal disease model. Intriguingly, lymphocytes are critical for controlling acute norovirus replication while simultaneously contributing to disease severity, likely reflecting their dual role as targets of viral infection and key components of the host response.

There are currently no antivirals available to treat infection with enterovirus A71 (EV-A71) or any other enterovirus. The extensively studied capsid binders select rapidly for drug-resistant variants. We here explore whether the combination of two direct-acting enterovirus inhibitors with a different mechanism of action may delay or prevent resistance development to the capsid binders. To that end, the in vitro dynamics of resistance development to the capsid binder pirodavir was studied either alone or in combination with (i) a viral 2C-targeting compound (SMSK_0213), (ii) a viral 3C-protease inhibitor (rupintrivir) or (iii) a viral RNA-dependent RNA polymerase (RdRp) inhibitor [7-deaza-2’C-methyladenosine (7DMA)]. We demonstrate that combining pirodavir with either rupintrivir or the nucleoside analogue 7DMA delays the development of resistance to pirodavir and that no resistance to the protease or polymerase inhibitor develops. The combination of pirodavir with the 2C inhibitor results in a double-resistant virus population. The deep sequencing analysis of resistant populations revealed that even though resistant mutations are present in less than 30% of the population, this still provides the resistant phenotype.

Currently, there are only three FDA-approved drugs that inhibit human immunodeficiency virus (HIV) entry-fusion into host cells. The situation is even worse for enterovirus EV71 infection for which no antiviral therapies are available. We describe here the discovery of potent entry dual inhibitors of HIV and EV71. These compounds contain in their structure three or four tryptophan (Trp) residues linked to a central scaffold. Critical for anti-HIV/EV71 activity is the presence of extra phenyl rings, bearing one or two carboxylates, at the C2 position of the indole ring of each Trp residue. The most potent derivatives, 22 and 30, inhibit early steps of the replicative cycles of HIV-1 and EV-A71 by interacting with their respective viral surfaces (glycoprotein gp120 of HIV and the fivefold axis of the EV-A71 capsid). The high potency, low toxicity, facile chemical synthesis, and great opportunities for chemical optimization make them useful prototypes for future medicinal chemistry studies., This work was supported by the Spanish Plan Nacional (Subprograma Retos) [projects SAF2015-64629-C2-1-R and SAF2015-64629-C2-2-R (MINECO/FEDER)], the Spanish “Ministerio de Ciencia, Innovación y Universidades” (project PID2019-104070RB-C21), and by the Spanish Agencia Estatal Consejo Superior de Investigaciones Cientifí cas (CSIC, Projects CSIC-PIE-201980E100 and CSIC-PIE- 201980E028), “The Centers of Excellence” of the KU Leuven (EF-05/15 and PF-10/18), EU FP7 (FP7/2007−2013) Project EUVIRNA (Grant 408 Agreement 264286), EU FP7 SILVER (Contract HEALTH-F3-2010-260644), a grant from the Belgian Interuniversity Attraction Poles (IAP) Phase VIIP7/ 45 (BELVIR), and the EU FP7 Industry-Academia Partnerships and Pathways Project AIROPICO. The Spanish MEC/MINECO is also acknowledged for grants to B.M.-G. and O.M.-M. and the China Scholarship Council (CSC) (grant 201403250056) for a grant to L.S. S.H. received a grant from the Pennsylvania Department of Health using Tobacco CURE Funds. We also thank Charlotte Vanderheydt, Sandra Claes, and Evelyne Van Kerckhove for helping with the processing of the antiviral data. This work has been awarded with the Janssen (XVIII call) and Esteve (XIX call) prizes within the “Prizes for Young Researchers of the Spanish Society of Medicinal Chemistry (SEQT).

OBJECTIVES: We report the use of reconstituted 3D human airway epithelium cells (HuAECs) of bronchial origin in an air-liquid interface to study respiratory syncytial virus (RSV) infection and to assess the efficacy of RSV inhibitors in (pre-)clinical development. METHODS: HuAECs were infected with RSV-A Long strain (0.01 CCID50/cell, where CCID50 represents 50% cell culture infectious dose in HEp2 cells) on the apical compartment of the culture. At the time of infection or at 1 or 3 days post-infection, selected inhibitors were added and refreshed daily on the basal compartment of the culture. Viral shedding was followed up by apical washes collected daily and quantifying viral RNA by RT-qPCR. RESULTS: RSV-A replicates efficiently in HuAECs and viral RNA is shed for weeks after infection. RSV infection reduces the ciliary beat frequency of the ciliated cells as of 4 days post-infection, with complete ciliary dyskinesia observed by day 10. Treatment with RSV fusion inhibitors resulted in an antiviral effect only when added at the time of infection. In contrast, the use of replication inhibitors (both nucleoside and non-nucleoside) elicited a marked antiviral effect even when the start of treatment was delayed until 1 day or even 3 days after infection. Levels of the inflammation marker RANTES (mRNA) increased ∼200-fold in infected, untreated cultures (at 3 weeks post-infection), but levels were comparable to those of uninfected cultures in the presence of PC786, an RSV replication inhibitor, suggesting that an efficient antiviral treatment might inhibit virus-induced inflammation in this model. CONCLUSIONS: Overall, HuAECs offer a robust and physiologically relevant model to study RSV replication and to assess the efficacy of antiviral compounds. ispartof: JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY vol:73 issue:7 pages:1823-1829 ispartof: location:England status: published

Individual virus particles have long been accepted as the infectious unit during cellular infection and host-to-host transmission. In this issue of Cell Host & Microbe, Santiana et al. (2018) uncover vesicle-cloaked rotavirus and norovirus clusters in feces of infected hosts that are more infectious than free virus particles during fecal-oral transmission.

We have previously reported a new class of dendrimers with tryptophan (Trp) residues on the surface that show dual antiviral activities against HIV and enterovirus EV71. The prototype compound of this family is a derivative of pentaerythritol with 12 peripheral Trp groups and trivalent spacer arms. Here a novel series of dendrimers with divalent and tetravalent branched arms, instead of the trivalent ones present on the prototype, has been synthesized and its activity against HIV, EV71 and a panel of 16 different viruses and other pathogens has been determined. Convergent or divergent approaches have been used for the synthesis of these compounds. Our findings demonstrate that only compounds with tetravalent branched arms showed the same anti-HIV and anti-EV71 activity of the prototype (low micromolar) and even gain significant antiviral activity against new pathogens such as HSV-2, adenovirus-2, human corona virus and respiratory syncytial virus, being the first members of the Trp dendrimer family that showed activity against those viruses. As the prototype, these compounds also showed low-nanomolar activity against a representative EV71 clinical isolate. Experimental work carried on to determine the mode of action of the most potent IIa, containing tetravalent branched arms, demonstrated that it interacts with the viral envelopes of HIV, EV71 and HSV-2 and thus may prevent virus attachment to the host cell. These results support the interest of this new series of Trp dendrimers and qualify them as useful prototypes for the development of novel inhibitors of viral entry with broad antiviral spectrum., This work has been supported by the Spanish Ministerio de Economía, Industria y Competitividad (MINECO) [project SAF2015- 64629-C2-1-R (MINECO/FEDER)], the Spanish Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC, Project CSIC201680E079), “The Centers of Excellence” of the KU Leuven (EF- 05/15 and PF-10/18), EU FP7 (FP7/2007–2013) Project EUVIRNA (Grant 408 Agreement 264286), EU FP7 SILVER (Contract HEALTH-F3- 2010-260644), a grant from the Belgian Interuniversity Attraction Poles (IAP) Phase VII–P7/45 (BELVIR) and the EU FP7 Industry-Academia Partnerships and Pathways Project AIROPICO. The Spanish MEC/ MINECO is also acknowledged for a grant to B.M.G and the China Scholarship Council (Grant 201403250056) for a grant to L.S. We also thank Charlotte Vanderheydt, Caroline Collard, Kim Donckers, Sandra Claes and Evelyne Van Kerckhove for help with the processing of the antiviral data and Arnaud Boonen and Sam Noppen with the generation of the SPR data.

International audience; Rhinoviruses (RVs) have been linked to exacerbations of many pulmonary diseases, thus increasing morbidity and/or mortality in subjects at risk. Unfortunately, the wide variety of RV genotypes constitutes a major hindrance for the development of Rhinovirus replication inhibitors. In the current investigation, we have developed a novel series of pyrazole derivatives that potently inhibit the Rhinovirus replication. Compounds 10e and 10h behave as early stage inhibitors of Rhinovirus infection with a broad-spectrum activity against RV-A and RV-B species (EC 50 < 0.1 μM). We also evaluate the dynamics of the emerging resistance of these promising compounds and their in vitro genotoxicity. Molecular docking experiments shed light on the pharmacophoric elements interacting with residues of the drug-binding pocket.

We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability.This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry.F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity).F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.

International audience; Rhinovirus (RV), member of the Enterovirus genus, is known to be involved in more than half of the common colds. Through advances in molecular biology, rhinoviruses have also been associated with exacerbations of chronic pulmonary diseases (e.g. asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis). In the current investigation, we develop a novel series of 4,5-dimethoxybenzyl derivatives that potently inhibits rhinovirus replication. Compound (S)-7f blocks RV-B14 replication with an EC50 value of 0.25 mu M and shows a low toxicity in HeLa cells (CC50 \\textgreater 271 mu M). Enantioseparation followed by an absolute configuration determination by a Mosher's method revealed the interest of enantiopure compounds. Molecular docking studies permitted the identification of key biological interactions within the drug-binding pocket and an in silico drug-like study revealed a good potential for the development of these derivatives. (C) 2017 Elsevier Masson SAS. All rights reserved.

Rhinoviruses are exclusive respiratory pathogens and the etiological agents of the common cold. These viruses are increasingly reported to cause exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Here, we review the role of rhinovirus infections in the pathogenesis of asthma and COPD and we discuss the current and potential future treatments. We propose that, in order to prevent exacerbations, the design of novel therapeutics should focus on directly acting antivirals but also include the design of drugs that simultaneously inhibit viral replication and alleviate symptoms of asthma and COPD. ispartof: F1000Research vol:6 pages:1860- ispartof: location:England status: published

We have shown that the circulating vaccine-derived polioviruses responsible for poliomyelitis outbreaks in Madagascar have recombinant genomes composed of sequences encoding capsid proteins derived from poliovaccine Sabin, mostly type 2 (PVS2), and sequences encoding nonstructural proteins derived from other human enteroviruses. Interestingly, almost all of these recombinant genomes encode a nonstructural 3A protein related to that of field coxsackievirus A17 (CV-A17) strains. Here, we investigated the repercussions of this exchange, by assessing the role of the 3A proteins of PVS2 and CV-A17 and their putative cellular partners in viral replication. We found that the Golgi protein acyl-coenzyme A binding domain-containing 3 (ACBD3), recently identified as an interactor for the 3A proteins of several picornaviruses, interacts with the 3A proteins of PVS2 and CV-A17 at viral RNA replication sites, in human neuroblastoma cells infected with either PVS2 or a PVS2 recombinant encoding a 3A protein from CV-A17 [PVS2-3A(CV-A17)]. The small interfering RNA-mediated downregulation of ACBD3 significantly increased the growth of both viruses, suggesting that ACBD3 slowed viral replication. This was confirmed with replicons. Furthermore, PVS2-3A(CV-A17) was more resistant to the replication-inhibiting effect of ACBD3 than the PVS2 strain, and the amino acid in position 12 of 3A was involved in modulating the sensitivity of viral replication to ACBD3. Overall, our results indicate that exchanges of nonstructural proteins can modify the relationships between enterovirus recombinants and cellular interactors and may thus be one of the factors favoring their emergence.

How the overlap between the hepatitis B virus (HBV) reverse transcriptase (RT) and HBV S antigen (HBsAg) genes modulates the extent of HBV genetic variability is still an open question, and was investigated here. The rate of nucleotide conservation (≤1 % variability) followed an atypical pattern in the RT gene, due to an overlap between RT and HBsAg (69.9 % nucleotide conservation in the overlapping region vs 41.2 % in the non-overlapping region; PP = 3.249×10−22]. The most conserved RT regions were located within the YMDD motif and the N-terminal parts of the palm and finger domains, critical for RT functionality. These regions also corresponded to highly conserved HBsAg domains that are critical for HBsAg secretion. Conversely, the genomic region encoding the HBsAg antigenic loop (where immune-escape mutations are localized) showed a sharp decrease in the extent of conservation (40.6 %), which was less pronounced in the setting of human immunodeficiency virus (HIV)-driven immune suppression (48.8 % in HIV–HBV co-infection vs 21.5 % in mono-infected patients; P = 0.020). In conclusion, the overlapping reading frame and the immune system appear to have shaped the patterns of RT and HBsAg genetic variability. Highly conserved regions in RT and HBsAg may deserve further attention as novel therapeutic targets.

Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed the NS5B polymerase genetic variability in circulating HCV genotypes/subtypes and its impact on the genetic barrier for the development of resistance to clinically relevant nucleoside inhibitors (NIs)/nonnucleoside inhibitors (NNIs). The study included 1,145 NS5B polymerase sequences retrieved from the Los Alamos HCV database and GenBank. The genetic barrier was calculated for drug resistance emergence. Prevalence and genetic barrier were calculated for 1 major NI and 32 NNI resistance variants (13 major and 19 minor) at 21 total NS5B positions. Docking calculations were used to analyze sofosbuvir affinity toward the diverse HCV genotypes. Overall, NS5B polymerase was moderately conserved among all HCV genotypes, with 313/591 amino acid residues (53.0%) showing ≤1% variability and 83/591 residues (14.0%) showing high variability (≥25.1%). Nine NNI resistance variants (2 major variants, 414L and 423I; 7 minor variants, 316N, 421V, 445F, 482L, 494A, 499A, and 556G) were found as natural polymorphisms in selected genotypes. In particular, 414L and 423I were found in HCV genotype 4 (HCV-4) ( n = 14/38, 36.8%) and in all HCV-5 sequences ( n = 17, 100%), respectively. Regardless of HCV genotype, the 282T major NI resistance variant and 10 major NNI resistance variants (316Y, 414L, 423I/T/V, 448H, 486V, 495L, 554D, and 559G) always required a single nucleotide substitution to be generated. Conversely, the other 3 major NNI resistance variants (414T, 419S, and 422K) were associated with a different genetic barrier score development among the six HCV genotypes. Sofosbuvir docking analysis highlighted a better ligand affinity toward HCV-2 than toward HCV-3, in agreement with the experimental observations. The genetic variability among HCV genotypes, particularly with the presence of polymorphisms at NNI resistance positions, could affect their responsiveness to NS5B inhibitors. A pretherapy HCV NS5B sequencing could help to provide patients with the full efficacy of NNI-containing regimens.

Summary Introduction The identification of novel reverse-transcriptase (RT) drug-resistance mutations is critical in predicting the probability of success to anti-HBV treatment. Furthermore, due to HBV-RT/HBsAg gene-overlap, they can have an impact on HBsAg-detection and quantification. Methods 356 full-length HBV-RT sequences from 197 drug-naive patients and 159 patients experiencing virological-breakthrough to nucleoside/nucleotide-analogs (NUCs) were analyzed. Mutants and wild-type HBs-antigens were expressed in HuH7-hepatocytes and quantified in cell-supernatants and cell-lysates by Architect HBsAg-assay. Results Ten novel RT-mutations (rtN53T-rtS78T-rtS85F-rtS135T-rtA181I-rtA200V-rtK212Q-rtL229V/F-rtM309K) correlated with specific NUC-treatments and classical drug-resistance mutations on divergent evolutionary pathways. Some of them reduced RT-binding affinity for anti-HBV drugs and altered S-antigen structure. Indeed, rtS78T (prevalence: 1.1% in drug-naive and 12.2% in adefovir-failing patients) decreased the RT-affinity for adefovir more than the classical adefovir-resistance mutations rtA181 T/V (WT:−9.63 kcal/mol, rtA181T:−9.30 kcal/mol, rtA181V:−7.96 kcal/mol, rtS78T:−7.37 kcal/mol). Moreover, rtS78T introduced a stop-codon at HBsAg-position 69, and completely abrogated HBsAg-quantification in both supernatants and cell-lysates, indicating an impaired HBsAg-secretion/production. Furthermore, the HBsAg-mutation sP217L, silent in RT, significantly correlated with M204V/I-related virological-breakthrough and increased HBsAg-quantification in cell-lysate. Conclusions Mutations beyond those classically known can affect drug-binding affinity of mutated HBV-RT, and may have potential effects on HBsAg. Their cumulative effect on resistance and HBV-pathogenicity indicates the importance of preventing therapeutic failures.

Background and Aims: Every year globally WHO reports nearly 20 million Hepatitis E virus (HEV) infections. The disease has potential to cause massive epidemics. The reservoir of HEV during interepidemic period in India is not well characterized, with evidences of deficient zoonotic links. Further, the sporadic cases usually lack history of contact with clinically overt HEV patients. In the present context the occurrence of subclinical HEV as a possible reservoir of infection in endemic region was evaluated. Methods: Blood samples were collected from 67 apparently-healthy individuals and 10 acute viral hepatitis (AVH) patients during two HEV outbreaks in North India. The serum samples were tested for anti-HEV IgM, IgG, HEV-IgG avidity-index, HEV viral-load and conventional-PCR followed by sequencing and phylogenetic analysis. Results: A total of 14 (20.89%) apparently-healthy individuals showed presence of both anti-HEV IgM and IgG. Based on HEVIgG avidity-index, 9 apparently-healthy individuals (64.28%) had secondary-exposure, 4 (28.57%) had primary-exposure, while one patient had intermediate-avidity. Subclinical subjects with primaryexposure had significantly higher anti-HEV IgM index as compared to secondary-exposure. Viral-load in clinically jaundiced patients was significantly higher as compared to subclinical subjects (p < 0.0001). Phylogenetic analysis showed HEV sequences retrieved from subclinical individuals (Genotype 1a) clustered along with AVH patients reported in the same outbreak suggesting matched origin. The significantly low viral-load in subclinical subjects hints towards the dose dependency for progression of clinical manifestation. Conclusions: We document subclinical passage of HEV occurs without producing clinically overt hepatitis goes un-noticed which, in turn helps maintaining the virus in nature possibly leading to its endemicity.

430 KEY GENETIC SIGNATURES IN THE WHOLE pre-S1/Pre-S2/S GENE CORRELATE WITH HBV-INDUCED CARCINOGENESIS BY AFFECTING HBsAg SECRETION AND RELEASE V. Svicher, M. Neumann-Fraune, C. Mirabelli, R. Salpini, V. Cento, V.-C. Di Maio, A. Bertoli, C. Alteri, M. Aragri, C. Gori, V. Micheli, G. Gubertini, P. Trimoulet, H. Fleury, J. Vecchiet, N. Iapadre, A. Barlattani, T. Mari, C. Pasquazzi, C. Sarrecchia, F. Ceccherini-Silberstein, M. Andreoni, M. Angelico, J. Verheyen, C.-F. Perno. University of Rome Tor Vergata, Rome, Italy; University of Cologne, Cologne, Germany; National Institute for Infectious Diseases ‘L. Spallanzani’, Rome, “L. Sacco” Hospital, Milan, Italy; Hopital Pellegrin Tripode, Bordeaux, France; “SS Annunziata” Hospital, Chieti, “S Salvatore” Hospital, L’Aquila, “S Giacomo” Hospital, “Regina Margherita” Hospital, “S Andrea” Hospital, Rome, Italy; University of Essen, Essen, Germany E-mail: valentina.svicher@uniroma2.it

Rhino- and enteroviruses are important human pathogens, against which no antivirals are available. The best-studied inhibitors are “capsid binders” that fit in a hydrophobic pocket of the viral capsid. Employing a new class of entero-/rhinovirus inhibitors and by means of cryo–electron microscopy (EM), followed by resistance selection and reverse genetics, we discovered a hitherto unknown druggable pocket that is formed by viral proteins VP1 and VP3 and that is conserved across entero-/rhinovirus species. We propose that these inhibitors stabilize a key region of the virion, thereby preventing the conformational expansion needed for viral RNA release. A medicinal chemistry effort resulted in the identification of analogues targeting this pocket with broad-spectrum activity against Coxsackieviruses B (CVBs) and compounds with activity against enteroviruses (EV) of groups C and D, and even rhinoviruses (RV). Our findings provide novel insights in the biology of the entry of entero-/rhinoviruses and open new avenues for the design of broad-spectrum antivirals against these pathogens., This study identifies a novel druggable pocket present in most enterovirus and rhinovirus particles, using this to develop broader-spectrum antivirals that inhibit enteroviruses from different groups, probably by interfering with viral RNA release.