Akt phosphorylation of HCV NS5B regulates polymerase activity and HCV infection

Hepatitis C virus (HCV) is a single-stranded RNA virus of positive polarity [ssRNA(+)] that replicates its genome through the activity of one of its proteins, called NS5B. This viral protein is responsible for copying the positive-polarity RNA genome into a negative-polarity RNA strand, which will be the template for new positive-polarity RNA genomes. The NS5B protein is phosphorylated by cellular kinases, including Akt. In this work, we have identified several amino acids of NS5B that are phosphorylated by Akt, with positions S27, T53, T267, and S282 giving the most robust results. Site-directed mutagenesis of these residues to mimic (Glu mutants) or prevent (Ala mutants) their phosphorylation resulted in a reduced NS5B in vitro RNA polymerase activity, except for the T267E mutant, the only non-conserved position of all those that are phosphorylated. In addition, in vitro transcribed RNAs derived from HCV complete infectious clones carrying mutations T53E/A and S282E/A were transfected in Huh-7.5 permissive cells, and supernatant viral titers were measured at 6 and 15 days post-transfection. No virus was rescued from the mutants except for T53A at 15 days post-transfection whose viral titer was statistically lower as compared to the wild type. Therefore, phosphorylation of NS5B by cellular kinases is a mechanism of viral polymerase inactivation. Whether this inactivation is a consequence of interaction with cellular kinases or a way to generate inactive NS5B that may have other functions are questions that need further experimental workMinisterio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 from Instituto de Salud Carlos III, and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). CP was supported by the Miguel Servet program of the Instituto de Salud Carlos III (CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CG-C was supported by the predoctoral contract PRE2018-083422 from MCIU. CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) was funded by the Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMS

Hepatitis E Virus Epidemiology in Industrialized Countries

To determine the prevalence of Hepatitis E virus (HEV) in industrialized nations, we analyzed the excretion of HEV strains by the populations of Spain, France, Greece, Sweden, and the United States. Twenty of 46 (43.5%) urban sewage samples collected in Barcelona from 1994 to 2002 tested positive for HEV. We identified 15 HEV strains, which were similar to two HEV isolates previously described in Barcelona in clinical samples and to strains from diverse geographic HEV-nonendemic areas. We also identified two HEV strains in sewage samples from Washington, D.C., and Nancy, France; these samples were also positive for Hepatitis A virus. In addition, we studied the role of pigs as a reservoir for HEV and identified one new swine HEV strain. Our results suggest that HEV may be more prevalent than previously considered in industrialized countries and that variants of the virus circulate simultaneously in one region

Epidemiologia molecular del virus de l'hepatitis E (VHE) en zones industrialitzades

[cat] El virus de l'Hepatitis E (VHE) és un virus que es transmet per via fecal-oral i causa hepatitis agudes. Tradicionalment les regions tropicals i subtropicals d'Àsia, Àfrica i Amèrica Central s'han considerat zones endèmiques pel VHE, on aquest és el responsable tant de brots epidèmics com de casos esporàdics d'hepatitis agudes. A regions industrialitzades, com Espanya, tradicionalment no endèmiques i lliures del virus, únicament es diagnostiquen alguns casos esporàdics d'infeccions pel VHE, associats majoritàriament a soques importades durant viatges a regions endèmiques. Malgrat els pocs casos diagnosticats, els valors de seroprevalença trobats en aquestes regions varien entre un 1 i un 5%, essent en algunes zones superiors. A més, en els últims anys s'estan identificant casos d'infeccions agudes pel VHE en àrees industrialitzades associats a soques diferents a les de regions endèmiques i que s'estan considerant soques autòctones d'aquestes regions. L'objectiu general d'aquesta tesi és l'estudi de l'epidemiologia molecular del VHE en regions industrialitzades, mitjançant la identificació de soques causants d'infeccions a la població i de possibles reservoris animals.S'analitzaren mostres d'aigua residual recollides a l'entrada d'una planta depuradora de la ciutat de Barcelona. Es detectà el genoma del virus en 24 de las 51 mostres analitzades (47,0%), 22 de les quals pertanyien al període entre desembre de 2000 i juny de 2002. S'identificaren 18 seqüències del VHE diferents. També s'analitzaren mostres d'aigua residual procedents de 4 regions considerades no endèmiques pel virus: Washington D.C. (EUA), Patras (Grècia), Umeå (Suècia) i Nancy (França), detectant-lo en 1 mostra de Washington i una de Nancy. Un estudi de diversitat va mostrar l'existència de múltiples soques del VHE infectant simultàniament la població.També s'analitzaren mostres de sèrum de pacients amb hepatitis agudes i IgG antiVHE en el moment de l'hepatitis. S'aïllà la soca causant de la infecció de 3 pacients, en un cas després d'un viatge a Etiòpia. L'estudi de marcadors d'infecció aguda pel VHE mostrà que els 3 pacients als quals se'ls hi havia detectat el genoma del virus també tenien nivells detectables d'IgM antiVHE. També s'identificaren casos amb possible diagnòstic d'hepatitis aguda pel VHE amb absència d'IgM antiVHE però amb increment dels nivells d'IgG antiVHE i disminució posterior, així com possibles coinfeccions con altres virus causants d'hepatitis.S'analitzaren mostres de sèrum i femta de porcs procedents de 3 granges comercials situades a Catalunya. A una d'elles es detectà una seroprevalença del 18,2%. Cap animal estudiat de 8 o menys setmanes havia seroconvertit en el moment de la presa de la mostra. S'aïllà la soca del VHE causant de la infecció (soca Por1) i s'observà que era molt similar a les soques identificades infectant la població de Barcelona. A les altres 2 granges no se detectaren infeccions pel VHE. Tampoc s'identificà cap soca del VHE d'origen boví a l'estudi preliminar realitzat.Finalment es va dur a terme un estudi per identificar els aminoàcids de l'epítop neutralitzant del VHE que podien ser potencialment importants per la conformació d'aquest epítop. S'estudià el comportament d'anticossos contra l'epítop neutralitzant de la soca Sar55 (genotip 1) davant la Mex14 (genotip 2) i pèptids mutats de Sar55 que contenien aa presents a Mex14. No s'observaren diferències en el reconeixement per part dels anticossos, confirmant l'existència d'epítops comuns a ambdues soques.[eng] The overall aim of this thesis is the study of the molecular epidemiology of the Hepatitis E virus (HEV) in industrialized countries, traditionally considered non-endemic for this virus. HEV is an important cause of sporadic and epidemic cases of acute hepatitis in endemic areas. In non-endemic regions sporadic cases are often associated to HEV strains imported from endemic areas, although autochthonous strains have also been isolated in industrialized countries, where the seroprevalence values vary from 1 to 5% or higher. Urban sewage samples from Barcelona (Spain), Washington D.C. (USA), Patras (Greece), Umeå (Sweden) and Nancy (France) were processed and tested by nested RT-PCR. HEV was detected in Barcelona (47.0% positives samples, with more than 18 different strains detected), Washington D.C. (20% positives samples) and Nancy (25% positive samples). A high diversity of strains was detected infecting simultaneously the population. The strains identified in these industrialized areas belonged mainly to genotype 3.Cases of acute hepatitis E were also detected in humans living in the area of Barcelona and caused by imported and autochthonous strains. The RNA of the virus was detected in patients with detectable levels of IgM antiHEV, although presumptive infections with only an increase and further decrease of the levels of IgG antiHEV were also identified.Moreover, HEV was detected in a swine herd located in the area of Barcelona, where the seropositive animals were older than 8 weeks. The strain infecting the animals belonged to genotype 3 and was very similar to the human HEV strains detected in humans and sewage in the same area. Finally an attempt to identify amino acids critical for the correct conformation of the neutralization epitope present in the capsid of the HEV was performed. Antibodies against Sar55 (genotype 1) were tested against Mex14 (genotype 2) and mutated peptides with the sequence of the neutralization epitope of Sar55 and some amino acids from Mex14. No differences were observed, confirming the existence of a common neutralization epitope in HEV strains from different genotypes

Hepatitis E Virus in Industrialized Countries: The Silent Threat

Hepatitis E virus (HEV) is the main cause of acute viral hepatitis worldwide. Its presence in developing countries has been documented for decades. Developed countries were supposed to be virus-free and initially only imported cases were detected in those areas. However, sporadic and autochthonous cases of HEV infection have been identified and studies reveal that the virus is worldwide spread. Chronic hepatitis and multiple extrahepatic manifestations have also been associated with HEV. We review the data from European countries, where human, animal, and environmental data have been collected since the 90s. In Europe, autochthonous HEV strains were first detected in the late 90s and early 2000s. Since then, serological data have shown that the virus infects quite frequently the European population and that some species, such as pigs, wild boars, and deer, are reservoirs. HEV strains can be isolated from environmental samples and reach the food chain, as shown by the detection of the virus in mussels and in contaminated pork products as sausages or meat. All these data highlight the need of studies directed to control the sources of HEV to protect immunocompromised individuals that seem the weakest link of the HEV epidemiology in industrialized regions

Akt Interacts with Usutu Virus Polymerase, and Its Activity Modulates Viral Replication

Usutu virus (USUV) is a flavivirus that mainly infects wild birds through the bite of Culex mosquitoes. Recent outbreaks have been associated with an increased number of cases in humans. Despite being a growing source of public health concerns, there is yet insufficient data on the virus or host cell targets for infection control. In this work we have investigated whether the cellular kinase Akt and USUV polymerase NS5 interact and co-localize in a cell. To this aim, we performed co-immunoprecipitation (Co-IP) assays, followed by confocal microscopy analyses. We further tested whether NS5 is a phosphorylation substrate of Akt in vitro. Finally, to examine its role in viral replication, we chemically silenced Akt with three inhibitors (MK-2206, honokiol and ipatasertib). We found that both proteins are localized (confocal) and pulled down (Co-IP) together when expressed in different cell lines, supporting the fact that they are interacting partners. This possibility was further sustained by data showing that NS5 is phosphorylated by Akt. Treatment of USUV-infected cells with Akt-specific inhibitors led to decreases in virus titers (>10-fold). Our results suggest an important role for Akt in virus replication and stimulate further investigations to examine the PI3K/Akt/mTOR pathway as an antiviral target

Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination

In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies

Characterization of the interaction between hepatitis C virus NS5B and the human oestrogen receptor alpha

[EN] The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is part of the viral replicative complex and plays a crucial role in HCV replication. It has been described that NS5B interacts with cellular proteins, and that interactions between NS5B and host proteins are crucial for viral replication. Some of the host factors involved in the HCV replication cycle include the oestrogen receptor alpha (ESR1), protein kinases (c-Src) and chaperones (Hsp70). In this report, we determine the requirements for the interplay between NS5B and the domain C of ESR1 (ESR1C) by using Forster Resonance Energy Transfer. NS5B-ESR1C and ESR1C-ESR1C interactions are dependent on ionic strength, indicating that contacts are mainly electrostatic. Additionally, NS5B residues involved in NS5B oligomerization were also essential for NS5B-ESR1C interaction. The study of the interactions among viral and host factors will provide data to establish innovative therapeutic strategies and the development of new antiviral drugsAuthors gratefully acknowledge Dr Watashi for providing oestrogen receptor domain C and derivative constructs. This work was supported by Consejeria de Educacion de Castilla La Mancha (grant no. PPII10-0243-6857) and Ministerio de Ciencia e Innovacion (BFU2010-18767). I. B. E. was supported by predoctoral fellowships from Fundacion para la Investigacion Sanitaria de Castilla-La Mancha (grant no. MOV-2006_JI/06).Hillung, J.; Ruiz-Lopez, E.; Bellon-Echeverria, I.; Clemente-Casares, P.; Mas, A. (2012). Characterization of the interaction between hepatitis C virus NS5B and the human oestrogen receptor alpha. Journal of General Virology. 93:780-785. https://doi.org/10.1099/vir.0.039396-0S78078593Behrens, S. E., Tomei, L., & De Francesco, R. (1996). 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Putative neutralization epitopes and broad cross-genotype neutralization of Hepatitis E virus confirmed by a quantitative cell-culture assay

Monolayers of Hep G2/C3A cells were inoculated with genotype 1 Hepatitis E virus (HEV) mixed with either anti-HEV or an appropriate control. After 5 or 6 days, cell monolayers were stained with anti-HEV and infected cells were identified by immunofluorescence microscopy and counted. Anti-HEV from vaccinated or infected rhesus monkeys neutralized the virus, as did mAbs that recognized epitopes on the C terminus of a recombinant vaccine protein. Antibodies were broadly cross-reactive, since convalescent serum from animals infected with any one of the four mammalian genotypes all neutralized the genotype 1 virus

Efectos sobre la salud de la contaminación de agua y alimentos por virus emergentes humanos

El desarrollo de tecnologías moleculares aplicadas a estudios ambientales ha permitido constatar que incluso en países altamente industrializados existe una alta prevalencia de virus en el medio ambiente, lo que causa un importante impacto en la salud pública e importantes pérdidas económicas, principalmente a través de la transmisión de virus por agua y alimentos. Concentraciones significativas de virus son detectadas en las aguas vertidas al ambiente y en los biosólidos generados en plantas de tratamiento de agua residual. En este trabajo se describen las características generales de la contaminaci ón ambiental por virus, principalmente por virus emergentes, analizándose con mayor profundidad los virus de la hepatitis E (VHE) y los poliomavirus humanos como los virus contaminantes ambientales de más reciente identificación en países industrializados. Se ha demostrado que existe una elevada prevalencia de los poliomavirus humanos, BK y JC, en agua residual en todos los paí- ses estudiados, lo que implica la potencial transmisión de los virus y de genes potencialmente cancerígenos por vía oral. Estudios recientes demuestran que el patrón epidemiológico de la infección por VHE en países industrializados es complejo y que una gran diversidad de cepas del VHE infecta simultáneamente a la población. El control de la contaminación viral del medio ambiente requiere la estandarización de técnicas moleculares y el desarrollo de un programa de vigilancia que permita valorar parámetros víricos y reducir la diseminación de las enfermedades establecidas y de las infecciones víricas emergentes