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ACE Score Identifies HBeAg-negative Inactive Carriers at a Single-point Evaluation,Regardless of HBV Genotype

Journal of Clinical and Translational Hepatology 2022年第6期10卷 1068-1076页

作者： Luisa Roade Mar Riveiro-Barciela Adriana Palom Francisco Rodríguez-Frías Marta Bes Ariadna Rando María Teresa Salcedo Rosario Casillas Elena Vargas-Accarino David Tabernero Silvia Sauleda rafael esteban María Buti Liver Unit Internal Medicine DepartmentHospital Universitari Vall d’HebronVall d’Hebron Barcelona Hospital CampusBarcelonaSpain Universitat Autònoma de Barcelona(UAB) Department of MedicineBarcelonaSpain Universitat Au-tònoma de Barcelona(UAB) Department of Biochemistry and Molecular BiologyBarcelonaSpain Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas(CIBERehd) Instituto de Salud Carlos IIIMadridSpain Liver Pathology Lab Biochemistry and Microbiology Departments(Clinical Laboratories)Hospital Universitari Vall d’HebronVall d’Hebron Barcelona Hospital CampusBarcelonaSpain Vall d’Hebron Institut de Recerca(VHIR) Liver Diseases GroupBarcelonaSpain Transfusion Safety Laboratory Banc de Sang i TeixitsServei Catalàde la SalutBarcelonaSpain De-partment of Pathology Hospital Universitari Vall d’HebronUniversitat Autònoma de BarcelonaBarcelonaSpain

Background and Aims:Hepatitis B virus(HBV)biomark-ers have been used for a better categorization of patients,even though the lack of simple algorithms and the impact of genotypes limit their *** aim was to assess the usefulness of noninvasive markers for the identification of HBV inactive carriers(ICs)in a single-point evaluation and to design a predictive model for their ***-ods:This retrospective-prospective study included 343 consecutive HBeAg-negative ***,analytical,and virological data were collected,and a liver biopsy was performed if *** were classified at the end of follow-up as ICs,chronic hepatitis B and gray zone.A pre-dictive model was constructed,and validated by 1000-boot-strap ***:After 39 months of follow-up,298 subjects were ICs,36 were chronic hepatitis B CHB,and nine were gray ***-nine(25.9%)individuals re-quired a liver *** HBV DNA hazard ratio(HR)6.0,p<0.001),HBV core-related antigen(HBcrAg)(HR 6.5,p<0.001),and elastography(HR 4.6,p<0.001)were inde-pendently associated with the IC *** ACE score(HBV DNA,HBcrAg,elastography),obtained by bootstrapping,yielded an area under the receiver operating characteris-tics(AUROC)of 0.925(95%CI:0.880-0.970,p<0.001)for identification of *** AUROC for genotype D was 0.95,0.96 for A,0.90 for E,and 0.88 for H/*** ACE score of<1 had a positive predictive value of 99.5%,and a score≤12 points had a diagnostic accuracy of 93.8%.Conclusions:Low baseline HBV DNA,HBcrAg,and liver stiffness were in-dependently associated with the IC phase.A score including those variables identified ICs at a single-point evaluation,and might be applied to implement less intensive follow-up strategies.

关键词： Hepatitis B virus Inactive carrier Liver stiffness HBV DNA Quan-titative HBsAg Core-related antigen.

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Quasispecies dynamics in main core epitopes of hepatitis B virus by ultra-deep-pyrosequencing

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World Journal of Gastroenterology 2012年第42期18卷 6096-6105页

作者： Maria Homs Maria Buti David Tabernero Josep Quer Alex Sanchez Noelia Corral rafael esteban Francisco Rodriguez-Frias Network Medical Research Center Hepatic and Digestive Diseases(CIBERehd)Institute of Health Carlos Ⅲ08036 BarcelonaSpain Department of Biochemistry Vall d'Hebron HospitalAutonomous University of Barcelona08035 BarcelonaSpain Department of Hepatology Vall d' Hebron HospitalAutonomous University of Barcelona08035 BarcelonaSpain Statistics and Bioinformatics Unit Research InstituteVall d'Hebron Hospital08035 BarcelonaSpain

AIM:To investigate the variability of the main immunodominant motifs of hepatitis B virus(HBV) core gene by ultra-deep-pyrosequencing(UDPS).METHODS:Four samples(2 genotype A and 2 genotype D) from 4 treatment-na ve patients were assessed for baseline *** additional samples from one patient(patient 4,genotype D) were selected for analysis:one sample corresponded to a 36-mo treatment-free period from baseline and the other to the time of viral breakthrough after 18 mo of lamivudine *** HBV region analyzed covered amino acids 40 to 95 of the core gene,and included the two main epitopic regions,Th50-69 and *** was carried out in the Genome Sequencer FLX system(454 Life Sciences,Roche).After computer filtering of UDPS data based on a Poisson statistical model,122 813 sequences were *** most conserved position detected by UDPS was analyzed by site-directed mutagenesis and evaluated in cell ***:Positions with highest variability rates were mainly located in the main core epitopes,confirming their role as immune-stimulating *** addition,the distribution of variability showed a relationship with HBV *** 1(genotype A) presented the lowest variability rates and patient 2(genotype A) had 3 codons with variability higher than 1%.Patient 3 and 4(both genotype D) presented 5 and 8 codons with variability higher than 1%,*** median baseline frequencies showed that genotype A samples had higher variability in epitopic positions than in the other positions analyzed,approaching significance(P = 0.07,sample 1 and P = 0.05,sample 2).In contrast,there were no significant differences in variability between the epitopic and other positions in genotype D ***,patient 1 presented a completely mutated motif from amino acid 64 to 67(E 64 LMT 67),which is commonly recognized by T helper ***,the variability observed in all 4 patients was particularly associated with the E 64 LMT 67 mo

关键词：肝炎病毒抗原表位焦磷酸测序准种 B型超深乙肝表面抗原位置分析

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New real-time-PCR method to identify single point mutations in hepatitis C virus

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World Journal of Gastroenterology 2016年第43期22卷 9604-9612页

作者： Qian Chen Irene Belmonte Maria Buti Leonardo Nieto Damir Garcia-Cehic Josep Gregori Celia Perales Laura Ordeig Meritxell Llorens Maria Eugenia Soria rafael esteban Juan Ignacio esteban Francisco Rodriguez-Frias Josep Quer Liver Unit Internal Medicine Lab. Malalties Hepàtiques Vall d’Hebron Institut Recerca-Hospital Universitari Vall d’ Hebron08035 Barcelona Spain Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas Instituto de Salud Carlos Ⅲ28029 madrid Spain Liver Pathology Unit Departments of Biochemistry and Microbiology Hospital Universitari Vall d’Hebron Autonomous University of Barcelona 08035 Bareelona Spain Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas del Instituto de Salud Carlos Ⅲ 28029 Madrid Spain Universitat Autònoma de Barcelona 08193 Bareelona Spain

AIM To develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus(HCV).METHODS In patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80 K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. Light Cycler methods, based on real-time PCR with sequencespecific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest. RESULTS Using nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10%(mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80 K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80 K was detected in 14.6% of G1 a patients and 0% of G1 b in our setting. CONCLUSION A fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This

关键词：丙肝病毒联系抵抗的氨基酸替换便宜测试单个点的变化 Q80K

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Naïve hepatitis B e antigen-negative chronic hepatitis B patients are at risk of carotid atherosclerosis:A prospective study

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World Journal of Gastroenterology 2021年第30期27卷 5112-5125页

作者： Mar Riveiro-Barciela Cristina Marcos-Fosch Fernando Martinez-Valle Fabrizio Bronte Olimpia Orozco Isidro Sanz-Pérez Daniele Torres Maria-Teresa Salcedo Salvatore Petta rafael esteban Antonio Craxi Maria Buti Department of Medicine of the UAB Hospital Universitari Vall d’HebronBarcelona 08035Spain Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas Instituto de Salud Carlos IIIMadrid 28029Spain Systemic Autoimmune Diseases Unit Internal Medicine DepartmentHospital Universitari Vall d'HebronBarcelona 08035Spain Sezione di Gastroenterologia Di.Bi.M.I.SUniversity of PalermoPalermo 90133Italy Pathology Department Hospital Universitari Vall d'HebronBarcelona 08035Spain

BACKGROUND There is an increased risk of atherosclerosis in patients with chronic hepatitis C or human immunodeficiency virus,but there is scarce data on hepatitis B virus *** hypothesis of this study is that hepatitis B virus infection increases the risk of carotid plaques and subclinical atherosclerosis in naïve hepatitis B e antigen(HBeAg)negative *** To assess the rate of carotid plaques and subclinical atherosclerosis in naïve HBeAg negative subjects in comparison with a cohort of healthy *** Prospective case-control collaborative study conducted in two tertiary *** hundred and two subjects prospectively recruited at the outpatient clinic were included from May 2016 to April 2017:201 naïve HBeAg-negative hepatitis B virus-infected[49 chronic hepatitis B(CHB)and 152 inactive carriers(ICs)]and 201 healthy *** and metabolic measures,liver stiffness and carotid Doppler ultrasound were *** atherosclerosis was established on an intima-media thickness increase of≥1.2 mm and/or the presence of carotid *** distributed quantitative variables were compared with the Student t test and those with a non-normal distribution with the Mann-Whitney U *** variables were compared between groups using theχ2 or Fisher exact *** Carotid plaques were found more often in CHB(32.7%)than ICs(17.1%)or controls(18.4%)(P=0.048).Subclinical atherosclerosis was also increased in CHB(40.8%)vsICs(19.1%)or controls(19.4%)(P=0.003).No differences in the risk of atherosclerosis were observed between controls and *** factors independently associated with the presence of carotid plaques were age[odds ratio(OR)1.43,P<0.001]and CHB(OR 1.18,P=0.004)and for subclinical atherosclerosis,age(OR 1.45,P<0.001),CHB(OR 1.23,P<0.001)and diabetes(OR 1.13,P=0.028).In the subset of young subjects(<50 years),carotid plaques(12.5%vs 1.1%,P=0.027)and subclinical atherosclerosis(12.5%vs 2.2%,P=0.058

关键词： Hepatitis B virus Carotid plaques Subclinical atherosclerosis Cardiovascular risk Endothelial dysfunction Intima-media thickness

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Analysis of hepatitis B virus preS1 variability and prevalence of the rs2296651 polymorphism in a Spanish population

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World Journal of Gastroenterology 2018年第6期24卷 680-692页

作者： Rosario Casillas David Tabernero Josep Gregori Irene Belmonte Maria Francesca Cortese Carolina González Mar Riveiro-Barciela Rosa Maria López Josep Quer rafael esteban Maria Buti Francisco Rodríguez-Frías Liver Pathology Unit Departments of Biochemistry and MicrobiologyHospital Universitari Vall d’HebronUniversitat Autònoma de BarcelonaBarcelona 08035Spain Liver Unit Liver Disease Laboratory-Viral HepatitisVall d’Hebron Institut Recerca-Hospital Universitari Vall d’HebronUniversitat Autònoma de BarcelonaBarcelona 08035Spain Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas(CIBERehd) Instituto de Salud Carlos IIIMadrid 28029Spain Roche Diagnostics SL Sant Cugat del Vallès 08174Spain Liver Unit Department of Internal MedicineHospital Universitari Vall d'HebronUniversitat Autònoma de BarcelonaBarcelona 08035Spain

AIM To determine the variability/conservation of the domain of hepatitis B virus(HBV) pre S1 region that interacts with sodium-taurocholate cotransporting polypeptide(hereafter, NTCP-interacting domain) and the prevalence of the rs2296651 polymorphism(S267 F, NTCP variant) in a Spanish population. METHODS Serum samples from 246 individuals were included and divided into 3 groups: patients with chronic HBV infection(CHB)(n = 41, 73% Caucasians), patients with resolved HBV infection(n = 100, 100% Caucasians) and an HBV-uninfected control group(n = 105, 100% Caucasians). Variability/conservation of the amino acid(aa) sequences of the NTCPinteracting domain,(aa 2-48 in viral genotype D) and a highly conserved pre S1 domain associated with virion morphogenesis(aa 92-103 in viral genotype D) were analyzed by next-generation sequencing and compared in 18 CHB patients with viremia > 4 log IU/mL. The rs2296651 polymorphism was determined in all individuals in all 3 groups using an in-house real-time PCR melting curve *** The HBV pre S1 NTCP-interacting domain showed a high degree of conservation among the examined viral genomes especially between aa 9 and 21(in the genotype D consensus sequence). As compared with the virion morphogenesis domain, the NTCPinteracting domain had a smaller proportion of HBV genotype-unrelated changes comprising > 1% of the quasispecies(25.5% vs 31.8%), but a larger proportion of genotype-associated viral polymorphisms(34% vs 27.3%), according to consensus sequences from Gen Bank patterns of HBV genotypes A to H. Variation/conservation in both domains depended on viral genotype, with genotype C being the most highly conserved and genotype E the most variable(limited finding, only 2 genotype E included). Of note, proline residues were highly conserved in both domains, and serine residues showed changes only to threonine or tyrosine in the virion morphogenesis domain. The rs2296651 polymorphism was not detected in any ***

关键词： Hepatitis B virus Hepatitis B virus preS1 region sodium-taurocholate co-transporting polypeptide NTCP-interacting domain virion morphogenesis domain SNP rs2296651 next-generation sequencing real-time PCR melting curves

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Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy

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World Journal of Gastroenterology 2018年第19期24卷 2095-2107页

作者： Carolina González David Tabernero Maria Francesca Cortese Josep Gregori Rosario Casillas Mar Riveiro-Barciela Cristina Godoy Sara Sopena Ariadna Rando Marcal Yll Rosa Lopez-Martinez Josep Quer rafael esteban Maria Buti Francisco Rodríguez-Frías Liver Pathology Unit Departments of Biochemistry and MicrobiologyHospital Universitari Vall d’HebronUniversitat Autònoma de BarcelonaBarcelona 08035Spain Liver Unit Liver Disease Laboratory-Viral HepatitisVall d’Hebron Institut Recerca-Hospital Universitari Vall d’HebronUniversitat Autònoma de BarcelonaBarcelona 08035Spain Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas(CIBERehd) Instituto de Salud Carlos IIIMadrid 28029Spain Roche Diagnostics SL Sant Cugat del Vallès 08174Spain Liver Unit Department of Internal MedicineHospital Universitari Vall d'HebronUniversitat Autònoma de BarcelonaBarcelona 08035Spain

AIM To detect hyper-conserved regions in the hepatitis B virus(HBV) X gene(HBX) 5' region that could be candidates for gene *** The study included 27 chronic hepatitis B treatmentnaive patients in various clinical stages(from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeA g-negative and HBeA g-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/m L, the HBX 5' end region [nucleotide(nt) 1255-1611] was PCRamplified and submitted to next-generation sequencing(NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences(haplotypes) obtained by NGS. Conservation at the HBx protein amino acid(aa) level was also *** NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample(2487-9279, IQR 2817). In 14/27 patients(51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region(nt 1255-1286) and the other in the 5' end coding region(nt 1519-1603). This last region coded for a conserved amino acid region(aa 63-76) that partially overlaps a Kunitz-like *** Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.

关键词： Hepatitis B virus Hepatitis B X gene Hepatitis B X protein Gene therapy Next-generation sequencing HBV conserved regions Small interference RNA

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Characterization of hepatitis B virus X gene quasispecies complexity in mono-infection and hepatitis delta virus superinfection

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World Journal of Gastroenterology 2019年第13期25卷 1566-1579页

作者： Cristina Godoy David Tabernero Sara Sopena Josep Gregori Maria Francesca Cortese Carolina González Rosario Casillas Mar?al Yll Ariadna Rando Rosa López-Martínez Josep Quer Gloria González-Aseguinolaza rafael esteban Mar Riveiro-Barciela Maria Buti Francisco Rodríguez-Frías Liver Pathology Unit Departments of Biochemistry and Microbiology Hospital Universitari Vall d’Hebron Universitat Autònoma de Barcelona Liver Unit Liver Disease Laboratory-Viral Hepatitis Vall d’Hebron Institut Recerca-Hospital Universitari Vall d'Hebron Universitat Autònoma de Barcelona Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) Instituto de Salud Carlos Ⅲ Roche Diagnostics SL Centro de Investigación Médica Aplicada (CIMA) Universidad de Navarra Liver Unit Department of Internal MedicineHospital Universitari Vall d’Hebron Universitat Autònoma de Barcelona (UAB)

Hepatitis delta virus(HDV) seems to strongly suppress hepatitis B virus(HBV)replication, although little is known about the mechanism of this interaction. Both these viruses show a dynamic distribution of mutants, resulting in viral quasispecies. Next-generation sequencing is a viable approach for analyzing the composition of these mutant spectra. As the regulatory hepatitis B X protein(HBx) is essential for HBV replication, determination of HBV X gene(HBX)quasispecies complexity in HBV/HDV infection compared to HBV monoinfection may provide information on the interactions between these two *** To compare HBV quasispecies complexity in the HBX 5' region between chronic hepatitis delta(CHD) and chronic HBV mono-infected *** Twenty-four untreated patients were included: 7/24(29.2%) with HBeAgnegative chronic HBV infection(CI, previously termed inactive carriers), 8/24(33.3%) with HBeAg-negative chronic hepatitis B(CHB) and 9/24(37.5%) with CHD. A serum sample from each patient was first tested for HBV DNA *** HBX 5' region [nucleotides(nt) 1255-1611] was then PCR-amplified for subsequent next-generation sequencing(MiSeq, Illumina, United States). HBV quasispecies complexity in the region analyzed was evaluated using incidencebased indices(number of haplotypes and number of mutations), abundancebased indices(Hill numbers of order 1 and 2), and functional indices(mutation frequency and nucleotide diversity). We also evaluated the pattern of nucleotide changes to investigate which of them could be the cause of the quasispecies *** CHB patients showed higher median HBV-DNA levels [5.4 logIU/mL,interquartile range(IQR) 3.5-7.9] than CHD(3.4 logIU/mL, IQR 3-7.6)(P = n.s.)or CI(3.2 logIU/mL, IQR 2.3-3.5)(P < 0.01) patients. The incidence and abundance indices indicated that HBV quasispecies complexity was significantly greater in CI than CHB. A similar trend was observed in CHD patients, although only Hill numbers of order 2 showed

关键词： Hepatitis B virus Hepatitis delta virus Hepatitis B X gene Next-generation sequencing Viral quasispecies Hepatitis B virus-hepatitis delta virus interaction

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Conservation and variability of hepatitis B core at different chronic hepatitis stages

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World Journal of Gastroenterology 2020年第20期26卷 2584-2598页

作者： Marcal Yll Maria Francesca Cortese Mercedes Guerrero-Murillo Gerard Orriols Josep Gregori Rosario Casillas Carolina González Sara Sopena Cristina Godoy Marta Vila David Tabernero Josep Quer Ariadna Rando Rosa Lopez-Martinez rafael esteban Mar Riveiro-Barciela Maria Buti Francisco Rodríguez-Frías Liver Pathology Unit Departments of Biochemistry and MicrobiologyHospital Universitari Vall d'HebronUniversitat Autònoma de BarcelonaBarcelona 08035Spain Liver Unit Liver Disease Laboratory-Viral HepatitisVall d'Hebron Institut Recerca-Hospital Universitari Vall d'HebronUniversitat Autònoma de BarcelonaBarcelona 08035Spain Department of Microbiology Hospital Universitari Vall d'HebronUniversitat Autònoma de BarcelonaBarcelona 08035Spain Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas Instituto de Salud Carlos ⅢMadrid 28029Spain Liver Unit Department of Internal MedicineHospital Universitari Vall d'HebronUniversitat Autónoma de BarcelonaBarcelona 08035Spain

BACKGROUND Since it is currently not possible to eradicate hepatitis B virus(HBV)infection with existing treatments,research continues to uncover new therapeutic *** core protein,encoded by the HBV core gene(HBC),intervenes in both structural and functional processes,and is a key protein in the HBV life *** this reason,both the protein and the gene could be valuable targets for new therapeutic and diagnostic ***,alterations in the protein sequence could serve as potential markers of disease *** To detect,by next-generation sequencing,HBC hyper-conserved regions that could potentially be prognostic factors and targets for new *** Thirty-eight of 45 patients with chronic HBV initially selected were included and grouped according to liver disease stage[chronic hepatitis B infection without liver damage(CHB,n=16),liver cirrhosis(LC,n=5),and hepatocellular carcinoma(HCC,n=17)].HBV DNA was extracted from patients’plasma.A region between nucleotide(nt)1863 and 2483,which includes HBC,was amplified and analyzed by next-generation sequencing(Illumina Mi Seq platform).Sequences were genotyped by distance-based discriminant *** and intergroup nt and amino acid(aa)conservation was determined by sliding window *** presence of nt insertion and deletions and/or aa substitutions in the different groups was determined by aligning the sequences with genotype-specific consensus *** Three nt(nt 1900-1929,2249-2284,2364-2398)and 2 aa(aa 117-120,159-167)hyper-conserved regions were shared by all the clinical *** groups showed a similar pattern of conservation,except for five nt regions(nt 1946-1992,2060-2095,2145-2175,2230-2250,2270-2293)and one aa region(aa 140-160),where CHB and LC,respectively,were less conserved(P<0.05).Some group-specific conserved regions were also observed at both nt(2306-2334 in CHB and 1935-1976 and 2402-2435 in LC)and aa(between aa 98-103 in CHB and 28-30 and 5

关键词： Hepatitis B virus Hepatitis B core gene Next-generation sequencing Genetic conservation Amino acid substitution Gene therapy Small interfering RNA

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Cross-sectional evaluation of circulating hepatitis B virus RNA and DNA: Different quasispecies?

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World Journal of Gastroenterology 2021年第41期27卷 7144-7158页

作者： Selene Garcia-Garcia Maria Francesca Cortese David Tabernero Josep Gregori Marta Vila Beatriz Pacín Josep Quer Rosario Casillas Laura Castillo-Ribelles Roser Ferrer-Costa Ariadna Rando-Segura Jesús Trejo-Zahínos Tomas Pumarola Ernesto Casis rafael esteban Mar Riveiro-Barciela Maria Buti Francisco Rodríguez-Frías Clinical Biochemistry Research Group Department of BiochemistryVall d'Hebron Institut Recerca-Hospital Universitari Vall d'HebronUniversitat Autònoma de BarcelonaBarcelona 08035Spain Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas Instituto de Salud Carlos IIIMadrid 28029Spain Liver Unit Liver Disease Laboratory-Viral HepatitisVall d'Hebron Institut Recerca-Hospital Universitari Vall d'HebronVall d’Hebron Barcelona Hospital CampusBarcelona 08035Spain Liver Pathology Unit Departments of Biochemistry and MicrobiologyHospital Universitari Vall d’HebronUniversitat Autònoma de BarcelonaBarcelona 08035Spain Department of Microbiology Hospital Universitari Vall d'HebronVall d'Hebron Barcelona Hospital CampusBarcelona 08035Spain Liver Unit Department of Internal MedicineHospital Universitari Vall d'HebronUniversitat Autónoma de BarcelonaBarcelona 08035Spain

BACKGROUND Different forms of pregenomic and other hepatitis B virus(HBV)RNA have been detected in patients’*** circulating HBV-RNAs may be useful for monitoring covalently closed circular DNA activity,and predicting hepatitis B eantigen seroconversion or viral rebound after nucleos(t)ide analog *** on serum HBV-RNA quasispecies,however,is *** is therefore important to develop methodologies to thoroughly analyze this quasispecies,ensuring the elimination of any residual *** circulating HBV-RNA quasispecies may facilitate achieving functional cure of HBV *** To establish a next-generation sequencing(NGS)methodology for analyzing serum HBV-RNA and comparing it with DNA *** Thirteen untreated chronic hepatitis B patients,showing different HBV-genotypes and degrees of severity of liver disease were enrolled in the study and a serum sample with HBV-DNA>5 Log10 IU/mL and HBV-RNA>4 Log10 copies/mL was taken from each ***-RNA was treated with DNAse I to remove any residual DNA,and the region between nucleotides(nt)1255-1611 was amplified using a 3-nested polymerase chain reaction protocol,and analyzed with ***/conservation and complexity was compared between HBV-DNA and RNA *** No HBV-DNA contamination was detected in cDNA samples from HBV-RNA *** quasispecies complexity showed heterogeneous behavior among *** Rare Haplotype Load at 1%was greater in DNA than in RNA quasispecies,with no statistically significant differences(P=0.1641).Regarding conservation,information content was equal in RNA and DNA quasispecies in most nt positions[218/357(61.06%)].In 102 of the remaining 139(73.38%),HBV-RNA showed slightly higher *** window analysis identified 4 hyper-conserved sequence fragments in each quasispecies,3 of them coincided between the 2 quasispecies:nts 1258-1286,1545-1573 and *** 2 hyper-variable sequence fragments also coincid

关键词： Hepatitis B virus RNA Hepatitis B X gene Quasispecies Next-generation sequencing Quasispecies conservation Quasispecies complexity

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Molecular epidemiology and putative origin of hepatitis C virus in random volunteers from Argentina

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World Journal of Gastroenterology 2013年第35期19卷 5813-5827页

作者： Noemí del Pino José Raúl Oubia Francisco Rodríguez-Frías Juan Ignacio esteban María Buti Teresa Otero Josep Gregori Damir García-Cehic Silvia Camos María Cubero Rosario Casillas Jaume Guàrdia rafael esteban Josep Quer Apunlab Laboratories Institute of Medical Microbiology and Parasitology (IMPAM) UBA-CONICET Department of MicrobiologyParasitology and Immunology Faculty of Medicine University of Buenos Aires CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos Ⅲ and Biochemistry Department Hospital Universitari Vall d’Hebrón (HUVH) CIBERehd del Instituto de Salud Carlos Ⅲ Autonomous University of Barcelona and Liver Unit Internal Medicine Lab. Malalties Hepatiques Vall d’Hebron Institut de Recerca (VHIR) Hospital Universitari Vall d’Hebrón (HUVH) Unitat de Diagnostic Molecular HUVH Liver Unit Internal Medicine Lab Malalties Hepatiques VHIR-HUVH Roche Diagnostics SL CIBERehd del Instituto de Salud Carlos Ⅲ Liver Unit Internal Medicine Lab MalaltiesHepatiques VHIR-HUVH Liver Unit and Biochemistry Department HUVH

AIM:To study the subtype prevalence and the phylogenetic relatedness of hepatitis C virus(HCV)sequences obtained from the Argentine general population,a large cohort of individuals was ***:Healthy Argentinian volunteers(n=6251)from 12 provinces representing all geographical regions of the country were *** parents or legal guardians of individuals younger than 18 years provided informed written consent for *** corresponding written permission from all municipal authorities was obtained from each city or town where subjects were to be *** RNA reverse transcription-polymerase chain reaction products were sequenced and phylogenetically *** 5’untranslated region(5’UTR)was used for RNA detection and initial genotype *** NS5B polymerase region,encompassing nt 8262-8610,was used for ***:An unexpectedly low prevalence of HCV infection in the general population(0.32%)was *** data contrasted with previous studies that reported rates ranging from 1.5%to 2.5%,mainly performed in selected populations of blood donors or vulnerable *** latter values are in keeping with the prevalence reported by the 2007 Argentinian HCV Consensus(approximately 2%).HCV subtypes weredistributed as follows:1a(25%),1b(25%),2c(25%),3a(5%),and 2j(5%).Two isolates ascribed either to genotype 1(5%)or to genotype 3(5%)by 5’UTR phylogenetic analysis could not be *** 1a sequences comprised a highly homogeneous population and clustered with United States ***1b sequences represented a heterogeneous population,suggesting that this genotype might have been introduced from different *** subtype 2c sequences clustered close to the 2c reported from Italy and Southern ***:HCV has a low prevalence of 0.32%in the studied general population of *** pattern of HCV introduction and transmission in Argentina appears to be a consequence of multiple events and different

关键词： Hepatitis C virus NS5B subtyping Molecular epidemiology Hepatitis C virus Argentina Hepatitis C virus 5’ untranslated region

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