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| 1 | Characterization of hepatitis B virus X gene quasispecies complexity in mono-infection and hepatitis delta virus superinfection显示文摘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 viruses.AIM To compare HBV quasispecies complexity in the HBX 5' region between chronic hepatitis delta(CHD) and chronic HBV mono-infected patients.METHODS 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 levels.The 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 complexity.RESULTS 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 statistically significant differences(CHB2.81, IQR 1.11-4.57 vs CHD 8.87, 6.56-11.18, P = 0.038). There were no significant differences in the functional indices, but CI and CHD patients also showed a trend towards greater complexity than CHB. No differences were found for any HBV quasispecies complexity indices between CHD and CI patients. G-to-A and C-to-T nucleotide changes, characteristic of APOBEC3 G, were higher in CHD and CI than in CHB in genotype A haplotypes, but not in genotype D. The proportion of nt G-to-A vs A-to-G changes and C-to-T vs T-to-C changes in genotype A and D haplotypes in CHD patients showed no significant differences. In CHB and CI the results of these comparisons were dependent on HBV genotype.CONCLUSION The lower-replication CHD and CI groups show a trend to higher quasispecies complexity than the higher-replication CHB group. The mechanisms associated with this greater complexity require elucidation. | 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 | 2019 | World Journal of Gastroenterology2019，25，13： | 6 |
| 2 | Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy显示文摘AIM To detect hyper-conserved regions in the hepatitis B virus(HBV) X gene(HBX) 5' region that could be candidates for gene therapy.METHODS 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 analyzed.RESULTS 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 domain.CONCLUSION 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. | 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 | 2018 | World Journal of Gastroenterology2018，24，19： | 6 |
| 3 | Analysis of hepatitis B virus preS1 variability and prevalence of the rs2296651 polymorphism in a Spanish population显示文摘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 analysis.RESULTS 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 participant.CONCLUSION In our CHB population, the NTCP-interacting domain was highly conserved, particularly the proline residues and essential amino acids related with the NTCP interaction, and the prevalence of rs2296651 was low/null. | 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 | 2018 | World Journal of Gastroenterology2018，24，6： | 5 |
| 4 | Cross-sectional evaluation of circulating hepatitis B virus RNA and DNA: Different quasispecies?显示文摘BACKGROUND Different forms of pregenomic and other hepatitis B virus(HBV)RNA have been detected in patients’sera.These 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 cessation.Data on serum HBV-RNA quasispecies,however,is scarce.It is therefore important to develop methodologies to thoroughly analyze this quasispecies,ensuring the elimination of any residual HBV-DNA.Studying circulating HBV-RNA quasispecies may facilitate achieving functional cure of HBV infection.AIM To establish a next-generation sequencing(NGS)methodology for analyzing serum HBV-RNA and comparing it with DNA quasispecies.METHODS 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 patient.HBV-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 NGS.Variability/conservation and complexity was compared between HBV-DNA and RNA quasispecies.RESULTS No HBV-DNA contamination was detected in cDNA samples from HBV-RNA quasispecies.HBV quasispecies complexity showed heterogeneous behavior among patients.The 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 variability.Sliding 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 1575-1604.The 2 hyper-variable sequence fragments also coincided:nts 1311-1344 and 1461-1485.Sequences between nts 1519-1543 and 1559-1587 were only hyper-conserved in HBV-DNA and RNA,respectively.CONCLUSION Our methodology allowed analyzing HBV-RNA quasispecies complexity and conservation without interference from HBV-DNA.Thanks to this,we have been able to compare both quasispecies in the present study. | 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 | 2021 | World Journal of Gastroenterology2021，27，41： | 1 |
| 5 | Endothelial Dysfunction Correlates with Liver Fibrosis in Chronic HCV Infection显示文摘 | Michele Barone Maria Teresa Viggiani Annabianca Amoruso Serafina Schiraldi Annapaola Zito Fiorella Devito Francesca Cortese Michele Gesualdo Natale Brunetti Alfredo Di Leo Pietro Scicchitano Marco Matteo Ciccone Eiji Sakai | 2015 | Gastroenterology Research and Practice2015，，： | 1 |
| 6 | Conservation and variability of hepatitis B core at different chronic hepatitis stages显示文摘BACKGROUND Since it is currently not possible to eradicate hepatitis B virus(HBV)infection with existing treatments,research continues to uncover new therapeutic strategies.HBV 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 cycle.For this reason,both the protein and the gene could be valuable targets for new therapeutic and diagnostic strategies.Moreover,alterations in the protein sequence could serve as potential markers of disease progression.AIM To detect,by next-generation sequencing,HBC hyper-conserved regions that could potentially be prognostic factors and targets for new therapies.METHODS 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 analysis.General and intergroup nt and amino acid(aa)conservation was determined by sliding window analysis.The presence of nt insertion and deletions and/or aa substitutions in the different groups was determined by aligning the sequences with genotype-specific consensus sequences.RESULTS 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.All 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 51-54 in LC)levels.No differences in insertion and deletions frequencies were observed.An aa substitution(P79 Q)was observed in the HCC group with a median(interquartile range)frequency of 15.82(0-78.88)vs 0(0-0)in the other groups(P<0.05 vs CHB group).CONCLUSION The differentially conserved HBC and HBV core protein regions and the P79 Q substitution could be involved in disease progression.The hyper-conserved regions detected could be targets for future therapeutic and diagnostic strategies. | 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 | 2020 | World Journal of Gastroenterology2020，26，20： | 0 |
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