Nucleos(t)ide analogues altered quasispecies composition of hepatitis B virus (HBV)-resistant mutations in serum HBV DNA and serum HBV RNA.

Serum hepatitis B virus (HBV) RNA is a new serological indicator reflecting viral replication with good clinical application prospects. This study aimed to clarify the dynamic changes of serum HBV RNA levels and the quasispecies of HBV RNA virus-like particles in nucleos(t)ide analogues (NAs)-experienced chronic hepatitis B (CHB) patients harboring NAs-resistant mutations and their identifiable effects on NAs resistance. We included CHB patients who were on long-term NAs treatment and with HBV DNA rebound. The longitudinally dynamics of serum HBV RNA levels were quantitatively detected, and the quasispecies differences between serum HBV DNA and serum HBV RNA were compared by high-throughput sequencing. The effect of NAs concentration pressure on altering the resistance mutations quasispecies proportion of HBV DNA and HBV RNA in cell supernatant was analyzed in vitro. A total of 447 serum samples from 36 CHB patients treated with NAs were collected. The median follow-up period was 47 months (about 4 years), and the longest follow-up period was 117 months (about 10 years). Our results showed that HBV RNA could reflect virological breakthrough in 23 (64%, 23/36) patients, and serum HBV RNA rebound earlier than HBV DNA in 12 (52%, 12/23) patients. However, serum HBV RNA remained at a consistently high level and did not fluctuate significantly with the HBV DNA rebound in 6 of 36 patients. In addition, serum HBV RNA was not consistently detectable in 7 of the 36 patients, and their serum HBV RNA was undetectable even after HBV DNA had rebounded. The proportion of drug-resistant mutations in HBV DNA was higher than that of HBV RNA by high-throughput sequencing. The results of in vitro experiments showed that the viral strains with drug-resistant mutation in HBV DNA in cell supernatants gradually become the dominant strains with the increase of NAs concentrations. Serum HBV RNA levels can reflect virological breakthrough in most NAs- treated CHB patients, but there are certain limitations. NAs alter the quasispecies composition of serum HBV DNA and serum HBV RNA, resulting in a higher detection rate of drug-resistant mutations in serum HBV DNA than in serum HBV RNA.

[Clinical emergence features and implications of hepatitis B virus rtA181T mutation].

OBJECTIVE: To determine the mutational profile and clinical implications of the viral reverse-transcriptase (rt)A 181T mutation in hepatitis B virus (HBV) through population-based analysis of clinical samples. METHODS: Serum samples from 3, 013 patients who visited The 302 Hospital (Beijing, China) were investigated.HBV DNA was extracted and HBV mutations and genotypes were determined by direct sequencing.Recombinant plasmids harboring the rtA181T/sW172* mutant or wild type sequence were constructed and transfected into the HepG2 cell line. The levels of HBsAg in culture supernatants were compared and statistically analyzed. RESULTS: The incidence of rtA181T across the study population was 4.1% (165/3, 013), and most of the rtAl 81T-positive patients had received adefovir and/or lamivudine.Forty percent (66/165) of the rtA 181T cases were single mutants and treatment responsive, 46.1% (76/165) included the adefovir-resistant mutation rtA 181 V/N236T, 12.1% (20/165) included the lamivudine-resistant mutation rtM204V/rtM2041, and 1.8% (3/165) included multidrug-resistant mutations.Interestingly, 73.9% (122/165) of the rtA181T-positive samples were detected with co-existing wild-type nucleotides at the site. The rates of HBV/C to HBV/B were 92.1% to 7.9% in the rtA181T-positive patients, but 82.1% to 17.9% in the rtA181T-negative paticnts (P less than 0.01).Almost all (98.2%; 129/165) of the rtA181T led to sW172*, while only 1.8% of the rtA181T (3/165) led to sW172L or sW172S.HBsAg secretion in vitro was reduced from the rtA181T/ sW172* strain, but there was no significant difference observed in the average serum HBsAg and HBV DNA levels of patients who carried or did not carry the mutant. CONCLUSION: The HBV rtA181T mutation is closely associated with adefovir and lamivudine exposure.rtA181T may led to sW172*, culminating in suppression of HBsAg secretion.However, co-existence of the mutant with wild-type sequences was common among our patient population, suggesting that the mutation had little impact on serum HBsAg and HBV DNA levels across the clinical study population.

The rtL229 substitutions in the reverse transcriptase region of hepatitis B virus (HBV) polymerase are potentially associated with lamivudine resistance as a compensatory mutation.

BACKGROUND: It remains unclear whether hepatitis B virus (HBV) reverse-transcriptase (RT) rtL229 substitutions influence HBV drug resistance. OBJECTIVE: The study was to investigate the association of HBV rtL229 substitutions with viral resistance to lamivudine (LAM). STUDY DESIGN: Entire HBV RT genes were amplified by nested PCR and sequenced from sera of 6000 nucleos(t)ide analog-experienced patients with chronic HBV infection. The incidence and clinic relevance of rtL229 substitutions were analyzed. Replication-competent viral amplicons which harbored HBV genomes of wild-type, rtM204I, or rtM204I in conjunction with various rtL229 substitutions (rtL229F/W/M/V) were constructed. The amplicons were transfected into HepG2 cells for phenotyping of replication capacity and susceptibility to nucleos(t)ide analogs. RESULTS: The rtL229 substitutions were detected in 6.57% (394/6000) of patients. Individual substitution incidences were 2.77%, 0.97%, 0.83% and 0.55% for rtL229V, rtL229F, rtL229M and rtL229W, respectively. The incidence of rtL229 substitutions was significantly higher in LAM-experienced patients (341/4220, 8.1%) than in LAM-naïve patients (53/1780, 3.0%), and were independently associated with genotypic LAM resistance (77.9% vs. 21.2%, OR 8.806, 95%CI 6.345-12.223) and low viral replication (HBV DNA <1000IU/mL) (4.60% vs. 24.2%, OR 0.478, 95%CI 0.254-0.898). Representative cases follow-up showed that rtL229F developed subsequent to rtM204I emergence during LAM treatment and regressed with rtM204I after LAM withdrawal. Functionally, rtL229F did not confer reduced susceptibility to LAM, but could restore replication capacity of rtM204I strain. CONCLUSION: The rtL229 substitutions were potentially associated with LAM resistance in Chinese patients and rtL229F had characteristics of a compensatory mutation of rtM204I mutant.

Characterization of hepatitis B virus genotypes/subgenotypes in 1,301 patients with chronic hepatitis B in North China.

BACKGROUND: There is still a paucity of data on hepatitis B virus (HBV) subgenotype prevalence in North China based on sequencing of large-size samples. In addition, whether HBV genotypes impact drug-resistance-associated and HBV e antigen (HBeAg)-loss-associated mutations in patients with chronic hepatitis B (CHB) is still under investigation. This study aimed to disclose clinical prevalence of HBV genotypes/subgenotypes in North China and the clinical implications of HBV genotype classification in respect to HBeAg loss and drug-resistant occurrence. METHODS: Sera were collected from 1301 nucleos(t)ide analog-experienced CHB patients. Viral DNA was extracted and used as template for HBV genome amplification by nested PCR. DNA sequencing was performed for the analysis of HBV genotypes/subgenotypes, drug-resistance-associated mutations in polymerase gene and HBeAg-loss-associated mutations in precore/basal core promoter (BCP) regions. RESULTS: HBV/B, HBV/C, and HBV/D were detected in 190 (14.6%), 1096 (84.2%), and 15 (1.2%) patients, respectively. HBV/B2 (182/190), HBV/C2 (1069/1096), and HBV/D1 (12/15) were predominant subgenotypes within individual genotypes. By contrast, C2 prevalence is relatively lower in Beijing area (77.2%) than in other north areas (84.9% - 87.4%). HBV/C-infected patients had an older age and a lower serum albumin level but similar HBV DNA and alanine aminotransferase (ALT) levels compared to HBV/B-infected patients. HBV/C infection had a higher incidence of lamivudine-resistant mutations rtM204I/V (44.9% vs. 30.2%, P < 0.01) and BCP mutations A1762T+G1764A (65.8% vs. 40.0%, P < 0.01) compared with HBV/B infection. CONCLUSIONS: C2 is the most prevalent HBV subgenotype followed by B2 in CHB patients in North China; and HBV genotype prevalence is influenced by immigrant population. HBV/C infection is likely to have longer disease duration and severer liver functional impairment and might be more susceptible to develop lamivudine resistance compared to HBV/B infection.

Features and clinical implications of hepatitis B virus genotypes and mutations in basal core promoter/precore region in 507 Chinese patients with acute and chronic hepatitis B.

BACKGROUND: The association of hepatitis B virus (HBV) genotypes and basal core promoter (BCP) and precore (PC) mutations with the clinical characteristics is increasingly recognized. OBJECTIVE: To investigate virologic features and clinical implications of HBV genotypes, BCP and PC mutations between large-size patients with acute hepatitis B (AHB) and chronic hepatitis B (CHB). STUDY DESIGN: One hundred and eighty-two AHB patients and 325 CHB patients were investigated. HBV genotypes and BCP/PC mutations were determined by direct sequencing. Mutations at 10 interested sites of the BCP/PC region were compared between the two groups of patients. RESULTS: AHB patients had a significantly higher ratio of genotype B to C than CHB patients (37.4-62.6% vs. 16.6-83.4%, P<0.001). The prevalence of BCP/PC wild-type virus was 60.4% in AHB patients in contrast to 28.9% in CHB patients. Significantly lower prevalence of A1762T, G1764A, G1896A, and G1899A but higher prevalence of T1758C was found in AHB patients. Interestingly, T1758C and A1762T/G1764A appeared mutual restraint. Genotype B virus had lower BCP mutation frequency and similar PC mutation frequency compared to genotype C virus. AHB patients with BCP/PC mutant virus had higher viral load, whereas CHB patients with BCP/PC mutant virus had lower viral load and elevated alanine aminotransferase, in comparison with those with the wild-type virus. CONCLUSION: Patients with genotype B virus, BCP/PC wild-type virus or T1758C mutant virus were more susceptible to develop AHB, whereas high prevalence of the BCP/PC mutations was associated with CHB development.

[Multiple-site analysis of HBV drug-resistant mutations in 340 patients with chronic hepatitis B].

OBJECTIVES: To analyze HBV drug-resistant mutations against nucleos(t)ide analogues at 12 reported sites in 340 patients with chronic hepatitis B. METHODS: Serum HBV DNA was extracted and a nested PCR assay was employed for the reverse transcriptase (RT) gene amplification. Direct sequencing of PCR product was performed. The significance of detected mutations was analyzed in view of clinical data of the patients. RESULTS: Drug-resistant mutations were detected in 68 patients taking lamivudine (LAM), 10 taking adefovir (ADV), 8 taking entecavir, and 1 taking telbivudine (LdT). M204V and M204I were the most common LAM-resistant mutations. The former usually emerged with L180M while the latter often emerged alone. N236T +/- A181 substitution was the most frequently seen ADV-resistant mutation. ETV-resistant mutations occurred on the basis of LAM-resistant mutations and T184 change was the most common form. LdT-resistance was observed as M204I. Interestingly, these drug-resistant mutations were detected in a few patients who had not been treated with nucleos(t)ide analogues. CONCLUSION: Detection of HBV drug-resistant mutations at multiple sites of the viral RT gene is valuable for discovering and verifying drug resistance and thus is very helpful in planning anti-HBV therapy.

Identification of genes upregulated by recombinant interferon-alpha in HepG2 cells by suppressive subtractive hybridization analysis.

BACKGROUND: Interferon-alpha (IFN-alpha) is an important cytokine with multiple functions, but the target genes transactivated by IFN-alpha remain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-alpha. To isolate the gene transcripts specifically upregulated by IFN-alpha in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-alpha protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-alpha (rIFN-alpha, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-alpha as driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed significant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-alpha was constructed successfully. rIFN-alpha upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc finger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-alpha can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-alpha.

[Screening and identification of proteins interacting with HCV NS4A via yeast double hybridization in leukocytes and gene cloning of the interacting protein].

OBJECTIVE: To screen proteins interacting with HCV NS4A protein in leukocytes by yeast-double hybridization. METHODS: The bait plasmid pGBKT7-NS4A was transformed into yeast AH109 was transformed, and the expressing of the fusion protein was identified by SDS-page. The transformed yeast was mated with yeast Y187 containing leukocytes cDNA library plasmid in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, analysis was conducted by bioinformatics. And, the gene encoding the interesting protein was cloned, and back-cross was performed. RESULTS: Forty-five colonies were sequenced, among them, 29 colonies were human calcium modulating cyclophilin ligand (CAML). The gene encoding CAML was cloned, and the interaction between NS4A and CAML was ensured. CONCLUSION: Seven kinds of proteins interacting with NS4A in leukocytes were successfully screened and the results brought some new clues for studying the pathogenesis of HCV.

[Transcriptional inhibitory effect of hepatitis B virus X protein on the expression of p53 tumor suppression gene].

BACKGROUND: To investigate the transcriptional inhibitory role of hepatitis B virus X protein on the expression of p53 tumor suppression gene. METHODS: The promoter sequence of the p53 tumor suppression gene was identified and amplified by bioinformatics and polymerase chain reaction (PCR). The recombinant reporter gene expression vector pCAT3-p53p was constructed and transfected into the hepatoblastoma cell line HepG2 and cotransfected with pcDNA3.1 (-)-X by Fugene 6 transfection reagents. The chloramphenicol acetyl transferase (CAT) activity was detected by enzyme-linked immunosorbent assay (ELISA). The expression of p53 mRNA was further detected by RT-PCR with or without HBV X protein. RESULTS: The reporter vector pCAT3-p53p has been successfully constructed and identified and the p53 promoter could cis-activate the transcription of the CAT gene. The relative expression level of CAT gene in HepG2 cells cotransfected with pCAT3-p53p and pcDNA3.1 (-)-X was lower than the control, and the inhibitory rate was approximately 78%, which indicate that HBV X protein could transcriptionally inhibit the activity of p53 promoter. After transfected with pcDNA3.1 (-)-X, the expression of p53 mRNA was lower than the control. CONCLUSION: HBV X protein could transcriptionally inhibit the expression of p53 tumor suppression gene, which might be a possible molecular mechanism responsible for the development of HBV-associated hepatocellular carcinoma.

[Study of the down-regulating effect of hepatitis C virus NS5A protein on NACA promoter].

OBJECTIVE: To investigate the effect of hepatitis C virus (HCV) non-structure protein NS5A on the activity of calcium-regulating protein alpha subunit of nascent polypeptide-associated complex (NACA) promoter. METHODS: HepG2 cell plasmid pCAT3-NACA, containing NACA promoter, was transfected alone or cotransfected with pcDNA3.1(-)-NS5A, and chloramphenicol acetyl transferase (CAT) enzyme activity was assayed by enzyme-linked immunoassay (ELISA). RESULTS: The CAT activity in the pcDNA3.1(-)-NS5A cotransfection group was 20.7% of the CAT activity in the pCAT3-NACA group. CONCLUSION: HCV non-structural protein NS5A has a down-regulating effect on the promoter of NACA gene.

[Screening of binding proteins to interferon-alpha promoter DNA by phage display technique].

OBJECTIVE: To investigate the interferon alpha regulation mechanisms by screening binding proteins of interferon alpha promoter by phage display. METHODS: PCR product of interferon-alpha promoter was incubated with a phage display cDNA library that expressed a library of human liver proteins on the surface of bacteriophage T7. Unbound phages were washed off and the phages bound to the interferon alpha promoter were amplified. Positive plaques were amplified by PCR and cloned into a pGEM-Teasy vector in order to perform DNA sequence analysis and subsequent computer blasting analysis. RESULTS: Positive phage-displayed proteins binding with interferon alpha promoter were enriched after five rounds of biopanning. We found that the following proteins were relevant to interferon alpha: mitochondrial ribosomal protein, chromosome clone, fibrinogen A alpha polypeptide, enoyl coenzyme A hydratase short chain, eukaryotic translation elongation factor 1 alpha, PI-3-kinase-related kinase SMG-1-like, xeroderma pigmentosum C group, and Homo sapien activity-dependent neuroprotector (ADNP). CONCLUSION: Many proteins with different functions could bind with interferon alpha promoter.