Dongping Xu

Association of hepatitis B virus mutations in basal core promoter and precore regions with severity of liver disease : an investigation of 793 Chinese patients with mild and severe chronic hepatitis B and acute-on-chronic liver failure

ObjectiveTo investigate the features of hepatitis B virus (HBV) basal core promoter/precore (BCP/PC) mutations and genotypes in a large number of mild/severe chronic hepatitis B (CHB-M/CHB-S), and acute-on-chronic liver failure (ACLF) patients and analyze the clinical implications of the virologic features.Patients and methodsSera of 793 (325 CHB-M, 170 CHB-S, and 298 ACLF) patients admitted to or who had visited Beijing 302 Hospital from January 2005 to December 2008 were collected and successfully amplified for the HBV BCP/PC and a 1225-bp-long S/Pol (nt 54–1278) gene regions. Biochemical and serological parameters and HBV DNA level were routinely performed. Viral DNA was extracted and subjected to a nested PCR. Genotypes/subgenotypes were determined based on complete genomic sequence or on analysis of the 1225-bp-long S/Pol-gene sequence. HBV genotyping was performed by direct PCR sequencing followed by molecular evolutionary analysis of the viral sequences. A P value of <0.05 (two-sided) was considered to be statistically significant.ConclusionsOur findings suggest that CHB patients infected with BCP/PC mutant viruses are more susceptible to severe hepatitis and ACLF than those with the BCP/PC wild-type virus and that ACLF patients with PC mutant viruses have an increased risk of death. As such, the HBV PC mutation is a potential predictive indicator of ACLF outcome.

Evolution and suppression of HBV strains with multidrug resistance to lamivudine, adefovir dipivoxil and entecavir in a patient with chronic hepatitis B

Here, we report a case of multidrug resistance in a patient with chronic hepatitis B. The patient sequentially received lamivudine, adefovir dipivoxil and entecavir, and subsequently developed single-, double- and triple-drug-resistant HBV strains. We consider this case report important because it documents, for the first time, that triple-drug-resistant HBV strains identified in a clinical setting were suppressible by lamivudine add-on adefovir dipivoxil when tenofovir disoproxil fumarate was not available.

Investigation into drug-resistant mutations of HBV from 845 nucleoside/nucleotide analogue-naive Chinese patients with chronic HBV infection.

BACKGROUNDnThis study aimed to clarify the clinical significance of drug-resistant HBV in nucleoside/nucleotide analogue (NA)-naive Chinese patients with chronic HBV infection in real clinical practice.nnnMETHODSnA total of 845 NA-naive patients who were admitted to Beijing 302 Hospital between July 2007 and March 2012 were included in the study. HBV drug-resistant mutations were examined by direct sequencing of the viral reverse transcriptase gene and verified by clonal sequencing. Phenotypic analysis of viral replication capacity and drug susceptibility were performed by measuring viral replicative intermediate level in 1.1-mer mutant or wild-type HBV amplicon-transfected HepG2 cells in absence or presence of serially diluted drugs.nnnRESULTSnDrug-resistant mutations were detected in 2.01% (17/845) of the patients by direct sequencing, including 15 with lamivudine-resistant mutations (rtM204V, rtM204I), one with adefovir-resistant mutation (rtA181V), and one with both lamivudine- and adefovir-resistant mutations (rtA181V, rtM204I). Clonal sequencing identified 13 drug-resistant HBV strains: rtL80I+M204I, rtL80I+M204V, rtL180M+M204I, rtL180M+M204V, rtM204I, rtM204V, rtL80I+L180M+M204I, rtL80I+L180M+M204V, rtA181V, rtA181V+M204I, rtA181T+N236T, rtA181V+N236T and rtN236T. Phenotypic analysis showed that two pre-existing lamivudine-resistant strains (rtL80I+M204I, rtL180M+M204V) had >1,000-fold resistance to lamivudine, and one pre-existing adefovir-resistant strain (rtA181V+N236T) had 15.4-fold resistance to adefovir compared with the wild-type strain. A follow-up study showed that the presence of pre-existing rtM204I strain in one patient increased from 20% at baseline to 85% after 13 months of entecavir treatment with corresponding recession of wild-type strain in the viral pool.nnnCONCLUSIONSnThe incidence of drug-resistant HBV mutations was low in NA-naive Chinese HBV-infected patients. Pre-existing mutants had similar resistance characteristics to those from NA refractory patients.

Increased occurrence of mutant rtI233V of HBV in patients receiving adefovir therapy.

BACKGROUNDnThe study aimed to clarify whether the rtI233V substitution affects adefovir (ADV) resistance.nnnMETHODSnA total of 18,419 patients from Beijing 302 Hospital were investigated. HBV complete reverse transcriptase region of the polymerase was screened by direct sequencing and verified by clonal sequencing if necessary. Replication-competent wild-type and mutant HBV genomic amplicons were transfected into HepG2 cells for phenotypic analysis of viral replication capacity and drug susceptibility.nnnRESULTSnThe rtI233V substitution was detected in 38/5,344 (0.71%) ADV-treated patients and in 8/13,075 patients without receiving ADV (P<0.001). Eight patients with rtI233V ± rtA181V/rtN236T had virological breakthrough in the clinical course of ADV treatment. Phenotypic analysis showed that rtI233V mutants from patient 1 and patient 2 exhibited 1.57-fold and 1.51-fold decreased susceptibility to ADV, respectively, compared to wild-type virus; by contrast, rtN236T and rtI233V+N236T mutants from patient 1 had 6.82-fold and 5.28-fold decreased susceptibility to ADV. rtI233V, rtN236T and rtI233V+N236T mutants had 97.5%, 30.2% and 69.7% of replication capacity compared to wild-type virus in the absence of antivirals and all remained susceptible to lamivudine, entecavir and tenofovir. Viral replication capacity correspondingly decreased after eliminating rtI233V from rtI233V+N236T mutant and was restored after introducing rtI233V into rtN236T mutant. In clinical practice, switching to entecavir rescue therapy suppressed HBV DNA to an undetectable level for both patients.nnnCONCLUSIONSnrtI233V usually emerged in ADV-treated patients with little impact on ADV susceptibility but it effectively restored replication capacity of the rtN236T mutant, suggesting that rtI233V may partly serve as a compensatory mutation associated with ADV resistance.

Screening and identification of a novel adefovir dipivoxil resistance associated mutation, rtN236V, of HBV from a large cohort of HBV-infected patients.

BACKGROUNDnThe study aimed to clarify whether rtN236V mutation of HBV derived from adefovir dipivoxil (ADV)-refractory patients was associated with drug resistance.nnnMETHODSnA total of 18,419 patients from Beijing 302 Hospital were investigated. HBV complete reverse transcriptase region of polymerase was screened by direct sequencing and verified by clonal sequencing if necessary. Replication-competent wild-type and mutant HBV genomic amplicons were constructed, transfected into HepG2 cells and cultured in the presence or absence of serially diluted nucleoside/nucleotide analogues. Intracellular HBV replicative intermediates were quantitated for calculating the 50% effective concentration of drug.nnnRESULTSnrtN236V was detected in six ADV-refractory patients; signature ADV-resistant mutations rtA181V and rtN236T were detected in 1,311 patients. rtN236V mutants emerged predominantly with virological breakthrough in the clinical course of the six patients. Phenotypic analysis of the mutants from two patients was performed. rtN236V mutants from patient 1 and patient 2 exhibited 3.90-fold and 3.10-fold decreased susceptibility to ADV, respectively, compared to the wild-type virus; by contrast, rtN236T mutants from the patients had 4.50-fold and 4.75-fold decreased susceptibility, respectively. Both mutants had a relatively lower viral replication capacity compared to wild-type virus in the absence of antivirals and remained susceptible to lamivudine, entecavir and tenofovir disoproxil fumarate. In clinical practice, switching to entecavir rescue therapy suppressed HBV DNA to an undetectable level and normalized alanine aminotransferase level for both patients.nnnCONCLUSIONSnrtN236V was a novel infrequently occurring ADV-resistance-associated mutation. It conferred a moderate resistance to ADV with relatively lower natural replication capacity.

Comparison of Detection Rate and Mutational Pattern of Drug-Resistant Mutations Between a Large Cohort of Genotype B and Genotype C Hepatitis B Virus-Infected Patients in North China

The study aimed to investigate the association of prevalent genotypes in China (HBV/C and HBV/B) with HBV drug-resistant mutations. A total of 13,847 nucleos(t)ide analogue (NA)-treated patients with chronic HBV infection from North China were enrolled. HBV genotypes and resistant mutations were determined by direct sequencing and confirmed by clonal sequencing if necessary. HBV/B, HBV/C, and HBV/D occupied 14.3%, 84.9%, and 0.8% across the study population, respectively. NA usage had no significant difference between HBV/B- and HBV/C-infected patients. Lamivudine-resistant mutations were more frequently detected in HBV/C-infected patients, compared with HBV/B-infected patients (31.67% vs. 25.26%, pu2009<u20090.01). Adefovir- and entecavir-resistant mutation detection rates were similar, but the mutational pattern was different between the two genotypes. For adefovir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtA181u2009V (HBV/C 5.29% vs. HBV/B 1.36%, pu2009<u20090.01) and a lower detection rate of rtN236T (2.70% vs. 6.54%, pu2009<u20090.01). For entecavir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtM204u2009V/I+T184 substitution or S202G/C (3.66% vs. 2.16%, pu2009<u20090.01) and a lower detection rate of rtM204u2009V/I+M250u2009V/I/L substitution (0.67% vs. 1.46%, pu2009<u20090.01). Multidrug-resistant mutations (defined as coexistence of mutation to nucleoside and nucleotide analogues) were detected in 104 patients. HBV/C-infected patients had a higher detection rate of multidrug-resistant mutation than HBV/B-infected patients (0.83% vs. 0.35%, pu2009<u20090.05). The study for the first time clarified that HBV/C-infected patients had a higher risk to develop multidrug-resistant mutations, compared with HBV/B-infected patients; and HBV/C- and HBV/B-infected patients had different inclinations in the ETV-resistant mutational pattern.

Drug-Resistant and Genetic Evolutionary Analysis of Influenza Virus from Patients During the 2013 and 2014 Influenza Season in Beijing.

The study aimed to analyze drug resistance and mutations and genetic evolution of influenza A and influenza B viruses during the 2013 and 2014 influenza season in Beijing, China. RNA was extracted from pharyngeal or nasal swabs of 28 patients, and determination of influenza genotypes was performed by using real-time reverse-transcription polymerase chain reaction. Influenza A virus samples were sequenced with the neuraminidase (NA) gene and M2 matrix protein gene to determine the NA inhibitor (NAI) resistance and amantadine resistance mutations, and influenza B virus samples were sequenced with the NA gene and hemagglutinin (HA) gene to analyze NAI resistance mutations. As a result, the enrolled subjects consisted of 19 patients with the A(H1N1)pdm09 subtype, four with A(H3N2) subtype and five with influenza B virus. All of the 23 samples with influenza A viruses harbored amantadine resistance mutation S31N in M2 matrix protein. V241I, a compensatory NAI resistance mutation, was detected in all of the 19 A(H1N1)pdm09 viruses. No other NAI resistance mutation was observed in both influenza A and B viruses. The NA gene of the five influenza B virus strains was classified as B-Victoria lineage, while the HA gene of five strains was classified as B-Yamagata lineage. In summary, all influenza A viruses from patients in Beijing in the 2013-2014 season were resistant to amantadine agent. Both influenza A and B viruses kept sensitive to NAIs. Lineage recombination was detected in influenza B virus strains and may impair the efficacy of influenza vaccination.

Risk of hepatitis B reactivation in HBsAg-negative/HBcAb-positive patients with undetectable serum HBV DNA after treatment with rituximab for lymphoma: a meta-analysis

BackgroundHepatitisxa0B surface antigen (HBsAg)-negative/hepatitisxa0B core antibody (HBcAb)-positive patients with undetectable serum hepatitisxa0B virus (HBV) DNA have experienced and resolved hepatitis B virus (HBV) infection. Lymphoma patients with resolved HBV infection have high risk of HBV reactivation when treated with robust immunosuppressive agents, but the reported rate varies extensively between different studies. This study aims to estimate the risk of HBV reactivation in HBsAg-negative/HBcAb-positive patients receiving rituximab-containing chemotherapy for lymphoma.MethodsDatabases were searched for papers published in English until 8 August 2016. The pooled risk of HBV reactivation was estimated using a random-effects model.ResultsData from 15 studies were retrieved, including a total of 1312 HBsAg-negative/HBcAb-positive lymphoma patients treated with rituximab-containing chemotherapy. The results revealed HBV reactivation rate of 9.0xa0% [95xa0% confidence interval (CI) 0.05–0.15]. In subgroup analysis, the reactivation rates for prospective and retrospective studies were 17xa0% (I2xa0=xa087.3xa0%; 95xa0% 0.08–0.39, pxa0<xa00.001) and 7xa0% (I2xa0=xa043.1xa0%; 95xa0% CI 0.05–0.11, pxa0=xa00.07), respectively.ConclusionsThis meta-analysis confirms a measurable and potentially substantial risk of HBV reactivation in HBsAg-negative/HBcAb-positive patients with rituximab treatment for lymphoma. Prophylactic use of anti-HBV agents should be seriously considered for such patients.

Chinese herbal extract Su-duxing had potent inhibitory effects on both wild-type and entecavir-resistant hepatitis B virus (HBV) in vitro and effectively suppressed HBV replication in mouse model

ABSTRACT This study aimed to investigate anti‐HBV effect and major active compounds of Su‐duxing, a medicine extracted from Chinese herbs. HBV‐replicating cell lines HepG2.2.15 (wild‐type) and HepG2.A64 (entecavir‐resistant) were used for in vitro test. C57BL/6 mice infected by adeno‐associated virus carrying 1.3 mer wild‐type HBV genome were used for in vivo test. Inhibitory rates of Su‐duxing (10 &mgr;g/mL) on HBV replicative intermediate and HBsAg levels were 75.1%, 51.0% in HepG2.2.15 cells and 65.2%, 42.9% in HepG2.A64 cells. The 50% inhibitory concentration of Su‐duxing and entecavir on HBV replicative intermediates had 0.2‐fold and 712.5‐fold increase respectively for entecavir‐resistant HBV compared to wild‐type HBV. Su‐duxing and entecavir combination showed a better anti‐HBV effect than each single of agents. Mice treated with Su‐duxing (45.0 mg kg−1 d−1 for 2 weeks) had 1.39 log10 IU/mL decrease of serum HBV DNA, and 48.9% and 51.7% decrease of serum HBsAg and HBeAg levels. GeneChip and KEGG analysis proposed that anti‐HBV mechanisms included relief of HBx stability and viral replication, deregulation of early cell cycle checkpoints, and induction of type I interferon. Quantitative RT‐PCR verified that CCNA2, ATF4, FAS and CDKN1A expression levels had significant difference between Su‐duxing‐treated and control groups. Six active compounds (Matrine, Oxymatrine, Chlorogenic acid, Sophocarpine, Baicalein, and Wogonin) against HBV were identified in Su‐duxing. Greater anti‐HBV effects were observed in some compound pairs compared to each single compound. In conclusion, Su‐duxing had potent inhibitory effects on both wild‐type and entecavir‐resistant HBV. Its effects were associated with coordinated roles of active compounds in its composition. HighlightsChinese herb extracts Su‐duxing had potent inhibitory effect on both wild‐type and entecavir‐resistant HBV.Su‐duxing had superiority to entecavir in persistent inhibition of HBV activities in mice model.Mechanisms may involve HBx stability and replication relief, early cell cycle checkpoint deregulation, and IFN induction.Six active compounds were identified and greater anti‐HBV effects were observed in some compound pairs than single one.

Hepatitis B virus rtA181T/sW172non-stop mutation may increase resistance fold to adefovir- and entecavir-resistant mutants compared to rtA181T/sW172* mutation

ABSTRACT The study aimed to characterize rtA181T/sW172stop (*) and rtA181T/sW172non‐stop mutations of hepatitis B virus (HBV). Total of 22,009 patients who visited Beijing 302 Hospital from 2007 to 2016 were enrolled. These patients all received nucleos(t)ide analogues (NAs) treatment and their serum samples were collected for sequence analysis of HBV reverse‐transcriptase (RT) and S regions. The rtA181T mutation was detected in 5.37% (1182/22,009) of the patients samples. The rtA181T‐causative sW172*, sW172non‐stop (sW172 L/S), and mixed sW172*/non‐stop mutations occupied 82.91%, 7.70%, and 9.39%, respectively. The patients with rtA181T/sW172non‐stop mutants had a higher HBV DNA level compared to those with rtA181T/sW172* mutants. 44.33% (524/1182) rtA181T‐positive samples were detected with signature drug‐resistant mutations, including 325 with adefovir‐resistant mutation rtA181V/N236T, 57 with lamivudine‐resistant mutation rtM204V/I, 99 with entecavir‐resistant mutation rtM204V/I plus rt184/202/250 substitution(s), and 43 with multidrug‐resistant mutation rtA181V/N236T + rtM204V/I ± rt184/202/250 substitution(s). The rtA181T/sW172non‐stop mutation had a higher ratio of coexistence with adefovir‐resistant mutation compared to rtA181T/sW172* mutation (42.86% vs. 24.59%, P < 0.05). rtA181T/sW172S + rtN236T and rtA181T/sW172L + rtN236T mutants exhibited higher HBV DNA production and adefovir resistance fold than that of rtA181T/sW172* + rtN236T mutant (98.02% and 85.5% vs. 42.1% in HBV DNA production, and 7.38‐fold and 5.49‐fold vs. 3.69‐fold in half maximal effective concentration of wild‐type strain); rtA181T/sW172L + rtS202G + rtM204V strain exhibited higher HBV DNA production and entecavir resistance fold than that of rtA181T/sW172* + rtS202G + rtM204V strain (50.98% vs. 34.49%, 524.00‐fold vs. 69.33‐fold). In conclusion, rtA181T/sW172non‐stop mutation may increase resistance fold of adefovir‐ and entecavir‐resistant mutants compared to rtA181T/sW172* mutation and might influence clinical presentation of NAs‐treated patients. HIGHLIGHTSThe rtA181T mutation was detected in 5.37% (1182/22,009) of NAs‐experienced chronic HBV‐infected patients.rtA181T‐causative sW172*, sW172non‐stop, and sW172*/non‐stop mutations occupied 82.91%, 7.70%, and 9.39%, respectively.rtA181T/sW172non‐stop mutation had a higher ratio with adefovir‐resistant mutation compared to rtA181T/sW172* mutation.Patients with rtA181T/sW172non‐stop mutation had a higher HBV DNA level than those with rtA181T/sW172* mutation.rtA181T/sW172non‐stop mutation increased resistance of ADV and ETV‐resistant mutants compared to rtA181T/sW172* mutation.