BACKGROUND:Whether rtS106C+H126Y+D134E/rtS106C+H126Y+D134E+L269I (rtCYE/rtCYEI) mutations in the hepatitis B virus (HBV) reverse-transcriptase (RT) region are associated with tenofovir disoproxil fumarate (TDF) resistance is controversial. AIM:To evaluate the presence of the rtCYE/rtCYEI mutations in a large cohort of Chinese patients with chronic HBV infection. METHODS:A total of 28236 patients who underwent drug resistance testing at the Fifth Medical Center of Chinese PLA General Hospital from 2007 to 2019 were enrolled. All patients received nucleoside/nucleotide analogues (NAs) therapy, and serum samples were collected for sequence analysis of the HBV RT domain with mutation analysis. RESULTS:The detection rates of a single mutation of rtS106C, rtH126Y, rtD134E, and rtL269I were 8.21%, 3.20%, 2.55% and 61.49% in 23718 genotype C patients, and 1.31%, 1.76%, 0.21%, and 92.33% in 4266 genotype B patients, respectively. The combined mutations of rtCYE/rtCYEI were only detected in 12 genotype C patients, accounting for 0.042% of all patients. These 12 patients had received NA treatments except TDF before testing. Among them, 6 patients had coexisting rtCYE/rtCYEI and lamivudine-resistance mutations, and 2 patients had coexisting rtCYE/rtCYEI and adefovir-resistance mutations. Compared with the wild-type (WT) strain, the replication capacity of rtCYE/rtCYEI mutants from representative patients decreased by 41.1%-71.8%, and TDF susceptibility reduced by less than 2-fold, but rtCYEI+rtA181V/N236T mutants exhibited a 6.2-/9.9-fold decrease in TDF susceptibility. Molecular modeling showed that rtCYE/rtCYEI mutants had a slight decrease in binding energy to TDF compared to the WT strain. In the clinic, emergence of the rtCYE/rtCYEI mutations was not specifically associated with TDF treatment. CONCLUSION:HBV rtCYE/rtCYEI mutations have a limited effect on TDF susceptibility and are not sufficient to cause TDF resistance.

BACKGROUND Liuweiwuling Tablet (LWWL) is a Chinese patent medicine approved for the treatment of chronic inflammation caused by hepatitis B virus (HBV) infection. Previous studies have indicated an anti-HBV effect of LWWL, specifically in terms of antigen inhibition, but the underlying mechanism remains unclear. AIM To investigate the potential mechanism of action of LWWL against HBV. METHODS In vitro experiments utilized three HBV-replicating and three non-HBV-replicating cell lines. The in vivo experiment involved a hydrodynamic injection-mediated mouse model with HBV replication. Transcriptomics and metabolomics were used to investigate the underlying mechanisms of action of LWWL. RESULTS In HepG2.1403F cells, LWWL (0.8 mg/mL) exhibited inhibitory effects on HBV DNA, hepatitis B surface antigen and pregenomic RNA (pgRNA) at rates of 51.36%, 24.74% and 50.74%, respectively. The inhibition rates of LWWL (0.8 mg/mL) on pgRNA/covalently closed circular DNA in HepG2.1403F, HepG2.2.15 and HepG2.A64 cells were 47.78%, 39.51% and 46.74%, respectively. Integration of transcriptomics and metabolomics showed that the anti-HBV effect of LWWL was primarily linked to pathways related to apoptosis (PI3K-AKT, CASP8-CASP3 and P53 pathways). Apoptosis flow analysis revealed that the apoptosis rate in the LWWL-treated group was significantly higher than in the control group (CG) among HBV-replicating cell lines, including HepG2.2.15 (2.92% ± 1.01% vs 6.68% ± 2.04%, P < 0.05), HepG2.A64 (4.89% ± 1.28% vs 8.52% ± 0.50%, P < 0.05) and HepG2.1403F (3.76% ± 1.40% vs 7.57% ± 1.35%, P < 0.05) (CG vs LWWL-treated group). However, there were no significant differences in apoptosis rates between the non-HBV-replicating HepG2 cells (5.04% ± 0.74% vs 5.51% ± 1.57%, P > 0.05), L02 cells (5.49% ± 0.80% vs 5.48% ± 1.01%, P > 0.05) and LX2 cells (6.29% ± 1.54% vs 6.29% ± 0.88%, P > 0.05). TUNEL staining revealed a significantly higher apoptosis rate in the LWWL-treated group than in the CG in the HBV-replicating mouse model, while no noticeable difference in apoptosis rates between the two groups was observed in the non-HBV-replicating mouse model. CONCLUSION Preliminary results suggest that LWWL exerts a potent inhibitory effect on wild-type and drug-resistant HBV, potentially involving selective regulation of apoptosis. These findings offer novel insights into the anti-HBV activities of LWWL and present a novel mechanism for the development of anti-HBV medications.

Chronic hepatitis B (CHB) remains a major world's most serious public health issues. Despite the remarkable effect of nucleos(t)ide analogues (NAs) in inhibiting hepatitis B virus (HBV) deoxyribonucleic acid (DNA) as the first-line drug, there are several limitations still, such as poor antigen inhibition, drug resistance, low-level viremia, restricting patients' functional cure. Due to the constraints of NAs, traditional medicines, such as traditional Chinese medicine (TCM), have become more prevalently used and researched in the clinical treatment of CHB as complementary alternative therapies. As a consequence, the review focuses on the background based on HBV's life cycle as well as the NAs' limitations, progress based on direct and indirect pathway of targeting HBV of TCM, and challenges of TCM. We found TCMs play an increasingly important role in anti-HBV. In a direct antiviral way, they regulate HBV infection, replication, assembly, and other aspects of the HBV life cycle. As for indirect way, TCMs can exert anti-HBV effects through targeting the host, including immune regulation, apoptosis, autophagy, oxidative stress, etc. Especially, TCMs have the advantages of strong antigenic inhibition compared to NAs. Specifically, we can combine the benefits of TCMs in strong HBV antigen inhibition with the benefits of NAs in targeted antiviral effects, in order to find a suitable combination of "TCM + NAs" to contribute to Chinese knowledge of the realisation of the "global elimination of HBV by 2030" goal of the World Health Organization.

Background: HBV infection is still one of the serious public health problems in the world. NAs as the first line drugs effectively inhibit viral replication, whereas the suppressive effects are much weaker on HBV antigen expression. Chinese patent medicine Liuweiwuling tablet (LWWL) has been used for chronic hepatitis B (CHB) patients for more than 10 years as an adjuvant treatment. Objective. This study aimed to assess the efficacy and safety of LWWL combined with NAs in treating CHB by meta-analysis. Methods. Systematic searches for randomized controlled trials (RCT) were conducted in four databases (up to December 2020) without language restrictions. Data were analyzed using Cochrane RevMan software version 5.3.0 and Stata software version 13.1, and reported as weight mean difference (WMD) or odds ratio (OR) with 95% confidence intervals (CIs). The primary outcomes were negative conversion rate of serum HBV DNA and HBeAg. The secondary outcomes were improvement of liver function including ALT and AST, and adverse drug reactions. The quality of evidence was evaluated according to criteria from the Cochrane risk of bias. Results. A total of 201 potentially relevant publications were identified, and 20 RCTs were eligible for inclusion. For the negative rate of HBV DNA (OR ​= ​2, 95%CI:1.64–2.45, P ＜0.05) and HBeAg (OR ​= ​1.83, 95%CI:1.46–2.30, P＜0.05), there were both statically significant benefit in favor of LWWL combined with NAs. In addition, Meta-analysis also showed that LWWL combined with NAs significantly reduced ALT (WMD ​= ​−17.73, 95%CI: 21.37∼-14.09, P ​< ​0.01), AST (MD ​= ​-23.49, 95%CI: −30.52∼-16.45, P ​< ​0.05), and increased the normalization rate of ALT (OR ​= ​3.89, 95%CI:2.82–5.36, P ​< ​0.01). Conclusion. The negative conversion rates of HBV DNA, HBeAg, and improvement of liver function in patients treated with LWWL combined with NAs were significantly higher than those treated with NAs alone, and no serious ADRs were identified. This work provides a reference to the evidence, for the beneficial effects of LWWL combined with NAs in treating CHB.

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.

目的 通过体外实验评价板蓝根Isatidis Radix及活性成分4-羟基-1H-吲哚-3-甲醛对乙型肝炎病毒(hepatitis B virus,HBV)的抑制作用,并探索其基于调控视黄酸诱导基因蛋白Ⅰ-线粒体抗病毒信号蛋白(retinoic acid-inducible gene Ⅰ-mitochondria antiviral signaling protein,RIG-Ⅰ-MAVS)信号通路的抗病毒机制.方法 采用CCK-8方法在HBV稳定复制细胞系HepG2.2.15细胞分析板蓝根毒性,选择安全药物浓度进行抗HBV实验研究,计算药物对HBVDNA、乙型肝炎E抗原(HBeAg)、乙型肝炎表面抗原(HBsAg)的抑制率,评价板蓝根抗病毒药效;采用Western blotting方法检测板蓝根对宿主免疫相关RIG-Ⅰ-MAVS信号通路中关键信号蛋白RIG-Ⅰ、MAVS、干扰素调节因子7(interferon regulated-factor 7,IRF7)、磷酸化IRF7(phosphorylated IRF7,p-IRF7)表达水平的影响.基于TCMSP数据库检索板蓝根中的活性成分,使用Schr?dinger软件将板蓝根活性成分与RIG-Ⅰ、MAVS、IRF7蛋白进行分子对接,初步筛选板蓝根中的抗HBV潜在活性成分,选定分子对接结果中与靶标蛋白结合能力较强的活性成分进行抗HBV活性评价并对其作用机制进行探索.结果 板蓝根对HepG2.2.15细胞中HBVDNA、HBeAg、HBsAg均具有明显的抑制作用(P＜0.05、0.01、0.001),抑制率分别为54.67％、23.29％、66.62％;板蓝根可上调RIG-Ⅰ、MAVS、IRF7、p-IRF7蛋白的表达,对RIG-I的上调作用最为显著(P＜0.001);分子对接结果显示,4-羟基-1H-吲哚-3-甲醛与RIG-Ⅰ、MAVS、IRF7蛋白具有较强的亲和力,结合能分别为-5.218、-6.525、-6.813kJ/mol;4-羟基-1H-吲哚-3-甲醛对 HepG2.2.15 细胞的 HBVDNA、HBeAg、HBsAg 的抑制率分别为 33.87％、28.14％、46.14％,可促进RIG-Ⅰ、MAVS、IRF7、p-IRF7蛋白的表达.结论 板蓝根及活性成分4-羟基-1H-吲哚-3-甲醛均可抑制HBV DNA、HBeAg、HBsAg的分泌,可能通过上调RIG-Ⅰ-MAVS信号通路发挥抗HBV作用.

目的:基于体外实验和网络药理学方法探究淫羊藿(epimedii folium,EF)醇提物对乙型肝炎病毒(hepatitis B virus,HBV)的抑制作用及潜在机制.方法:使用EF醇提物处理野生型HBV稳定复制细胞系HepG 2.2.15细胞,采用CCK8法进行药物毒性评价;应用PCR-荧光探针"一管法"、ELISA法检测乙型肝炎病毒脱氧核糖核酸(HBV DNA)、乙型肝炎表面抗原(HBsAg)和乙型肝炎E抗原(HBeAg)水平,计算抑制率;利用TCMSP,GeneCards数据库筛选EF主要活性成分及HBV相关靶点,借助Cytoscape软件构建药物-活性成分-潜在疾病靶点网络,对核心靶点运用DAVID数据库进行GO和KEGG富集分析.结果:EF醇提物单独用药对HepG 2.2.15细胞的半数毒性浓度(CC50)值为1.21 mg·mL-1;对HBV DNA最大抑制率为59.54％,对HBsAg和HBeAg最大抑制率分别为63.07％和45.73％(P<0.05);对照药替诺福韦酯(tenofovir disoprox-il fumarate,TDF)对HBsAg和HBeAg均未见明显抑制作用(抑制率<20.00％).EF醇提物联合TDF对HBV DNA的最大抑制率为93.66％,对HBsAg和HBeAg的最大抑制率分别为54.97％和61.28％(P<0.05).通过网络药理学分析共筛选出EF 23个候选活性成分及104个治疗HBV潜在靶点;GO富集分析得到624个条目,包括生物过程442个、细胞组分73个、分子功能109个;KEGG分析主要聚集在癌症通路、PI3 K-Akt、脂质与动脉粥样硬化、人类乳头瘤病毒感染、乙型肝炎等信号通路.结论:EF可有效抑制野生型HBV复制,和TDF联合用药有协同抑制作用,尤其对抗原抑制效果显著;EF抗HBV具有多成分多靶点的作用特点,预测可以通过多条信号通路发挥作用,为后续深入探讨EF治疗HBV感染作用机制提供了理论基础.

Methods:A total of 111 patients in total from different disease phases were recruited, including 21 in immune-tolerant (IT) phase, 49 in immune-clearance (IC) phases, 29 in immune-control or low replicative (LR) phase, and 12 in reactivation phases. Serum HBV RNA, anti-HBc, HBcrAg, and intrahepatic covalently closed circular DNA (cccDNA) were quantified and each of these indicator's correlation with liver inflammation was analyzed.Results:HBeAg-positive individuals had significant higher serum levels of HBV RNA and HBcrAg than those who were HBeAg negative, similar to that of serum HBV DNA. Comparatively, HBV RNA (r =0.79, P < 0.01) and HBcrAg (r =0.78, P < 0.01) had almost same higher overall correlation with the cccDNA, as that of HBV DNA (r =0.81, P < 0.01). Serum anti-HBc level (r = -0.52, P < 0.05) is negatively correlated with cccDNA level at IT phase rather than the other three phases. When set the cutoff value at 4.00 log10 IU/mL, serum anti-HBc showed potential to indicate liver inflammation, with AUC as 0.79 and the specificities as 78.85% for HBeAg positive, and with AUC as 0.72 and the specificities as 62.16% for HBeAg-negative patients, respectively.Conclusions:In treatment-naïve patients, levels of serological markers HBV RNA and HBcrAg could mirror intrahepatic cccDNA level, but were not superior to HBV DNA level. Serum anti-HBc level had certain potential to be used as a predicting marker for liver inflammation.

Multidrug-resistance hepatitis B virus (MDR HBV), defined as those with mutations resistant to both nucleoside analogs lamivudine/telbivudine/entecavir (LAM/LdT/ETV) and nucleotide analog adefovir (ADV), has potential to cause treatment difficulty. To clarify clinical prevalence and virological features of MDR HBV, we investigated serum samples from 28,236 chronic HBV-infected patients with treatment of nucleoside/nucleotide analogs. All patients underwent resistance testing in the Fifth Medical Center of Chinese PLA General Hospital between 2007 and 2019. MDR mutations were screened by direct sequencing; MDR strains (with mutations co-located on the same viral genome) were verified by clonal sequencing (>= 20 clones/sample) and subjected to phenotypic analysis if necessary. MDR mutations were detected in 0.81% (229/28,236) patients. MDR strains were verified in 83.0% (190/229) of MDR mutation-positive patients. As ETV-resistance mutation (ETVr) had additional mutation(s) on LAMr conferring more resistance, MDR mutations fell into LAMr + ADVr and ETVr + ADVr subsets. Sixteen mutation patterns of MDR strains were verified, including eight with LAMr + ADVr and eight with ETVr + ADVr. Refractory to sequential therapies of LAM/LdT/ETV and ADV were closely linked with MDR HBV development. Ten representative MDR strains (five LAMr + ADVr and five ETVr + ADVr) tested all had decrease in replication capacity compared to wild-type strains and decrease extent was positively related with the number of primary resistance on viral genome. Compared to ADV + ETV, TDF/TDF + ETV showed higher inhibitory rates on MDR HBV, especially for the five ETVr + ADVr strains (74.5%-97.6% vs. 60.2%-79.5%, all P < 0.05). This study significantly extends the knowledge on MDR HBV and has clinical implications for resistance management.

Liuweiwuling Tablet (LWWL) is a licensed Chinese patent medicine (approval number: Z20060238) included in the national health insurance for anti-inflammation of chronic HBV infection, whereas its anti-HBV effect remains clarification. The study aimed to clarify its antiviral effect and related mechanisms. HepG2.2.15 cells (wild-type HBV-replicating cells) and HepG2. A64 cells (entecavir-resistant HBV-replicating cells) were used for in vitro test. Hydrodynamic injection-mediated HBV-replicating mouse model was used for in vivo test. Active compounds and related mechanisms for antiviral effect of LWWL were analyzed using network pharmacology and transcriptomics. The inhibition rates of LWWL (0.8 mg/ml) on HBV DNA, HBsAg, and pgRNA were 57.06, 38.55, and 62.49% in HepG2.2.15 cells, and 51.57, 17.57, and 53.88% in HepG2. A64 cells, respectively. LWWL (2 g kg−1 d−1 for 4 weeks)-treated mice had 1.16 log10 IU/mL decrease of serum HBV DNA, and more than 50% decrease of serum HBsAg/HBeAg and hepatic HBsAg/HBcAg. Compared to tenofovir control, LWWL was less effective in suppressing HBV DNA but more effective in suppressing HBV antigens. Thirteen differentially-expressed genes were found in relation to HBV-host interaction and some of them were enriched in interferon (IFN)-β pathway in LWWL-treated HepG2.2.15 cells. CD3+CD4+ T-cell frequency and serum IFN-γ were significantly increased in LWWL-treated mice compared to LWWL-untreated mice. Among 26 compounds with potential anti-HBV effects that were predicted by network pharmacology, four compounds (quercetin, luteolin, wogonin, and kaempferol) were experimentally confirmed to have antiviral potency. In conclusion, LWWL had potent inhibitory effect on both wild-type and entecavir-resistant HBV, which might be associated with increasing IFN-β and IFN-γ production.

目的:研究舒肝宁注射液对人肝星状细胞LX2(HSC-LX2)细胞生长的影响,探讨其抗纤维化机制.方法:使用舒肝宁注射液处理HSC-LX2细胞,用细胞计数试剂盒-8(CCK8)法测定细胞的吸光度值,计算细胞增殖率.荧光定量聚合酶链式反应(PCR)法和免疫印迹法分别检测Ⅰ型胶原蛋白、Ⅲ型胶原蛋白和转化生长因子-β(TGF-β)的mRNA和蛋白水平.流式细胞术检测细胞周期分布及细胞凋亡率.结果:与未用药组比较,低、中、高浓度的舒肝宁注射液处理HSC-LX2细胞120 h对细胞增殖的抑制率分别为14.27％,16.37和18.67％(P<0.05).舒肝宁注射液处理组细胞Ⅰ型胶原蛋白和Ⅲ型胶原蛋白的mRNA和蛋白水平较未用药组明显降低(P<0.05).舒肝宁注射液阻滞细胞周期在G2/M期,诱导细胞的早期和晚期凋亡,最大凋亡率达到2.69％,诱导凋亡作用呈浓度依赖性(P<0.05).结论:舒肝宁注射液通过阻滞细胞周期进而抑制细胞增殖,同时促进细胞早期和晚期凋亡,下调Ⅰ型胶原蛋白和Ⅲ型胶原蛋白mRNA和蛋白表达水平,从而起到抗纤维化作用.

目的 研究复方肃毒星提取物(简称肃毒星)体外对乙型肝炎病毒(HBV)复制模板共价闭合环状DNA(cccDNA)的抑制作用.方法 通过细胞计数试剂盒CCK-8检测肃毒星在恩替卡韦耐药HBV稳定复制细胞系(HepG2.A64)、野生HBV稳定复制细胞系(HepG2.2.15和HepAD38)以及肝癌细胞系(HepG2)中的毒性作用;选择安全有效的高、中、低浓度肃毒星分别处理3个细胞系,采用实时荧光定量PCR法检测细胞内HBV cccDNA水平.以高斯荧光素酶微环cccDNA(mc-cccDNA)转染HepG2细胞,同时选择高、中、低浓度的肃毒星在不同时间点作用转染mc-cccDNA的HepG2细胞,荧光素酶检测试剂盒分析相对荧光强度,计算对cccDNA的抑制率,评价cccDNA转录活性.结果 肃毒星在HepG2.A64、HepG2.2.15、HepAD38和HepG2细胞中的半数毒性浓度分别为27.01μg/ml、29.36μg/ml、31.20μg/ml和52.80μg/ml.肃毒星对HepG2.A64、HepG2.2.15和HepAD38细胞中HBV cccDNA有抑制作用,最佳作用时间为5 d,5 d最大抑制率分别为(84.24±2.1)％、(52.02±4.74)％和(47.16±6.69)％,与未用药处理组差异有统计学意义(P均<0.05).肃毒星对转染HepG2细胞后mc-cccDNA转录活性的最佳抑制率为(48.44±4.54)％,抑制作用优于干扰素对照组(P<0.05),略弱于特异性敲低mc-cccDNA的pLV-CRISPR处理对照组(P<0.05),差异有统计学意义.结论 肃毒星对恩替卡韦耐药型和野生型细胞系HBV cccDNA的复制及转录均有很好的抑制作用,为慢性乙型肝炎功能学治愈新药的开发提供参考.

The study aimed to characterize the prevalence and virological features of the rtA181S + T184I + M204I mutant in a large cohort of patients with chronic HBV infection. In total, 22,009 nucleoside/nucleotide analog-treated patients who underwent resistance testing at the Fifth Medical Center of Chinese PLA General Hospital between 2007 and 2016 were enrolled. Serum samples were collected for HBV reverse-transcriptase gene sequencing. Phenotypic analysis of the viral replication capacity and drug susceptibility was performed. The rtA181S mutation was detected in 0.82% (180/22,009) of samples. rtA181S-positive patients had significantly higher lamivudine (LAM), adefovir (ADV), and entecavir (ETV) exposure than rtA181S-negative patients. Of 180 rtA181S-positive patients, 42 had no coexistent resistance mutations, 34 had coexisting LAM-resistance mutation (LAMr), 17 had coexisting ADV-resistance mutation (ADVr), and 86 had coexisting ETV-resistance mutation (ETVr), and one had ADVr + ETVr. rtA181S + T184I + M204I occurred in 79.1% (68/86) of patients with rtA181S + ETVr and 37.8% (68/180) of all rtA181S-positive patients. Longitudinal analysis of the clinical course of resistant mutant evolution for four representative cases showed that rtA181S + T184I + M204I developed in all patients who had received LAM/telbivudine ± ADV and was receiving ETV or ADV + ETV. Compared with wild-type, the rtA181S + T184I + M204I mutant had 53.7% lower replication capacity and >1000-, 3.9-, and 383.3-fold greater LAM, ADV, and ETV resistance, respectively, but remained sensitive to tenofovir. Artificial elimination of rtA181S from the rtA181S + T184I + M204I mutant restored viral susceptibility to ADV but decreased viral replication capacity. Our study presented the first evidence that HBV rtA181S + T184I + M204I mutation had features of multidrug-resistance that contributed to resistance to both nucleoside and nucleotide analogs.

Background: Occult HBV infection (OBI) is of great concern due to their complicated diagnosis and potential for public transmission. Objective: The study aimed to determine the clinical prevalence of OBI and if viral immune escape-associated mutations contribute to the occurrence of OBI. Study design: A total of 91,037 HBV-infected patients with different related illnesses who were admitted to the Fifth Medical Center of Chinese PLA General Hospital from January 2005 to December 2017 were tested for OBI. Serum samples from 62 patients with OBI manifestations (OBI patients) and 124 matched non-OBI patients were sequenced for possible immune escape-associated mutations within the major hydrophilic region of HBV S protein. HBsAg and HBV DNA levels in representative viral strains were measured. Results: Of the 91,037 tested patients, 487 (0.53 %) were negative for HBsAg but positive for HBV DNA and were defined as OBI patients. The prevalence in different illness categories varied. Immune escape-associated mutations were more frequently detected in OBI patients than in non-OBI patients (59.68 % vs. 35.48 %, P < 0.01), as did the coexistence of multiple mutations (43.55 % vs. 22.58 %, P < 0.01). Specifically, the prevalence rates of sT118 K, sK122R, and sV168A were increased in OBI patients. Strains with sK122R mutants (sK122R, sK122R + D144E, sK122R + C121R + D144E, and sK122R + F134L + D144E) from a follow-up OBI patient all showed significantly lower levels of HBsAg production than a wild-type strain. Conclusions: The study clarified the clinical prevalence of OBI, verified the influence of immune escape-associated mutations, and identified the role of the sK122R mutation in multiple OBI patients.

[Abstract] Objective To analyze the mutation characteristics of immune escape-associated mutations in the main hydrophilic region (MHR) of HBsAg in patients with drug-resistant mutations in HBV RT. Methods The clinical data were analyzed from 6917 patients with C-genotype HBV infection attending to the hospital from July 2007 to August 2017. And these patients were treated with nucleoside/nucleotide analogs (NAs) and received drug resistance tests. The detection rate of drug-resistant mutations in HBV RT was determined and the mutation characteristics of MHR immune escape-related mutations in HBsAg between drug-resistant mutations of HBV RT group and wild-type of HBV RT group were compared. Results Classical drug-resistant mutations in HBV resistant mutations group were higher than those in RT wild-type group ( P <0.05). Conclusions Patients with HBV RT resistant mutations have a higher detection rate of MHR immune escape mutations in HBsAg, which suggested that MHR immune escape related mutations were closely related to HBV RT drug-resistant mutations.

Objective To analyze the clinical occurrence and phenotypic characteristics of rtA181S-related novel multidrug-resistance mutation in reverse transcription region of hepatitis B virus (HBV). Methods The clinical data and sequence information of 16 443 patients with chronic HBV infection who received nucleos(t)ide analogues (NAs)-resistance testing at the original PLA 302 Hospital from 2012 to 2017 were retrospectively analyzed. rtA181S-related mutation patterns were analyzed and cloning-sequencing (≥20 clones/sample) was performed on the mutation samples of rtA181S+T184I+M204I with the highest detection rate. Phenotypic analysis was performed to evaluate the viral replication capacity and drug susceptibility. Results The rtA181S mutation was detected in 0.75% (124/16 443) of the patients. Among them, 21 patients were detected with coexistence of lamivudine (LAM)-resistance mutation and 74 patients were detected with coexistence of entecavir-resistance (ETVr) mutation. The rtA181S+T184I+M204I novel mutation accounted for 77.0% (57/74) of the rtA181S+ETVr mutation. Dynamic clinical data analysis showed that the rtA181S+T184I+M204I mutation emerged after adefovir dipivoxil (ADV), ETV, and LAM/telbivudine (LdT) treatment, accompanied by virological breakthrough or inadequate virological response. Compared to wild-type strain, rtA181S+T184I+M204I mutant had 53.7% decreased replication capacity, over 1000-, 3.9-, and 383.3-fold increased LAM, ADV, and ETV resistance, respectively, and remained sensitive to tenofovir (TDF). Conclusions rtA181S+T184I+M204I mutation is a novel multidrug-resistance mutation, which is related to the ADV, ETV or LAM/LdT sequential or combined treatment. TDF-based rescue therapy should be considered for patients harboring this mutation in clinical practice. DOI: 10.11855/j.issn.0577-7402.2020.06.10

BACKGROUND It is unclear whether immune escape-associated mutations in the major hydrophilic region of hepatitis B virus surface antigen (HBsAg) are associated with nucleoside/nucleotide analog resistance. AIM To evaluate the association between immune escape-associated mutations and nucleoside/nucleotide analog resistance mutations. METHODS In total, 19440 patients with chronic hepatitis B virus infection, who underwent resistance testing at the Fifth Medical Center of Chinese PLA General Hospital between July 2007 and December 2017, were enrolled. As determined by sequence analysis, 6982 patients harbored a virus with resistance mutations and 12458 harbored a virus lacking resistance mutations. Phenotypic analyses were performed to evaluate HBsAg production, replication capacity, and drug-induced viral inhibition of patient-derived drug-resistant mutants with or without the coexistence of sA159V. RESULTS The rate of immune escape-associated mutation was significantly higher in 9 of the 39 analyzed mutation sites in patients with resistance mutations than in patients without resistance mutations. In particular, these mutations were sQ101H/K/R, sS114A/L/T, sT118A/K/M/R/S/V, sP120A/L/Q/S/T, sT/I126A/N/P/S, sM133I/L/T, sC137W/Y, sG145A/R, and sA159G/V. Among these, sA159V was detected in 1.95% (136/6982) of patients with resistance mutations and 1.08% (134/12,458) of patients lacking resistance mutations (P < 0.05). The coexistence of sA159V with lamivudine (LAM) and entecavir (ETV)-resistance mutations in the same viral genome was identified during follow-up in some patients with drug resistance. HBsAg production was significantly lower and the replication capacity was significantly higher, without a significant difference in LAM/ETV susceptibility, in sA159V-containing LAM/ETV-resistant mutants than in their sA159V-lacking counterparts. CONCLUSION In summary, we observed a close link between the increase in certain immune escape-associated mutations and the development of resistance mutations. sA159V might increase the fitness of LAM/ETV-resistant mutants under environmental pressure in some cases.

目的 分析多种肝脏疾病中隐匿性HBV感染(occult HBV infection,OBI)检出率,并探讨OBI患者HBV S基因主要亲水区(major hydrophilic region,MHR)免疫逃逸相关突变特点.方法 回顾性分析2005年1月—2017年12月就诊于中国人民解放军总医院第五医学中心的91037例HBV感染住院患者临床资料,筛选出OBI患者并扩增其HBV S基因序列,分析其HBV S基因MHR免疫逃逸相关突变特点.结果 91037例住院患者中OBI总检出率为0.53％(487/91037),急性乙型肝炎患者中OBI检出率最高(9.26％,130/1404),肝硬化患者中OBI检出率最低(0.26％,78/29921).62例OBI患者组与124例非OBI患者组相比,OBI患者组MHR免疫逃逸相关突变总体检出率显著高于非OBI患者组(59.68％vs.35.48％;P<0.05);OBI患者组MHR多个免疫逃逸相关突变的联合检出率显著高于非OBI患者组(43.55％vs.22.58％;P<0.05);其中,sT118K、sK122R和sV168A 3种单点突变的检出率显著高于非OBI患者组.结论 本研究显示临床HBV感染患者中有较高的OBI检出率,而且不同肝脏疾病中OBI检出率不同.此外,HBV S基因MHR的免疫逃逸相关突变与临床实践中OBI的发生密切相关.

目的 分析RT耐药突变患者乙肝表面抗原(HBsAg)主要亲水区(MHR)免疫逃逸相关突变的特点.方法回顾性分析2007年7月-2017年8月于解放军总医院第五医学中心接受NAs抗病毒治疗并行耐药检测的6917例C基因型CHB患者的临床资料,并根据是否发生耐药突变分为HBV RT耐药突变组与HBV RT野生组,比对两组HBsAg MHR免疫逃逸相关突变的特点.结果经抗病毒治疗的CHB患者HBV RT耐药相关突变总体检出率为37.37％(2585/6917);且HBV RT耐药突变组HBsAg MHR总体突变率显著高于HBV RT野生组(30.00％vs.17.13％,P<0.05);HBV RT耐药突变组患者的sQ101K/R/H、sS114T/A/L、sT/I126S/N/A、sG130N/R/K/A、sM133T/I、sS143T/L、sA159V/G、sE164D/G等单点突变检出率均高于HBV RT野生组(P<0.05).结论HBV RT耐药突变患者具有更高的HBsAg MHR免疫逃逸相关突变检出率,提示MHR免疫逃逸相关突变与HBV RT耐药突变密切相关.

Background and Aims: Entecavir (ETV) resistance of hepatitis B virus (HBV) conventionally requires rt184, 202, or 250 mutations plus lamivudine-resistance mutation (rtM204V/I ± L180M). This study aimed to clarify whether rtL180M+A181C+M204V mutations may contribute to HBV ETV resistance. Methods: Serum samples were collected from 22,009 patients who underwent resistance testing in Beijing 302 Hospital from 2007 to 2016. HBV reverse transcriptase (RT) gene was screened by direct sequencing and verified by clonal sequencing. Phenotypic analysis was performed for evaluating replication capacity and drug susceptibility. Results: Classical ETV-resistance mutations of HBV were detected in 1252 patients who were receiving ETV therapy. The rtA181C mutation was detected with rtL180M+M204V mutations in 18 lamivudine-experienced ETV-treated patients, and the emergence of the mutations was associated with virological breakthrough or inadequate virological response to ETV. Patient-derived representative rtA181C-containing mutants, rtL180M+A181C+M204V, rtL180M+A181C+M204V+M250V, and rtL180M+A181C+S202G+M204V, exhibited 45.7%, 25.9%, and 25.0% replication capacity and 85.6-, 356.1-, and 307.1-fold decreased susceptibility to ETV respectively compared to the wild-type strain, while the three mutants remained sensitive to tenofovir (TDF). Artificial elimination of rtA181C largely restored the rtL180M+A181C+M204V mutant’s sensitivity to ETV. Molecular modelling of viral RT binding to ETV showed that the rtL180M+A181C+M204V mutant had a less stable conformation compared to rtL180M+M204V mutant. In clinical practice, undetectable serum HBV DNA was achieved in two of five longitudinally followed rtA181C-positive patients who received switching-to TDF therapy, but not in the other three who received add-on adefovir therapy during observation. Conclusions: Both clinical and experimental data support rtL180M+A181C+M204V as a novel non-classical ETV-resistance mutation pattern.