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.

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.

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.

HepatologyVolume 73, Issue 5 p. 2075-2076 Correspondence Letter to the Editor: Why Serum Hepatitis B Virus (HBV) DNA Has Higher Frequency of rtM204I/V Mutation Than Serum HBV RNA in the Same Individual? Hao Liao Ph.D., orcid.org/0000-0001-8827-2884 Intervention and Cell Therapy Center, Peking University Shenzhen Hospital, Shenzhen Peking University–The Hong Kong University of Science and Technology Medical Center, Shenzhen, ChinaSearch for more papers by this authorJie Wang Ph.D., Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, ChinaSearch for more papers by this authorYan Liu Ph.D., Institute of Infectious Diseases, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, ChinaSearch for more papers by this authorJunhui Chen Ph.D., Intervention and Cell Therapy Center, Peking University Shenzhen Hospital, Shenzhen Peking University–The Hong Kong University of Science and Technology Medical Center, Shenzhen, ChinaSearch for more papers by this authorDongping Xu Ph.D., Institute of Infectious Diseases, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, ChinaSearch for more papers by this authorFengmin Lu Ph.D., Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Center of Precision Medicine, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, ChinaSearch for more papers by this author Hao Liao Ph.D., orcid.org/0000-0001-8827-2884 Intervention and Cell Therapy Center, Peking University Shenzhen Hospital, Shenzhen Peking University–The Hong Kong University of Science and Technology Medical Center, Shenzhen, ChinaSearch for more papers by this authorJie Wang Ph.D., Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, ChinaSearch for more papers by this authorYan Liu Ph.D., Institute of Infectious Diseases, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, ChinaSearch for more papers by this authorJunhui Chen Ph.D., Intervention and Cell Therapy Center, Peking University Shenzhen Hospital, Shenzhen Peking University–The Hong Kong University of Science and Technology Medical Center, Shenzhen, ChinaSearch for more papers by this authorDongping Xu Ph.D., Institute of Infectious Diseases, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, ChinaSearch for more papers by this authorFengmin Lu Ph.D., Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Center of Precision Medicine, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, ChinaSearch for more papers by this author First published: 09 August 2020 https://doi.org/10.1002/hep.31514 Supported by the National 13th Five-Year Special Grand Project for Infectious Disease (2017ZX10302201-008) and Guangdong Basic and Applied Basic Research Foundation (2019A1515110615). Potential conflict of interest: Nothing to report. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume73, Issue5May 2021Pages 2075-2076 RelatedInformation

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.

HepatologyVolume 74, Issue 3 p. 1720-1721 Correspondence Letter to the Editor: The Differences Between the Reverse Transcriptional Efficiency of HBV Pregenomic RNA and Transcriptional Efficiency of HBV Covalently Closed Circular DNA Hao Liao Ph.D., Hao Liao Ph.D. orcid.org/0000-0001-8827-2884 Intervention and Cell Therapy Center, Peking University Shenzhen Hospital, Shenzhen Peking University–The Hong Kong University of Science and Technology Medical Center, Shenzhen, ChinaSearch for more papers by this authorYan Liu Ph.D., Yan Liu Ph.D. Department of Infectious Diseases, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, ChinaSearch for more papers by this authorJun Wang Ph.D., Jun Wang Ph.D. Peking University Ditan Teaching Hospital, Beijing, ChinaSearch for more papers by this authorGuanxun Cheng Ph.D., Guanxun Cheng Ph.D. Intervention and Cell Therapy Center, Peking University Shenzhen Hospital, Shenzhen Peking University–The Hong Kong University of Science and Technology Medical Center, Shenzhen, ChinaSearch for more papers by this authorDongping Xu Ph.D., Dongping Xu Ph.D. Department of Infectious Diseases, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, ChinaSearch for more papers by this authorJunhui Chen Ph.D., Junhui Chen Ph.D. Intervention and Cell Therapy Center, Peking University Shenzhen Hospital, Shenzhen Peking University–The Hong Kong University of Science and Technology Medical Center, Shenzhen, ChinaSearch for more papers by this authorFengmin Lu Ph.D., Fengmin Lu Ph.D. Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Center for Precision Medicine, Academy of Medical Sciences, Zhengzhou University, Henan, ChinaSearch for more papers by this author Hao Liao Ph.D., Hao Liao Ph.D. orcid.org/0000-0001-8827-2884 Intervention and Cell Therapy Center, Peking University Shenzhen Hospital, Shenzhen Peking University–The Hong Kong University of Science and Technology Medical Center, Shenzhen, ChinaSearch for more papers by this authorYan Liu Ph.D., Yan Liu Ph.D. Department of Infectious Diseases, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, ChinaSearch for more papers by this authorJun Wang Ph.D., Jun Wang Ph.D. Peking University Ditan Teaching Hospital, Beijing, ChinaSearch for more papers by this authorGuanxun Cheng Ph.D., Guanxun Cheng Ph.D. Intervention and Cell Therapy Center, Peking University Shenzhen Hospital, Shenzhen Peking University–The Hong Kong University of Science and Technology Medical Center, Shenzhen, ChinaSearch for more papers by this authorDongping Xu Ph.D., Dongping Xu Ph.D. Department of Infectious Diseases, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, ChinaSearch for more papers by this authorJunhui Chen Ph.D., Junhui Chen Ph.D. Intervention and Cell Therapy Center, Peking University Shenzhen Hospital, Shenzhen Peking University–The Hong Kong University of Science and Technology Medical Center, Shenzhen, ChinaSearch for more papers by this authorFengmin Lu Ph.D., Fengmin Lu Ph.D. Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Center for Precision Medicine, Academy of Medical Sciences, Zhengzhou University, Henan, ChinaSearch for more papers by this author First published: 04 March 2021 https://doi.org/10.1002/hep.31786 Supported by the National 13th Five-Year Special Grand Project for Infectious Disease (2017ZX10302201-001) and Project funded by China Postdoctoral Science Foundation (2019TQ0206). Potential conflict of interest: Nothing to report. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume74, Issue3September 2021Pages 1720-1721 RelatedInformation

Objective To profile transcriptome dynamics in the entecavir resistant hepatitis B virus (ETV-r HBV) stably transfected cell line, and provide fundamental data for future studies to understand mechanisms of infection using this cell line. Methods ETV-r HBV stably transfected cell lines (HepG2.A64) and the corresponding isogenic cell lines (HepG2) were used to profile the transcriptome (n=3). We used DESeq2 software to analyze the sequencing results to identify differentially expressed genes with |log2 Fold Change|>2, padj<0.05. GO and KEGG functional enrichment analysis of differential genes were performed by clusterProfiler software, and then the transcriptional and protein levels of related genes in the DNA repair pathway were detected by RT-qPCR and Western blotting. Results A total of 613 differentially expressed genes were identified in the cell lines before and after stable ETV-r HBV transfection, of which 401 were up-regulated and 212 down-regulated (padj<0.05). GO and KEGG enrichment analysis showed that differentially expressed genes were mainly involved in the inflammatory response, PI3K/Akt signal pathway, PPAR signal pathway, NF-κB signal pathway and immune-related biological processes. Through the analysis of the relevant genes in the DNA repair pathway and the verification by RT-qPCR and Western blotting, it was found that after stable transfection of ETV-r HBV, the mRNA expression of RAD52 and XRCC2 genes in the DNA repair pathway decreased by 68.96%±7.59% and 69.58%±6.32%, respectively, and the protein expression decreased by 69.93%±3.88% and 26.47%±12.7%, respectively, and the differences were statistically significant (P<0.05). Conclusion Compared with the original background cell line HepG2, a lot of genes are differentially expressed at the transcription level in HBV stable cell line, and the DNA repair-related genes in the HepG2. A64 cell line had been decreased significantly. DOI: 10.11855/j.issn.0577-7402.2021.05.04

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.

We read with great interest the article entitled \"HBV RNA profiles in chronic hepatitis B patients under different disease phases and anti-viral therapy\" authored by Mak et al(1). The authors proposed that the reverse transcription efficiency in HBeAg-positive patients was significantly higher than that in HBeAg-negative patients, based on the RNA: DNA ratio in the two groups of patients (0.79 vs. 0.53; p\u003c 0.001). We had found that a higher ratio of serum HBV RNA to HBV DNA was inversely related with reverse transcription efficiency of HBV pgRNA(2), and HBeAg-positive patients had a lower reverse transcription efficiency of HBV pgRNA than HBeAg-negative patients did. We here suggest that the ratio of serum HBV DNA: (HBV DNA + HBV RNA) is more reliable to assess the reverse transcriptional efficiency of pgRNA. The medians of ratio in our cohort were 0.56 (n = 54), 0.56 (n = 23), 0.69 (n = 64) and 0.66 (n = 4), for patients with HBeAg-positive chronic infection, HBeAg-positive chronic hepatitis, HBeAg-negative chronic infection, and HBeAg-negative chronic hepatitis, respectively. Inconsistent with the report by Mak et al., our data showed that the reverse transcriptional efficiency was lower in HBeAg-positive patients than in HBeAg-negative patients (0.56 vs. 0.68; p\u003c 0.001). A schematic diagram for illustrating the efficiencies of covalently closed circular DNA (cccDNA) transcription and reverse transcription of pgRNA are shown in Figure 1.

Hepatitis B virus (HBV) infection is a global epidemic. The main transmission route of chronic HBV infection is from mother to child, yet the mechanisms underlying HBV intrauterine infection remain unclear. In the present study, the effect and the mechanism underlying hepatitis B virus X antigen (HBxAg) on HBV replication and EGFR activation in trophoblasts was investigated. Serum samples from pregnant women with HBV infection were used to infect trophoblasts and HBxAg expression was detected using ELISA. HBV plasmids carrying either full length hepatitis B virus X (HBx) or HBx with a deletion mutation (ΔHBx) were transfected into trophoblasts and expression levels of HBV DNA, hepatitis B e-antigen and pregenomic (pg)RNA, and structural maintenance of chromosomes (Smc) 5/6 were assessed. The association between HBx and EGFR promoters was characterized using a luciferase reporter assay and EGFR/PI3K/phosphorylated (p)-AKT expression and apoptosis rate were also monitored. The results of the present study indicated that HBxAg expression increased with the increasing titre of HBV DNA (P<0.05). Compared with the wild-type group, the amount of HBV DNA in the supernatant and cells was significantly reduced (P<0.05) in the ΔHBx group and the intracellular HBeAg and pgRNA levels were also significantly decreased (P<0.05). In addition, Smc5/6 expression was also significantly decreased (P<0.05) when the intracellular HBx protein was expressed compared with mock-transfected cells. Co-transfection of HBx and EGFR promoter plasmids in JEG-3 and HTR-8 cells significantly elevated EGFR promoter driven luciferase expression relative to the control group (P<0.01). In EGFR overexpressing cells, the expression of PI3K/p-AKT was significantly increased, whereas the apoptosis rate was significantly decreased (P<0.05). These results were reversed in the EGFR-knockdown group. In conclusion, the present study demonstrated that HBx promotes HBV replication in trophoblasts via downregulation of Smc5/6, activates the EGFR promoter and inhibits trophoblast apoptosis via the PI3K/p-AKT downstream signalling pathway, thereby increasing the risk of HBV intrauterine infection.

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.

INTRODUCTION:Adefovir plus entecavir (ADV+ETV) rescue therapy in ETV-resistant patients with chronic hepatitis B virus (HBV) infection is suboptimal in some patients. This study aims to elucidate the evolutionary characteristics of drug-resistant HBV mutants and their association with clinical responses in such patients.METHODOLOGY:Thirty-seven ETV-resistant patients were enrolled, among whom twelve had an inadequate virological response to ADV+ETV rescue therapy. The clonal sequence (³ 20 clones/sample) of HBV reverse transcriptase gene was performed to identify the resistance mutations. Phenotypic analysis was performed to evaluate the replication capacity and drug susceptibility of the mutants.RESULTS:ETV-resistant mutants were continuously detected in 10 of the 12 patients, and multidrug-resistant (MDR) mutants, including a novel strain (rtL180M+A181V+T184A+S202G+M204V), were detected in two patients. Seven of the 12 patients who subsequently received tenofovir (TDF)-based therapy for 38 (23-60) months all achieved undetectable HBV DNA after treatment, and ETV-resistant mutants converted to wild-type in the four patients' samples. In contrast, the other five patients who did not achieve an adequate virological response had remaining of ETV-resistant mutants. The novel MDR strain exhibited multiple resistances to LAM, ADV, and ETV, and 11.2-fold lower susceptibility to TDF.CONCLUSIONS:This study is the first to demonstrate that MDR HBV mutations may contribute to the poor efficacy of ADV+ETV combination therapy in ETV-resistant patients. Moreover, a novel MDR HBV strain was identified. Our results indicate that a TDF-based rescue therapy would be effective for the treatment of the refractory cases.

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.

肝细胞癌(hepatocellular carcinoma,HCC)是常见的消化系统恶性肿瘤之一,其具有起病隐袭、早期症状不明显、进展迅速等特点,一经发现已发展至中晚期,给患者生命健康带来严重威胁.近年来,随着新型生物标志物的发现和检测技术的构建与应用,HCC的诊断、疗效及预后评估效果有了显著提升,大大提高了HCC患者生存质量.本文就近年来HCC的诊断、疗效及预后标志物的研究进展作一综述,重点分析HCC不同标志物的功能作用,为HCC标志物的临床应用提供重要依据.

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.