拉米夫定耐药慢性乙型肝炎患者HBV多聚酶区基因突变分析

目的探讨拉米夫定（LAM）耐药慢性乙型肝炎（CHB）患者多聚酶区序列突变特点。方法收集63例接受拉米夫定治疗且耐药的CHB患者的临床资料,采用PCR产物直接测序法检测HBVP基因多聚酶区序列耐药变异。结果 63例诊断为耐药的患者中,51例患者检测到LAM相关的HBV多聚酶区基因突变,其中rtM204V/I变异46例（73.0%）,rtL180M变异25例（39.7%）,rtV173L/M变异5例（7.9%）,rtQ214E变异1例（1.6%）,rtS213T变异2例（2.%）12,rtV207L/M/I变异2例（3.2%）,rtA181T变异3例（4.8%）,rtT184I/S/M变异1例（1.6%）。结论对拉米夫定治疗患者的耐药检测除HBV多聚酶区常见的rtLl80M和rtM204V/I位点变异外,还应考虑其他位点的耐药变异。     

拉米夫定治疗慢性乙型肝炎患者YMDD变异及其伴随变异的初步研究

目的 探讨拉米夫定治疗慢性乙型肝炎过程中HBV P基因YMDD变异及其伴随变异的分布情况.方法:以聚合酶链反应(PCR)扩增93例慢性乙型肝炎患者血清HBV P区,对PCR产物直接测序,检测YMDD及相关变异.结果:93份血清标本中,发生YMDD变异者为60例.变异及伴随变异:1型(rtL180M rtM204Ⅴ)变异26例(43%),2型(rtM204Ⅰ)14例(23.3%),rtL180M rtM204Ⅰ变异8例(13.3%),rtV173L rtL180M rtM204Ⅴ变异8例(13.3%),rtV173L rtM204Ⅰ变异和单纯rtM204Ⅴ部分变异各2例(3.3%).结论:拉米夫定治疗慢性乙型肝炎过程中,部分忠者可发生HBV YMDD变异和(或)其它位点伴随变异.     

重度慢性乙型肝炎患者的病毒耐药突变检测

目的：探讨石家庄地区重度慢性乙型肝炎患者HBV耐药突变的状况。方法对51例2012年1月至2013年12月在石家庄市五医院就诊的重度慢性乙型肝炎患者进行HBV P区基因序列测定。结果51例重度慢性乙型肝炎患者中,25例存在HBV P区发生突变,突变率为49·．0％。突变率在HBeAg阳性组和阴性组之间无显著差别（ P ＞0．05）。 HBV P区突变组血清HBV DNA水平显著高于未突变组（ P ＜0．05）,ALT、AST和TBIL在突变组也显著升高（ P ＜0．05）。对25例存在HBV P区基因突变患者进行突变模式分析,居于前四位的突变模式的总和占所有突变的72％。突变模式主要为rtM204I／V单独突变以及联合rtL180M和（或）其他突变位点、rtA181V／T单独突变以及联合rtN236T或rtM250V／L突变。结论重度慢性乙型肝炎患者服用拉米夫定及阿德福韦酯等进行抗病毒治疗过程中,应实时监测HBV P区耐药突变状况,及时调整用药。     

扬州地区2013年—2016年乙型肝炎病毒逆转录酶区基因突变与多位点变异基因耐药的特征及其对策

目的:研究扬州地区经核苷(酸)类似物治疗疗效不佳的慢性乙型肝炎(CHB)患者发生乙型肝炎病毒(HBV)耐药基因突变的特征、多位点变异的发生率、影响因素及其对策。方法:选取2013年1月—2016年12月间就诊并经核苷(酸)类似物治疗6月以上且HBV-DNA阳性患者182例资料,采用核酸扩增技术,通过测序和检测耐药突变的变异特点,分析核苷(酸)类药物对HBV耐药相关位点,并比较近4年多位点变异的发生率有无统计学差异。结果:182例患者中,检出基因耐药103例,未检出79例;在11个经典耐药位点中,检测到8个耐药位点,未检出rtA194T、rtI233V、rtM250V等3个位点的变异;其中单个位点突变以rtM204I的突变率为最高(65.31%),其次为rtA181T;2个位点突变以rtL180M合并rtM204I突变为主(占42.86%),3个及其以上突变主要以rtL180M及rtM204V(或rtM204I)突变的基础上合并rtI169T、rtT184A、rtA181C、rtA181V、rtS202S、rtV173L等位点的突变;比较2013年—2016年4年间,3个及其以上位点突变的年发生率经比较其差异无统计学意义(P>0.05)。结论:扬州地区CHB患者接受核苷(酸)类药物治疗中,发生耐药变异的相关位点种类多,模式复杂;3个及其以上位点突变(多位点变异)与初治未使用高基因屏障核苷类似物及拉米夫定、阿德福韦酯、恩替卡韦不规则联用或序贯治疗相关;规范、优化核苷(酸)类药物抗HBV治疗能有效控制多位点变异的发生率,治疗中应密切监测耐药相关位点的变异。     

石家庄地区乙型肝炎病毒基因分型及P区耐药基因突变分析

目的:分析石家庄地区乙型肝炎病毒(HBV)基因型分布以及核苷(酸)类似物耐药基因突变状况。 方法:对2011年1月-2013年12月在石家庄市五医院就诊的遵医嘱口服核苷(酸)类似物6个月以上的354例慢性乙型肝炎患者进行HBV P区基因序列测定。结果:HBV B型为17例(4.8%),C型为337例(95.2%);共检出突变182例,在B型中检测出有10例突变(58.8%),在C型中检测出有172例突变(51.1%),两组间突变率无显著差别;182例HBV P区存在突变的病例中以rtL180M+rtM204I/V/S联合突变最多,占突变比例24.2%;其次是rtM204I/V/S单位点突变,占突变比例20.3%,这两种突变模式的比例达到44.5%;第3位是rtL180M+rtM204I/V/S 再加上其他一个或多个位点的突变,占突变比例12.6%。结论:石家庄地区HBV基因型以C型为主;核苷(酸)类似物耐药基因突变以拉米夫定和替比夫定耐药最常见。     

核苷(酸)类似物耐药相关位点分析

目的:分析慢性乙型肝炎患者HBV逆转录酶基因与核苷(酸)类似物耐药相关位点的变异情况与临床耐药的关系。方法:分析不同核苷(酸)类似物的耐药相关突变情况及不同核苷(酸)类似物耐药的突变形式。结果:拉米夫定耐药突变中以M204V/I最常见,可伴随L180M突变,阿德福韦耐药中以N236T±A181位碱基替换为主;恩替卡韦耐药突变发生在拉米夫定耐药基础上;替比夫定的耐药突变为M204I;替诺福韦的变异位点以194位碱基替换为主(rtA194T)。结论:检测HBV逆转录酶基因多位点耐药相关突变,有助于临床及时发现乙型肝炎患者是否存在HBV耐药,从而合理进行抗病毒治疗。     

核苷和核苷酸类药物在慢性乙型肝炎病毒感染者中的应用及耐药突变分析

目的 分析慢性乙型肝炎病毒(hepatitis B virus,HBV)感染者核苷和核苷酸类药物(nucleoside and nucleotide analogs,NAs)发生耐药后的突变模式,为规范抗病毒治疗和耐药管理提供参考价值。方法 选取2010年1月—2014年12月发生耐药突变的375例慢性HBV感染者的临床资料,采用实时荧光定量PCR方法对耐药患者血清HBV聚合酶基因逆转录酶区进行扩增,对PCR产物进行直接测序。结果 拉米夫定(lamivudine,LAM)耐药组和阿德福韦酯(adefovir dipivoxil,ADV)耐药组基因型耐药伴生物化学突破的构成比均高于恩替卡韦(entecavir,ETV)耐药组(χ²=12.111,P<0.001;χ²=7.992,P=0.005)。253例LAM耐药者中有16种突变类型,单位点突变134例(52.96%),以rtM204I 最多见,多位点突变119例(47.04%),以rtLl80M+M204V最多见;在rtM204突变模式中,rtM204I为单位点突变模式的主要位点(χ²=154.555,P<0.001),rtM204V为多位点突变模式的主要位点(χ²=4.317,P=0.038)。88例ADV耐药者中有24种突变类型,单位点突变61例(69.32%),以rtA181T最多见,多位点突变27例(30.68%),以rtL180M+rtM204V+rtA181T最多见;无论是单位点突变模式还是多位点突变模式,相对于rtN236T,rtA181T均是主要突变位点(χ²=42.749,P<0.001;χ²=6.033,P=0.014)。34例ETV耐药者中有5种突变类型,均为多位点突变,以rtL180M+rtM204V+rtS202I/G最多见;相对于rtT184,rtS202为主要突变位点(χ²=5.882,P=0.015)。ADV耐药者突变模式的复杂性最高,其次为LAM耐药者,最低为ETV耐药者。结论 NAs的耐药突变复杂多样,尤其是ADV,应接受和执行优选和优化治疗策略,以实现预防耐药,减少或避免挽救治疗。     

拉米夫定治疗变异后慢性乙型肝炎患者血清IFN-γ和IL-10的测定及临床意义

目的评估拉米夫定治疗的慢性乙型肝炎患者在治疗后及病毒变异后血清中Th1型[γ干扰素（IFN-γ）]和Th2型[白细胞介素（IL）-10]细胞因子水平,探讨拉米夫定治疗乙型肝炎病毒变异后的细胞因子动态变化。方法选取拉米夫定治疗而发生病毒变异及未变异的慢性乙型肝炎患者各20例,另选取20例健康人作为对照,检测患者血清IFN-γ、IL-10的水平。结果在治疗后6个月时,未变异组血清IFN-γ、IL-10水平与变异组相比差异无统计学意义（P〉0.05）;但在病毒变异后,变异组IFN-γ水平均低于未变异组,IL-10水平则高于未变异组,差异有统计学意义（P〈0.01）。结论拉米夫定抗病毒治疗出现变异后,机体细胞因子表达发生改变。     

HBV YMDD外基因变异对核苷类耐药影响的临床研究

目的 探讨YMDD变异阴性的拉米夫丁耐药患者体内HBV逆转录酶RT区基因是否有变异.方法 采用荧光PCR技术检测拉米夫丁耐药患者HBV YMDD基因情况,用PCR产物直接测序分析RT区基因序列.结果 7份标本均测出YMDD阳性,无发生YMDD变异,2例发生rtL80V/I变异,1例发生rtV173L,其余均无发生RT区基因变异.结论 部分拉米夫丁临床耐药现象的出现可能与HBV RT区基因变异无关.     

阿德福韦酯治疗拉米夫定耐药慢性乙型肝炎22例

目的 观察阿德福韦酯治疗拉米夫定耐药慢性乙型肝炎的临床疗效. 方法 慢性乙型肝炎42例,随机分为两组,对照组继续服用拉米夫定治疗,治疗组口服阿德福韦酯,治疗前后检测HBV-DNA定量、HBVM、肝功能变化. 结果 治疗组血清HBV-DNA水平下降迅速、ALT持续下降,肝功能改善显著,与对照组比较差异有极显著性（P＜0.01）. 结论 阿德福韦酯可使耐药变异株迅速消失,HBV-DNA水平下降,显著改善肝功能,是拉米夫定治疗变异耐药后有效、安全的替代药物.     

Establishment of drug-resistant HBV small-animal models by hydrodynamic injection

In antiviral therapy of hepatitis B virus (HBV) infection, drug resistance remains a huge obstacle to the long-term effectiveness of nucleoside/tide analogs (NAs). Primary resistance mutation (rtM204V) contributes to lamivudine (LAM)-resistance, and compensatory mutations (rtL180M and rtV173L) restore viral fitness and increase replication efficiency. The evaluation of new anti-viral agents against drug-resistant HBV is limited by the lack of available small-animal models. We established LAM-resistance HBV replication mice models based on clinical LAM-resistant HBV mutants. Double (rtM204V+rtL180M) or triple (rtM204V+rtL180M+rtV173L) lamivudine-resistant mutations were introduced into HBV expression vector, followed by hydrodynamic injection into tail vein of NOD/SCID mice. Viremia was detected on days 5, 9, 13 and 17 and liver HBV DNA was detected on day 17 after injection. The serum and liver HBV DNA levels in LAM-resistant model carrying triple mutations are the highest among the models. Two NAs, LAM and entecavir (ETV), were used to test the availability of the models. LAM and ETV inhibited viral replication on wild-type model. LAM was no longer effective on LAM-resistant models, but ETV retains a strong activity. Therefore, these models can be used to evaluate anti-viral agents against lamivudine-resistance, affording new opportunities to establish other drug-resistant HBV small-animal models.Key words: Drug-resistance, Animal models, Hepatitis B virus, Hydrodynamic injection     

The main hepatitis B virus (HBV) mutants resistant to nucleoside analogs are susceptible in vitro to non-nucleoside inhibitors of HBV replication

Long-term treatment of chronic hepatitis B with nucleos(t)ide analogs can lead to the emergence of HBV resistant mutants of the polymerase gene. The development of drugs with a different mode of action is warranted to prevent antiviral drug resistance. Only a few non-nucleosidic molecules belonging to the family of phenylpropenamides (AT-61 & AT-130) and heteroaryldihydropyrimidines (BAY41-4109) can prevent RNA encapsidation or destabilize nucleocapsids, respectively. The sensitivity of the main nucleos(t)ide analog- resistant mutants to these inhibitors was evaluated in vitro. HepG2 stable cell lines permanently expressing wild type (WT) HBV or the main HBV mutants resistant to lamivudine and/or adefovir (rtL180M + rtM204V, rtV173L + rtL180M + rtM204V, rtM204I, rtL180M + rtM204I, rtN236T, rtA181V, rtA181V + rtN236T, rtA181T, rtA181T + rtN236T) were treated with AT-61, AT-130 or BAY-41 4109. Analysis of intracellular encapsidated viral DNA showed that all mutants were almost as sensitive to these molecules as WT HBV; indeed, the fold-resistance ranged between 0.7 and 2.3. Furthermore, the effect of a combination of either AT-61 or AT-130 with BAY41-4109, and the combination of these compounds with tenofovir was studied on wild type HBV as well as on a lamivudine and an adefovir-resistant mutant (rtL180M + M204V and rtN236T, respectively). These combinations of compounds resulted in inhibition of viral replication but showed slight antagonistic effects on the three HBV species. Based on this in vitro study, BAY-41 4109, AT-61 and AT-130 molecules that interfere with capsid morphogenesis are active against the main lamivudine- and adefovir-resistant mutants. These results suggest that targeting nucleocapsid functions may represent an interesting approach to the development of novel HBV inhibitors to prevent and combat drug resistance.     

Dynamics of hepatitis B virus resistance to entecavir in a nucleoside/nucleotide-naïve patient

The genotypic evolution of HBV quasi-species was analyzed in a nucleoside/nucleotide-na?ve patient who developed resistance to entecavir. The lamivudine resistant quasi-species (rtM204V+/-rtL180M), absent at baseline, were emerged as early as 48 weeks after entecavir administration. Entecavir-resistant quasi-species (rtM204V+/-rtL180M plus S202G) were found after week 112 and gradually became the predominant mutations afterwards. The lamivudine- and entecavir-resistant mutations emerged closely in combination with the rtV207L, rtA222T, rtP237T or rtI163V substitutions. Our results indicated that the lamivudine-resistant mutations were developed first and may serve as a prequisite for subsequent entecavir-resistant mutations in this nucleoside/nucleotide-na?ve patient.     

Changes in different regions of hepatitis B virus gene in hepatitis B 'e' antigen-negative patients with chronic hepatitis B: the effect of long-term lamivudine therapy

BACKGROUND: Lamivudine therapy for chronic hepatitis B has been associated with changes in different regions of the hepatitis B virus nucleotide sequence. AIM: To study changes in the sequences of polymerase and precore/core promoter regions of hepatitis B virus, before and during 5 years of therapy with lamivudine. METHODS: Eighty consecutive samples were taken from 10 chronic hepatitis B 'e' antigen-negative patients. RESULTS: Nine patients carried hepatitis B virus precore mutations during the study. Before therapy, wild type was replaced by A1896 in two (20%) cases. During treatment, A1896 reverted transitory to wild type in five cases (50%) and in one case wild type was replaced by A1896. The continuous detection of precore mutations during therapy was associated with a lower response rate. YMDD mutations were observed in nine cases and both, L180M and M204V/I mutations were simultaneously detected in six cases. About 75% of the patients with M204V mutations were responders and none with M204I or mixed pattern sustained response. CONCLUSION: Hepatitis B 'e' antigen-negative patients exhibit changes in the precore regions both spontaneously and under lamivudine therapy, the transitory reversion to wild type being most frequently witnessed. Patients carrying M204V mutations are more likely to respond to therapy. If, in further studies, these results are confirmed some patients with YMDD mutations could benefit from prolonging the duration of lamivudine therapy.     

核苷类药与慢性HBV感染者病毒P基因准种及变异特点的关系

目的观察核苷类药治疗不同时间段慢性HBV感染患者血清乙肝病毒聚合酶(HBV P)基因准种变化及变异特点。方法运用焦磷酸测序仪器配套的软件Assay Design SW在目的区域两端保守区域设计PCR引物及测序引物,采用套式PCR方法检测血清中HBV DNA,按照Pyro-Mark ID遗传分析系统用SNP模式进行PCR产物(HBV P基因相关位点)的焦磷酸测序和突变频率检测。108例慢性HBV感染患者中用药史明确者同期测定HBV DNA、HBV标志物、ALT。结果108例患者中,61例发生变异,变异模式以经典突变L180M、M204V/I、A181V/T、N236T突变为主,有少量V173L、S202G、T184G突变;其中40例用药史明确患者中,18例发生变异,发生变异者耐药突变型在样本中所占比例为20%-100%,HBV P基因变异可以在HBV DNA、ALT发生突变前、中、后检出。结论焦磷酸测序可以快速检测HBV P基因变异;变异株突变频率变化可初步反映HBVP基因准种异质性;HBV P基因变异模式、突变频率与核苷类药物敏感性密切相关。