拉米夫定治疗中乙型肝炎病毒基因序列变异特点及其准种研究

目的研究拉米夫定治疗中乙型肝炎病毒（HBV）逆转录酶区核苷酸序列变异、特点、含量及其与基因型和病毒载量的关系。方法普通DNA测序法检测拉米夫定治疗的117份慢乙肝患者血清HBV逆转录酶区基因序列及其基因型;其中99份用TaqMan法定量HBVDNA;64份用焦磷酸测序（Pyrosequencing）检测YMDD基序中碱基的频率。结果HBVYMDD变异组中C型43例,B型10例,A／B混合型1例;YMDD不变异组中C型54例,B型8例,D型1例,HBVDNA含量平均对数值在变异组和不变异组分别为：6．5699和6．6165;YMDD变异与其基因型及病毒载量无统计学意义;rtL180M位点变异与rtM204I／V位点变异高度相关;并且HBV野生株与变异株均同时存在。结论结合两种测序方法可以用来研究拉米夫定治疗中HBV基因序列变异情况及对YMDD耐药株进行定量。     

HIV-1拉米夫定(3TC)耐药毒株的体外诱导培育和鉴定

目的 通过体外传代培养，将我国HIV-1药物敏感毒株诱导为拉米夫定耐药毒株。方法 在MT4细胞-中国HIV-1B’亚型CNHN24毒株培养系统中加入0．008μmol／L拉米夫定，逐渐增加药物浓度进行传代和培养，每隔3～5代测定半数抑制浓度（IC50），并用RT-PCR法扩增plo区基因，进行基因型耐药性分析。将表型和基因型耐药结果与敏感株进行比较。结果 培养6代后，IC50由野生毒株的0．018／μmol／L逐渐提高到0．15μmol／L；第7代时，IC50突然增加到大于2048／μmol／L；pol区的184位氨基酸由甲硫氨酸（M）突变为异亮氨酸（I）。去除药物继续培养5代，IC50仍维持〉2048／μmol／L，pol区184位氨基酸仍为异亮氨酸，没有发生回复突变。结论 在国内首次用中国HIV-1毒株诱导培育出可能稳定传代的3TC耐药株，可用于HIV-1耐药性的研究和HIV-1药物的药效学评价。该耐药株已申请国家专利．     

阿德福韦治疗拉米夫定耐药慢性乙肝患者的疗效

目的拉米夫定是临床上最早应用于慢性乙型肝炎治疗的抗病毒药物,但是随着治疗时间的延长,有部分患者会对拉米夫定产生耐药性,从而影响了临床治疗的效果,而对耐药患者的治疗成为临床上亟待解决的问题。本研究选用阿德福韦联合拉米夫定和阿德福韦单药治疗拉米夫定耐药的慢性乙型肝炎,以观察阿德福韦的疗效和安全性。方法选取经拉米夫定治疗后产生YMDD耐药的慢性乙型肝炎患者62例,将患者随机分成两组:A组采用阿德福韦(10mg,每日1次口服)联合拉米夫定(0.1g,每日1次口服)治疗72周;B组采用阿德福韦单药治疗72周。治疗期间分别于治疗的第12周、24周、48周、72周检测乙肝病毒标志物检测(HBV-M),HBV DNA定量、肝功能,并记录。结果第12、24、48、72周时A组ALT复常率为59.3%、71.9%、78.1%、87.5%,B组为33.3%、36.7%、40.0%、53.3%;A、B组的HBV-DNA阴转率分别为56.3%、62.5%、71.9%、90.6%;30.0%、33.3%、40.0%、53.3%;A组HBe Ag阴转率、HBe Ab转换率为50.0%、65.6%、75.0%、90.6%;B组为20.0%、26.7%、46.7%、66.7%。应用SPSS 19.0统计软件进行统计学分析,判定为差异有统计学意义(P<0.05)。结论 1阿德福韦联合拉米夫定的疗效,要明显优于阿德福韦单药治疗;2提示阿德福韦能有效抑制拉米夫定耐药株的复制,阿德福韦联合拉米夫定和阿德福韦单药治疗对拉米夫定耐药的慢性乙型肝炎都具有较强的抗病毒作用;3阿德福韦联合拉米夫定治疗拉米夫定耐药的慢性乙型肝炎可以降低耐药的发生。     

阿德福韦酯单药及其联合拉米夫定治疗拉米夫定耐药型HBeAg阳性慢性乙型肝炎的疗效比较

目的比较阿德福韦酯（ADV）单药及其联合拉米夫定治疗拉米夫定耐药型HBeAg阳性慢性乙型肝炎（chronic hepatitis B,CHB）的临床疗效。方法对24例ADV单药及28例ADV联合拉米夫定治疗拉米夫定耐药型HBeAg阳性慢性乙型肝炎患者的疗效进行回顾性分析,比较两组患者HBVDNA、ALT水平及HBV DNA检测不到率、ALT复常率的差异。结果两组患者的性别、年龄、治疗前的HBV DNA及ALT水平差异均无统计学意义。两组的HBV DNA、ALT水平在治疗48周、72周时分别与同组治疗前比较均有明显降低（P〈0．05）。治疗48周时,联合组的HBV DNA检测不到率虽高于单药组,但差异无统计学意义（50％vs25％,P〉0．05）;ALT复常率两组间无明显差别（67．9％vs75％,P〉0．05）。治疗72周时,联合组的HBV DNA检测不到率为68％,要显著高于单药组的33．3％（P〈0．05）;联合组的ALT复常率为93％,显著高于单药组的70．8％（P〈0．05）。结论ADV单药或联合拉米夫定均是治疗拉米夫定耐药HBeAg阳性CHB的有效方法,联合治疗的疗效要优于单药治疗。     

PCR-RFLP结合克隆测序监测乙肝病毒拉米夫定耐药突变株的产生

目的 建立PCR结合酶切的方法监测慢性乙型肝炎患者体内拉米夫定耐药突变株的产生,并与PCR产物克隆后测序相结合。了解此方法的可靠性和可行性,同时用此方法筛查50例应用拉米夫定治疗的慢性乙型肝炎患者中耐药株的发生情况。方法 拉米夫定治疗的慢性乙型肝炎患者50例,治疗时间9个月-24个月,设计错配PCR结合限制性片段长度多态性方法,快速检测患者体内乙型肝炎病毒（HBV）酪氨酸-蛋氨酸-天门冬氨酸-天门冬氨酸（Tyrosine-Methionine-Aspartic acid-Aspartic acid,YMDD)变异株的发生情况,对筛检耐药株阳性的标本应用PCR产物克隆后测序加以证实。结果 在50例服用拉米夫定患者中发现9斧正患者在用药超过9个月时出现拉米夫定耐药突变株（YMDD变异株）,其中YIDD变异5例,YVDD变异4例,后者有3例合并有1526M突变。结论 本方法在检测YMDD变异方面具人快速简便的特点,经克隆后测序证实,具有较好的可靠性。     

拉米夫定引起HBV基因突变研究进展

拉米夫定 (lamivudine)为核苷酸类似物 ,主要用于抗病毒治疗 ,长期使用可引起乙肝病毒基因突变 ,突变主要在聚合酶基因YMDD模序 ,导致耐药病毒株 ,影响临床治疗。目前临床检测突变的手段是建立在PCR技术基础上的方法。     

北京地区多重耐药的腹泻病原菌的构成及耐药特点

目的 感染性腹泻是全球性的公共卫生问题,三代头孢菌素(third-generation cephalosporins, TGCs)是严重腹泻患者的首选治疗,监测医院与腹泻有关耐TGCs的腹泻病原菌的增长趋势和耐药特点,为本地区的流行病学研究及临床合理用药提供依据。方法 收集北京地区的传染病医院2011～2020年腹泻患者大便标本进行培养,筛选致病菌经生化及血清学进一步鉴定到种或群,并以纸片扩散法测定抗菌药物的敏感性;分析耐TGCs的腹泻病原菌的特点。结果 10年间共分离出沙门菌、志贺菌、气单胞菌、类志贺毗邻单胞菌、大肠埃希菌和弧菌等腹泻病原菌2 111株,其中268株耐TGCs,占整个肠道致病菌的12.70%,产生率由2011年的2.71%上升至2020年的29.29%,以致泻大肠埃希菌(39.57%)和气单胞菌(21.55%)最高,对四代头孢菌素、氟喹诺酮类、氯霉素、复方新诺明的耐药率在29%～83%,对磷霉素的耐药率最低8.27%。耐TGCs常表现多重耐药,同时耐喹诺酮类、氯霉素、复方新诺明3类抗生素为主。结论 耐TGCs的腹泻病原菌上升迅速,耐药广泛,不同种属的耐药性不同,应加强...     

艾滋病合并慢性乙型肝炎感染患者对拉米夫定耐药的研究CSCD

目的了解拉米夫定（3TC）治疗艾滋病（AIDS）/慢性乙型肝炎（CHB）合并感染患者对3TC的耐药情况。方法收集广州市第八人民医院2005年至2010年接受含3TC方案治疗的AIDS/CHB合并感染患者的资料,检测患者抗病毒治疗前和治疗12个月、24个月、36个月、48个月和60个月时HBV DNA含量,对HBV DNA阳性患者,扩增HBV逆转录酶区,分析HBV变异情况。结果185例患者符合入选标准,治疗前均未发现3TC耐药。治疗过程中,有29例患者检出3TC耐药,其中7例患者在两个不同的随访点检出耐药。29例患者中,有9例治疗前HBV DNA阴性,3例患者是治疗12个月时检出耐药。与非耐药患者相比,耐药患者HBeAg阳性的比例更高（65.5%比33.3%,P＝0.001）,基线HBV DNA水平更高（7.49拷贝/ml比6.59拷贝/ml,P＝0.017）,使用3TC时间明显延长（40个月比26.5个月,P〈0.001）。3TC耐药模式有四种：rtM204I （n＝7）、rtL180M （n＝2）、rtL180M＋rtM204V/I（n＝23）和rtL80V＋rtM204I （n＝4）。结论本地区AIDS/CHB合并感染患者对3TC耐药率低。治疗前HBV DNA阴性患者亦可较早出现耐药。     

基因芯片技术对乙型肝炎病毒拉米夫定耐药基因变异检测的临床应用

目的通过对应用拉米夫定治疗患者HBV基因耐药性变异的长期监测，验证基因芯片技术的临床应用前景。方法针对HBV3个主要的拉米夫定耐药性相关突变设计探针，制成HBV耐药寡核苷酸芯片，选取51例应用拉米夫定进行治疗的乙型肝炎患者分别采用基因芯片和传统测序的方法对其进行24个月的监测，观察HBV基因变异情况。结果39％的患者在用药2年内发生了耐药性突变，使用基因芯片与传统的测序方法得到的结果一致，并且可以在早期方便检测出混合感染。结论应用基因芯片技术可以准确，高效的检测出耐药基因变异，具有临床应用价值。     

Sequence Analysis of the RNA Polymerase Gene of Foot-and-Mouth Disease Virus Serotype Asia1

The complete nucleotide (nt.) sequence of the RNA polymerase (3D) gene and 81 nt. in the 3-untranslated region of foot-and-mouth disease virus (FMDV) serotype Asia1 (IND63/72) was determined and compared with the sequence of other FMDV serotypes. The 3D genomic region was 1410 nt. long encoding 470 amino acids with an inframe stop codon (TAA) at nt. position 1411–1413. The deduced amino acid sequence of the protein showed 8 conserved motifs as reported in other picornaviruses, 2 of which are 100% identical across the serotypes. Antigenic regions in the polymerase protein were predicted and found to be located at the N-terminus of the protein. The phylogenetic analysis showed that the FMD viruses were segregated into different clusters based on geographical origin; the Asia1 virus did not cluster tightly with any of the geographical groups.     

Cloning and Sequence Analysis of a Non-Structural Gene of an Aquareovirus

The nucleotide and deduced amino acid sequence of genome segment 11 encoding a nonstructural protein of an aquareovirus strain SBR have been determined. Nucleotide sequence analysis showed that the genome segment 11 of SBR virus is 780 nucleotides long and contains a major open reading frame that codes for a polypeptide of 236 amino acids with a predicted molecular weight of 25,504 Da. The second reading frame of genome segment 11 was 480 nucleotides long and codes for a polypeptide of 145 with a predicted molecular weight of 15,715 Da. The genome segment 11 contains 24 nontranslated nucleotides at the 5′-end and 48 nontranslated nucleotides at the 3′-end. This gene codes for two nonstructural polypeptides NS29 and NS15. Comparison of the deduced amino acid sequence of this gene with the published sequences of other members of the family Reoviridae indicated no sequence relatedness. This revised version was published online in July 2006 with corrections to the Cover Date.     

Correlation between pretreatment viral sequences and the emergence of lamivudine resistance in hepatitis B virus infection

The emergence of amino acid or nucleotide substitutions leads to lamivudine resistance in hepatitis B virus (HBV) infected patients. The aim of this study was to investigate whether viral sequences help predict the emergence of lamivudine resistance. The study subjects comprised 59 consecutive patients infected with HBV treated with daily therapy of 100 mg lamivudine. Among those, 32 patients with adequate pretreatment serum preservation were investigated for the correlation between viral amino acid substitutions and the appearance of lamivudine resistance with consideration of clinical background by determining dominant HBV full open reading frames. Viral resistance to lamivudine emerged in 28 of 59 patients (47%) in a median period of 2.45 years. Sequence comparisons of HBV genomes between patients who later developed lamivudine resistance and patients who did not revealed the existence of significant differences between the two groups in the pre‐S1 84 (P = 0.042), pre‐S2 1 (P = 0.017) and 22 (P = 0.015), and polymerase tp 95 (P = 0.046), judged by a log‐rank test. Viral sequence analyses revealed the presence of amino acid substitutions in HBV pre‐S1 and pre‐S2 that may be associated with the emergence of lamivudine resistance during chronic HBV infection. J. Med. Virol. 84:1360–1368, 2012. © 2012 Wiley Periodicals, Inc.     

Identification of Mycobacterial Species by Comparative Sequence Analysis of the RNA Polymerase Gene (rpoB)

For the differentiation and identification of mycobacterial species, the rpoB gene, encoding the beta subunit of RNA polymerase, was investigated. rpoB DNAs (342 bp) were amplified from 44 reference strains of mycobacteria and clinical isolates (107 strains) by PCR. The nucleotide sequences were directly determined (306 bp) and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Comparative sequence analysis of rpoB DNAs provided the basis for species differentiation within the genus Mycobacterium. Slowly and rapidly growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. Pathogenic Mycobacterium kansasii was easily differentiated from nonpathogenic M. gastri; this differentiation cannot be achieved by using 16S rRNA gene (rDNA) sequences. By being grouped into species-specific clusters with low-level sequence divergence among strains of the same species, all of the clinical isolates could be easily identified. These results suggest that comparative sequence analysis of amplified rpoB DNAs can be used efficiently to identify clinical isolates of mycobacteria in parallel with traditional culture methods and as a supplement to 16S rDNA gene analysis. Furthermore, in the case of M. tuberculosis, rifampin resistance can be simultaneously determined.     

Cloning and Sequence Analysis of the Spike Gene of Porcine Epidemic Diarrhea Virus Chinju99

The spike (S) gene of the porcine epidemic diarrhea virus (PEDV) Chinju99 which was previously isolated in Chinju, Korea was cloned and sequenced to aid in the development of genetically engineered vaccines and diagnostic reagents against PEDV. The nucleotide sequence encoding the entire S gene open reading frame (ORF) of Chinju99 was 4152 bases long encoding 1383 amino acids. It consisted of 1001 adenine (24.1%), 849 cytosine (20.4%), 877 guanine (21.1%) and 1425 thymine (34.3%) residues. The Chinju99 S ORF nucleotide sequence was 94.5% homologous with that of the Br1/87 and CV777 strains, respectively. The Chinju99 S protein had 92.8% amino acid identity with that of Br1/87 and CV777, respectively. The amino acid sequence contained 27 potential sites for asparagine (N)-linked glycosylation and there was a stretch of highly hydrophobic residues at position 1325–1350.     

Cloning and Sequence Analysis of the Nucleocapsid Gene of Porcine Epidemic Diarrhea Virus Chinju99

The nucleocapsid (N) gene of the porcine epidemic diarrhea virus (PEDV) Chinju99 which was previously isolated in Chinju, Korea was cloned and sequenced to establish the information for the development of genetically engineered diagnostic reagents. Also, sequences of the nucleotides and deduced amino acids of the Chinju99 N gene were analyzed by alignment with those of CV777 and Br1/87. The nucleotide sequence encoding the entire N gene open reading frame (ORF) of Chinju99 was 1326 bases long and encoded a protein of 441 amino acids with predicted M _r of 49 kDa. It consisted of 405 adenine (30.5%), 293 cytosine (22.1%), 334 guanines (25.2%) and 294 thymines (22.2%) residues. The Chinju99 N ORF nucleotide sequence was 96.5% and 96.4% homologous with that of the CV777 and Br1/87, respectively. The Chinju99 N protein revealed 96.8% amino acid identity with that of Br1/87 and CV777, respectively. The amino acid sequence contained seven potential sites for threonine (T)- or serine (S)-linked phosphorylation by each protein kinase C and casein kinase II.     

Sequence Analysis of the Gene Encoding the Capsid Protein of the Snow Mountain Human Calicivirus

Snow Mountain virus (SMV) is the reference strain for serotype 3 as determined by immune electron microscopy of the human caliciviruses that are associated with epidemic gastroenteritis. In order to establish the genetic relationship of its capsid protein with those from other human caliciviriuses, the sequence of the open reading frame 2 (ORF2) encoding the SMV capsid protein was determined. The SMV ORF2 sequence was 1626 nucleotides in length and the deduced protein of 542 amino acids had a calculated molecular weight of 59.2 kD. The SMV capsid sequence showed approximately 48 and 77% amino acid sequence identity with the capsid proteins of the Norwalk (serotype 1) and Hawaii (serotype 2) human calicivirus reference strains, respectively, a finding consistent with its serotypic distinctiveness. Furthermore, the predicted amino acid sequence of the SMV capsid was found to share highest sequence identity (98%) with the Melksham human calicivirus in database searches. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sequence Analysis of the NSP4 Gene from Human Rotavirus Strains Isolated in the United States

Two major and one minor genotype of the rotavirus NSP4 gene have been described. The sequences of 29 NSP4 genes from rotavirus isolates obtained in the United States during the 1996–1997 rotavirus season (types P[8]G1, P[8]G9, P[4]G2 and P[6]G9) and 10 strains isolated during previous rotavirus seasons (types P[8]G1 and P[4]G2) were determined. All NSP4 genes from strains with short E types (6 P[4]G2, 4 P[6]G9) belonged to genotype NSP4A, whereas all 19 strains with long E types (16 P[8]G1, 3 P[8]G9) had NSP4 genes of genotype NSP4B. Genetic variation within genotypes was low (2.3% for both NSP4A and NSP4B), confirming that the NSP4 genes are highly conserved. Nonetheless, at least two distinct sub-lineages could be detected within each genotype: strains isolated in the same year, regardless of geographic location, were more closely related or even identical at the deduced amino acid level; strains isolated in different years were more distinct. Thus, geographic distance did not affect genetic distance. Northern hybridization analysis with NSP4A and NSP4B total gene probes failed to detect any unusual combinations of the VP6 and NSP4 genes in 31 additional isolates from the 1996–1997 rotavirus season.     

Characteristics of lamivudine-resistant hepatitis B virus (HBV) strains with and without breakthrough hepatitis in patients with chronic hepatitis B evaluated by serial HBV full-genome sequences

Lamivudine therapy often causes breakthrough of hepatitis B virus (HBV) DNA and breakthrough hepatitis. The aim of this study was to determine the viral factors that relate to HBV-DNA breakthrough with and without breakthrough hepatitis. Among 82 patients with chronic hepatitis B (CHB) who received lamivudine at a dose of 100 mg daily for more than 24 months, 23 patients had HBV-DNA breakthrough induced by a lamivudine-resistant mutant. Of these 23 patients, 16 had breakthrough hepatitis and 7 had only HBV-DNA breakthrough. Serial HBV-DNA full-genome sequences during therapy were examined in 10 (7 had breakthrough hepatitis and 3 did not) of these 23 patients by direct sequencing. Mutations in the S region were examined by cloning in representative patients. There were no significant differences in the baseline clinical backgrounds and virus marker between patients with and without breakthrough hepatitis. The HBV amino acid substitutions at breakthrough hepatitis were identical to those at HBV-DNA breakthrough. Cloning analysis revealed that monoclonal mutational strain appeared at breakthrough and no such mutations existed at baseline. Regarding HBV amino acid substitutions in the polymerase region, S region, X region, and precore-core region with breakthrough compared to baseline, there was no significant differences of the numbers of amino acid substitution between breakthrough hepatitis and non-breakthrough hepatitis. There were no common amino acid changes in patients with breakthrough hepatitis. Although monoclonal lamivudine-resistant strain emerged at HBV-DNA breakthrough in patients with CHB, there were no common amino acid changes, suggesting viral factor may have insignificant role in breakthrough hepatitis.