Polymerase chain reaction can resolve some undefined cases of Hepatitis B virus antigenic subtyping

HBsAg subtypes were defined by means of adsorbed polyclonal antisera; however, HBsAg subtyping is currently usually carried out with monoclonal antibodies (Mab). We developed a complementary subtyping method based on the polymerase chain reaction. Reference samples belonging to all known HBsAg subtypes could be detected and grouped into four different categories (ayw1/ayw4/ayr, ayw2/ayw3, adw2/adrq+/adrq-, adw4). Thirteen HBsAg-positive serum samples previously subtyped as ad by means of monoclonal antibodies fell into the adw2/adrq+/adrq- group, as well as 13 ay samples into the ayw2/ayw3 group. These results could be confirmed by means of reference polyclonal antisera in nine ad cases (all adw2) and in seven ay cases (all ayw3); the remaining seven were below the detection limit of the polyclonal assay. Four samples which were not recognized by any of the d/y subtype-specific Mab were shown to contain ayw2/ayw3 sequences. Only one contained sufficient HBsAg to be confirmed as ayw3 by means of reference antisera. Three of five sera showing simultaneous reactivity both for d and y-specific Mab were classified as adw4 by PCR, as was one by reference polyclonal antisera. The y-specific monoclonal antibody cross-reacted with the adw4 subtype. Single adw2 sequences were amplified in one of the remaining two cases, as well as single ayw2/ayw3 sequences in the other, suggesting that they showed true coexistence of two strains of different subtype, only one of which was in active replication state. It is concluded that the method described is useful in the solution of some undefined cases obtained with the monoclonal-based assays. © 1994 W iley-Liss, Inc.     

Hepatitis B virus DNA in sera of virus carriers positive exclusively for antibodies to the hepatitis B core antigen

The prevalence of serum HBV DNA in individuals positive for anti-HBc alone was determined by the polymerase chain reaction in two groups with endemic HBV infection from Canton (group A) and Hainan (group B), provinces of China. Twenty-one out of 294 individuals in group A (7.2%) and 193 out of 1995 in group B (9.7%) were positive for anti-HBc but negative for other markers of ongoing or past HBV infection (HBsAg and anti-HBs). HBV DNA was detected in 6/21 sera in group A (28.6%) and 68/193 in group B (35.2%) in their initial serum specimen. One of the six HBV-DNA-positive individuals in group A became negative after 6 months and four of the 58 positive in group B became negative at 4 years of follow-up. All of the individuals remained positive for anti-HBc and negative for anti-HBs, but one of them became positive for HBsAg on follow-up. None of the anti-HBc- and HBV-DNA-positive subjects had symptoms of liver diseases. They were, therefore, defined as chronic asymptomatic HBV carriers with undetectable HBsAg. This type of carrier should be added to the typical HBsAg-positive carrier, who constitutes about 10-15% of the general Chinese population, to give a more complete estimate of asymptomatic HBV carriers in China.     

Characterization of RNA-dependent RNA Polymerase Activity of CSFV NS5B Proteins Expressed in Escherichia coli

The full-length NS5B protein, and the truncated NS5B proteins of classical swine fever virus (CSFV) resulted from deletion of 24, 36, 65 or 82, amino acid residues at the C terminal were expressed in Escherichia coli cells and purified with a C-terminal hexahistidine tag. In addition to the full-length NS5B protein, those truncated NS5B proteins with deletion of 24, 36, or 65 amino acid residues were demonstrated to have RNA-dependent RNA polymerase (RdRp) activity, which was not found in the truncated NS5B proteins with deletion of 82 amino acid residues. Analysis of the template specificity of CSFV RdRp was done containing the different NS5B proteins with RdRp activity. It was shown that the template specificity of the enzyme was not strict with NS5B proteins truncated, suggesting that the C terminal of CSFV NS5B protein was involved in the template specificity of the enzyme. Site-directed mutagenesis of and prediction of the secondary structure of 3 terminal sequence of the template indicated that the cytidines at 3 terminus and the correct secondary structure of the template were essential to initiation of RNA synthesis by RdRp. Oxidation of the hydroxyl groups of the RNA template revealed that both the de novo initiation mechanism and the template-priming mechanism preference might be employed by the CSFV RdRp.     

Hepatitis B Viral Surface Mutations in Patients with Adefovir Resistant Chronic Hepatitis B with A181T/V Polymerase Mutations

The hepatitis B virus (HBV) polymerase gene has overlapping reading frames with surface genes, which allows to alter the amino acid codon of the surface genes. In adefovir (ADV) treated chronic hepatitis B patients carrying rtA181T/rtA181V mutations, overlap with surface gene mutations such as sW172stop/sL173F has been reported. However, the clinical consequences of such surface mutations have not been determined. The aim of this study was to determine the surface gene sequence in ADV-resistant patients carrying the A181T/V mutation and to describe the clinical significance. Of the 22 patients included in this study, 13 were ADV-resistant with rtA181T/V mutations (polymerase mutation group, Group P) and nine were antiviral treatment-naïve (control group, Group C). The Pre-S1 gene mutation, V60A, was detected in 11 patients (Group P=8, Group C=3). A start codon mutation in the Pre-S2 gene was found in five patients (Group P=3, Group C=2). An S gene mutation, sA184V, was found in nine patients, all of whom were in group P. Although sW172stop and sL173F mutations were detected, reduced HBsAg titer was not observed. Further study of these mutations and their clinical implications are needed.     

高灵敏度乙肝HBV DNA检测在乙肝诊疗中的应用及临床意义

目的探讨高灵敏度乙肝HBV DNA检测在乙肝诊疗中的应用及临床意义。方法收集23例普通荧光定量PCR(常规)检测HBV DNA为阴性患者的血清,采用高灵敏度乙肝HBV DNA检测,将检测数据进行整理,与其临床实验室资料相结合,进行综合分析,归纳总结。结果23例国产乙肝荧光定量 PCR检测均为阴性患者,使用高灵敏度乙肝HBV DNA检测结果为13例阳性（最低检测限20IU/mL）,阴性10例,阳性检出率为56.52%,显然高灵敏度乙肝 HBV DNA检测的灵敏度高于常规检测方法。结论高灵敏度乙肝HBV DNA检测,其检测敏感性远高于常规检测方法,能检测到体内低水平的 HBV DNA,结合患者其他实验室资料及用药史综合分析,对乙型肝炎诊断和治疗具有重要的指导意义。     

荧光定量聚合酶链反应检测慢性乙肝患者HBV DNA的意义

目的探讨荧光定量聚合酶链反应(FQ-PCR)检测慢性乙肝患者血清乙型肝炎病毒(HBV)脱氧核糖核苷酸(DNA)的临床意义。方法回顾性分析248例慢性乙肝患者的资料,均采用FQ-PCR技术检测血清HBV DNA,检测乙肝病毒标志物(HBV-M)并对比不同HBV-M患者血清HBV DNA水平;对比不同病情患者血清HBV DNA水平;对比不同HBV DNA表达患者外周血T淋巴细胞亚群水平及异常率,分析患者血清HBV DNA水平与外周血T淋巴细胞亚群水平的关系。结果不同HBV-M患者血清HBV DNA水平对比:HBsAg+HBeAg+HBcAb>HBsAg+HBeAg>HBsAg+HBsAb+HBcAb>HBsAg+HBeAb>HBsAb+HBeAb+HBcAb>HBcAb/HBsAb+HBeAb/HBeAb+HBcAb,除HBcAb、HBsAb+HBeAb、HBeAb+HBcAb血清HBV DNA水平差异无统计学意义(P>0.05),其余每2样本比较差异均有统计学意义(P<0.05);不同病情患者血清HBV DNA水平对比:重度病情患者>中度病情患者>轻度病情患者(P<0.05);不同HBV DNA表达患者CD3+、CD4+、CD4+/CD8+对比,HBV DNA阴性患者>低拷贝患者>高拷贝患者(P<0.05),CD3+、CD4+、CD4+/CD8+异常率对比,HBV DNA阴性患者<低拷贝患者<高拷贝患者(P<0.01);本组患者血清HBV DNA水平与外周血CD3+、CD4+、CD4+/CD8+均呈负相关(r=-0.789、-0.812、-0.706,P=0.012、0.007、0.001)。结论在慢性乙肝患者中FQ-PCR检测血清HBV DNA水平与HBV-M、病情和外周血T淋巴细胞亚群水平均有密切关系。