Diagnostic impact of the genetic variability of the hepatitis B virus surface antigen gene

The genetic variability of hepatitis B virus (HBV) represents a challenge for the sensitivity of immunologic and molecular based assays. Genotyping studies show that the genetic diversity of HBV is very high even in industrialized countries. The analytical sensitivity of HBsAg and anti-HBs assays may be dependent on HBV genotype or subtype and could possibly lead to false negative results in samples with low-level HBsAg. It is possible that the recognition of genotypes E and F may be impaired. Immunoassays based on polyclonal capture antibody show the highest sensitivity for the recognition of recombinant mutants or serum samples harboring mutant forms of HBsAg. However, they do not guarantee full sensitivity, especially for the detection of the G145R mutation and amino acid insertions or substitutions in positions 120-123. Detection of HBsAg needs to be improved by the introduction of new HBsAg assays able to recognize so far described S gene mutants and with a lower detection threshold than current immunoassays in order to detect smallest amounts of HBsAg in low level carriers. There is also a need for more complete epidemiological data on the prevalence of HBsAg mutants especially for G145R and assays for the (differential) screening of mutants need to be developed and evaluated.     

Genetic variability and evolution of hepatitis E virus

Hepatitis E virus (HEV) is the sole member of the genus Hepevirus in the family Hepeviridae. HEV is transmitted primarily by the fecal-oral route, and water-borne epidemics are characteristic of hepatitis E in many developing countries in Asia, Africa and Latin America where sanitation conditions are suboptimal. Accumulating lines of evidence indicate that HEV-associated hepatitis also occurs domestically among individuals in industrialized countries, that there are animal reservoirs of HEV such as domestic pigs and wild boars, and that hepatitis E is a zoonosis. Based on the extensive genomic variability among HEV isolates, HEV sequences have been classified into four genotypes: genotype 1 consists of epidemic strains in developing countries in Asia and Africa; genotype 2 has been described in Mexico and several African countries; genotype 3 HEV is widely distributed and has been isolated from sporadic cases of acute hepatitis E and/or domestic pigs in many countries in the world, except for countries in Africa; and genotype 4 contains strains isolated from humans and/or domestic pigs exclusively in Asian countries. This paper reviews current knowledge on the genomic variability, geographic distribution and zoonotic aspects of HEV as well as the clinical significance of genotype and evolution of HEV.     

Comparing the immunogenicity of hepatitis B virus S gene variants by DNA immunization.

DNA immunization was used to compare the immunogenicity of hepatitis B virus S gene variants. Four recombinant plasmid DNAs containing the full-length virus genome with different S gene inserts were used to immunize BALB/c and C57/BL/6 mice. These inserts were cloned from 129L (residue 129, glutamine to leucine), 129H (residue 129, glutamine to histidine) 145R (residue 145, glycine to arginine) variants and the wild-type virus. The titer of hepatitis B virus core antibodies (anti-HBc) in immunized mice was used as the control for the efficiency of DNA immunization. Serum hepatitis B surface antibody (anti-HBs) titer and cytokines induced in splenocytes stimulated with hepatitis B surface antigen (HBsAg) were monitored as specific immune responses induced by different plasmid DNAs. 129L DNA induced significantly lower anti-HBs antibodies (IgG, IgG1, and IgG2a) and less interferon-gamma, compared to those in mice immunized with the 129H variant and the wild-type HBV DNA (p < 0.05). Computer modeling showed that a change from glutamine to leucine at 129 residue led to higher hydrophobicity and could result in decreased immunogenicity. Results indicate that DNA immunization can be used to compare the humoral and cellular immunogenicity among different HBV S variants.     

Genetic variability of hepatitis C virus in South Egypt and its possible clinical implication

Egypt is one of the countries with very high rates of hepatitis C virus (HCV) related morbidity and mortality. However, little is known about geographical and clinical differences in genetic variability of HCV in Egypt. Using direct sequencing and phylogenetic analysis of partial core/E1 and NS5B regions of the HCV genome, HCV genotype/subtype was determined in 129 HCV‐infected patients residing in three governates in south Egypt: Assuit, Sohag, and Qena. According to clinical stage of infection, patients were categorized into four groups: asymptomatic carriers, n = 16; chronic hepatitis C patients, n = 36; liver cirrhosis, n = 54; and hepatocellular carcinoma (HCC), n = 23. Genotype 4a was detected in 80.6%, whereas 1g, 4l, 4n, 4o, 4f, and 4m were identified in 7.7%, 4.7%, 3.9%, 1.6%, 0.8%, and 0.8% of cases, respectively. The prevalence of 4a differed regionally; from 88.5% (in Sohag) to 64% (in Assuit, P = 0.002). Genotypes 4l and 4n had a higher prevalence in Assuit (12.8%, 10.3%) than Sohag (0%, 0%; P ≤ 0.011). Difference in clinical features of determined genotypes/subtypes was observed; more carriers of non‐4a variants (4l and 4n, 4f, or 4m) had chronic hepatitis compared to carriers of 4a (53.3% vs. 23.1%, P = 0.025), while more patients with 4a had liver cirrhosis (45.2% vs. 13.3%, P = 0.023). Two HCV‐4o strains were isolated in this study, both from patients with HCC. In conclusion, geographical diversity of HCV was revealed in this study in southern Egypt. A further case–control study is required to confirm the trends of differential pathogenicity of HCV subtypes, indicated by this study. J. Med. Virol. 81:1015–1023, 2009. © 2009 Wiley‐Liss, Inc.     

Genetic variability and molecular evolution of hepatitis A virus

Hepatitis A virus (HAV), the causative agent of type A viral hepatitis, was first identified about three decades ago. Recent findings have shown that HAV possess several characteristics that make it unique among the family Picornaviridae, particularly in terms of its mechanisms of polyprotein processing and virion morphogenesis. HAV circulates in vivo as distributions of closely genetically related variants referred to as quasispecies. HAV exploits all known mechanisms of genetic variation to ensure its survival, including mutation and recombination. Only one serotype and six different genetic groups (three humans and three simian) have been described. HAV mutation rate is significantly lower as compared to other members of the family Picornaviridae. The mode of evolution appears, at least in part, to contribute to the presence of only one known serotype.     

Variability of the S gene of hepatitis B virus in southeastern China

In a study of 315 HBV specimens obtained from southern China, 240 (76.9%) were assigned to genotype B, 72 (22.9%) were genotype C, two (0.6%) were genotype A and one (0.3%) was genotype D. Statistical analysis revealed that variables such as age, gender, HBV vaccination rate, hepatitis anamnesis rate, anti-HBs and HBeAg prevalence and virus load were insignificant between genotype B (n = 240) and genotype C cases (n = 72) (P > 0.05). However, the frequency of amino acid (aa) substitutions in the major hydrophilic region (MHR; aa 99–169) and the putative HLA class I-restricted cytotoxic T lymphocyte (CTL) epitope region of the S gene, as well as the overlapping polymerase/RT region (aa 32–212), were significantly higher in genotype C group than genotype B (P < 0.001). These results suggest that the higher variability within genotype C carriers may account for the pathogenic potential.     

Analysis of hepatitis B virus X gene phylogeny, genetic variability and its impact on pathogenesis: implications in Eastern Indian HBV carriers

HBx genetic variability was explored in the Eastern Indian population with low HCC incidence. DNase I sensitive HBV DNA was detected in 53% samples, which differed significantly between clinical groups (P < 0.001). HBV genotypes A (Aa/A1), C (Cs/C1) and D (D1, D2, D3, D5) were detected in 37.5%, 18.7% and 43.7% samples respectively. Population specific signature HBx residues A₃₆, V₈₈, S₁₀₁ in Aa/A1 and residues P₄₁, Q₁₁₀ in D5 were detected. Mutations T₁₂₇, M₁₃₀ and I₁₃₁ were detected in 66.7%, 91% and 75% of genotype A, C and D5 samples respectively. Very low occurrence of HCC associated mutations (V₅M/L, P₃₈S, and H₉₄Y) and absence of C-terminal deletions were observed. Our study shows that HBV genotype associated clinically important HBx variations may evolve and act distinctly in different geo-ethnic populations. Further studies on HBx functions from the perspective of genetic variability are essential for the better understanding of the clinical significance of HBV.     

Hepatitis B virus genotype E surface antigen detection with different immunoassays and diagnostic impact of mutations in the preS/S gene

The major neutralizing epitope, the “a” determinant of the hepatitis B virus (HBV) genotype E surface antigen (HBsAg) is most divergent from that of genotype A, which is used for preparing monoclonal antibodies used in commercially available HBV reagents. To evaluate the performance of the latest generation of HBsAg detection assays with respect to genotype E HBsAg. Three commercial assays were evaluated using sera from 200 Nigerian patients compared to the preS/S sequence of DNA positive samples. Out of 200 samples, 61 and 103 gave concordant positive and negative results between the three HBsAg assays. Of 36 samples with discordant results, 35 were confirmed negative by neutralisation. One of the three assays showed significantly high rate of false positives (29 of 35). DNA positive samples with no detectable HBsAg or reduced HBsAg detection signals (<75% of mean signal obtained with HBsAg positive samples) revealed several mutations (V14A, F46S, N48T, L49R, I49T, D51G, A53V, P54L, Q82P, F83C, L127P, A184V, T189I, S204N, V224A), mostly outside the a-determinant. Several of these mutations are found as wild type nucleotides normally in genotype A and only exceptionally in genotype E. All three assays showed comparable sensitivities for genotype E HBsAg detection (98.4–100%) but differed considerably in specificity (84–99%). Failure to detect HBsAg antigen and differences in signal intensity were mainly associated with mutations in the preS/S gene outside the “a” determinant.     

Genotypes and S-gene variability of Mexican hepatitis B virus strains

The genotypes and subtypes of 15 Mexican hepatitis B virus strains were determined by sequencing and phylogenetic analysis of the small S-gene. The most predominant strains were found to be divergent genotype/subtype F/adw4 strains (66.6%), followed by A/adw2 (20.0%), D/ayw3 (6.7%), and G/adw2 (6.7%). The S-genes of the Mexican genotype F strains and two Nicaraguan strains described previously formed a subcluster with more than 4% divergence from the other strains within this genotype. The Mexican strains within genotypes A and D showed the highest homology with strains from Europe and the United States. Ten amino acid substitutions not described previously were found in the S-genes of strains from nine chronic carriers, whereas the S gene in strains from six acute hepatitis B patients were highly conserved as compared to their respective genotypes. One genotype F strain from an HBsAg positive chronic carrier had a T to A mutation at position 647, forming a translational stop at codon 216. Two genotype F strains from HBsAg negative chronic carriers had a Val180 instead of an Ala found in the other genotype F strains. This study shows that a divergent genotype F predominates in Mexican strains analyzed, which presented amino acid substitutions not reported previously outside the a determinant.     

Hepatitis B virus X gene variability in French-born patients with chronic hepatitis and hepatocellular carcinoma

The polymorphism of the hepatitis B virus (HBV) X gene from patients born in Lorraine has been studied in serum samples from 22 HBV infected patients, 14 presenting with chronic hepatitis and 8 with hepatocellular carcinoma (HCC). Subtypes adw and ayw represented 21 of the 22 sequenced isolates. The sequence of the X gene of HBV strains from these patients differed from the ones of Far East origin by A to T(1678) and G to A(1759) changes for subtype ayw and C to T(1792) for adw. The expression of the preC region, as indicated by the detection of HBe antigen (HBeAg), was not observed in 11 patients. In 6 patients (3 HCC and 3 non HCC), the absence of HBeAg could be related to a stop codon at position 28. For the 5 remaining patients, the precore stop mutation at codon 28 was not evidenced but 3 out these 5 patients had mutations at nt 1764 and nt 1766 in the promoter of the preC/C gene. These two mutations were also observed in 2 patients with HBeAg, indicating that they are not implicated in the suppression of expression of this gene. Independently of the serotype, two main differences were noted between aminoacid (aa) sequences of chronic hepatitis and HCC related strains: first, twice as many aa changes were found in HCC patients than in chronic hepatitis B carriers (mean of aa changes per patient 4.1 vs. 2.0) and second, we found apparition of polar aa in HCC patients. Most mutations already described in patients from the Far East with HCC have been found in strains of patients from Lorraine. The changes K130M and V131I, considered as "hot spot mutations," were found in strains of HCC patients carrying an ayw subtype of the HBV genome but not in the ones of chronically infected patients. In contrast, strains of the adw subtype had these two changes in the two groups of patients. However when considering the 22 sequenced genes, these hot spot mutations were associated with HCC (P = 0.034).     

乙型肝炎病毒S基因密码子最佳化及在毕赤酵母中的高效表达

目的 制备适合酵母表达的乙型肝炎病毒S基因,在毕赤酵母中高效表达重组乙型肝炎病毒S蛋白.方法 选用酵母偏爱的同义密码子替换野生型S基因的酵母稀有密码子,采取基因搭桥法及递归式聚合酶链反应,制备合成基因,捕入酵母表达载体pPICZB.携带有合成S基因的重组质粒转化毕赤酵母菌株KM71 H,经甲醇诱导表达乙型肝炎病毒S蛋白.结果 酶切电泳及DNA测序证实合成的S基因正确克隆到表达载体中;聚丙烯酰胺凝胶电泳及免疫印迹显示优化后的乙型肝炎病毒S基因在毕赤酵母中的重组蛋白表达量远高于野生型基因.结论 密码子优化的S基因能明显提高重组S蛋白在毕赤酵母表达系统中的表达量.     

乙型肝炎病毒基因分型及临床应用研究

目的了解常州地区乙型肝炎病毒基因型分布特征,探讨其基因型与肝功能损伤、病毒复制水平及对拉米夫定疗效的关系. 方法采用巢式聚合酶链反应 (nest-PCR), 扩增乙型肝炎病毒S基因区, 用末端标记方法对PCR产物标记并直接测序, 测序结果和GenBank中登录的标准基因型序列相比较. 结果对该地区146份不同HBV感染者血清HBV DNA进行了基因分型,B型51份 (34.9%),C型95份(65.1%),未发现B、C以外其他基因型;丙氨酸转氨酶(ALT)水平分别为383.8±335.7IU和364.3±333.7 IU,(t=0.335,P>0.05)、HBV DNA含量分别为107.795±1.22和107.69±1.19拷贝/毫升(t=0.138,P>0.05)、HBeAg 阳性数分别为36/51和64/95,(χ2=0.159,P>0.05);104例慢性乙型肝炎中B型为43例、C型为61例,28例肝硬化和肝癌患者检出B型4例、C型24例,二组比较χ2=7.65,P<0.01;23例B基因型患者和45例C基因型患者接受48周以上拉米夫定治疗,48周后反跳者B型为18例,C型为14例,χ2=13.49,P<0.001.结论本地区HBV DNA基因型为B型和C型;二种基因型丙氨酸转氨酶水平、病毒复制水平和HBeAg表达水平差异均无显著性;C基因型与肝硬化和肝癌关系密切;拉米夫定对C基因型患者的疗效强于B型.     

Genotypes and clinical phenotypes of hepatitis B virus in patients with chronic hepatitis B virus infection

Genotype C of hepatitis B virus (HBV) has been shown to be associated with a poor clinical outcome, compared to genotype B. To explore the clinical phenotypes, with special reference to the seroconversion of hepatitis B e antigen (HBeAg) and frequency of acute exacerbation between patients infected with HBV genotypes B and C, a cohort of 272 Taiwanese patients with chronic HBV infection was analyzed. According to the status of HBeAg at enrollment and frequency of acute exacerbation during the follow-up period, five groups of patients with distinct clinical phenotypes were categorized. Of the 272 HBV carriers, 185 (68%) were infected with HBV genotype B and the remaining 87 (32%) were infected with genotype C. Among them, 150 (55%) were positive for HBeAg and patients with genotype C infection tended to have a higher positive rate of HBeAg than those with genotype B infection (63 versus 51%). Genotype B was more prevalent than genotype C in different groups of HBV carriers. However, the prevalence of genotype C in patients with multiple episodes of acute exacerbation who failed to have HBeAg seroconversion was significantly higher than in all 272 patients (50 versus 32%, P = 0.025), in those with HBeAg seroconversion after only one episode of acute exacerbation (50 versus 12%, P = 0.01), or in those negative for HBeAg at enrollment and without acute exacerbations (50 versus 23%, P = 0.002). In conclusion, patients with genotype C infection have a more aggressive clinical phenotype than do those with genotype B infection, which contributes to the former group's progressive liver disease and poor clinical outcomes.     

乙型肝炎病毒新的免疫逃避株的S基因序列分析

目的分析乙型肝炎患者体内ＨＢＶＳ基因的变异情况及对乙型肝炎的免疫预防的现实意义。方法采用巢式聚合酶链反应（ＰＣＲ）、Ｍ１３噬菌体克隆和核苷酸序列分析方法对一例乙型肝炎免疫失败儿童患者体内ＨＢＶＳ基因序列进行分析。结果发现该患儿所感染ＨＢＶ的Ｓ基因上有一个重要的点突变,即第５５１位碱基由野生型的Ａ变为Ｇ。该突变使乙型肝炎表面抗原（ＨＢｓＡｇ）１３３位氨基酸由甲硫（ＡＴＧ）变为缬（ＧＴＧ）。这一氨基酸替换恰恰发生在ＨＢｓＡｇ中和性α抗原决定簇区段（ａａ１２４～ａａ１４７）内。结论鉴于该患者接种乙型肝炎疫苗,其血清呈抗－ＨＢｓ阳性和ＨＢｓＡｇ阴性,推测其体内的ＨＢＶ为一个新的疫苗诱导的免疫逃避株     

Genetic variability in the coat protein gene of Potato virus S isolates and distinguishing its biologically distinct strains

The complete coat protein (CP) nucleotide sequences of 13 Potato virus S (PVS) isolates from Australia and three from Europe were compared to those of 37 others. On phylogenetic analysis, the Australian sequences were in PVS^O sub-clades III and IV, and the European isolates were in sub-clades I and VII. The European isolates invaded Chenopodium spp. systemically, but eight Australian isolates did not. Amino acid sequence differences at the N-terminal ends of the CPs were unrelated to the ability to invade Chenopodium spp. systemically. The acronym PVS^O–CS is suggested for isolates that invade Chenopodium spp. systemically but are not within clade PVS^A.     

Mutations in the S gene region of hepatitis B virus genotype D in Golestan Province-Iran

Mutations of HBsAg especially within the “a” determinant could alter the antigenicity of the protein causing failure of HBsAg neutralization and escaping from the host’s immune system, resulting in active viral replication and liver disease. This project aimed to investigate mutation in the S gene region of HBV infected patients in Golestan Province-Iran. HBV-DNA extractions from plasma and PCR of 100 patients were performed. Direct sequencing and alignment of S gene were applied using reference sequence from Gene Bank database. All isolates were belonged to genotype D, subgenotype D1, subtype ayw2. Overall 92 point mutations occurred. Of them, 40 (43.47%) were missense and 52 (56.52%) were silent. Mutations were detected in 95 cases (95%). Five of 40 mutations (12.5%) occurred in “a” determinant and 13 (32.5%), 17 (42.5%), and 2 (5%) were seen in antigenic epitope regions of B cell, CD₄ ⁺ and CTL, respectively. Frame shift mutations were seen in 22 cases (22%). 14% of mutations occurred at Major Hydrophilic Region(MHR) area which P120T/S and R122K/T substitutions were the most frequent ones (4%). Mutation in G145R of the S gene was observed in one case. A large number of MHR mutants are in association with failure of HBsAg detection, vaccine, and immunotherapy escape. This study showed “a” determinant S gene mutations in HBV infected people with HBsAg positivity in Golestan Province-Iran. The rate of mutation in our study was 95%. Collectively, the results of this project exhibited that most of mutations were clustered in CD₄ ⁺ antigenic epitopes.