X region mutations of hepatitis B virus related to clinical severity

Chronic hepatitis B virus (HBV) infection remains a major health problem, with more than 240 million people chronically infected worldwide and potentially 650000 deaths per year due to advanced liver diseases including liver cirrhosis and hepatocellular carcinoma (HCC). HBV-X protein (HBx) contributes to the biology and pathogenesis of HBV via stimulating virus replication or altering host gene expression related to HCC. The HBV X region contains only 465 bp encoding the 16.5 kDa HBx protein, which also contains several critical cis-elements such as enhancer II, the core promoter and the microRNA-binding region. Thus, mutations in this region may affect not only the HBx open reading frame but also the overlapped cis-elements. Recently, several types of HBx mutations significantly associated with clinical severity have been described, although the functional mechanism in most of these cases remains unsolved. This review article will mainly focus on the HBx mutations proven to be significantly related to clinical severity via epidemiological studies.

Investigation of immune escape-associated mutations of hepatitis B virus in patients harboring hepatitis B virus drug-resistance mutations

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.

Precore/core region mutations of hepatitis B virus related to clinical severity

Despite the availability of an effective vaccine, hepatitis B virus (HBV) infection remains a major health problem, with more than 350 million chronically infected people worldwide and over 1 million annual deaths due to cirrhosis and liver cancer. HBV mutations are primarily generated due both to a lack of proofreading capacity by HBV polymerase and to host immune pressure, which is a very important factor for predicting disease progression and therapeutic outcomes. Several types of HBV precore/core (preC/C) mutations have been described to date. The host immune response against T cells drives mutation in the preC/C region. Specifically, preC/C mutations in the MHC class II restricted region are more common than in other regions and are significantly related to hepatocellular carcinoma. Certain mutations, including preC G1896A, are also significantly related to HBeAg-negative chronic infection. This review article mainly focuses on the HBV preC/C mutations that are related to disease severity and on the HBeAg serostatus of chronically infected patients.

Clinical implications of hepatitis B virus mutations:Recent advances

Hepatitis B virus(HBV)infection is a major cause of acute and chronic hepatitis,and of its long-term complications.It is the most variable among DNA viruses,mostly because of its unique life cycle which includes the activity of error-prone enzyme,reverse transcriptase,and the very high virion production per day.In last two decades,numerous research studies have shown that the speed of disease progression,reliability of diagnostic methods and the success of antiviral therapy and immunization are all influenced by genetic variability of this virus.It was shown that mutations in specific regions of HBV genome could be responsible for unwanted clinical outcomes or evasion of detection by diagnostic tools,thus making the monitoring for these mutations a necessity in proper evaluation of patients.The success of the vaccination programs has now been challenged by the discovery of mutant viruses showing amino acid substitutions in hepatitis B surface antigen(HBsAg),which may lead to evasion of vaccine-induced immunity.However,the emergence of these mutations has not yet raised concern since it was shown that they develop slowly.Investigations of HBV genetic variability and clinical implications of specific mutations have resulted in significant advances over the past decade,particularly in regard to management of resistance to antiviral drugs.In the era of drugs with high genetic barrier for resistance,on-going monitoring for possible resistance is still essential since prolonged therapy is often necessary.Understanding the frequencies and clinical implications of viral mutations may contribute to improvement of diagnostic procedures,more proper planning of immunization programs and creating the most efficient therapeutic protocols.

Hepatitis B virus genotypes and lamivudine resistance mutations in Jordan

AIM:To investigate and identify prevalent hepatitis B virus(HBV) genotypes and to explore lamivudine-resistant mutations among treated and untreated patients in Jordan.METHODS:A total of 107 cases with chronic hepatitis B were recruited from different medical centers in Jordan.Serological tests were preformed for all cases using a microparticle enzyme immunoassay.HBV Genotyping was performed for 70 cases using Line probe genotyping assay.The YMDD mutations were explored for 20 cases(4 were lamivudine naive) using the INNO-LiPA HBV DR assay.RESULTS:Genotype D was the only detected genotype.A total of 6 YMDD mutations were detected in 5 treated patients(31%) while one mutation was detected in the naive patients.Seventeen percent of cases were positive for HBeAg and had statistically significant higher levels of serum aminotransferases.CONCLUSION:HBV genotype D appears to be the only circulating type in Jordanian patients.The YMDD mutations were detected in 31% of lamivudine-treated cases with similar patterns to those found in the literature.We also found a relatively low prevalence of HBeAg expression among examined cases(17%).Awareness of these serologic,genotypic and resistance patterns might help in the formulation of management plans and for predicting clinical outcomes.Further larger scale studies are needed to confirm our results and to examine possible associations among clinical,serologic,and genetic patterns of HBV infections in Jordan.

Hepatitis B virus mutations potentially conferring adefovir/ tenofovir resistance in treatment-naive patients

Anti-hepatitis B virus(HBV)therapy leads to the emer- gence of mutant viral strains during the treatment of chronic hepatitis B with nucleos(t)ides analogues. The existence of HBV variants with primary antiviral resistance may be important for treatment choice. We studied two patients with chronic HBV infection by sequencing the HBV polymerase gene.They had adefovir-and tenofovir-related mutations in the viral polymerase,although they had never been treated. These mutations were rtV214A/rtN238T in one patient and rtA194T in the other.Thus,mutations in untreated patients deserve cautious surveillance.These data indicate that mutations that can theoretically confer adefovir or tenofovir resistance may emerge in treatmentnaive patients.

Lamivudine resistance mutations in patients infected with hepatitis B virus genotype D

AIM: To determine the distribution of viral genotypes for primary or acquired lamivudine resistance. METHODS: A total of 283 patients with chronic hepatitis B virus (HBV) infection (245 patients with chronic hepatitis B and 38 inactive hepatitis B surface antigen carriers) were included in the study. The HBV geno-type was determined by using quantitative real-time polymerase chain reaction and sequence analysis, and tyrosine-methionine-aspartate-aspartate (YMDD) motif mutations were determined using the reverse transcriptase hybridization method. RESULTS: Lamivudine resistance was determined in a total of 25 (10.7%) chronic hepatitis B patients. Eight subjects (4%) had primary resistance to lamivudine, and 17 (53.1%) had secondary resistance to lamivudine. Genotype D, which was isolated from 267 of the patients with chronic HBV infection, was the dominant genotype in Turkey. CONCLUSION: Identification of YMDD motif mutations should have a positive impact on the selection of proper antiviral medication for patients, even for those who are nucleoside nave.

Association of core promoter mutations of hepatitis B virus and viral load is different in HBeAg(+) and HBeAg(-) patients

AIM:To identify the prevalence of hepatitis B e antigen (HBeAg) and to assess the association of hepatitis B virus (HBV) core promoter mutations and viral load in Indonesian patients.METHODS:Sixty-four patients with chronic hepatitis,65 with liver cirrhosis and 50 with hepatocellular carcinoma were included in this study.HBeAg and hepatitis B e antibody (HBeAb) tests were performed using enzyme-linked immunosorbent assay and the mutations were analyzed by sequencing.Viral load was measured by real-time polymerase chain reaction.RESULTS:Of 179 patients,108 (60.3%) were HBeAg(-) and 86 (79.6%) of these HBeAg(-) patients had been seroconverted.The A1896 mutation was not found in HBeAg(+) patients,however,this mutation was detected in 70.7% of HBeAg(-) patients.This mutation was frequently found when HBeAg was not expressed (87.7%),compared to that found in HBeAg seroconverted patients (65.1%).The A1899 mutation was also more prevalent in HBeAg(-) than in HBeAg(+) patients (P=0.004).The T1762/A1764 mutation was frequently found in both HBeAg(+) and HBeAg(-) patients,however,the prevalence of this mutation did not significantly differ among the two groups (P=0.054).In HBeAg(+) patients,the T1762/A1764 mutation was correlated with lower HBV DNA (P < 0.001).The A1899 mutation did not correlate with HBV DNA (P=0.609).In HBeAg(-) patients,the T1762/A1764 mutation alone was not correlated with HBV DNA (P=0.095),however,the presence of either the T1762/A1764 or A1896 mutations was associated with increased HBV DNA (P < 0.001).CONCLUSION:The percentage of HBeAg(-) patients is high in Indonesia,and most of the HBeAg(-) patients had been seroconverted.The A1896 mutation was most likely the major cause of HBeAg loss.The T1762/A1764 mutation alone was associated with lower viral loads in HBeAg(+) patients,but not in HBeAg(-) patients.

A mutation of the start codon in the X region of hepatitis B virus DNA in a patient with non-B,non-C chronic hepatitis

There are cases of hepatitis involving occult hepatitis B virus(HBV)infection in which,even though the HB surface antigen(HBsAg)is negative,HBV-DNA is detected by a polymerase chain reaction(PCR).We con-ducted a sequence analysis of the entire HBV region in a case of non-B non-C chronic hepatitis in a 46-yearold female.A diagnosis of non-B non-C chronic hepatitis was made.Although HBV markers,such as HBs antibody(anti-HBs),anti-HBc,HBeAg and anti-HBe,were negative,HBV-DNA was positive.Nested PCR was performed to amplify the precore region of HBV-DNA and all remaining regions by long nested PCR.Sequence analysis of the two obtained bands was conducted by direct sequencing.Compared with the control strains,the ATG(Methionine)start codon in the X region had mut ated to GTG(Valine).It is assumed that a mutation at the start codon in the X region may be the reason why HBV markers are negative in some cases of hepatitis that involve occult HBV infection.

Relation between hepatitis B virus genotypes and gene mutation of basic core promoter in Li nationality

Objective：To investigate the relation between hepatitis B virus（HBV） genotypes and the double mutation of A-to-T nucleotide（nt） 1762 and G-to-A nt 1764 in basic core promoter（BCP T1762/A1764） in patients of the Li nationality. Methods：Subjects were 125 HBV DNA positive patients that belong to the Li nationality on Hainan Island. HBV DNA genotype was determined by real time fluorimetry polymerase chain reaction. BCP T1762/A1764 mutation was performed using the direct sequencing method. Results：The prevalence rates of genotype B, genotype C, genotype D, genotype C and D mixed infection（genotype C＋D） and genotype B and D mixed infection （genotype B ＋C） were 31.20%, 53.60%, 12.00%, 2.40% and 0.80% respectively. Mutation frequencies in patients infected with HBV genotype C（58.21%） were significantly higher than in those infected with other genotypes （P 〈 0.01 ）. The serum viral load of the patients with genotype C（5.74_＋ 1.21） was also higher than that of those with genotype B（P 〈0.01）. Conclusion：The major genotypes in the Li nationality were genotype C and genotype B. The infection of genotype D and mixed infection also occurred in the Li nationality. Genotype C HBV has a higher replication rate, and the different degrees of pathogenecity among HBV genotypes may be related to BCP T1762/ A1764 mutation frequency.

Visualization of Gene Mutation Complicated Pattern of Hepatitis B Virus Based on Cellular Automata

Hepatitis B virus shows instantaneous and high rate mutations in biological experiments, some sorts of which affect the efficiency of virus replication greatly through enhancing or depressing the viral replication, while others have no influence at all. Taking advantage of prominent features of cellular automata, we simulate the effect of hepatitis B virus gene mutation on its replication efficiency. The computer simulation results demonstrate the feasibility of our novel model by comparing with the results of biological experiments.

EFFECTS OF MUTATIONS IN THE POLYMERASE GENE OF HEPATITIS B VIRUS GENOME ON LAMIVUDINETHERAPY

Objective To explore the offects of mutations in tyrosine-methionine-aspartic acid-aspartic acid (YMDD) motif of the polymerase in the hepatitis B virus (HBV) genome on lamivudine antiviral therapy. Methods Partial HBV DNA segment containing the YMDD motif in the P gene wes obtained through amplifi- cation by polymerase chain reaction (PCR )from 19 chronic hepatitis B patients with serum HBV DNA positive at the 48th week treatment with lamivudine and subjected to automatic sequencing. Influences of vartants with YMDD mutations on lamivudine therapy were seen by observing the dynamic changes of serum HBV DNA and ALT levels. Results Serum HBV DNA breakthrough was found in 3 out of 10 individuals with detection of the YMDD mutations at the 48th week and in 5 at the 52th week, 2 of the 5 patients accompanied by serum ALT re-elevation, whereas of 9 subjects without YMDD mutations, 2 experenced an HBV DNA breakthrough at the 48th week and 1 of them had a conversion from HBV DNA positive DNA positive to negative at the 52th week. Patients with detectable HBV DNA level had a fluctuating level of serum ALT all time during the treatment. Conclusion Detection of mutations in the YMDD motif of polymerase gene in HBV genome during the lamivudine therapy will be helpful to monitoring its therapeutic outcomes.

Quantitative evaluation of hepatitis B virus mutations and hepatocellular carcinoma risk:a meta-analysis of prospective studies

Background： The temporal relationship between hepatitis B virus （HBV） mutations and hepatocellular carcinoma （HCC） remains unclear. Methods： We conducted a meta-analysis including cohort and nested case-control studies to prospectively examine the HCC risk associated with common variants of HBV in the PreS, Enhancer Ⅱ, basal core promoter （BCP） and precore regions. Pertinent studies were identified by searching PubMed, Web of Science and the Chinese Biological Medicine databases through to November 2014. Study-specific risk estimates were combined using fixed or random effects models depending on whether significant heterogeneity was detected. Results： Twenty prospective studies were identified, which included 8 cohort and 12 nested case-control studies. There was an increased risk of HCC associated with any PreS mutations with a pooled relative risk （RR） of 3.82 [95% confidence interval （CI）： 2.59-5.61]. The pooled-RR for PreS deletion was 3.98 （95% CI： 2.28-6.95）, which was higher than that of PreS2 start codon mutation （pooled-RR=2.63, 95% CI： 1.30-5.34）. C1653T in Enhancer Ⅱ was significantly associated with HCC risk （pooled-RR=l.83; 95% CI： 1.21-2.76）. For mutations in BCP, statistically significant pooled-RRs of HCC were obtained for T1753V （pooled- RR=2.09; 95% CI： 1.49-2.94） and AI762T/G1764A double mutations （pooled-RR=3.11; 95% CI： 2.08- 4.64）. No statistically significant association with HCC risk was observed for G1896A in the precore region （pooled-RR=0.77; 95% CI： 0.47-1.26）. Conclusions： This study demonstrated that PreS mutations, C1653T, T1753V, and A1762T/G1764A, were associated with an increased risk of HCC. Clinical practices concerning the HCC risk prediction and diagnosis may wish to focus on patients with these mutations.

In vitro resistance to interferon of hepatitis B virus with precore mutation

AIM: Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFN-α), which results in an efficient reduction of the viral load only in 20-40% of treated patients. Mutations at HBV precore prevail in different clinical status of HBV infection. The roles of precore mutation in the progression of chronic hepatitis and interferon sensitivity are still unknown. The aim of this study was to explore if there was any relationship between HBV precore mutation and sensitivity to interferon in vitro. METHODS: HBV replication-competent recombinant constructs with different patterns of precore mutations were developed. Then the recombinants were transiently transfected into hepatoma cell line (Huh7) by calcium phosphate transfection method. With or without IFN, viral products in culture medium were collected and quantified 3 d after transfection. RESULTS: We obtained 4 recombinant constructs by orientation-cloning 1.2-fold-overlength HBV genome into pUC18 vector via the EcoRI and Hind lll and PCR mediated site-directed mutagenesis method. All the recombinants contained mutations within precore region. Huh7 cells transfected with recombinants secreted HBsAg and HBV particles into the cell culture medium, indicating that all the recombinants were replication-competent. By comparing the amount of HBV DNA in the medium, we found that HBV DNA in medium reflecting HBV replication efficiency was different in different recombinants. Recombinants containing precore mutation had fewer HBV DNAs in culture medium than wild type. This result: showed that recombinants containing precore mutation had lower replication efficiency than wild type. HBV DNA was decreased in pUC18-HBV1.2-WT recombinants after IFN was added while others with precore mutations were not, indicating that HBV harboring precore mutation was less sensitive to IFN in cell culture system. CONCLUSION: These data indicate that HBV harboring precore mutation may be resistant to IFN in vitro.

Hepatitis B virus mutations related to liver disease progression of Korean patients

Hepatitis B virus(HBV)infection is a global health problem and more than 350 million people worldwide are chronic carriers of the virus.Despite the recent dramatic decline in HBV chronic patients through successful programs of hepatitis B surface antigen vaccination,South Korea is still recognized as an endemic area of HBV infection.HBV infections in South Korea exhibit several distinct features in epidemiologic and clinical aspects.In this review paper,we summarize the distinct HBV mutation patterns related to clinical severity and the molecular epidemiologic traits in Korean chronic patients based on previous reports.Generally,several lines of evidence,including our previous results,have led to the conclusion that a combination of the exclusive predominance of genotype C2,which is prone to mutations,the high prevalence of basal core promoter double mutations,and the presence of distinct immune responses against HBV proteins in the Korean population may generate the distinct HBV variants rarely or not encountered in other areas,which results in distinct clinical manifestations in Korean chronic patients.This may provide a novel insight into the relationships between clinical severity,HBV genotype distribution,and HBV naturally occurring variants.

A sensitive method to detect the hepatitis B virus mutations by using solid phase PCR on oligonucleotide array

A sensitive method based on solid phase PCR on oligonucleotide array was established to detect two point mutations 1896G-A and 1901G-A in hepatitis B virus (HBV) DNA, in which 6 probes including these two point mutations were immobilized on modified glass slides through 5' terminal linker, while the 3' terminal was made to be free. The mutated loci were designed to locate on the last nucleotides of 3' terminal respectively, and the positive control probes lacked the last nucleotide of 3' terminal in comparison with the probes used for detection. Probes fixed on oligonucleotide array were also the solid phase amplification primers. One pair of liquid primers was used to amplify the short template product from whole HBV DNA. Using target DNA as template, the solid primers were extended under the action of Taq DNA polymerase and incorporated with Cy-3dCTP as marker. During the thermal cycling reaction, probes served as solid phase amplification primers and amplification products bound to the oligonucleotide array, which could be visualized by incorporation with fluorescent dyes. After amplification, the oligonucleotide array was washed, performed with laser scanning, and then used for quantitative analysis to obtain the information for mutations. The hybridization results were compared with DNA sequencing. It was demonstrated that in case of sample A, the ratios of fluorescence intensities in wide type and in the mutated types of 1896G-A and 1901G-A mutations in HBV were 3.81:1 and 1:3.79 respectively, while, in case of sample B, those were 1:2.89 and 1:3.03 respectively, indicating the presence of point mutations in these two loci. These results correlated to those obtained from DNA sequencing analysis in which the fluorescence intensity ratios in wide type and in the mutated types of 1996G-A and 1901D-A mutations in HBV were 1.26:1 and 1.67:1 respectively. From the above observations, it is evident that the method using solid phase PCR based on oligonucleotide array appears to be a sensitive and promising way to detect mutations with low-density.

Hepatitis B virus genetic mutations and evolution in liver diseases

Hepatitis B virus(HBV)belongs to the genus Orthohepadnavirus of the Hepadnaviridae family and is approximately 3.2 kb in length.Owing to a lack of proofreading capacity during reverse transcription and a high replication rate,HBV exhibits as quasispecies.To detect the genetic mutations of HBV,many methods with different sensitivities and throughputs were developed.According to documentary records,HBV mutation and evolution were important vial parameters in predicting disease progression and therapeutic outcome.In this review,we separately discussed the correlation between HBV genomic mutations in four open reading frames and liver disease progression.Since some of the results were controversial from different laboratories,it remains to be seen whether functional analyses will confirm their role in modifying the course of infection.

Biological impacts of "hot-spot" mutations of hepatitis B virus X proteins are genotype B and C differentiated

AIM: To investigate the biological impacts of 'hot-spot'mutations on genotype B and C HBV X proteins (HBx).METHODS: Five types of'hot-spot' mutations of genotype B or C HBV X genes, which sequentially lead to the amino acid substitutions of HBx as I127T, F132Y, K130M+V131I,I127T+K130M+V131I, or K130M+V131I+F132Y, respectively,were generated by means of site-directed mutagenesis.To evaluate the anti-proliferative effects, HBx or related mutants' expression vectors were transfected separately to the Chang cells by lipofectamine, and the cells were cultured in hygromycin selective medium for 14 d, drug-resistant colonies were fixed with cold methanol, stained with Giemsa dyes and scored (increase of the colonies indicated the reduction of the anti-proliferation activity,and vice versa). Different types of HBx expression vectors were co-transfected separately with the reporter plasmid pCMVβ to Chang cells, which were lysed 48 h post-transfection and the intra-cellular β-galactosidase activities were monitored (increase of the β-galactosidase activities indicated the reduction of the transactivation activity, and vice versa). All data obtained were calculated by paired-samples t-test.RESULTS: As compared to standard genotype B HBx,mutants of I127T and I127T+K130M+V131I showed higher transactivation and anti-proliferative activities, while the mutants of F132Y, K130M+V131I, and K130M+V131I+F132Y showed lower activities. As compared to standard genotype C HBx, I127T mutant showed higher transactivation activity, while the other four types of mutants showed no differences. With regard to anti-proliferative activity,compared to standard genotype C HBx, F132Y and K130M+V131I mutants showed lower activities, and K130M+V131I +F132Y mutant, on the other hand, showed higher activity,while the mutants of I127T and I127T+K130M+V131I showed no differences.CONCLUSION: 'Hot-spot' mutations affect the antiproliferation and transactivation activities of genotype B and/or C HBx, and the biological impacts of most 'hot-spot' mutations on HBx are genotype B and C differentiated.

Clonal evolution of hepatitis B virus polymerase gene mutations during lamivudine-adefovir combination treatment

AIM:To identify hepatitis B virus polymerase gene mutations during antiviral therapy using lamivudineadefovir sequential monotherapy followed by lamivudine-adefovir combination therapy.METHODS:The patient cohort included four adult chronic hepatitis B patients who had undergone sequential monotherapy,first with lamivudine(LMV) and then,after developing viral breakthrough,with adefovir(ADV) therapy.All of the patients had non-response or viral breakthrough after LMV-ADV sequential monotherapy,which resulted in the switching of their antiviral regimen to LMV-ADV combination therapy.Eleven serum samples from the four patients who showed non-response to rescue LMV-ADV combination therapy were collected sequentially at a time before the antiviral treatment and then during the LMV monotherapy,ADV monotherapy,and LMV-ADV combination therapy.For the genotypic analysis,the whole 1310-bp polymerase gene region was amplified,cloned and sequenced.RESULTS:All patients had been previously treated with 100 mg of LMV once daily for a 15-to 26-mo period.The emergence of resistance mutations to LMV,such as rtM204V/I and/or rtL180M,were found in all patients.Their antiviral regimens were switched to ADV monotherapy as the second line treatment.All patients had viral breakthrough or non-response after the LMV-ADV sequential monotherapy.ADV-resistant mutations were detected after 13 to 19 mo of LMV-ADV sequential monotherapy.The rtA181V/T mutations were predominantly identified during the ADV treatment in the LMV-resistant patients.Twenty-seven of 38 clones were combined with an amino acid change at rt181;three clones had mutations in rt236 and one clone had a combined mutation.The rtA181V/T mutations were not suppressed by the LMV-ADV combination therapy.Thirty-nine of 64 clones showed an rtA181V/T mutation and six clones showed combined mutations in rt181 and rt236.Mutations in rt204 re-emerged during the combination treatment.The rt181 and rt204 mutations did not co-exist in one clone.CONCLUSION:Add-on lamivudine therapy with adefovir for adefovir resistance may not suppress the pre-existing adefovir-resistant mutation that develops during lamivudine-adefovir sequential monotherapy.

the rtA181 Mutation Produces Similar Clinical and Cellular Characteristics in Patients infected with Hepatitis B Virus Genotypes B and C

Objective To investigate the association between HBV genotypes and characteristics of rt A181 mutation. Methods Total of 85 chronic hepatitis B(CHB) patients who appeared rt A181 mutation after nucleos(t)ide analogs(NAs) therapy were enrolled in this study. Levels of serum ALT, AST, HBV DNA and HBs Ag titers were monitored during therapy. HBV reverse transcriptase genes were amplified and sequenced to identify genotypes and resistance mutations. Virions and HBs Ag in Hep G2 cell with rt A181 mutation were also compared between genotypes B and C. Results The majority of sera contained HBV genotypes B(15.7%) and C(84.3%). There were no significant difference of rt A181 mutant patterns between genotypes(P > 0.05). After emergence of rt A181 mutation, serum ALT, AST, HBV DNA levels and HBs Ag titers were decreased than that at baseline(P < 0.05), while these characteristics were not different between genotypes B and C(P > 0.05). In cellular experiment, there were no significant differences between genotypes B and C not only in HBV virions but also in HBs Ag titres(P > 0.05). Conclusions No differences of clinical characteristics and cellular results were found in rt A181 mutation of HBV genotypes B and C.