Study For Clinical Characteristics And Significances Of Mutations In The Basal Core Promoter And Precore Regions And Reverse-transcriptase Domain Of Hepatitis B Virus

Background:Hepatitis B virus (HBV) infection leads to a wide spectrum of clinical manifestations,ranging from asymptomatic carrier state, acute hepatitis B (AHB), mild or severe chronichepatitis B (CHB-M, CHB-S), liver cirrhosis (LC), hepatocellular carcinoma (HCC), toacute-on-chronic liver failure (ACLF). In China, HBV infected chronically approximate93million people, of whom hepatitis B-related LC patients account for4million, and about280thousand patients died of HCC each year. Hepatitis B-related ACLF is reported to result in morethan20thousand deaths annually. The development of hepatitis B-related ACLF may beassociated with stronger immune response. HBV gene mutation may be one of the key factorscontributing to abnormal immune response.HBV is a highly variable DNA virus. Like retrovirus, HBV replicates its genome via anRNA intermediate using reverse transcription. However, due to the lack of the rigorousproofreading function of reverse transcriptase, errors in HBV DNA replication occur at a muchhigher rate than other DNA viruses. HBV is also a highly replicable virus and all possiblenucleotide (nt) mutations might emerge per site per day in HBV genome. In addition, the maintemplate for viral transcription, HBV covalently closed circular DNA (cccDNA), has a longhalf-life and stably exists in the infected hepatocyte nuclear, resulting in persistent infection. Innatural infection, wild-type HBV strains predominantly exist in the viral quasispecies pool.HBV has been classified into at least eight genotypes (≥8%) and multiple subgenotypes (≥4%)according to the differences in HBV entire genomic sequences or small surface gene sequences.Virologic features vary in different genotype HBV and may associate with the clinical outcomesof liver diseases.The most common hotspot mutations in HBV genome are located in the basal corepromoter (BCP) and precore (PC) regions. Hepatitis B e antigen (HBeAg), encoded by precoremRNA, is considered to be an immune tolerogen and its expression is transcriptionally regulatedby BCP. Therefore mutations in the two regions may abrogate or decrease the translation ofHBeAg, leading to the increase of the viral replication capacity in vitro and evasion of the hostimmune response. On the other hand, as HBeAg and hepatitis B core antigen (HBcAg) sharingthe same immune epitopes, mutations in PC region may enhance the immune attack on HBcAgof the infected hepatocytes when lacking of HBeAg. This may be associated with the diseaseprogression and the severity of hepatitis B.Several studies have reported that BCP/PC mutations may be associated with acute fulminant hepatic failure, while some other studies have debated on this. HBV genotypes mayinfluence the incidence of BCP/PC mutations and complicate the result analysis. Additionally,there is still a paucity of data on the association of HBV BCP/PC mutations and genotypefeatures with ACLF occurrence. The first part of this study systemically investigated the clinicalimplications and virologic features of HBV BCP/PC mutations and genotypes in a large numberof AHB, CHB-M, CHB-S and ACLF patients, which will help understand the virologicmechanisms of chronicity and severity of hepatitis B.Currently, nucleos(t)ide analogs (NAs) are the commonly used anti-HBV therapy drugs inclinic, including three nucleoside analogs, i.e. lamivudine (LAM), entecavir (ETV), andtelbivudine (LdT); and two nucleotide analogs, i.e. adefovir dipivoxil (ADV) and tenofovirdisoproxil fumarate (TDF), and the former four ones have been approved for clinical use inChina. These medications act primarily through blocking reverse transcription of pregenomicRNA to HBV DNA. However, they are unable to eradicate HBV infection because of thepersistence of HBV cccDNA in the infected hepatocytes. High virus rebound and/or hepaticflare will occur once the therapy is discontinued. Herein, a long-term antiviral therapy is neededfor sustained suppression of HBV DNA replication.However, drug pressure may select fitness mutants with a survival advantage compared towild-type virus or make minute quantity of preexisting mutants in viral quasispecieses poolacquire selectively amplified during long-term antiviral treatment of NAs. Drug-resistant HBVmutants may lead to treatment failure and progression of liver diseases, which has been anintractable issue in clinical practice. The resistance mutations are located in the HBV RT domain.The primary resistance mutations can directly decrease the viral susceptibility to NAs and thesecondary or compensatory mutations can restore or enhance the replication capacity of resistantmutants. Some proposed mutations may be associated with ADV resistance, although it is stillcontroversial. As more antiviral strategies are available for anti-HBV therapy, the risks andpatterns of drug-resistance and cross-resistance are higher. In addition, nonoptimal sequentialuse of NAs may promote the development of multidrug resistant HBV.To date, data on HBV resistance mutations are largely derived from some clinical trials andcohorts with relatively fixed NAs schedules, limited samples and drug resistance profiling.However, the NAs schedules are more complex in real clinical practice resulting in specificregularity in HBV drug resistance profile and there is a paucity of data on resistance profile inChinese patients. Therefore, the second part of this study investigated the HBV genotypicresistance profiles in a large number of NAs-experienced Chinese patients with chronic HBVinfection based on our previously-established HBV resistance database. The analysis of clinicaland experimental data from a rare and novel multidrug resistance (LAM, ADV and ETV) patientreveals the dynamic evolution and suppression of the multidrug resistant HBV strains underlong-term antiviral treatment. The results will provide new information on HBV genotypicresistance profiles in real clinical practices and may have important clinical implications for HBV drug resistance management in China.Objective:(1) To investigate the features of HBV BCP/PC mutations and genotypes in a large numberof AHB, CHB-M, CHB-S and ACLF patients, and uncover the clinical implications of thevirologic features and help understand the virologic mechanisms of chronicity and severity ofhepatitis B.(2) To investigate the HBV genotypic resistance profiles in a large number ofNAs-experienced Chinese patients with chronic HBV infection and to further analyze a dynamicevolution and suppression of HBV strains with rare and novel multidrug resistance to LAM,ADV and ETV in a patient and provide new information on HBV genotypic resistance profilesin real clinical practices and help standardise HBV drug resistance management in China.Methods:(1) Serum samples from182patients with AHB,325patients with CHB-M,170patientswith CHB-S, and298patients with ACLF were collected and HBV DNA was extracted. TheHBV sequences of695base pair (bp)-long BCP/PC gene and1225bp-long RT/S gene weredetermined by direct sequencing method after nested polymerase chain reaction (PCR)amplification which has been improved by our group. HBV genotype assignment was based onphylogenetic analysis of the RT/S sequence using MEGA4software. Ten sites of interest wereanalyzed based on their clinical or potential clinical significance suggested in previouspublications, namely, nt1753,1754,1758,1762,1764,1766,1768in BCP region and nt1862,1896,1899in PC region using Vector NTI Suite software.(2) Serum samples were collected from1803NAs-experienced Chinese patients withchronic HBV infection and HBV DNA was extracted.1225bp-long HBV RT full gene wasamplified by a high-sensitive direct PCR sequencing. Mutations at15locations (including rt80,rt84, rt173, rt180, rt181, rt184, rt194, rt202, rt204, rt214, rt215, rt217, rt233, rt236, rt250) in theRT domain were analyzed. Furthermore, serially dynamic serum samples were collected fromone CHB patient who successively experienced116-month NAs antiviral treatment. The PCRproducts of HBV RT gene were directly sequenced and also cloned into the pGEM-Teasy vectorfor clonal sequencing (more than20clones per sample per time-point). The dynamicallyevolutionary regulation, phenotypic resistance characteristics and efficient treatment of thenovel multidrug-resistant HBV strains was analyzed.Results:(1) AHB patients had a significantly higher ratio of genotype B to C and a higherprevalence of BCP/PC wild-type virus than CHB patients. Significantly lower prevalence ofA1762T, G1764A, G1896A, and G1899A but higher prevalence of T1758C was found in AHBpatients. Interestingly, T1758C and A1762T/G1764A appeared mutual restraint. Compared to genotype C virus, genotype B virus had a lower BCP mutation frequency and a similar PCmutation frequency. AHB patients harboring four basic BCP/PC mutations had similar alanineaminotransferase (ALT) levels, while CHB patients harboring PC mutations had higher ALTlevels. AHB patients harboring basic BCP+/PC+mutations had higher viral loads, while lowerviral loads in CHB patients with the same mutations. AHB patients had a higher percentage ofHBeAg negativity than CHB patients and the percentage was even higher in those harboringbasic BCP+/PC+mutations. In CHB patients, accumulation of BCP/PC mutations wasaccompanied with escalation of HBeAg negativity.(2) Compared to CHB patients, ACLF patients had a significantly higher mutationfrequency of T1753V (C/A/G), A1762T, G1764A, and C1766T in the BCP region and G1862T,G1896A, and G1899A in the PC region. Moreover, the frequencies of A1762T, G1764A andG1896A hotspot mutations and the average substitution number at the10sites of interest of theviral sequences increased in a stepwise manner in the order of CHB-M＜CHB-S＜ACLFpatients. Genotype B virus had significantly lower BCP mutation frequencies and higher PCmutation frequencies than genotype C virus. Different from the BCP mutants, the PC mutantsshowed a strong positive influence on HBeAg seroconversion for all three groups of patients.Notably, ACLF patients infected with the PC mutants had a significantly higher mortality thanthose infected with the wild-type viruses.(3) Drug-resistant mutations were detected in560of the1803patients, including214of490patients who received LAM,35of428patients who received ADV,5of18patients whoreceived LdT monotherapy and306of794patients who received various sequential/combinedNA therapies. No drug-resistant mutations were detected in the73patients who received ETVmonotherapy. ADV resistant mutations were detected in36of381patients who received LAMand then switched-to ADV in contrast to one of82patients who received ADV add-on LAM.ETV-resistant mutations were detected not only in LAM-and ETV-treated patients but also inLAM-treated ETV-naive patients. Double mutations rtM204I and rtL180M were detected morefrequently in genotype C than in genotype B virus, and patients infected with this mutant hadhigher alanine aminotransferase levels than those infected with mutant containing the rtM204Isubstitution alone. Notably, multidrug-resistant HBV strains were identified in eight patients.(4) A representative CHB patient who received116months of successively antiviraltherapy from June,2002was followed up. He experienced10months of LAM plus interferon(IFN)-α2b combination therapy,23months of LAM monotherapy,13months of ADVmonotherapy,12months of ETV monotherapy,12months of ADV plus interferon (IFN)-α2bcombination therapy,6months of ADV monotherapy, and40months of ADV plus LAMcombination therapy. HBV DNA maintained undetectable level and normalized ALT level wasachieved after serial fluctuation.Clonal sequencing showed that the multidrug-resistant HBV strains in viral quasispeciesdynamically evolved from initial wild-type strain, to LAM-resistant rtM204I strains, ADV-resistant rtA181V and rtA181T strains, LAM-resistant rtL180M+rtM204V strains,ETV-resistant rtL180M+rtS202G+rtM204V strains, ADV-resistant rtN236T±rtA181T strains,multidrug-resistant rtL180M+rtA181V+rtS202G+rtM204V+rtN236T and rtL180M+rtS202G+rtM204V+rtN236T strains, and finally to ETV-resistant rtL180M+rtS202G+rtM204V strains.Conclusions:(1) Patients infected genotype B virus, BCP/PC wild-type virus or BCP T1758C mutantHBV are more likely to develop AHB. In contrast, the development of CHB is closelyassociated with the accumulation of BCP/PC mutations.(2) HBV BCP/PC mutations are positively associated with the progression of CHB. CHBpatients with BCP/PC mutants are prone to develop CHB-S or ACLF. Notably, ACLF patientswith PC mutants have a higher risk of mortality.(3) Although only four NAs are approved for clinical use in China, there are more than40antiviral schedules in real clinical practice, adding the complexity and diversity of drug-resistantmutational patterns in China. The long-term use of NAs with nonoptimal responses promotesarising of the drug-resistant and multidrug-resistant HBV, which brings larger challenges toclinical treatment. The data on HBV genotypic resistance profiles in this study may havesignificant implications for HBV drug resistance management in China.(4) The emergence of multidrug-resistant HBV is based on complex single-anddouble-drug-resistant viruses in viral quasispecies. This is the first time that multidrug-resistantHBV strains were identified in a clinical case and were suppressible by LAM plus ADV whenTDF was not available in China, which provide reference for management of multidrug-resistantHBV in China.