Background and Aims Lamivudine (LAM) is still widely used for anti-HBV therapy in China. The study aimed to clarify whether a newly-found rtM204Q mutation from patients was associated with the drug resistance. Methods HBV complete reverse-transcriptase region was screened by direct sequencing and verified by clonal sequencing. Replication-competent plasmids containing patient-derived 1.1mer mutant or wild-type viral genome were constructed and transfected into HepG2 cells. After cultured with or without serially-diluted antiviral drugs, intracellular HBV replicative intermediates were quantitated for calculating the 50% effective concentration of drug (EC50). Results A total of 12,000 serum samples of 9,830 patients with chronic HBV infection were screened. rtM204Q mutation was detected in seven LAM-refractory patients. By contrast, rtM204I/rtM204V mutations were detected in 2,502 patients' samples. The rtM204Q emerged either alone or in concomitance with rtM204I/rtM204V, and all were accompanied with virologic breakthrough in clinical course. Clonal sequencing verified that rtM204Q mutant was predominant in viral quasispecies of these samples. Phenotypic analysis showed that rtM204Q mutant had 89.9% of replication capacity and 76-fold increased LAM EC50 of the concomitant wild-type strain. By contrast, rtM204I mutant in the sample had lower replication capacity and higher LAM resistance (46.3% and 1396-fold increased LAM EC50 of the wild-type strain) compared to rtM204Q mutant. rtM204Q mutant was susceptible to adefovir dipivoxil (ADV) in vitro and ADV/ADV+LAM rescue therapy in clinic. Conclusion rtM204Q is suggested to be a novel LAM-resistance-associated mutation. It conferred a moderate resistance with higher competent natural replication capacity compared to rtM204I mutation.
References
[1]
World Health Organization (2012) Hepatitis B fact sheets. Available: http://www.who.int/mediacentre/factsheet？s/fs204/en/. Updated on July, 2013. Accessed on January 1, 2013.
[2]
Nebbia G, Peppa D, Maini MK (2012) Hepatitis B infection: current concepts and future challenges. QJM 105: 109–113 doi:10.1093/qjmed/hcr270.
[3]
Ma H, Jia J (2013) Why do I treat HBeAg-positive chronic hepatitis B patients with a nucleoside analogue. Liver Int 33 Suppl 1133–136 doi:10.1111/liv.12065.
Chao DC, Hu KQ (2013) Update on rescue therapies in patients with lamivudine-resistant chronic hepatitis B. Drug Des Devel Ther. 7: 777–788 doi:10.2147/DDDT.S33947.
[6]
Soriano V, McMahon B (2013) Strategic use of lamivudine in the management of chronic hepatitis B. Antiviral Res. doi:10.1016/j.antiviral.2013.08.026.
[7]
Zoulim F, Locarnini S (2013) Optimal management of chronic hepatitis B patients with treatment failure and antiviral drug resistance. Liver Int Suppl 1: 116–124. doi:10.1111/liv.12069.
[8]
Locarnini S, Mason WS (2006) Cellular and virological mechanisms of HBV drug resistance. J Hepatol 44: 422–431 doi:10.1016/j.jhep.2005.11.036.
[9]
Lai CL, Dienstag J, Schiff E, Leung NW, Atkins M, et al. (2003) Prevalence and clinical correlates of YMDD variants during lamivudine therapy for patients with chronic hepatitis B. Clin Infect Dis. 36: 687–696 doihttp://dx.doi.org/10.1086/368083.
[10]
Yuen LK, Locarnini SA (2009) Genetic variability of hepatitis B virus and response to antiviral treatments: searching for a bigger picture. J Hepatol 50: 445–448 doi:10.1016/j.jhep.2008.12.005.
[11]
Patterson SJ, George J, Strasser SI, Lee AU, Sievert W, et al. (2011) Tenofovir disoproxil fumarate rescue therapy following failure of both lamivudine and adefovir dipivoxil in chronic hepatitis B. Gut. 60: 247–254 doi:10.1136/gut.2010.223206.
[12]
Chinese Society of Infectious Diseases and Parasitology, Chinese Society of Hepatology (2000) Management scheme of diagnostic and therapy criteria of viral hepatitis. Zhonghua Gan Zang Bing Za Zhi (Chinese J Hepatol) 8: 324–329.
[13]
Xu D, Fu J, Jin L, Zhang H, Zhou C, et al. (2006) Circulating and liver resident CD4+CD25+ regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B. J Immunol 177: 739–747. PMID: 16785573.
[14]
Liu Y, Wang C, Zhong Y, Li X, Dai J, et al. (2011) Genotypic resistance profile of hepatitis B virus (HBV) in a large cohort of nucleos(t)ide analogue-experienced Chinese patients with chronic HBV infection. J Viral Hepat 18: e29–39 doi:–10.1111/j.1365–2893.2010.01360.x.
[15]
Xu Z, Liu Y, Liu L, Li X, Bai S, et al. (2013) Association of interferon-gamma induced protein 10 promoter polymorphisms with the disease progression of hepatitis B virus infection in Chinese Han population. PLoS One 8: e72799 doi:10.1371/journal.pone.0072799.
[16]
Zoulim F (2006) In vitro models for studying hepatitis B virus drug resistance. Semin Liver Dis 26: 171–180 doi:10.1055/s-2006-939759.
[17]
Ji D, Liu Y, Li L, Xu Z, Si LL, et al. (2012) The rtL229 substitutions in the reverse transcriptase region of hepatitis B virus (HBV) polymerase are potentially associated with lamivudine resistance as a compensatory mutation. J Clin Virol 54: 66–72 doi:10.1016/j.jcv.2012.02.003.
[18]
Yang H, Westland CE, Delaney WE4th, Heathcote EJ, Ho V, et al. (2002) Resistance surveillance in chronic hepatitis B patients treated with adefovir dipivoxil for up to 60 weeks. Hepatology 36: 464–467 doi:10.1053/jhep.2002.34740.
[19]
Lok AS, Zoulim F, Locarnini S, Bartholomeusz A, Ghany MG, et al. (2007) Antiviral drug-resistant HBV: standardization of nomenclature and assays and recommendations for management. Hepatology 46: 254–265 doi:10.1002/hep.21698.
[20]
Lai CL, Gane E, Liaw YF, Hsu CW, Thongsawat S, et al (2007) Telbivudine versus lamivudine in patients with chronic hepatitis B. N Engl J Med. 357: 2576–2588 doi:10.1056/NEJMoa066422.
[21]
Bozdayi AM, Uzunalimo？lu O, Türkyilmaz AR, Aslan N, Sezgin O, et al. (2003) YSDD: a novel mutation in HBV DNA polymerase confers clinical resistance to lamivudine. J Viral Hepat 10: 256–265 doi:–10.1046/j.1365–2893.2003.00435.x.
[22]
Frost S, Nijhuis M, Schuurman R (2000) Evolution of lamivudine resistance in human immunodeficiency virus type 1 infected individuals the relative roles of drift and selection. J Virol 74: 6262–6268 doi:–10.1128/JVI.74.14.6262–6268.2000.
[23]
Sarafianos SG, Das K, Clark AD Jr, Ding J, Boyer PL, et al. (1999) Lamivudine (3TC) resistance in HIV reverse transcriptase involves steric hindrance with beta-branched amino acids. Proc Natl Acad Sci USA 96: 10027–10032 doi:10.1073/pnas.96.18.10027.
[24]
Gao HQ, Boyer PL, Sarafianos SG, Arnold E, Hughes SH (2000) The role of steric hindrance in 3TC resistance of human immunodeficiency virus type-1 reverse transcriptase. J Mol Biol 300: 403–418 doi:10.1006/jmbi.2000.3823.
[25]
Ono-Nita SK, Kato N, Shiratori Y, Masaki T, Lan KH, et al. (1999) YMDD motif in hepatitis B virus DNA polymerase influences on replication and lamivudine resistance: A study by in vitro full-length viral DNA transfection. Hepatology 29: 939–945 doi:10.1002/hep.510290340.
[26]
Schildgen V, Ziegler S, Tillmann RL, Schildgen O (2010) Novel mutation in YMDD motif and direct neighbourhood in a child with chronic HBV-infection and clinical lamivudine and adefovir resistance - a scholarly case. Virol J 7: 167 doi:10.1186/1743-422X-7-167.
[27]
van B？mmel F, Trojan J, Deterding K, Wedemeyer H, Wasmuth HE, et al. (2012) Evolution of adefovir-resistant HBV polymerase gene variants after switching to tenofovir disoproxil fumarate monotherapy. Antivir Ther 17: 1049–1058 doi:10.3851/IMP2307.
[28]
Wang Y, Liu Y, Xu Z, Si L, Chen L, et al. (2012) Phenotypic characteristics of a novel mutation rtM204Q in the reverse-transcriptase domain of hepatitis B virus isolated from a chronic hepatitis B patient receiving lamivudine treatment. Med J Chin PLA 37: 446–450.
[29]
Ghany MG, Doo EC (2009) Antiviral resistance and hepatitis B therapy. Hepatology 49: S174–S184 doi:10.1002/hep.22900.
[30]
Kay A, Zoulim F (2007) Hepatitis B virus genetic variability and evolution. Virus Res 127: 164–176 doi:10.1016/j.virusres.2007.02.021.
[31]
Lacombe K, Boyd A, Gozlan J, Lavocat F, Girard PM, et al. (2010) Drug-resistant and immune-escape HBV mutants in HIV-infected hosts. Antivir Ther 15: 493–497 doi:10.3851/IMP1495.
[32]
Rodriguez-Frías F, Tabernero D, Quer J, Esteban JI, Ortega I, et al. (2012) Ultra-deep pyrosequencing detects conserved genomic sites and quantifies linkage of drug-resistant amino acid changes in the hepatitis B virus genome. PLoS One 7: e37874 doi:10.1371/journal.pone.0037874.
[33]
Thai H, Campo DS, Lara J, Dimitrova Z, Ramachandran S, et al. (2012) Convergence and coevolution of hepatitis B virus drug resistance. Nat Commun 3: 789 doi:10.1038/ncomms1794.
[34]
Yatsuji H, Noguchi C, Hiraga N, Mori N, Tsuge M, et al. (2006) Emergence of a novel lamivudine-resistant hepatitis B virus variant with a substitution outside the YMDD motif. Antimicrob Agents Chemother 50: 3867–3874 doi:10.1128/AAC.00239-06.
[35]
Osiowy C, Villeneuve JP, Heathcote EJ, Giles E, Borlang J (2006) Detection of rtN236T and rtA181V/T mutations associated with resistance to adefovir dipivoxil in samples from patients with chronic hepatitis B virus infection by the INNO-LiPA HBV DR line probe assay (version 2). J Clin Microbiol 44: 1994–1997 doi:10.1128/JCM.02477-05.
[36]
Gerolami R, Bourliere M, Colson P, Halfon P, Borentain P, et al. (2006) Unusual selection of rtA181V HBV mutants cross-resistant to adefovir following prolonged lamivudine monotherapy: report of two cases. Antivir Ther 11: 1103–1106. PMID: 17302381.
