In this study, we identified a novel lamivudine-resistant strain of HBV with an intact YMDD motif in a patient who received long-term lamivudine therapy. YMDD mutants were not detected even by a sensitivity-enhanced detection method, which was reported previously by our group (6
). The double nucleotide substitutions (GG to TA) induced amino acid substitutions in both polymerase (rtA181T) and HBs antigen (HBs W172L). One might assume that the compliance of the patient was poor. However, the patient was very punctual and confirmed that he took lamivudine with perfect compliance.
Our study demonstrated that the rtA181T mutation reduced the susceptibility to lamivudine 3.0- to 3.9-fold in vitro (Table ). Furthermore, we also confirmed lamivudine resistance of this mutant strain in vivo using human hepatocyte-chimeric mice. The amino acid substitution in the reverse transcriptase (RT) domain is similar to that reported previously (7
). However, in contrast to our results, the mutant strains in the latter reports emerged with or after those with the mutation in the YMDD motif (YIDD or YVDD) and took over them (34
). There are two additional differences between the substitutions we identified and those described by Yeh et al. (34
), as detailed below.
Firstly, the HBs antigen was prematurely terminated in the mutant strain reported by Yeh et al. (34
). In this regard, a similar amino acid substitution of the B domain of the polymerase FLLA motif in woodchuck hepatitis virus (WHV) treated with lamivudine was reported (15
). The HBs antigen in these WHV mutant strains also had premature stop codons. These findings suggest that the mutant strains of HBV and WHV cannot replicate and spread by themselves because of the lack of HBs antigen. Such strains are thought to replicate by using in vivo-supplied HBs antigen from wild-type strains as helper antigens. In contrast, the novel strain identified in this study had no premature termination of the HBs gene. The in vitro study suggested that the strain had a replication ability similar to that of the wild type. Furthermore, we also showed that the strain infected and reached a high viral load in human hepatocyte-chimeric mice. Although the inoculum contained only a small amount of wild-type strain (one of 12 clones), all clones obtained from mouse serum were mutant strains (rtA181T). Considering these results and the fact that the index patient showed high viral titers after breakthrough (more than 7.6 log copies/ml), this mutant strain can spread and replicate by itself and has strong replicative ability.
Secondly, the substitutions identified in this study appeared with nucleotide and amino acid substitutions in the spacer region of the polymerase (S331C). There are only a few studies that reported the function of the spacer domain (19-21
), leaving the biological significance of this region unknown. The substitution in the spacer region reappeared with the A181T mutation in the RT domain in the index patient after the patient restarted lamivudine therapy. Although our study showed no significant contribution of this mutation to drug resistance (Fig. and ; Table ), the significance of the mutation in this region (fingers in the HBV polymerase homology model [8
]) should further be investigated.
Recently, the amino acid substitutions rtA181T and rtA181V were reported to emerge with resistance against adefovir (11
). Tillmann et al. (29
) reported one case in which the virus developed the rtA181T mutation during famciclovir breakthrough. The A556T mutation of WHV, analogous to the rtA181T mutation of HBV, has been reported to be associated with lamivudine resistance (15
). These results indicate that the amino acid substitutions at position 181 may associate with resistance against many nucleoside analogues, including lamivudine, famciclovir, and adefovir. Although our in vitro study indicated that the rtA181T mutant had no resistance against adefovir and the animal study showed that combination therapy with lamivudine and adefovir effectively reduced the virus load in woodchucks (15
), such combination therapy did not produce sufficient suppression of HBV in the index patient (Fig. ). The amino acid substitution at position 181 has to be further analyzed with regard to resistance to anti-HBV drugs.
The rtA181T mutation detection system using RFLP PCR developed in this study is a useful tool, as we were able to distinguish the wild type from all mutants with nucleotide substitutions in a given region. The system also enabled us to monitor the fluctuation of the wild-type/mutant ratio during therapy against HBV (Fig. and ). The incidence of rtA181T mutants with an intact YMDD motif is rare in Japanese patients with chronic HBV infection treated with lamivudine. Interestingly, 4 of the 39 (10%) patients who developed lamivudine breakthrough and were positive for YMDD mutants were found to have small amounts of rtA181T mutant strains. Different from the previous report (34
), the mutants did not take over another strain and were not preceded by exacerbation. We have to monitor these patients carefully for further population change of mutants and for exacerbation of hepatitis.
A recent study reported that the prevalence of genotype A HBV infection is increasing in Japan and that the incidence of disease chronicity is higher than for other genotypes (26
). It is thus expected that an increasing number of the sexually active population will receive nucleoside analogue therapy against HBV and multiple mutant strains can potentially emerge and spread along with long-term treatment. There is an increasing possibility of emergence of novel mutants resistant to multiple anti-HBV drugs. The importance and significance of the rtA181 mutations, including the novel mutant strain identified in this study, should be investigated further to develop more useful treatment strategies.