More than 350 million people worldwide are chronically infected with hepatitis B virus (HBV), and those are at high risk to ultimately develop severe liver disease, including cirrhosis, hepatocellular carcinoma, and liver failure. Several studies revealed significant correlations between viral quasispecies evolution and antiviral efficacy. However, previous studies have focused mainly on the evolution of the reverse transcriptase region with known mutation sites but not other regions which might have potential co-effects on the viral quasispecies evolution. At present, the dynamic changes of HBV quasispecies heterogeneity within the context of nucleoside-analogue treatment particularly in the precore/core region have not been well defined. This study was conducted to clarify the long-term viral evolutionary behavior in patients receiving sequential nucleoside-analogue therapy. Thirteen patients as the study group had dual resistances after lamivudine (LAM) and entecavir (ETV) treatment. The control group included 14 patients who had LAM resistance, but responded to ETV. Serum samples were collected at six time points, namely (I) before LAM treatment, (II) six months after LAM treatment, (III) virologic breakthrough after LAM treatment, (IV) before ETV treatment, (V) six months after ETV treatment, (VI) virologic breakthrough after ETV treatment. The controls were matched to the study cases on the characteristics of age, sex, and HBV genotype. HBV precore/core region was amplified from extracted serum by the first- and second-round PCR primers. Amplicon of HBV gene was then cloned into pGEM T Easy Vector and transinfected into the E. coli. At least 20 colonies with HBV insert were selected and sequenced directly for each sample. All the sequences were used for analysis regarding viral diversity, evolutionary rate, positive and negative selection and phylogenetic analysis. The results showed that, after occurrence of LAM resistance (LAMr), patients progressing ETV resistance (ETVr) showed increasing viral diversity and non-synonymous (dN) substitution. At the time point V, patients progressing ETVr had higher number dN substitution of core protein than patients responding to ETV treatment (P<0.015). Furthermore, the evolutionary rates of patients progressing ETVr were significantly low than those of patients responding to ETV treatment. Increased dN values and lower evolutionary rate may be associated with better fitness under antiviral pressure.
In conclusion, viral quasispecies of increased diversity and dN substitution, and lower evolutionary rate might be used to predict the occurrence of ETVr.

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