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Serum hepatitis B virus (HBV) RNA is detected during treatment with nucleoside analogue as a consequence of interrupted reverse transcription (RT) and unaffected replicative intermediates. The presence of serum HBV RNA in chronic HBV patients is confirmed by using ribonuclease digestion. Serum HBV RNA is differentially inhibited by interferon, but not by nucleoside analogue. The inhibitory effect of interferon on HBV RNA replicative intermediates may potentiate the suppression of HBV replication. Clinical significance of serum HBV RNA includes: (I) reflect the antiviral potency of nucleoside analogue;(II) predictor of early emergence of viral mutation during lamivudine therapy; (III) independently predict initial virologic response or earlier HBV suppression during nucleoside analogue therapy; (IV) predict HBV reactivation after discontinuation of nucleoside analogue. Thus, serum HBV RNA might be useful to optimize treatment efficacy in patients with chronic HBV, including shifting of oral antivirals or conversion to immunomodulatory agent i.e., interferon. Furthermore, serum HBV RNA levels correlate better with serum quantitative HBsAg (qHBsAg) than with serum HBV DNA levels. The predictive role of serum HBV RNA in long-term treatment effects of nucleoside analogue needs further study.