Abstract
BackgroundHepatocytes infected by hepatitis B virus (HBV) produce different HBV RNA species, including pregenomic RNA (pgRNA), which is reverse transcribed during replication. Particles containing HBV RNA are present in serum of infected individuals, and quantification of this HBV RNA could be clinically useful.MethodsIn a retrospective study of 95 patients with chronic HBV infection, we characterised HBV RNA in serum in terms of concentration, particle association and sequence. HBV RNA was detected by real-time PCR at levels almost as high as HBV DNA.ResultsThe HBV RNA was protected from RNase and it was found in particles of similar density as particles containing HBV DNA after fractionation on a Nycodenz gradient. Sequencing the epsilon region of the RNA did not reveal mutations that would preclude its binding to the viral polymerase before encapsidation. Specific quantification of precore RNA and pgRNA by digital PCR showed almost seven times lower ratio of precore RNA/pgRNA in serum than in liver tissue, which corresponds to poorer encapsidation of this RNA as compared with pgRNA. The serum ratio between HBV DNA and HBV RNA was higher in genotype D as compared with other genotypes.ConclusionsThe results suggest that HBV RNA in serum is present in viral particles with failing reverse transcription activity, which are produced at almost as high rates as viral particles containing DNA. The results encourage further studies of the mechanisms by which these particles are produced, the impact of genotype, and the potential clinical utility of quantifying HBV RNA in serum.
Highlights
Hepatocytes infected by hepatitis B virus (HBV) produce different HBV RNA species, including pregenomic RNA, which is reverse transcribed during replication
HBV RNA in serum correlated with HBV DNA in serum and pregenomic RNA (pgRNA) in the liver As shown in Fig. 1a, serum levels of HBV DNA and HBV RNA correlated with a Spearman’s rho of 0.93 and an overall R2 of 0.83, and with a similar slope for HBeAg positive (0.77) and negative (0.81) samples
The HBV RNA levels correlated with pgRNA in liver tissue, but with weaker correlations for HBeAg negative patients (Fig. 1b)
Summary
Hepatocytes infected by hepatitis B virus (HBV) produce different HBV RNA species, including pregenomic RNA (pgRNA), which is reverse transcribed during replication. Quantification of HBV DNA in serum is the main marker for predicting the risk of cirrhosis and HCC [2], and HBV DNA levels are measured to monitor the response to antiviral treatment [1]. The cccDNA episome of HBV in the nucleus of infected hepatocytes is transcribed to several mRNAs during the viral replication One of these transcripts, the pregenomic RNA (pgRNA), is reverse transcribed to HBV DNA by the viral polymerase during the formation of new viral capsids in the cytoplasm of the hepatocytes before release of the enveloped virions, but HBV RNA containing particles can be detected in serum [8].
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