The levels of circulating pgRNA strongly correlate with intrahepatic pgRNA and cccDNA amounts both in HBV mono-infected and in HBV/HDV coinfected humanized mice

In chronic hepatitis B infection, not only HBV DNA-containing particles but also HBV RNA is detected in serum of infected patients. We recently showed that pgRNA amounts detected in serum of HBV-infected human liver chimeric uPA/SCID/beige (USB) mice strongly correlated with intrahepatic pgRNA levels and with cccDNA amounts (Giersch, J Hepatol. 2016).

Aim:

To investigate if a hepatitis delta coinfection (HDV), which is frequently associated with the suppression of HBV viremia and lower average cccDNA contents in coinfected chimeric mice (Lütgehetmann, Hepatol. 2012), affects serum HBV RNA levels.

Methods:

Virological parameters in serum and liver were determined in HBV/HDV co-infected USB mice (n = 54) and HBV mono infected USB mice (n = 23) by qPCR, immunofluorescence and RNA in situ hybridization (RNA ISH). We quantified serum pgRNA levels by qPCR after DNase treatment of nucleic acids extracted from serum samples.

Results:

Levels of serum pgRNA strongly correlated with intrahepatic pgRNA loads not only in HBV mono-infected mice but also in HBV/HDV co-infected mice (Spearman r = 0.9; p < 0.0001 and r = 0.9, p < 0.0001, respectively), which harbored a wide range of intrahepatic HBV infection levels (0.02 – 721 cccDNA/ng liver DNA) and of HDV loads (1 – 7.6 × 10E4 HDV RNA/ng liver RNA). Of note, RNA ISH showed lower pgRNA amounts in human hepatocytes expressing high HDV RNA levels, suggesting that HDV may hinder HBV replication in a subset of coinfected cells. However, we found a good correlation between intrahepatic cccDNA contents and serum pgRNA levels both in HBV monoinfected mice and in HBV/HDV coinfected mice. In the HDV superinfection setting, when all hepatocytes are already HBV positive, we observed a median drop of HBV DNA (-0.9 log) and pgRNA (-0.4 log) viremia 6 weeks after HDV infection, at a time when HDV developed the highest titer. The lack of strong HDV-mediated suppression of HBV replication, may explain the maintenance of a good correlation between cccDNA and pgRNA in serum.

Conclusions:

The clear relationship determined between pgRNA in serum and intrahepatic pgRNA and cccDNA amounts in both HBV monoinfected and HBV/HDV coinfected mice indicates that the presence of both viruses within the liver of humanized immune deficient mice did not cause a substantial impairment of cccDNA activity. Thus, quantification of circulating pgRNA may serve as a useful serum maker to estimate the intrahepatic pgRNA amounts and hence activity of HBV also in the setting of HBV/HDV coinfection.