Hepatitis B virus nucleic acids associated with human peripheral blood mononuclear cells do not originate from replicating virus

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Abstract
There have been numerous reports suggesting that human peripheral blood mononuclear cells (PBMCs) can be productively infected with human hepatitis B virus (HBV). We therefore examined whether the PBMCs can be used to establish an in vitro infection system for HBV. Freshly purified PBMCs were incubated with HBV with or without mitogen stimulation. Successful infection was tested using a newly developed PCR method that can differentiate between the relaxed circular (RC) DNA of the virus inoculum and the covalently closed circular (CCC) DNA which is formed only after successful virus entry. This method enables virus uptake to be proven even if the infection is abortive because there is no gene expression because of the lack of liver specific gene expression factors. All attempts to detect CCC DNA after incubation of PBMCs with HBV failed. On the contrary, CCC DNA could easily be detected in infected liver or after in vitro infection of primary human hepatocytes. Because this result appeared to be contradictory to the published data, we analyzed PMBCs isolated from infected patients. We could confirm that HBV DNA and RNA are associated with these cells. However, even after restimulation with mitogens, we could only detect RC DNA. Moreover, we could also demonstrate that viral RNA is present in free virus. Apparently, a certain amount of defective particles do not reverse transcribe the packaged pregenomic RNA. In summary we found no evidence that PMBCs can be infected with HBV and conclude that all previous observations can be explained by adsorbed virus.