HEPATITIS-B VIRUS NUCLEIC-ACIDS ASSOCIATED WITH HUMAN PERIPHERAL-BLOOD MONONUCLEAR-CELLS DO NOT ORIGINATE FROM REPLICATING VIRUS

There have been numerous reports suggesting that human peripheral bloo d mononuclear cells (PBMCs) can be productively infected with human he patitis B virus (HEV), We therefore examined whether PBMCs can be used to establish an in vitro infection system for HBV, Freshly purified P BMCs were incubated with HBV with or without mitogen stimulation, Succ essful infection was tested using a newly developed PCR method that ca n differentiate between the relaxed circular (RC) DNA of the virus ino culum and the covalently closed circular (CCC) DNA which is formed onl y after successful virus entry,This method enables virus uptake to be proven even if the infection is abortive because there is no gene expr ession because of the lack of liver specific gene expression factors, All attempts to detect CCC DNA after incubation of PBMCs with HBV fail ed, On the contrary, CCC DNA could easily be detected in infected live r or after in vitro infection of primary human hepatocytes, Because th is result appeared to be contradictory to the published data, we analy zed PMBCs isolated from infected patients. We could confirm that HBV D NA and RNA are associated with these cells, However, even after restim ulation 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 PBMCs c an be infected with HBV and conclude that all previous observations ca n be explained by adsorbed virus.