Abstract
The determinants of the maintenance of chronic hepadnaviral infection are yet to be fully understood. A long-standing unresolved argument in the hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral infection, cell-to-cell spread of hepadnavirus is at least very inefficient (if it occurs at all), virus superinfection is an unlikely event, and chronic hepadnavirus infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. Superinfection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced hepatocellular carcinomas (HCCs), were superinfected with another WHV strain, WHVNY. Six weeks after the superinfection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY superinfection was demonstrated by using WHV strain-specific PCR assays and (i) finding WHVNY relaxed circular DNA in the serum samples collected from all superinfected animals during weeks one through six after the superinfection, (ii) detecting replication-derived WHVNY RNA in the tissue samples of the livers and HCCs collected from three superinfected woodchucks, and (iii) finding WHVNY DNA replication intermediates in tissues harvested after the superinfection. The results are consistent with the occurrence of continuous but inefficient hepadnavirus cell-to-cell spread and superinfection during chronic infection and suggest that the replication space occupied by the superinfecting hepadnavirus in chronically infected livers is limited. The findings are discussed in the context of the mechanism of chronic hepadnavirus infection. IMPORTANCE: This study aimed to better understand the determinants of the maintenance of chronic hepadnavirus infection. The generated data suggest that in the livers chronically infected with woodchuck hepatitis virus, (i) hepadnavirus superinfection and cell-to-cell spread likely continue to occur and (ii) the virus spread is apparently inefficient, which is consistent with the interpretation that a limited number of cells in the livers facilitates the spread of hepadnavirus. The limitations of the cell-to-cell virus spread most likely are mediated at the level of the cells and do not reflect the properties of the virus. Our results further advance the understanding of the mechanism of chronic hepadnavirus infection. The significance of the continuous but limited hepadnavirus spread and superinfection for the maintenance of the chronic state of infection should be further evaluated in follow-up studies in order to determine whether blocking the virus spread would facilitate the suppression of chronic hepadnavirus infection.