Superinfection with Woodchuck Hepatitis Virus Strain WHVNY of Livers Chronically Infected with Strain WHV7

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 ( Worldwide, approximately four hundred million people are chronic carriers of human hepatitis B virus (HBV), which is a prototype hepadnavirus. Chronic HBV infection is a main risk factor of hepatocellular carcinoma (HCC), and it is associated with more than 50% of all known cases of HCC (1-14). During the early stage of hepadnaviral infection, virtually the entire liver becomes infected with a hepadnavirus within several weeks. However, during chronic infection, areas of apparently hepadnavirusfree hepatocytes were observed in HBV carrier humans and chimpanzees and also in woodchucks chronically infected with woodchuck hepatitis virus (WHV), which is closely related to HBV. The hepadnavirus-free hepatocytes tested negative for (i) the hepadnavirus core antigen (by immunostaining) and (ii) the viral DNA (by in situ hybridization) regardless of the ongoing viremia. In HBV carrier humans, up to 90% of all hepatocytes could appear to be HBV free. Some of the virus-free hepatocytes form foci of altered hepatocytes (FAH) with morphology similar to that of liver cancer cells and are considered premalignant. In addition, the cells of hepadnavirus-induced HCCs often were reported to be virus free as well (1,4,(15)(16)(17)(18)(19)(20)(21)(22)(23). In contrast, several previous studies reported the presence of hepadnaviral , making the issue still controversial. The observations of apparent hepadnavirus-free HCC cells and hepatocytes, along with the analysis of the kinetics of the emergence of drug-resistant HBV mutants, laid the foundation for a long-standing unresolved argument in the HBV field that during chronic infection, cell-to-cell spread of a hepadnavirus is at least very inefficient (if it occurs at all), and superinfection is an un-

Infection Patterns Induced in Naive Adult Woodchucks by Virions of Woodchuck Hepatitis Virus Collected during either the Acute or Chronic Phase of Infection

“…In a separate study, we compared strains WHV7 and WHVNY and concluded that the two strains were quite similar in terms of their replication parameters and the ability to induce productive acute infection in naive adult woodchucks (16). This result was consistent with the interpretation that the observed limited efficiency of WHVNY superinfection in vivo (15) was not mediated by the properties of WHVNY virions but rather reflected the properties of hepatocytes residing in livers chronically infected with WHV7. In another study, we compared the infectivity of different types of HDV virions that differed only by the nature of the envelope proteins which coated the virions.…”

“…After the resuspension, 40 l of each DNA sample was incubated at 37°C overnight in a 500-l reaction mixture, which contained 1ϫ NEB (New England BioLabs) buffer 4, 1 mM ATP, 40 g/ml of RNase A, and 50 U of plasmid-safe ATP-dependent DNase (Epicentre). Upon completion of the incubation, cccDNA was further isolated by extraction with phenol and a second extraction with a phenolchloroform mixture followed by ethanol precipitation (15). The measurements of cccDNA amounts were conducted using qPCR.…”

“…The 3= end of the probe contained Black Hole Quencher 1 (BHQ_1). The positions of the primers and probes throughout the paper are shown based on the sequence of strain WHV7 (15). qPCR was performed using the TaqMan gene expression mastermix on a 7500 Real-Time PCR system (Applied Biosystems) as previously described (19).…”

“…Liver tissue samples first were homogenized in glass grinders using 0.6 to 1.5 ml of TE buffer (10: 10) (10 mM Tris-HCl pH 7.6, 10 mM EDTA) (15). To isolate total DNA, 390 l of TE buffer (10:10) and 600 l of a buffer containing 10 mM Tris-HCl, pH 7.6, 10 mM EDTA, 0.2% SDS, and 4 mg/ml Pronase were added to 210 l of the resulting homogenate.…”

“…Recently, we showed that woodchuck livers chronically infected with strain WHV7 can be superinfected in vivo with a different WHV strain, WHVNY. Our results suggested (i) that hepadnavirus cell-to-cell spread and superinfection continue during chronic hepadnavirus infection and (ii) that spread and superinfection, while being limited, still may represent the determinants of maintenance of chronic infection (15). In a separate study, we compared strains WHV7 and WHVNY and concluded that the two strains were quite similar in terms of their replication parameters and the ability to induce productive acute infection in naive adult woodchucks (16).…”