Inhibition of duck hepatitis B virus replication by 9-(2-phosphonylmethoxyethyl)adenine, an acyclic phosphonate nucleoside analogue

Abstract

The use of regimens that use nucleoside analogues for the treatment of chronic hepatitis B virus infection is often limited because of their high relapse rates. This is thought to be due to the persistence of virus in nonhepatocyte reservoirs and/or the viral covalently closed circular (CCC) DNA species in the nucleus of infected hepatocytes. We have evaluated the novel nucleoside analogue 9-(2-phosphonylmethoxyethyl)adenine (PMEA) in the duck model of hepatitis B. Eight Pekin-Aylesbury ducks congenitally infected with the duck hepatitis B virus (DHBV) were treated with PMEA at a dosage of 15 mg/kg of body weight/day via the intraperitoneal route for 4 weeks. At the end of the treatment period, four animals were killed and the remainder were monitored for a further 4-week drug-free period before analysis. The results were compared with those for eight age-matched, untreated controls. The levels of viremia, the total intrahepatic DHBV load, and CCC DNA, viral RNA, and protein levels were measured by Southern hybridization, Northern hybridization, and immunoblotting of the appropriate specimen, respectively. Viral proteins and DNA were also measured by immunohistochemistry (IHC) and in situ hybridization (ISH) of sections of liver and pancreatic tissue. PMEA treatment reduced the viremia to undetectable levels, while the total viral DNA load in the liver was reduced by 95% compared to the control level. Viral RNA and protein levels decreased by approximately 30%. ISH and IHC confirmed the PMEA-related intrahepatic changes and established that the amount of virus in bile duct epithelial cells (BDEC) was reduced by 70% during therapy. During the follow-up period all parameters of active virological replication returned to those for the age-matched controls. PMEA had no significant effect upon the number of virus-infected islet or acinar cells in the pancreas. PMEA at a dosage of 15 mg/kg/day has potent activity against DHBV found within hepatocytes and BDEC and inhibits DHBV replication in BDEC.

FIG. 1

Diagrammatic representation of the experimental treatment strategy and number of animals in each treatment arm. LFT, liver function test.

FIG. 2

DHBV DNA dot blot hybridization results for serum samples from four ducks treated for 4 weeks with PMEA and then kept for a further 4-week follow-up period without treatment compared with those for serum samples from age-matched untreated controls. The viremia levels indicate the marked suppression of DHBV DNA during PMEA therapy and relapse on treatment cessation compared to the DHBV DNA levels for control animals. Pre, pretreatment; lanes 1 to 7, weeks from start of treatment.

FIG. 3

Summary of the results of PMEA therapy on intrahepatic expression of the major markers of active DHBV replication in vivo. Densitometry of autoradiographs was used to estimate the amounts of DHBV DNA, CCC DNA, RNA, and proteins. The mean value for the age-matched control was defined as 100%. vge, viral genome equivalents.

FIG. 4

Southern blot hybridization analysis of the total DHBV DNA from liver tissue (A) and DHBV DNA extracted from liver tissue by the CCC DNA enrichment procedure (B) from control, PMEA-treated, and followed-up animals. (A) The amount of total DHBV DNA within the liver was reduced during therapy in all treated animals and returned to pretreatment (pre-Rx) levels on the cessation of therapy. The bands corresponding to the migration of relaxed circular (RC), double-stranded linear (DSL), and single-stranded (SS) DNAs, as determined by comparison with the molecular mass markers (lane MW), are labelled. (B) A reduction of viral CCC DNA levels was seen during PMEA therapy, but relapse occurred in the follow-up period. Bands corresponding to the migration of the CCC, relaxed circular, and double-stranded linear DNAs are labelled. The Southern analysis with the same samples reextracted two more times was repeated, generating similar profiles.

FIG. 5

Immunohistochemical studies with untreated age-matched control and PMEA-treated duck livers. (A) Staining for DHBpreS1Ag with livers from untreated controls shows that all hepatocytes and the majority of BDEC (arrow) are positive. By the end of PMEA treatment (B), all of the hepatocytes and most of the BDEC are negative for DHBpreS1Ag, with relapses in both cell types during the follow-up period (C). Magnification, ×400.