Duck hepatitis B virus research
C.J. Burrell, A.R. Jilbert, M. Qiao, I. Kotlarski, E. Bertram, M. Triyatni, B. Lee, M. Arens, D. Miller, C. Scougall, K. Davies, J. Botten
The laboratory is funded by the NH&MRC to study duck hepatitis B virus (DHBV) infection of Pekin ducks as a model for hepatitis B virus (HBV) infection of humans. Several projects are proceeding independently but with extensive collaboration.
The first project involves use of the DHBV infection model to study vaccination strategies using yeast-derived, DNA-based and recombinant antigen vaccines. The project aims to examine how ducks which have been previously infected with DHBV or vaccinated with virus surface and core protein vaccines are protected against subsequent infection. Significant progress includes (i) detailed characterisation of virus stocks; (ii) development of serological assays to detect DHBV surface antigen and antibodies to DHBV surface and core antigens; (iii) characterisation of the removal rate and the site of uptake of DHBV inoculated into control and immune ducks; (iv) studies of the time course of DHBV infection in neonatal and adult ducks after inoculation of different virus doses; (iv) cloning of the Australian strain of DHBV (AusDHBV) and expression of DHBV surface proteins in yeast for use as vaccines; (v) use of DNA based vaccination of DHBV PreS/S, S and core proteins as an alternative approach to recombinant vaccines.
The second project involves collaborative studies with Professor I. Kotlarski and Dr E. Bertram to establish in-vitro assays to detect cell mediated immune responses (CMI) in ducks. As a first step reagents and methods were developed to identify and purify T and B lymphocytes, thrombocytes and duck antigen presenting cells (APC) from duck peripheral blood mononuclear cells (PBMC) and lymphatic tissues. Lymphocyte stimulation and lymphokine assays were then developed and optimal in-vitro proliferative responses of duck nylon wool purified-PBMC to lectins PHA and ConA were obtained by co-culturing with homologous red blood cells and APC. Supernatants from PHA-stimulated duck PBMC and spleen cells were shown to contain lymphokines with an IL-2 like activity capable of maintaining proliferation of duck and chicken, but not mouse lymphoblasts.
These assays are being applied to detect virus antigen-specific lymphocyte responses to DHBV infection and to understand: (i) the factors involved in the resolution of acute transient DHBV infections (ii) the age and dose-related effects on the outcome of DHBV infection. Recent studies from the laboratory have shown that one and seven-day-old ducks inoculated with a constant low dose of DHBV develop persistent infection whilst older 14-42-day-old ducks develop transient infection. The effect of virus load on the outcome of infection was demonstrated as increasing the dose of inoculated virus 25-fold resulted in persistent infection in one, seven and 14-day-old ducks. Understanding the mechanism(s) that result in resolution of DHBV infection and for the age-related effect on outcome are important for designing and testing approaches to help resolve infection in persistently HBV-infected patients.
The third project aims to study the regulation of plasma virus levels in chronic DHBV infection by determining the rate of turnover, and the sites and mechanisms of removal of virus from the bloodstream. These same principles of virus turnover apply to a number of persistent virus infections, for example, HIV and HCV. The project also seeks evidence for super-infection of chronically infected ducks with different virus strains. Co- or superinfection of susceptible cells would allow study of a number of classical outcomes of dual virus infection in vitro and in vivo, including recombination, interference, pseudotyping and immune selection. We hope ultimately to use this information to design and evaluate possible ways to eradicate chronic infection by use of antisense or other vector mediated forms of therapy. Current work includes: Cloning of the genome of the Australian strain of DHBV (AusDHBV); creation of a pool of the USA strain of DHBV by intrahepatic inoculation of plasmid DNA; establishment of detection systems for USA DHBV in presence of AusDHBV; Purification and iodination of AusDHBV for studies of virus removal.
Ongoing collaborative studies with Dr W Mason, Fox Chase Cancer Center, Philadelphia, USA have included studies of the antiviral effects of Lamivudine in chronic woodchuck hepatitis virus infection and studies of the antiviral effects of 2’-CDG and 524W in vivo in congenitally DHBV-infected ducks.
A fourth project aimed to establish and validate one-step growth conditions of DHBV infection of primary duck hepatocytes (PDH) and to use this in-vitro model to define and characterise the early molecular events in DHBV replication. Establishment of one-step growth conditions for DHBV in PDH included characterisation of a purified virus inoculum and optimisation of high multiplicity infection. Synchronised infection was achieved using a multiplicity of infection of 640 DHBV DNA-containing virions/cell that resulted in more than 90 per cent of PDH expressing core antigen by day 5 p.i. Studies of early events in DHBV replication in this in vitro model, indicated that internalisation of the input viral DNA and its localisation to the nucleus occurred within 4 hr. However, both covalently closed circular (ccc) DNA and single-stranded (ss) DNA were not detected until 48 hr suggesting that there was a delay between DHBV DNA localisation to the nucleus and formation of cccDNA. Translocation of nucleocapsids from the particulate to soluble nuclear fractions was found to be relatively slow being only 50 per cent complete by 48 hr p.i. This project is the first study to formally demonstrate one-step growth conditions in hepadnavirus replication, and thus allows direct study of the early molecular events in hepadnavirus replication in individual infected cells.