| The human hepatitis B virus (HBV) and Duck Hepatitis B Virus (DHBV) belong to the same virus family with similar structural characteristics and replication processes. Chronic DHBV infection can be achieved by inoculating one-day-old ducklings with DHBV. The ducklings then develop a persistent viremia, which is accompanied by liver histological changes. The main purpose of the experiments presented in this thesis was to design, synthesize and test the inhibitory effect of an antisense poly-2'-O-(2,4-dinitrophenyl)-oligoribonucleotide, complementary to direct repeat sequence of DHBV DNA polymerase mRNA (2468--2487), on DHBV infection.; In this work, a procedure was developed to synthesize antisense oligoRNA by using synthetic DNA templates. Then, the purified oligoRNA was derivatized with 2,4-dinitrofluorobenzene (FDNB) to make poly-DNP-RNA. The antisense poly-DNP-RNA's resistance to RNase, its hybridization, and its sequence-independent inhibition of the reverse transcriptase activity of DHBV DNA-polymerase were demonstrated. The optimal DNP/base ratio for use in the antisense strategy was also determined.; In the subsequent in vivo studies, DHBV-infected ducks were randomly assigned into three groups, nine ducks were treated with antisense, four ducks with sense and two ducks with random sequence of poly-DNP-RNA. The data showed that the antisense poly-DNP-RNA completely inhibited duck viremia in all treated ducks. The viremia did not come back in these ducks after 8 months. Only one out of four ducks in the sense group showed the inhibition, and both ducks in the random group maintained their viremia. Virus concentrations in the infected ducks were determined by quantitative-competitive PCR. In a repeat of these experiments, eight infected ducks were subjected to the same treatment. After 45 days of treatment with the antisense poly-DNP-RNA, followed by 2 weeks of recession, PCR and microscopic examination showed that viral DNA had disappeared from the livers of infected ducks, and that the histology of the damaged liver (filled with fat granules) had returned to normal.; These results show that the antisense inhibitor used in these studies is a very effective therapeutic agent for treating DHBV infection. Presumably, similar poly-DNP-RNAs can be designed and synthesized for the treatment on HBV. |
