The Study On Quasispecies And Mutation Of HBV Precore In Liver Tissue And Serum With Fulminant Hepatitis B Patients

Hepatitis B is a serious disease that is endemic in many parts of the world. Approximately 350 million individuals world wide are carriers of hepatitis B virus (HBV). Fulminant hepatitis B is uncommon, occurring in about 1% of patients with asymptomatic carrier. The pathogenesis of fulminant hepatitis B is not well understood so far, virus factor and host factor may also plays an important role. Many cases focus on the relationship between precore mutant and fulminant hepatitis B. Hepatitis B virus quasispecies have been detected in patients with chronic hepatitis B. In order to elucidate the relationship between HBV precore mutation and fulminant hepatitis B, this study compared quasispecies of HBV precore and nucleotide mutation in liver tissue and serum .Method: (1) Extraction of HBV DNA from liver tissue and serum. (2) Amplification of HBV precore by the nest polymerase chain reaction (nest PCR). (3) The PCR products were cloned using a T-A cloing kit. (4) Amplification of the objective fragments using inner primers. (5) Using single-strand conformational polymorphism/ heteroduplex analysis (SSCP/HDA) electrophoretic detect different clonetypes. (6) Different clonetypes were sequenced. (7) Analysis the results by DNA star.Results: (1) The first patient, there were no distinct diffence on quasispecies in liver tissue and serum, quasispecies were 15 in liver tissue, 17 in serum. The second patient have 17 clonetypes in liver tissue and in serum respectivly. The third patient have 14 and 8 clonetypes respectivly, in liver tissue was more the quasispecies than in serum. (2) There were 42 points mutation of HBV precore in 72 sequenced clones . This study find 5 hypervariable point: 1896G→A(41/72), 1846A→T(18/72), 1856C→T, 1858T→C( 10/72), 1898G→A(6/72). Mutations at nucleotide 1898, double mutation at nucleotide 1856 and 1858 coexist in liver tissue of the third patient, 1896 stop codon mutant was not found. Deletion 1825T, mutation 1828C→T,1829 C→T.1831C→A were observed in DR1 region .Conclusion: (1) There were high prevalence of 1896 mutantation in HBeAg-negative fulminant hepatitis B patients. Fulmiannt hepatitis B correlated little with at position 1896 mutant. (2) HBV precore region extracted from fulminant hepatitis B patients have many points mutation which have 5 hypervariable point, the other points mutation have individually feature. (3) The composition of quasispecies were different in liver tissue and serum. This study revealed that the poorer prognosis, the higher nucleotide divergence rate in liver tissue and serum.