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The Detection and Analysis of Natural Mutations Associated with Drug Resistance of HBV Covalently Closed Circular DNA

Author: WangZuo
Tutor: ZuoXiaoHui
School: Second Military Medical University
Course: Internal Medicine
Keywords: Hepatitis virus Beta ccc DNA Polymerase chain reaction ( PCR ) Natural variation cccDNA Clone Quasispecies
CLC: R512.62
Type: Master's thesis
Year: 2011
Downloads: 53
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Abstract


Not antiviral therapy of chronic HBV infection, intrahepatic HBV ccc DNA resistant sites mutation detection confirmed antiviral therapy of chronic HBV infection, intrahepatic HBV ccc DNA variation of the resistance-associated sites understand The intrahepatic HBV mutant virus strains and the similarities and differences in the blood. Methods ① selected 85 cases without antiviral treatment of chronic HBV infection, specimens from liver tissue and serum. (2) using the QIAamp? DNA Mini Kit kit and directly boiling method of extraction of the liver tissue, the serum of HBV-related gene. ③ to ATP degradation plasmid DNA-dependent enzyme (PSAD) the digested purified liver biopsy in HBV DNA. Quantitative digestion intrahepatic HBV DNA load before and after as well as serum HBV DNA level. (4) detection of serum hepatitis B virus serum immune markers and serum liver function biochemical parameters (ALT), to analyze the relationship between its content with HBV. ⑤ application of the laboratory design cross double notch primer line nested polymerase chain reaction (PCR) amplification of HBV ccc DNA, two pairs of non-cross-notch primer line nested PCR amplification relaxation ring DNA (rc DNA) . All primers amplified fragment covers lamivudine, adefovir dipivoxil, entecavir common known drug sites. The the ⑥ direct sequencing of nested PCR DNA product the sequencing detection, observed variations in resistance-associated mutation sites. The ⑦ comparative analysis of intrahepatic HBV ccc DNA gene sequence of HBV rc DNA and serum HBV DNA. Results of intrahepatic HBV ccc DNA level, intrahepatic HBV DNA levels, and serum HBV DNA between the three was a positive correlation (P lt; 0.05); liver function indicators ALT levels and HBV DNA load between no correlation (P gt ; 0.05); the of HBV ccc of DNA resistant Loci positive in two cases (2.4%): 204 point mutation the one case rtM204I (45), in 85 patients, 214 point mutation in one case (49). In another instance sequencing profiles showed mixed peaks (85). And also found the same mutation in the serum and liver tissue HBV DNA. Conclusions without antiviral treatment of chronic HBV infection, intrahepatic HBV ccc DNA found resistance-associated locus variation, confirmed the presence of drug-resistant HBV ccc DNA sites of natural variation. HBV ccc of DNA resistant sites clonal analysis of the natural variation of the virus strains of the target gene fragment the purpose of understanding the proportion of mutant and wild-type strain of HBV ccc DNA resistance-associated sites of natural variation in vivo in patients with HBV. Methods ① variation on the first part of the test specimen (serum before and after digestion intrahepatic HBV-related gene) cloned template. Nested PCR method with the first part. (2) in accordance with the specific steps of cloning kit (purified, connection, conversion, etc.) to complete the operation. Observation cloning results, each specimen were randomly picked 50 monoclonal colonies, and direct sequencing. ③ sequencing results to compare and analyze, to understand the proportion of mutant and wild-type strains of each specimen. Results ① 204 points clone analysis: the intrahepatic HBV ccc DNA ATG / the ATT / ATC = 10/38/2 of intrahepatic HBV rc DNA ATG / the ATT / ATC = 3/45/2, serum HBV DNA ATG / ATT / the ATC = 0/50/0; ② 214-bit point cloning analysis: the intrahepatic HBV ccc DNA GTA / GCT / GTT = 20/29/1 of intrahepatic HBV rc DNA GTA / GCT = 5/45, the serum of HBV DNA GTA / GCT = 10 / 40; ③ The 85 mixed peaks specimens clone analysis results show that the majority of drug-related Loci nonsense mutations found only in 50 cases of intrahepatic HBV ccc DNA target gene fragment cloned one cases of 181 points GCT become ACT (rtA181T), was found in 50 cases of clones of the target gene fragments in serum HBV DNA and one case of 250 points ATG becomes the GTG (rtM250V). Conclusion intrahepatic ccc DNA resistance-related sites to natural variation occurs in patients with strain of the virus in the body for mixed mutants and the wild-type strain, the proportion of mutant and wild-type strains of intrahepatic HBV ccc DNA rc DNA and serum HBV DNA inconsistent. 85 specimens clone analysis prompted this HBV strains exist more nonsense mutation, but does not affect HBV gene expression.

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CLC: > Medicine, health > Internal Medicine > Infectious disease > Viral infections > Viral Hepatitis > Hepatitis B
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