| Background:Hepatitis B virus (hepatitis B virus, HBV) infection was distributed worldwide. Despite the hepatitis B vaccine has been populated nearly20years, there are still2billion people infected with HBV and240million people with chronic infection. Now China still has93million people chronically infected, of them about20million are chronic hepatitis B patients.The most important means to treat chronic hepatitis B is suppression HBV replication. There are mainly two kinds of these drugs:interferon and nucleos(t)ide analogues. As nucleos(t)ide analogues is easy to take, effective, and fewer adverse reactions, it has been accepted by more and more CHB patients. But long-term antiviral therapy could lead the emergence of drug-resistant virus strains. Nucleos(t)ide analogues resistant could cause serious consequences, such as increasing the progress of the patient’s condition and liver failure. Therefore, scholars have paid more attention to resistance-related researches.Resistant sites of nucleos(t)ide analogues are mostly locus in the reverse transcriptase (RT) A to D structure domain. Existing researches have showed that there were five pathways of viral resistance, of which there were two pathways related to the B domain rtA181T/V mutation. This mutation is a cross resistance site of nucleoside and nucleotide. Almost all nucleos(t)ide analogues have showed resistance to rtA181T/V mutation. Research on rtA181T/V variation has a profound meaning. HBV genome is short but efficient,67%of the genome is overlapping coding, and its sequence can be repeated coding1.5times. Mutations in the reverse transcriptase region can also lead to changes of surface antigen region. rtA181T variation can truncate the surface antigen and the rtA181V mutation can replace the amino acids of surface antigen. Whether there is difference between clinical and laboratory about this change of surface antigen?HBV reproduction and dissemination is effected by the host immune and drug pressure. Nucleos(t)ide analogues resistance mutation often occurred in the RT region. How about the corresponding changes in C domain?With the species of clinical nucleos(t)ide increasing, the resistance mutations also diversified and complicated. Combination resistance is a new difficulty in clinical. There are a lot of researches in this area, but most of these were done by direct sequencing and expansion. Thus we can’t distinguish the difference mutation sites with a virus or between different strains of the virus. So it is necessary to carry out a detailed analysis of the viral quasispecies by sequencing after cloning viral.Genotype may affect the condition and prognosis of disease. Genotype B and C are mainly genotypes in China. Whether there is a selection of rtA181T/V mutation for the two different genotypes. Whether there is a difference performance of rtA181T/V mutation between these two genotypes? Many questions should be carried out to provide a theoretical basis for clinical antiviral therapy.Chapter One:Analysis of the clinical features of hepatitis B virus reverse transcriptase A181mutationObjective:To explore the relationship of rtA181mutation and clinical antiviral medication, the clinical features of the rtA181mutation, the difference characteristic between the mutation patterns, the relationship between genotypes and rtA181mutation.Method:Select HBV rtA181mutation patients who accepted nucleos(t)ide analogues to antiviral therapy. Total85patients diagnosed to comply with the prevention and treatment of chronic hepatitis B Guide (2010Edition) were enrolled in the study. All clinical relevant data of patients during antiviral therapy were reviewed, and the liver function, surface antigen, e antigen, HBV DNA, genotypes of these patients were detected when resistant occurrence. Resistance sites were determined by sequencing of RT region after PCR.All data were analyzed by using statistical software SPSS13.0. Count data constitute were used R×C table test, the quantitative data were analyzed by using independent samples t test. Difference of P<0.05was considered statistically significant.Results:1. The mutation patterns of RT A181site were mainly eight kinds:A181T, A181V, A181T+N236T, A181V+N236T, A181T+A181V, A181T+M204I/V+L180M±V173L, A181V+M204I/V±L180M±V173L, A181T+N236T+M204I. Of them,84(98.82%) patients were used to receive adefovir therapy. A181T/V mutation in the adefovir treatment group was accounted for9, and in the lamivudine change adefovir treatment group was accounted for33, there was significant statistically difference (P=0.046); A181T/V+N236T mutation patterns in the two groups were16and11; there was a statistically significant difference (P<0.001). There was also significant difference in A181T/V+M204I/V mutation between two different treatment groups (P=0.016).2. Even rtA181mutation appearance, clinical indicators were still improved significantly. Liver function, ALT dropped from175±130U/L to8.2±49U/L (P <0.001); Viral replication status, HBV DNA decreased from5.97+1.26log10copies/ml to4.10±1.18log10copies/ml (P<0.001):virus surface protein, HBsAg decreased from3.66±0.36log10COI to3.50±0.45logio COI (P<0.001). But the e antigen status did not change when mutation appearance (P=0.973).3. Of85patients, there were13patients with genotype B and72patients with genotype C, no other genotypes were detected. In genotype B,4cases were rtA181V mutation and9were rtA181T mutation; in genotype C,40cases were rtA181T mutation,30cases were rtA181V mutation and2cases were rtA181T+rtA181V mutations. Statistics of the three groups was no difference (P=0.175).4. Grouping patients according to HBV genotype B and C, clinical features (ALT, HBsAg, HBV DNA, HBeAg, etc.) were no statistically significant difference not only before the antivirus but also at the emergence of drug mutation (P>0.05).5. Grouping patients according to different mutations, clinical features of the patients before antiviral treatment were no difference (P>0.05). At the emergence of drug mutation, rtA181T mutation group has less HBsAg representing than that at the rtA181V group (3.40±0.351gCOI vs.3.62±0.541gCOI,P=0.030).Conclusion:1. The rtA181mutation is mainly associated with adefovir medication. rtA181T/V mutation were more common in adefovir after lamivudine therapy patients, while A181T/V+N236T mutation were more common in adefovir treatment patients.2. The rtA181mutations were generally not leading to viral load rebound, and some patients could still get the treatment response.3. HBV genotype B and C may not affect rtA181mutation, and rt181T/V mutation was independent in HBV genotype distribution.4. The rtA181T and rtA181V mutation were similar in the clinical data before antiviral therapy, but HBsAg levels of rtA181T group were lower than that of rtA181V group.Chapter two:Virology characteristics of hepatitis B virus rtA181mutationObjective:To explore the quasispecies characteristics of HBV rtA181mutation virus, and investigate the pre-C gene mutation (G1896A) and basic C promoter mutation (A1762T/G1764A) in rtA181mutations.Method: 1. C gene was amplified in all specimens from nt1623to nt2076, analysis the BCP and pC mutation in rtA181mutation. Reverse transcriptase region of some specimens were amplificated, purified and inserted into pMD18T vector, and transformed into DH5a bacteria. For each sample, we selected more than18clones to sequence. After these, we assembled and splice sequence by the Vector NTI software. Chromatogram software, Bioedit software and MEGA4software were used to analyse sequence mutations.2. Count Data test was used to analyse pre-C and basic core promoter mutations. P values less than0.05is significant difference in all statistical. SPSS13.0statistical software was used for analysis.Results:1. We found such combinations in one strain of the virus:A181T+M204V、 A181T+N236T、A181V+N236T, A181T+V173L、L180M+181V and A181T+N236T+M250L. L180M+181T combination mutation was not found in these clones.2. The rtA181T mutant virus was account for14.3%to95.2%in all quasispecies. The proportion of rtA181V mutants in all viral quasispecie was60.0%to100.0%. The genome group distance and entropy were lower in rtA181V mutants. We also found some mutations such as rtA181D and rtA181L, which can not be detected in direct sequencing.3. The mutation of C gene core promoter A1762T and G1764A double mutation in rtA181T mutant group was significantly higher than that in rtA181V mutation group and CHB patients (P=0.009). While the pre-C G1896A point mutation did not show difference between the three groups (P=0.144).Conclusion:1. We identified some multi-drug resistant forms associated with rtA181T/V in one strain of the virus.2. The quasispecies of rtA181T mutant were often mixed with wild-type virus. Except rtA181T and rtA181V mutation, we also identified rtA181D and rtA181L mutation in all clones.3. BCP mutations appear higher probability in rtA181T mutation than rtA181V mutation group and CHB group.Chapter three:Construct different genotypes of1.24copies rtA181T/V mutation hepatitis B Virus PlasmidObjective:To further compare biological characteristics of rtA181mutation between the different genotypes, mutant plasmids of rtA181Tand rtA181V were constructed. And these mutation plasmids provide a necessary prerequisite for the next step.Method:1. The Ae-JPN, Ba-JPN58, C-JPNAT, D-US68kindly given by professor Mizokami are different genotypes of1.24copies HBV recombinant plasmids. We used these plasmids as templates, designed mismatch base primer by overlap extension PCR to achieve fragment of artificial directed mutagenesis RT region. After degest the wild type virus RT domain with restriction enzyme (Xbal I and Sph I), we recombined HBV with mutagenesis RT region. By this way, we obtained the different genotypes of rtA181T mutant and rtA181V mutant virus plasmids.2. After construct mutant plasmids, we used PCR primers M13F and M13R to amplified the sequence, used restriction enzyme EcoR I and HindⅢ to digested recombinant plasmid. After preliminary identification of the recombinant mutant plasmids,3clone of different genotypes rt181T/V mutant plasmid were sequenced to confirm mutant plasmids constructed correctly.Results:We built the different genotypes pUC-HBV1.24plasmid of rtA181T and rtA181V mutation. These mutation plasmids were identified by PCR, restriction enzyme digestion and nucleotide sequencing. pUC-HBV1.24-181T plasmid has mutation from FLLA motif to FLLT, while the pUC-HBV1.24-181V is mutated to FLLV. Compared with the wild-type plasmids, there was no other mutation point introduced in these plasmids.Conclusion: We used artificial mutagenesis and recombinant technology to successly built1.24-fold HBV plasmids that included rtA181T/V mutant of different genotypes.Chapter Four:In vitro characteristics of hepatitis B virus rtA181mutants between different genotypesObjective:To explore the biological characteristics of the rtA181T or rtA181V resistant mutant in A, B, C, D genotypes in vitro, and provide a theoretical basis for clinical antiviral therapy.Method:1. Endo-Free Plasmid extraction kit (OMEGA Company) were used to extracted Ae-Jpn, Ba-JPN58, C-JPNAT, US68four wild-type plasmid and its rtA181T/V mutant virus plasmids, pSEAP plasmid was extracted at the same time.2. HepG2human hepatoma cells were cultured in Dulbecco’s modified Eagle medium (DMEM). After24h of culture, cells were transiently transfected with5μg HBV DNA and0.1μg pSEAP by X-tremeGENE HP Transfection (Roche, Switzerland). Idling dye controls were set at the same time. The cell supernatants were harvested after48to72hours; HBsAg and HBV DNA were detected use commercial assay kits.3. To simulate the status of virus in host, we mixed the rtA181T mutation plasmids and wild-type plasmid, respectively0:1,1:0,1:1,1:9four different proportions, and transfected into HepG2cell. Cell supernatant HBsAg and HBV DNA levels were measured48to72hours after transfection.4. In order to balance transfection efficiency, SEAP kit was used to detect the expression of the secreted alkaline phosphatase (SEAP). Translation results were control by the wild-type plasmid of each genotype.5. Independent samples t test were used in the same genotype statistics. If heterogeneity of variance, Satterthwaite approximate t test were used. One-Way ANOVA Statistics were used between the different genotypes. HBV DNA was logarithmic transformation before performing statistical.Results:1. The rtA181T mutations affect the secretion of HBsAg significantly, HBsAg from cell culture supernatant were hardly detected, and they were only0.33%to2.22%of the wild-type. The rtA181V mutation showed moderate impact to HBsAg levels, and its HBsAg can be maintained80%compare to the wild-type. HBsAg were significant differences between rtA181T mutation and rtA181V mutation (P<0.001).2. The rtA181T mutations also affect the synthesis of the HBV particles, HBV particles in cell supernatant were not detected in most of the samples, it was at102copies/mL level even if it be detected. There were significant differences of the viral particles between rtA181T mutations and rtA181V mutations in each genotype.3. HBsAg of rtA181T mutations in the different genotypes have no statistically significant difference (F=1.563, P=0.272); while HBsAg level of rtA181V mutation between different genotypes have significant differences (F=6.261, P=0.017). HBsAg level in genotype B was lower than genotype A and D.4. HBV virus particle of rtA181V mutant between different genotypes showed differences (F=25.630, P<0.001), DNA level in genotype B and C were lower than genotype A and D.5. When the rtA181T mutant plasmid was mix transfected with the wild-type plasmid, the HBsAg level can be corrected. When the wild-type plasmid is10%, a small amount of HBsAg can be detected in the supernatant. When the wild-type plasmid is increased to50%, HBsAg levels can recover up to50%. Compare with different genotypes, HBsAg levels showed differences (F=4.799, P=0.034) at the condition of50%wild-type plasmid. Genotype A activity was relatively highest, while genotype D activity was relatively lowest. In the case of wild plasmid10%and0, HBsAg levels between the different genotypes have no significant difference (P>0.05).6. The expression of HBV DNA in the cell supernatant was approximation to HBsAg. With the increase of mix transfected wild-type plasmid, virus expression was gradually increased. When wild-type plasmid was50%, HBV DNA levels have reached40%or more. Mix transfections were not statistically different between different genotypes in different proportions (P>0.05).Conclusion:1. The rtA181T mutations have greater impact on virus surface antigen releasing and the synthesis of viral particles, but there was no significant difference between genotypes. The rtA181V mutant have moderate affected to HBV, while we can see differences between genotypes.2. Wild type plasmids can rescue rtA181T mutant biological defects; there was a slightly different in genotypes. |
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