| BackgroundHepatitis B virus (HBV) infection is a major public health problem worldwide. It was reported that there were2billion people infected, and approximately350million people were chronically infected. Currently in China, there are estimatedly93million people infectedwith HBV, and about20million of them are chronic hepatitis B patients.HBV infection is associated with a broad spectrum of liver injury, ranging from asymptomatic carrier state to acute or chronic hepatitis B (CHB), advanced liver disease, acute-on-chronic liver failure (ACLF) and hepatocellular carcinoma (HCC). ACLF was recently defined as an’acute hepatic insult manifesting as jaundice and coagulopathy, complicated within4weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease’. In China, ACLF is mostly caused by HBV infection. It represents a relatively small portion of CHB patients, but carries a high mortality of50-90%. Hepatitis B related ACLF (HB-ACLF) is always life-threathening; it needs high cost of treatment but with poor prognosis. To such a diease condition, till now the only clinically effective treatment is orthotopic liver transplantation, which is not often timely available due to its notorious lack of donor graft.It is speculated that both viral and host factors are involved in the pathogenesis of HBV related ACLF (HB-ACLF) although the exact pathways remain unclear. It has been reported that Bj (B1) subgenotype, basal core promoter (BCP), and pre-core (pre-C) mutations are associated with the development of fulminant hepatitis. In vitro studies showed that, these mutations could enhance HBV replication or reduce or abolish HBeAg expression, which plays an important role in the development of liver failure. However, other reports showed that there is no confirmative association between BCP/pre-C mutations and fulminant hepatitis. In China, ACLF casesare reported more frequently than fulminant hepatitis. The clinical manifestations of ACLF are different from those of fulminant hepatitis, which is normally derived ofacute HBV infection. Recently, several studies have investigated the relationship between HBV genotype and mutations and the development of ACLF, however tthe relevant results are not consistent among them. Ren et al., showed that genotype B, T1753V (C/A/G), A1762T, G1764A, G1896A, G1899A were more prevalent in patients with ACLF than the other CHB disease. Our previous study found that in patients with ACLF there was a significantly higher distribution ratio of genotype B to C, as compared with patients with the other CHB disease, and a significantly higher frequency of A1762T/G1764A, A1846T, and G1896A mutations, however only happened to patients with genotype B. Similar result that BCP/pre-C mutations, rather than genotype, were also reported in several other studies. In addition, Yan et al., showed that A1846T and G1896A and A1846T and C1913A/G were associated with ACLF in a longitudinal, study and a cross-sectional study, respectively, whereas there was no difference between the diseases regarding HBV genotype. Overall, it remains controversial whether HBV genotype B and which mutation are positively associated with the development of HB-ACLF. The major causative issue may be because most of these recent studies was performed based on the cohort from a single center, for example,some one is from Northern China (a genotype C predominant region) while anther one may be from Southern China (a genotype B predominant region). Given that there is the distinct distribution of HBV genotype and different mutation tendency in the BCP/pre-C regions of genotype B and C between different geographic areas within China mainland, a multi-center study is essentially necessary to clarify the exact association between the HBV viral genotype and mutations and the morbidity of HB-ACLF. In the other hand, these factors’relevant mechanism underlying liver disease progression was also unclear. It was ever reported in an in vitro study that PC/C mutations could enhance HBV replication capacity, or/and influence HBeAg expression, thereby maybe playing important roles in liver failure development. However, several other studies had showed that PC/C mutations could lead to no or only a marginal enhancement of HBV replication. So, the effect of PC/C mutations on HBV biological phenotype needs more systematic studies to confirm.To address the above issues, we collected serum samples from patients with HB-ACLF, severe-chronic hepatitis B (CHB-S), and mild-chronic hepatitis B (CHB-M); respectively, and based on their viral genetic profiling, we identifed the association between HBV genotype and mutations and the development of HB-ACLF; then we preliminarily explored the possible mechanism of these factors relevant to disease development, employing an in vitro specifically transfected cell model.Chapter One:Characterization of viral genetics in multi-center-cohort patients with hepatitis B related acute-on-chronic liver failureObjectiveTo analyse the serum virus characters of HB-ACLF, CHB-S, and CHB-M patients and elucidate the association between HBV genotype and mutations and the development of HB-ACLF, a multi-center study was performed.MethodsA total of522patients (from January2011to June2012) were enrolled from four different provinces acrossChina mainland as follows:Guangdong (n=215), Zhejiang (n=147), Sichuan (n=100), and Hubei (n=60), representing the south, east, west and central China, respectively. The cohort comprised231patients with mild-chronic hepatitis B (CHB-M),84with severe-chronic hepatitis B (CHB-S), and207with HB-ACLF. All patients had persistent sero-positive detection of hepatitis B surface antigen (HBsAg) for at least6months before enrollment.Diagnosis of CHB-M was based on clinical examination and liver functional tests, with mild liver disease activities which did not reach the criteria of CHB-S. CHB-S patients had severe liver disease symptoms, including obvious clinical manifestations and significant alterations in liver functional parameters. Based on the liver functional parameters, the diagnosis of CHB-S had to meet at least one of the following criteria:(1) serum total bilirubin (TBIL)>85.5μmol/L (i.e.,>5.0mg/dL); or (2) plasma prothrombin activity (PTA)>40%and≤60%; or (3) albumin (ALB)<32g/L. The HB-ACLF was defined as acute liver decompensation on the basis of CHB with mandatory jaundice (TBIL>171.0μmol/L [i.e.,>10.0mg/dL]) and coagulopathy (PTA<40%), and recently with development of complications. All the patients presented no evidence for HCC or other malignant liver disease along with no evidence for concomitant hepatitis virus (C or D), HIV infection, or autoimmune liver disease.HBV serological markers were detected by chemiluminescent assays (Abbott Laboratories, Chicago, IL, USA). Serum HBV DNA level was measured by Roche Light-Cycler Real-Time PCR system (based on fluorescence and quantitative analysis) with a detection range of1×103-1×109copies/m. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), TBIL, ALB, PTA and other biochemical parameters were determined by commercial kits. HBV DNA was extracted from200μl serum according to the manufacturer’s instructions of QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Hilden, Germany). The HBV S gene and BCP/PC/C gene regions were amplified, the PCR products were sequenced with ABI3730automated DNA sequencer. The HBV DNA sequences were aligned and phylogenetic trees were constructed using Mega5. The sequences of S gene were used for genotyping, and BCP/PC/C gene regions for analyzing frequency of mutations. All statistical tests were performed by using SPSS, version16.0(SPSS, Inc., Chicago, IL). Continuous variables were expressed as mean±SD. Differences in continuous data were evaluated using analysis of One-way ANOVA or Kruskal-Wallis H test appropriately, and categorical data were analyzed using the Pearson chi-square test or Fisher’s exact test. Forward stepwise binary logistic regression analysis (Pentry=0.05, Premoval=0.10) was used to determine independent factors for HB-ACLF. A two-sided P value<0.05was considered statistically significant.Results1) Demographic and clinical characteristics of the522patients. There was no significant difference in sex among three groups. The mean age was significantly different among the groups, and with the eldest age in HB-ACLF patients. The levels of ALT, AST, TBIL were significantly different among the groups, with a stepwise increase in the order of HB-ACLF> CHB-S> CBH-M; but the levels of ALB and HBV-DNA, the HBeAg positivity were decreased in a stepwise marine in the above order with a significant difference.2) Distribution of HBV genotypes in patients’ group. In patients with CHB-M, CHB-S, and HB-ACLF, HBV genotype B accounted for49.8%(115/231),52.4%(44/84), and79.2%(164/207), while genotype C accounted for50.2%(116/231),47.6%(40/84), and20.8%(43/207), respectively. The distribution of HBV genotypes was significantly different among the three patients groups, and genotype B was more frequent in patients with HB-ACLF than the other two groups, with a stepwise increase in the order of HB-ACLF> CHB-S> CBH-M.3) Distribution of HBV genotypes in the four provinces. Genotype B was highly prevalent in Sichuan and Hubei, while there is predominant occurrence of genotype C in Zhejiang and the comparable frequency of genotype B and C in Guangdong. When the prevalence of HBV genotypes were compared between patients with HB-ACLF and CHB, respectively, a higher frequency of genotype B was observed in HB-ACLF in the four centers, and in patients from Guangdong and Zhejiang provinces, there is a significant higher frequency of genotype B in HB-ACLF than the other CHB disease.4) Prevalence of BCP/pre-C/C mutations in patients’groups. The occurrences of A1762T/G1764A, A1846T, G1896A, C1913A/G, and A2159G mutations were high in the patients with the frequency of more than30%, except for that of the mutation A2159G. Prevalence of these mutations was significantly different among the three patients groups. A1762T/G1764A, A1846T, G1896A mutations were more frequent in patients with HB-ACLF than the other two groups, with a stepwise increase in the order of HB-ACLF> CHB-S> CBH-M. The occurrences of T1753V, C1766T, T1768A, G1862T, and G1899A mutations were low in the three patients groups, with no significant difference observed.5) The occurrences of A1846T, G1896A, and C1913A/G mutations were significantly higher in genotype B than that in genotype C patients; while the T1753V, A1762T/G1764A and C1766T mutations were more common in genotype C patients than that in genotype B patients; the frequency of T1768A, G1862T, G1899A and A2159G mutations were comparable in the two genotypes.6) Frequencies of BCP/pre-C/C mutations according to HBV genotype in patiens’groups. In genotype B infection patients, the occurrences of A1762T/G1764A, A1846T, and G1896A mutations were significantly different among the three patients groups, and the frequency of A1762T/G1764A and G1896A mutations was higher in patients with HB-ACLF than the other two groups. In genotype C infection patients, it was observed that except for the higher prevalence of A1762T/G1764A, A1846T, and G1896A mutations, the occurrences of C1913A/G and A2159G mutations were significantly different among the three patients groups, and all those mutations were more common in HB-ACLF patients.7) Changes of BCP/Pre-C/C mutation frequencies with increasing age. Over all, the frequencies of A1762T/G1764A, A1846T, and G1896A mutations collectively showed an incremental trend with aging in CHB patients irrespective of being infected with genotype B or C, whereas such a trend was not obvious in HB-ACLF patients. The mutation rates of these four sites were higher in HB-ACLF patients in most age groups (especially among patients aged<30years), as compared with CHB patients. The C1913A/G and A2159G mutations showed a higher prevalence in most age group infected HB-ACLF patients compared with CHB patients, although the occurrences of mutation did not increase with aging. In genotype B, the trend of A1762T/G1764A, A1846T, and G1896A mutation frequency in CHB group, and C1913A/G in HB-ACLF group were significantly different, respectively. However, in genotype C, no significant difference among age groups in patients groups was observed, respectively.8) Binary logistic regression analysis was performed to determine the independent factors associated with the development of HB-ACLF compared with CHB-M. The factors such as age of≥40years, genotype B, the presence of A1762T/G1764A, A1846T, and G1896A mutations were independently associated with the development of HB-ACLF.Conclusion1) HBV genotype B was significantly more common in HB-ACLF patients than in CHB-M or CHB-S patients, indicating viral genotype B was associated with the occurrence of HB-ACLF.2) The frequency of HBV BCP/PC/C mutations was different between genotype B and C. So that it was necessary to compare these mutation frequencies in patients’ groups with genotype B or C independently, to avoid the possible interference effect of genotype.3) The occurrences of A1762T/G1764A, A1846T, and G1896A mutations were significantly higher in patients with HB-ACLF than in patients with CHB-M, irrespective of HBV genotype B or C, indicating these mutations were strongly associated with HB-ACLF.4) Multivariate analysis showed HBV genotype B, A1762T/G1764A, A1846T, and G1896A mutations were independently associated with the development of HB-ACLF, providing further evidence to support above point. These virological factors might serve as possible molecular markers for prediction of the clinical outcomes of chronic HBV infection.Chapter two:Construction of recombinant plasmids specifically containing B/C genotypes with different mutation patterns of1.3copies hepatitis B virusObjective:To further observe the biological characteristics of mutations significantly associated with HB-ACLF, based on the results in Chapter one, different genotypes with different mutation pattern of1.3copies hepatitis B virus recombinant plasmids were constructed to provide the fundamental agents for further mimicking the relevant particular pathogenesis process.Methods:1) Using the HBV DNA extracted from patients serum, HBV gene sequence fragments A (nt1063-1982), B (nt2812-1084) and C (nt1063-2839) were amplified. Amplified fragments were inserted into pGEM-T Easy Vector and cloned in JM109competent cells to construct sub-clone (plasmid A, B and C); then using QuikChange(?) XL Site-Directed Mutagenesis Kit to complete site direct mutation, obtaining the mutation plasmid A, B and C. Plasmid A, B and C were digested with EcoT22I and EcoR I, EcoT22I and Eco065I, and Hind III and EcoO65I respectively, and then ligated into plasmid pUC19cleaved with EcoR I and Hind III, resulting in1.3copies hepatitis B Virus recombinant plasmids.2) We used restriction enzyme EcoR I and Hind III to digested recombinant plasmid for a preliminary identification; then direct sequencing was used to further confirm the constructed sequences of1.3copies hepatitis B Virus recombinant plasmids.ResultsWe built eight pUC-HBV1.3copies plasmids according to three virus strains from patients.The first strain included four plasmids with different mutation profile of nt1762/1764and nt1896; the second strainhad two plasmids with nt1846A or nt1846T, respectively; andthe third strain had nt2159A or nt2159G, respectively. Sequences of these constructed plasmids were identified by PCR, restriction enzyme digestion and nucleotide sequencing. Compared with the corresponding wild-type plasmids, there was no other mutation point introduced in these plasmids.ConclusionUsing recombinant technology and artificial mutagenesis method, we successfully construct plasmids particularly containing different genotypes with different mutation pattern of1.3copies hepatitis B virus.Chapter Three:In vitro characterization of cellular activity due to transfection with different genotypes with different mutation pattern of1.3copies hepatitis B virusObjectiveTo explore the biological characteristics of different mutation pattern of1.3copies hepatitis B virus plasmids in vitro, and get a glimpse of the possible mechanism of these factors influencing the outcome of CHB patients, for the purpose of providing any theoretical cues for understanding HB-ACLF development.Method 1) QIAGEN Hispeed midi kit was used to extract1.3copies hepatitis B Virus plasmids, and pSEAP plasmid was extracted at the same time.2) Huh7human hepatoma cells were cultured in in the complete Dulbecco’s modified Eagle medium (DMEM).1.5×106Huh7cells were seeded for each well in six-well plates. After24h of culture, cells were transiently transfected with2μg HBV DNA and0.0μg pSEAP by Lipofecatamine2000transfection reagent (Invitrogen), and controls were set at the same time.3) The supernatants were collected72hours after transient transfection, and detected using commercial assay kits. Cells were harvested at the same time. Southern blot was performed to detect HBV replication intermediately after DNA extraction. Transfection efficiency was evaluated by measuring the activity of cotransfectedpSEAP plasmid-expressing alkaline phosphatase.Results:1) Intracellular HBV replication capacity:compared to the corresponding wildtype strains, clones with different mutation pattern had no obvious effect on HBV replication.2) HBeAg level in supernatants:compared to the corresponding wildtype strains, clones with G1896A mutation solely or combined with A1762T/G1764A mutations could abolish HBeAg expression level, while clone with A1762T/G1764A mutations could reduce HBeAg expression level for65%or so; clone with A1846T mutation had no obvious effect on HBeAg expression level; clone with A2159G could elevate HBeAg expression level for about17%.Conclusions:1) Different mutation patterns do not have any apparent effect on HBV replication.2) G1896A or A1762T/G1764A mutation can abolish or reduce HBeAg expression level; A2159G could elevate HBeAg expression level; while A1846T mutation had no obvious effect on HBeAg expression level. |
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