[Show abstract][Hide abstract] ABSTRACT: Hepatitis B virus (HBV) chronically infects approximately 350 million people worldwide. The replication of HBV which genome is only 3.2 kb long relies heavily on host factors. Previous studies demonstrated that a highly expressed liver-specific microRNA (miRNA) miR-122 suppresses HBV expression and replication in multiple ways. In this study, we found that the miR-122 response elements in viral genome facilitate HBV expression and replication in miR-122 highly-expressed hepatocytes. Moreover, mutations in miR-122 response elements are correlated with viral loads and disease progression in HBV-infected patients. We next found that HBV mRNA with miR-122 response elements alone could lead to altered expression of multiple host genes by whole genome expression analysis. HBV mRNA-mediated miR-122 down-regulation plays a major role in HBV mRNA-induced differential gene expression. HBV mRNA could enhance viral replication via miR-122 degradation and the up-regulation of its target cyclin G1. Our study thereby reveals that under the unique condition of high abundance of miR-122 and viral mRNAs and much lower level of miR-122 target in HBV infection, HBV may have evolved to employ the miRNA-mediated virus and host mRNAs network for optimal fitness within hepatocytes.
[Show abstract][Hide abstract] ABSTRACT: Non-alcoholic steatoheaptitis (NASH), the critical stage of non-alcoholic fatty liver disease (NAFLD), is of chronic progression and can develop cirrhosis even hepatocellular carcinoma (HCC). However, non-invasive biomarkers for NASH diagnosis remain poorly applied in clinical practice. Our study aims at testing the accuracy of the combination of cytokeratin-18 M30 fragment (CK-18-M30), fibroblast growth factor 21 (FGF-21), interleukin 1 receptor antagonist (IL-1Ra), pigment epithelium-derived factor (PEDF) and osteoprotegerin (OPG) in diagnosing NAFLD and NASH.
179 patients with biopsy-proven NAFLD were enrolled as training group, 91 age- and gender-matched healthy subjects were recruited at the same time as controls. 63 other NAFLD patients were separately collected as validation group. 45 alcoholic fatty liver disease (AFLD) patients, 50 hepatitis B virus (HBV) patients, 52 hepatitis C virus (HCV) patients were also included. Serum biomarker levels were measured by enzyme-linked immunosorbent assay.
Serum levels of CK-18-M30, FGF-21, IL-1Ra and PEDF increased, while OPG decreased in a stepwise fashion in controls, non-NASH NAFLD patients and NASH patients (P < 0.01). The area under receiver-operating characteristics curve to diagnose NASH was 0.86 for CK-18-M30, 0.89 for FGF-21, 0.89 for IL-1Ra, 0.89 for PEDF and 0.89 for OPG. CK-18-M30 had 70% negative predictive value (NPV) and 79% positive predictive value (PPV) to diagnose NASH. A 5-step approach measuring CK-18-M30 followed by FGF21, IL-1Ra, PEDF and OPG gradually improved the NPV to 76% and PPV to 85%, which reached 80% and 76% respectively in the validation cohort.
Compared to single biomarker, stepwise combination of CK-18-M30, FGF-21, IL-1Ra, PEDF and OPG can further improve the accuracy in diagnosing NASH.
[Show abstract][Hide abstract] ABSTRACT: The study aimed to clarify whether the rtI233V substitution affects adefovir (ADV) resistance.
A total of 18,419 patients from Beijing 302 Hospital were investigated. HBV complete reverse-transcriptase region of the polymerase was screened by direct sequencing and verified by clonal sequencing if necessary. Replication-competent wild-type and mutant HBV genomic amplicons were transfected into HepG2 cells for phenotypic analysis of viral replication capacity and drug susceptibility.
The rtI233V substitution was detected in 38/5,344 (0.71%) ADV-treated patients and in 8/13,075 patients without receiving ADV (P <0.001). Eight patients with rtI233V ± rtA181V/rtN236T had virologic breakthrough in clinical course of ADV treatment. Phenotypic analysis showed that rtI233V mutants from patient 1 and patient 2 exhibited 1.57-fold and 1.51-fold decreased susceptibility to ADV respectively compared to wild-type virus; by contrast, rtN236T and rtI233V+N236T mutants from patient 1 had 6.82-fold and 5.28-fold decreased susceptibility to ADV. rtI233V, rtN236T and rtI233V+N236T mutants had 97.5%, 30.2% and 69.7% of replication capacity compared to wild-type virus in the absence of antivirals and all remained susceptible to lamivudine, entecavir and tenofovir. Viral replication capacity correspondingly decreased after eliminating rtI233V from rtI233V+N236T mutant and restored after introducing rtI233V into rtN236T mutant. In clinical practice, switching-to entecavir rescue therapy suppressed HBV DNA to an undetectable level for both patients.
rtI233V usually emerged in ADV-treated patients with little impact on ADV susceptibility but it effectively restored replication capacity of rtN236T mutant, suggesting that rtI233V may partly serve as a compensatory mutation associated with ADV resistance.
No preview · Article · Jun 2015 · Antiviral therapy
[Show abstract][Hide abstract] ABSTRACT: Early onset of hepatocellular carcinoma (HCC) (males and females under the age of 40 or 50 years old, respectively) has a significant prevalence and poor prognosis; however, few studies have reported the risk factors and development of HCC in such cases.
In this study, we retrospectively analyzed clinical, laboratory and demographic data from 588 treatment-naïve HCC patients with hepatitis B virus (HBV)-associated liver cirrhosis (LC) and 708 age-matched HBV-associated LC patients as control in Beijing 302 Hospital.
15.1% (89/588) of the HCC patients and 36.7% (181/708) of the LC patients were classified as early onset. Compared with age-matched LC controls, male gender (odds ratio (OR) = 2.09, P < 0.05), family history of HBV infection (OR = 2.45, P < 0.05) and alpha-fetoprotein (AFP) > 200 ng/ml (OR = 30.8, P < 0.05) were independent risk factors for early-onset HCC. Comparing late-onset LC controls, male gender (OR = 1.92, P < 0.05), age (OR = 1.04, P < 0.05), family history of HCC (OR = 2.06, P < 0.05), history of smoking (OR = 1.68, P < 0.05) and AFP > 200 ng/ml (OR = 12.0, P < 0.05) were associated with the development of naturally occurring HCC. Overall, male gender and AFP > 200 ng/ml is associated with HCC development across all ages, whereas a family history of HBV infection may identify younger HBV-associated LC patients at risk for HCC.
Our data suggest that a family history of HBV infection is a unique risk factor for naturally-occurring early-onset HCC patients with HBV-associated LC, who should be considered for intensive screening programs.
No preview · Article · Mar 2015 · International Journal of Clinical and Experimental Medicine
[Show abstract][Hide abstract] ABSTRACT: Background
Intrahepatic hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is the original template for HBV replication. The persistence of cccDNA is responsible for the recurrence of HBV infection. The detection of cccDNA can help the development of new antiviral drugs against HBV replication links, and reduce the resistance and recurrence as well as to discover extrahepatic HBV infection. In situ polymerase chain reaction (IS-PCR) can be used to determine the distribution and localization of cccDNA in liver tissues, but it is hampered by its low sensitivity and specificity. We developed a novel method to detect HBV cccDNA using rolling circle amplification (RCA) combined with IS-PCR.Methods
Biopsy liver tissues were obtained from 26 patients with HBV infection, including 10 chronic hepatitis B (CHB), 6 liver cirrhosis (LC) and 10 hepatocellular carcinoma (HCC) patients. Four pairs of primers were designed to mediating RCA for the first round amplification of HBV cccDNA specifically. The liver tissue sections from patients were treated by plasmid-safe ATP-dependent DNase (PSAD) prior to RCA. After RCA, HBV cccDNA was further amplified by a pair of selective primers labeled digoxigenin that target the gap region between the two direct repeat regions (DR1 and DR2) of the virus via IS-PCR.ResultsHBVcccDNA was expressed and located in hepatocyte nucleus in 19 patients (73.07%). Compared with the IS-PCR, the introduction of RCA increase the limit of detection. RCA combined with IS-PCR yielded strong positive signals in HCC liver tissue in spite of low copy number cccDNA (2 copies of target sequence per cell), meanwhile, no positive signal was detected via negative control.ConclusionsRCA combined with IS-PCR is an effective and practicable method which could detect the presence of low copy number of cccDNA sensitively and specifically, and reflect the relationship between cccDNA expression level and liver tissue pathological characteristics.
Preview · Article · Dec 2014 · BMC Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: To justify the clinical use of Traditional Chinese Medicine (TCM) in the treatment of influenza.
MEDLINE, EMBASE, Chinese Biomedical Literature Database, China National Knowledgeln-frastructure Database, China Science and Technology Journal Database, Wanfang Database and the Cochrane Database of Systematic Reviews were searched from the date of inception until January 1, 2013, for the literature on treatment of influenza with TCM.
A total of 7 randomized controlled trials were identified and reviewed. Of these trials, 2 compared a (modified) prescription of TCM with oseltacmivir and 5 compared a patent traditional Chinese drug with oseltamivir. Based on the Meta-analysis, compared to oseltamivir, the (modified) prescription had similar effect in defervescence [WMD = 5.66, 95% CI (- 32.02, 43.35), P = 0.77] and viral sheddingWMD = - 6.21, 95% CI (- 84.19, 71.76), P = 0.88], and the patent traditional Chinese drug also had similar effect in viral shedding [WMD = - 0.24, 95% CI (- 4.79, 4.31), P = 0.92] but more effective in defervescence [WMD = - 4.65, 95%CI (- 8.91, - 0.38),P = 0.03].
TCM has potential positive effects in the treatment of influenza.
No preview · Article · Oct 2014 · Journal of Traditional Chinese Medicine
[Show abstract][Hide abstract] ABSTRACT: Background:
This study aimed to clarify the clinical significance of drug-resistant HBV in nucleoside/nucleotide analogue (NA)-naive Chinese patients with chronic HBV infection in real clinical practice.
A total of 845 NA-naive patients who were admitted to Beijing 302 Hospital between July 2007 and March 2012 were included in the study. HBV drug-resistant mutations were examined by direct sequencing of the viral reverse transcriptase gene and verified by clonal sequencing. Phenotypic analysis of viral replication capacity and drug susceptibility were performed by measuring viral replicative intermediate level in 1.1-mer mutant or wild-type HBV amplicon-transfected HepG2 cells in absence or presence of serially diluted drugs.
Drug-resistant mutations were detected in 2.01% (17/845) of the patients by direct sequencing, including 15 with lamivudine-resistant mutations (rtM204V, rtM204I), one with adefovir-resistant mutation (rtA181V), and one with both lamivudine- and adefovir-resistant mutations (rtA181V, rtM204I). Clonal sequencing identified 13 drug-resistant HBV strains: rtL80I+M204I, rtL80I+M204V, rtL180M+M204I, rtL180M+M204V, rtM204I, rtM204V, rtL80I+L180M+M204I, rtL80I+L180M+M204V, rtA181V, rtA181V+M204I, rtA181T+N236T, rtA181V+N236T and rtN236T. Phenotypic analysis showed that two pre-existing lamivudine-resistant strains (rtL80I+M204I, rtL180M+M204V) had >1,000-fold resistance to lamivudine, and one pre-existing adefovir-resistant strain (rtA181V+N236T) had 15.4-fold resistance to adefovir compared with the wild-type strain. A follow-up study showed that the presence of pre-existing rtM204I strain in one patient increased from 20% at baseline to 85% after 13 months of entecavir treatment with corresponding recession of wild-type strain in the viral pool.
The incidence of drug-resistant HBV mutations was low in NA-naive Chinese HBV-infected patients. Pre-existing mutants had similar resistance characteristics to those from NA refractory patients.
No preview · Article · Jul 2014 · Antiviral therapy
[Show abstract][Hide abstract] ABSTRACT: The study aimed to clarify whether rtN236V mutation of hepatitis B virus (HBV) derived from adefovir dipivoxil (ADV)-refractory patients was associated with the drug resistance.
A total of 18,419 patients from Beijing 302 Hospital were investigated. HBV complete reverse-transcriptase region of polymerase was screened by direct sequencing and verified by clonal sequencing if necessary. Replication-competent wild-type and mutant HBV genomic amplicons were constructed, transfected into HepG2 cells and cultured in the presence or absence of serially-diluted nucleos(t)ide analogs. Intracellular HBV replicative intermediates were quantitated for calculating the 50% effective concentration of drug (EC50).
rtN236V was detected in six ADV-refractory patients; signature ADV-resistant mutations rtA181V and rtN236T were detected in 1,311 patients. rtN236V mutants emerged predominantly with virologic breakthrough in clinical course of the six patients. Phenotypic analysis of the mutants from two patients was performed. rtN236V mutants from patient 1 and patient 2 exhibited 3.90-fold and 3.10-fold decreased susceptibility to ADV respectively compared to the wild-type virus; by contrast, rtN236T mutants from the patients had 4.50-fold and 4.75-fold decreased susceptibility respectively. Both mutants had a relatively lower viral replication capacity compared to wild-type virus in the absence of antivirals and remained susceptible to lamivudine, entecavir and tenofovir disoproxil fumarate. In clinical practice, switching-to entecavir rescue therapy suppressed HBV DNA to an undetectable level and normalized alanine aminotransferase level for both patients.
rtN236V was a novel infrequently-occurred ADV-resistance-associated mutation. It conferred a moderate resistance to ADV with relatively lower natural replication capacity.
No preview · Article · Apr 2014 · Antiviral therapy
[Show abstract][Hide abstract] ABSTRACT: Lamivudine (LAM) is still widely used for anti-HBV therapy in China. The study aimed to clarify whether a newly-found rtM204Q mutation from patients was associated with the drug resistance.
HBV complete reverse-transcriptase region was screened by direct sequencing and verified by clonal sequencing. Replication-competent plasmids containing patient-derived 1.1mer mutant or wild-type viral genome were constructed and transfected into HepG2 cells. After cultured with or without serially-diluted antiviral drugs, intracellular HBV replicative intermediates were quantitated for calculating the 50% effective concentration of drug (EC50).
A total of 12,000 serum samples of 9,830 patients with chronic HBV infection were screened. rtM204Q mutation was detected in seven LAM-refractory patients. By contrast, rtM204I/rtM204V mutations were detected in 2,502 patients' samples. The rtM204Q emerged either alone or in concomitance with rtM204I/rtM204V, and all were accompanied with virologic breakthrough in clinical course. Clonal sequencing verified that rtM204Q mutant was predominant in viral quasispecies of these samples. Phenotypic analysis showed that rtM204Q mutant had 89.9% of replication capacity and 76-fold increased LAM EC50 of the concomitant wild-type strain. By contrast, rtM204I mutant in the sample had lower replication capacity and higher LAM resistance (46.3% and 1396-fold increased LAM EC50 of the wild-type strain) compared to rtM204Q mutant. rtM204Q mutant was susceptible to adefovir dipivoxil (ADV) in vitro and ADV/ADV+LAM rescue therapy in clinic.
rtM204Q is suggested to be a novel LAM-resistance-associated mutation. It conferred a moderate resistance with higher competent natural replication capacity compared to rtM204I mutation.
[Show abstract][Hide abstract] ABSTRACT: This study aimed to investigate the relationships of intrahepatic cccDNA with serum HBsAg and with HBV DNA in treatment-naive patients throughout acute and chronic HBV infection.
A total of 120 patients who had a liver biopsy were enrolled, including 19 with acute hepatitis B (AHB), and 101 patients with chronic HBV infection (CHB) of whom were 10 in immune-tolerant (IT) phase, 59 in immune-clearance (IC) phase, 8 in low-replicative (LR) phase, and 24 in HBeAg-negative hepatitis (ENH) phase. Intrahepatic cccDNA, serum HBsAg and serum HBV DNA levels were comparatively analyzed.
The median intrahepatic cccDNA levels were 0.18 4.80, 3.81, 0.22 and 0.97 copies/cell for patients with AHB, CHB-IT, CHB-IC, CHB-LR, and CHB-ENH, respectively. In AHB patients, intrahepatic cccDNA was positively correlated with serum HBsAg (r = 0.665, P = 0.003), as well as serum HBV DNA (r = 0.536, P = 0.022). In CHB patients, intrahepatic cccDNA was positively correlated with serum HBsAg in the IC phase (r = 0.392, P = 0.005), and with serum HBV DNA in the IC phase (r = 0.301, P = 0.036) and ENH phase (r = 0.588, P = 0.013). HBV replicative efficiency, defined as the ratio of serum HBV DNA to intrahepatic cccDNA, was obviously lower in AHB and CHB-LR patients than in CHB-IT, CHB-IC and CHB-ENH patients (0.70 and 0.53 vs. 1.12, 1.09 and 0.99, P<0.001, values were logarithmic transformed for analysis). In CHB-IC patients, HBV replicative efficiency was positively correlated with histological activity index of liver inflammation (r = 0.308, P = 0.009).
Serum HBsAg and HBV DNA levels may reflect the amount of active intrahepatic cccDNA in treatment-naive AHB and CHB-IC patients. Reduced intrahepatic cccDNA and HBV replicative efficiency may imply effective immune control of HBV infection.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis B virus (HBV) genotypes and subgenotypes may vary in geographical distribution and virological features. Previous investigations, including ours, showed that HBV genotypes B and C were respectively predominant in South and North China, while genotypes A and D were infrequently detected and genotype G was not found. In this study, a novel A/C/G intergenotype was identified in patients with chronic HBV infection in Guilin, a city in southern China. Initial phylogenetic analysis based on the S gene suggested the HBV recombinant to be genotype G. However, extended genotyping based on the entire HBV genome indicated it to be an A/C/G intergenotype with a closer relation to genotype C. Breakpoint analysis using the SIMPLOT program revealed that the recombinant had a recombination with a arrangement of genotypes A, G, A and C fragments. Compared with the HBV recombinants harboring one or two genotype G fragments found in Asian countries, this Guilin recombinant was highly similar to the Vietnam (98-99%) and Long An recombinants (96-99%), but had a relatively low similarity to the Thailand one (89%). Unlike those with the typical genotype G of HBV, the patients with the Guilin recombinant were seropositive for HBeAg. Moreover, a relatively high HBV DNA viral load (>2×10(6) IU/ml) was detected in the patients, and the analysis of viral replication capacity showed that the Guilin recombinant strains had a competent replication capacity similar to genotypes B and C strains. These findings can aid in not only the clarification of the phylogenetic origin of the HBV recombinants with the genotype G fragment found in Asian countries, but also the understanding of the virological properties of these complicated HBV recombinants.
[Show abstract][Hide abstract] ABSTRACT: Interferon-gamma induced protein 10 (IP-10) was suggested to be involved in liver injury in viral hepatitis. This study aimed to investigate the impact of the single nucleotide polymorphisms (SNP) G-201A (rs1439490) in IP-10 gene on disease progression of hepatitis B virus (HBV) infection.
The -201 SNP in IP-10 promoter was genotyped from 577 patients with different illness categories and 275 health controls; In vitro IP-10 promoter activity was compared between haplotype GG and AA homozygotes using luciferase reporter system in HepG2 cells. In vivo expression of IP-10 was compared between patients with -201 AA genotype and GG genotype.
The detected frequency of G-201A SNP was 17.8%, 25.3%, 26.6%, and 13.8% for patients with acute hepatitis B (AHB), patients with mild chronic hepatitis B (CHB-M), patients with severe chronic hepatitis B (CHB-S), and health controls, respectively. In vitro IP-10 promoter-driven luciferase activity in pGL3-Enhancer-201A transfected HepG2 cells was 1.43-fold higher than that in pGL3-Enhancer-201G transfected HepG2 cells (P<0.01). In vivo IP-10 transcriptional expression of peripheral blood mononuclear cells was 1.38-fold higher in patients with -201 AA genotype than in patients with -201 GG genotype (P<0.01).
G-201A in promoter region of IP-10 gene was associated with liver disease progression in patients with HBV infection through up-regulating IP-10 expression.
[Show abstract][Hide abstract] ABSTRACT: Mutations in the core protein (HBc) of hepatitis B virus (HBV) are associated with aggressive hepatitis and advanced liver diseases in chronic hepatitis B (CHB). In this study, we identified the L60V variation in HBc that generates a new HLA-A2-restricted CD8(+) T cell epitope by screening an overlapping 9-mer peptide pool covering HBc and its variants. The nonameric epitope V60 were determined by structural and immunogenic analysis. The HBc L60V variation is correlated with hepatic necroinflammation and higher viral levels, and it may be associated with poor prognosis in CHB patients. Immunization with the defined HBV epitope V60 peptide elicited specific cytotoxic T lymphocyte (CTL)-induced liver injury in HLA-A2(+)/HBV transgenic mice. In addition, in vitro and in vivo experiments both demonstrated that the HBc L60V variation facilitates viral capsid assembly and increases HBV replication. These data suggest that the HBc L60V variation can impact both HBV replication and HBV-specific T cell responses. Therefore, our work provides further dissection of the impact of the HBc L60V variation, which orchestrates HBV replication, viral persistence, and immunopathogenesis during chronic viral infection.
Full-text · Article · May 2013 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Objective:
To express T lymphocyte receptors (TCRs) on hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) mediated by retrovirus and investigate their binding affinity.
Peripheral blood mononuclear cells were isolated from acute hepatitis B patients with HLA-A2⁺; phenotype, and after induced, HBV-specific CTLs were sorted out followed by cloning and proliferating. Cell RNAs were extracted. The sequences of TCR's α and β chains were obtained by means of RT-PCR, 5'RACE and OVER-LAP PCR, for constructing TCR retrovirus vectors. Through retrovirus-mediated transduction, HBV-specific TCRs were expressed on Jurkat cells and CD8⁺; T cells from HLA-A⁺; healthy subjects.
Two paired TCR Vα and Vβ, respectively named α21β13 and α15β13, were obtained from one patient with acute hepatitis B and HLA-A2⁺;. The titers of packaged recombinant retroviruses were 1.5×10;-5.0×10⁵; IU/mL. Immunofluorescence staining by anti-Vβ13 TCR-PE targeting the specific TCR and HLA-A2 restricted epitope-specific pentamer showed a positive expression of reconstructed TCR on T cells. The positive cells accounted for 1.06%-2.25% for Vβ13 on Jurkat cells, 1.03%-2.06% for Vβ13 chain and 1.05%-1.12% for the epitope-specific pentamer on T cells from healthy HLA-A2⁺; subjects respectively. By contrast, only less than 0.05% cells from healthy HLA-A2⁻ ;subjects were positive for either Vβ13 or the pentamer.
TCRs on HBV-specific CTLs could be expressed by TCR gene transfer mediated by retrovirus, and they were proved with binding affinity to HLA-A2-restricted epitope.
No preview · Article · May 2013 · Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology
[Show abstract][Hide abstract] ABSTRACT: Lamivudine (LAM) resistance now poses a major problem in the management of patients with chronic hepatitis B virus (HBV) infection. We retrospectively collected clinical data on chronic HBV-infected patients who had developed LAM resistance under de novo LAM monotherapy and subsequently took nucleos(t)ide analogs as rescue strategy in our hospital. From initiation of rescue therapies to January 2012, incidence of antiviral drug resistance was 23.67%, 18%, 6.94% and 0% (P=0.007) in the group of switching to adefovir dipivoxil (ADV) monotherapy, switching to entecavir (ETV) monotherapy, adding on ADV and switching to combination of ADV and ETV. At month 12, the median levels of serum HBV DNA were respectively 9300IU/mL, 4648IU/mL, 2054IU/mL and 100IU/mL (P<0.001), and the cumulative rates of serum ALT normalization were respectively 75%, 84%, 93% and 100% (P=0.003). Additionally, the strategy of switching to ADV monotherapy induced more single rtA181T mutations. In conclusion, switching to ADV monotherapy has been widely used in real-world clinical practice in China, however, due to the high incidence of drug resistance, switching to neither ADV nor ETV monotherapy is optimal when LAM resistance occurs; combination of ADV and ETV is most effective, whereas the strategy of adding on ADV is rational for most of LAM-resistant Chinese patients with chronic hepatitis B.
Full-text · Article · Aug 2012 · Antiviral research
[Show abstract][Hide abstract] ABSTRACT: It remains unclear whether hepatitis B virus (HBV) reverse-transcriptase (RT) rtL229 substitutions influence HBV drug resistance.
The study was to investigate the association of HBV rtL229 substitutions with viral resistance to lamivudine (LAM).
Entire HBV RT genes were amplified by nested PCR and sequenced from sera of 6000 nucleos(t)ide analog-experienced patients with chronic HBV infection. The incidence and clinic relevance of rtL229 substitutions were analyzed. Replication-competent viral amplicons which harbored HBV genomes of wild-type, rtM204I, or rtM204I in conjunction with various rtL229 substitutions (rtL229F/W/M/V) were constructed. The amplicons were transfected into HepG2 cells for phenotyping of replication capacity and susceptibility to nucleos(t)ide analogs.
The rtL229 substitutions were detected in 6.57% (394/6000) of patients. Individual substitution incidences were 2.77%, 0.97%, 0.83% and 0.55% for rtL229V, rtL229F, rtL229M and rtL229W, respectively. The incidence of rtL229 substitutions was significantly higher in LAM-experienced patients (341/4220, 8.1%) than in LAM-naïve patients (53/1780, 3.0%), and were independently associated with genotypic LAM resistance (77.9% vs. 21.2%, OR 8.806, 95%CI 6.345-12.223) and low viral replication (HBV DNA <1000IU/mL) (4.60% vs. 24.2%, OR 0.478, 95%CI 0.254-0.898). Representative cases follow-up showed that rtL229F developed subsequent to rtM204I emergence during LAM treatment and regressed with rtM204I after LAM withdrawal. Functionally, rtL229F did not confer reduced susceptibility to LAM, but could restore replication capacity of rtM204I strain.
The rtL229 substitutions were potentially associated with LAM resistance in Chinese patients and rtL229F had characteristics of a compensatory mutation of rtM204I mutant.
No preview · Article · Mar 2012 · Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology
[Show abstract][Hide abstract] ABSTRACT: The mimotopes of viruses are considered as the good targets for vaccine design. We prepared mimotopes against multiple subtypes of influenza A and evaluate their immune responses in flu virus challenged Balb/c mice.
The mimotopes of influenza A including pandemic H1N1, H3N2, H2N2 and H1N1 swine-origin influenza virus were screened by peptide phage display libraries, respectively. These mimotopes were engineered in one protein as multi- epitopes in Escherichia coli (E. coli) and purified. Balb/c mice were immunized using the multi-mimotopes protein and specific antibody responses were analyzed using hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). The lung inflammation level was evaluated by hematoxylin and eosin (HE).
Linear heptopeptide and dodecapeptide mimotopes were obtained for these influenza virus. The recombinant multi-mimotopes protein was a 73 kDa fusion protein. Comparing immunized infected groups with unimmunized infected subsets, significant differences were observed in the body weight loss and survival rate. The antiserum contained higher HI Ab titer against H1N1 virus and the lung inflammation level were significantly decreased in immunized infected groups.
Phage-displayed mimotopes against multiple subtypes of influenza A were accessible to the mouse immune system and triggered a humoral response to above virus.
[Show abstract][Hide abstract] ABSTRACT: The study aimed to develop an effective method to quantitate HBV covalently closed circular DNA (cccDNA) using small section of formalin fixed paraffin-embedded (FFPE) liver biopsy.
Plasmid-safe ATP-dependent DNase (PSAD)-treated samples were subjected to rolling circle amplification (RCA) prior to real-time PCR mediated by cccDNA-selective primers. Human beta-actin gene was used as a reference control.
Compared to the classical method, i.e., PSAD digestion+real-time PCR, introduction of RCA increased the lower limit of detection for about 2 logs with good inter- and intra-assay reproducibility. HBV cccDNA was detected in 91.5% (119/130) of the FFPE samples. The cccDNA levels (copy/cell) between FFPE liver tissues and fresh frozen counterpart tissues were comparable. The median of cccDNA level in HBeAg-positive patients was higher than that in HBeAg-negative ones (52.60 vs. 31.25copies/cell, P<0.01). Intrahepatic cccDNA level was positively correlated with intrahepatic HBV total DNA level, but not obviously correlated with serum HBV DNA or alanine aminotransferase levels.
The method could sensitively and specifically quantitate intrahepatic HBV cccDNA in micro FFPE liver biopsy tissue for evaluation of HBV replication status in the liver.
No preview · Article · Oct 2011 · Clinica chimica acta; international journal of clinical chemistry