Huiying Rao

MicroRNAs‐372/373 promote the expression of hepatitis B virus through the targeting of nuclear factor I/B

MicroRNAs (miRNAs) play important roles in the posttranscriptional regulation of gene expression. Recent evidence has indicated the pathological relevance of miRNA dysregulation in hepatitis virus infection; however, the roles of microRNAs in the regulation of hepatitis B virus (HBV) expression are still largely unknown. In this study we identified that miR‐373 was up‐regulated in HBV‐infected liver tissues and that the members of the miRs‐371‐372‐373 (miRs‐371‐3) gene cluster were also significantly co‐up‐regulated in HBV‐producing HepG2.2.15 cells. A positive in vivo association was identified between hepatic HBV DNA levels and the copy number variation of the miRs‐371‐3 gene cluster. The enhanced expression of miRs‐372/373 stimulated the production of HBV proteins and HBV core‐associated DNA in HepG2 cells transfected with 1.3×HBV. Further, nuclear factor I/B (NFIB) was identified to be a direct functional target of miRs‐372/373 by in silico algorithms and this was subsequently confirmed by western blotting and luciferase reporter assays. Knockdown of NFIB by small interfering RNA (siRNA) promoted HBV expression, whereas rescue of NFIB attenuated the stimulation in the 1.3×HBV‐transfected HepG2 cells. Conclusion: Our study revealed that miRNA (miRs‐372/373) can promote HBV expression through a pathway involving the transcription factor (NFIB). This novel model provides new insights into the molecular basis in HBV and host interaction. (HEPATOLOGY 2011;)

Performance Comparison of the Versant HCV Genotype 2.0 Assay (LiPA) and the Abbott Realtime HCV Genotype II Assay for Detecting Hepatitis C Virus Genotype 6

ABSTRACT The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5′ untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1.

Serum HBV core‐related antigen is a good predictor for spontaneous HBeAg seroconversion in chronic hepatitis B patients

Early prediction of spontaneous hepatitis B virus e antigen (HBeAg) seroconversion is pivotal in the prevention of unnecessary drug prescription, corresponding financial burden, and adverse reactions. One hundred and thirteen chronic hepatitis B patients with HBeAg‐positive in the immune active phase were followed up for about 1.5 years. Patients were classified into two groups: spontaneous HBeAg seroconversion group (group A, n = 18) and non‐spontaneous HBeAg seroconversion group. Among the non‐spontaneous HBeAg seroconversion group, 35 patients were selected as controls (group B, n = 35). At week 12, there was a significant difference in hepatitis B core‐related antigen (HBcrAg) levels between the two groups (group A 4.32 ± 1.05 log10 kU/ml, and group B 5.16 ± 0.53 log10 kU/ml, P = 0.004), and this significance magnified at week 28. Only two variables, HBcrAg level and the reduction in the HBcrAg levels (ΔHBcrAg) at week 28 were enrolled, with the odds ratio of 4.19 and 0.21, respectively. The optimal cutoffs of HBcrAg levels and the ΔHBcrAg at week 28 were 4.90 and 2.00 log10 kU/ml, respectively. The positive predictive value and negative predictive value of HBcrAg levels at week 28 were 73.9% and 96.7%, respectively. The positive predictive value and negative predictive value of the ΔHBcrAg at week 28 were 76.2% and 93.8%, respectively. The measurement of HBcrAg is useful for monitoring the natural course of chronic hepatitis B virus infection. The dynamics of HBcrAg levels could accurately predict the spontaneous HBeAg seroconversion. J. Med. Virol. 89:463–468, 2017.

Real-world treatment patterns and clinical outcomes of HCV treatment-naive patients in China: an interim analysis from the CCgenos study

In China, chronic hepatitis C virus (HCV) infection represents a considerable healthcare burden. Although interferon‐based therapy has been the standard‐of‐care for many years, few long‐term, real‐life studies have assessed interferon‐based treatment in China. The objective of CCgenos follow‐up study was to analyze long‐term treatment patterns and outcomes in a cohort of treatment‐naïve, Han ethnic, patients with chronic HCV infection.

Survey of hepatitis B knowledge and stigma among chronically infected patients and uninfected persons in Beijing, China

Hepatitis B virus (HBV) infection carries substantial stigma in China. We surveyed HBV knowledge and stigma among chronic hepatitis B (CHB) patients and persons without HBV infection in Beijing, China.

Automated evaluation of liver fibrosis in thioacetamide, carbon tetrachloride, and bile duct ligation rodent models using second-harmonic generation/two-photon excited fluorescence microscopy

Animal models provide a useful platform for developing and testing new drugs to treat liver fibrosis. Accordingly, we developed a novel automated system to evaluate liver fibrosis in rodent models. This system uses second-harmonic generation (SHG)/two-photon excited fluorescence (TPEF) microscopy to assess a total of four mouse and rat models, using chemical treatment with either thioacetamide (TAA) or carbon tetrachloride (CCl4), and a surgical method, bile duct ligation (BDL). The results obtained by the new technique were compared with that using Ishak fibrosis scores and two currently used quantitative methods for determining liver fibrosis: the collagen proportionate area (CPA) and measurement of hydroxyproline (HYP) content. We show that 11 shared morphological parameters faithfully recapitulate Ishak fibrosis scores in the models, with high area under the receiver operating characteristic (ROC) curve (AUC) performance. The AUC values of 11 shared parameters were greater than that of the CPA (TAA: 0.758–0.922 vs 0.752–0.908; BDL: 0.874–0.989 vs 0.678–0.966) in the TAA mice and BDL rat models and similar to that of the CPA in the TAA rat and CCl4 mouse models. Similarly, based on the trends in these parameters at different time points, 9, 10, 7, and 2 model-specific parameters were selected for the TAA rats, TAA mice, CCl4 mice, and BDL rats, respectively. These parameters identified differences among the time points in the four models, with high AUC accuracy, and the corresponding AUC values of these parameters were greater compared with those of the CPA in the TAA rat and mouse models (rats: 0.769–0.894 vs 0.64–0.799; mice: 0.87–0.93 vs 0.739–0.836) and similar to those of the CPA in the CCl4 mouse and BDL rat models. Similarly, the AUC values of 11 shared parameters and model-specific parameters were greater than those of HYP in the TAA rats, TAA mice, and CCl4 mouse models and were similar to those of HYP in the BDL rat models. The automated evaluation system, combined with 11 shared parameters and model-specific parameters, could specifically, accurately, and quantitatively stage liver fibrosis in animal models.

Abnormal CD4 + T helper (Th) 1 cells and activated memory B cells are associated with type III asymptomatic mixed cryoglobulinemia in HCV infection

BackgroundMixed cryoglobulinemia (MC) in hepatitis C virus (HCV) infection is associated with abnormal immune responses mediated by T cells and B cells, while the relationships of different subsets of CD4 + T helper (Th) cells, B cells and associated cytokines with type III asymptomatic MC in HCV infection are poorly understood.MethodsFifty-four chronic hepatitis C (CHC) patients and 23 healthy controls (HCs) were enrolled in the study. Serum cryoglobulins were detected by cryoprecipitation. The types of cryoglobulin were determined by western blot. The phenotypes and frequencies of Th cell and B cell subsets were detected by flow cytometric analysis. The cytokines IFN-γ, IL-4, IL-17, IL-21, IL-22, and TGF-β were measured by enzyme-linked immunosorbent assay.ResultsTwenty-six CHC patients were detected with type III asymptomatic MC. The frequencies of Th2, Th17, follicular helper T (Tfh cells), Th22, and tissue-like B cells were significantly higher in CHC patients compared to HCs, while these cell subsets were not significantly different between CHC patients and HCV-related MC patients. The frequencies of Th1 and activated memory B cells increased in HCV-related MC patients compared to HCs, although the difference between the two cell subsets in CHC patients and HCs was not significant. The frequency of regulatory T cells (Treg cells) was higher in CHC patients than in HCV-related MC patients and HCs. Higher expressions of serum IFN-γ, IL-17, IL-21, and IL-22 were observed in CHC patients than in HCs, but the differences were not significantly different in CHC patients and HCV-related MC patients. The frequency of Th1 cells was associated with activated memory B cells in HCV-related MC patients, and the frequency of Th1 cells and activated memory B cells was closely related to HCV RNA in HCV-related MC patients.ConclusionsThe increased frequencies of Th17 cells, Tfh cells, Th22 cells, Treg cells, cytokines IL-17, IL-21, IL-22, and tissue-like B cells, were related to HCV infection but not type III asymptomatic MC. Higher frequencies of Th1 cells and activated memory B cells were associated with type III asymptomatic MC in HCV infection.

Antibodies Targeting Novel Neutralizing Epitopes of Hepatitis C Virus Glycoprotein Preclude Genotype 2 Virus Infection

Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1–6 was determined. We defined a domain comprising amino acids (aa) 192–221, 232–251, 262–281 and 292–331 of E1, and 421–543, 564–583, 594–618 and 634–673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444–463) and PUHI45 (aa 604–618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection.

Direct-acting Antiviral Agents Resistance-associated Polymorphisms in Chinese Treatment-naïve Patients Infected with Genotype 1b Hepatitis C Virus.

Background:It has been reported that several baseline polymorphisms of direct-acting antivirals (DAAs) agents resistance-associated variants (RAVs) would affect the treatment outcomes of patients chronically infected with hepatitis C virus (CHC). The aim of this study is to investigate the prevalence of DAAs RAVs in treatment-naïve GT1b CHC patients. Methods:Direct sequencing and ultra-deep sequencing of the HCV NS3, NS5A, and NS5B gene were performed in baseline serum samples of treatment-naïve patients infected with genotype 1b hepatitis C virus (HCVs). Results:One hundred and sixty CHC patients were studied. Complete sequence information was obtained for 145 patients (NS3), 148 patients (NS5A), and 137 patients (NS5B). Treatment-failure associated variants of DAAs were detected: 56.6% (82/145) of the patients presented S122G for simeprevir (NS3 protease inhibitor); 10.1% (14/148) of the patients presented Y93H for daclatasvir and ledipasvir (NS5A protein inhibitors); 94.2% (129/137) of the patients presented C316N for sofosbuvir (NS5B polymerase inhibitor). Nearly, all of the DAAs RAVs detected by ultra-deep sequencing could be detected by direct sequencing. Conclusions:The majority of genotype 1b CHC patients in China present a virus population carrying HCV DAAs RAVs. Pretreatment sequencing of HCV genome might need to be performed when patients infected with GT1b HCV receiving DAAs-containing regimens in China. Population sequencing would be quite quantified for the work.

CD4+CXCR5+ T cells activate CD27+IgG+ B cells via IL-21 in patients with hepatitis C virus infection.

BACKGROUND Chronic hepatitis C virus (HCV) infection causes the skewing and activation of B cell subsets, but the characteristics of IgG+ B cells in patients with chronic hepatitis C (CHC) infection have not been thoroughly elucidated. CD4+CXCR5+ follicular helper T (Tfh) cells, via interleukin (IL)-21 secretion, activate B cells. However, the role of CD4+CXCR5+ T cells in the activation of IgG+ B cells in CHC patients is not clear. METHODS The frequency of IgG+ B cells, including CD27-IgG+ B and CD27+IgG+ B cells, the expression of the activation markers (CD86 and CD95) in IgG+ B cells, and the percentage of circulating CD4+CXCR5+ T cells were detected by flow cytometry in CHC patients (n=70) and healthy controls (n=25). The concentrations of serum IL-21 were analyzed using ELISA. The role of CD4+CXCR5+ T cells in the activation of IgG+ B cells was investigated using a co-culture system. RESULTS A significantly lower proportion of CD27+IgG+ B cells with increased expression of CD86 and CD95 was observed in CHC patients. The expression of CD95 was negatively correlated with the percentage of CD27+IgG+ B cells, and it contributed to CD27+IgG+ B cell apoptosis. Circulating CD4+CXCR5+ T cells and serum IL-21 were significantly increased in CHC patients. Moreover, circulating CD4+CXCR5+ T cells from CHC patients induced higher expressions of CD86 and CD95 in CD27+IgG+ B cells in a co-culture system; the blockade of the IL-21 decreased the expression levels of CD86 and CD95 in CD27+IgG+ B cells. CONCLUSIONS HCV infection increased the frequency of CD4+CXCR5+ T cells and decreased the frequency of CD27+IgG+ B cells. CD4+CXCR5+ T cells activated CD27+IgG+ B cells via the secretion of IL-21.