Huagan tongluo Fang improves liver fibrosis via down-regulating miR-184 and up-regulating FOXO1 to inhibit Th17 cell differentiation.

BACKGROUND: The purpose of this research is to reveal the improvement effect and potential mechanism of Huagan tongluo Fang (HGTLF) on liver fibrosis. METHODS: A mouse model of liver fibrosis induced by CCl4 was established to analyze the effect of HGTLF on liver fibrosis. The expression changes of miRNA after HGTLF stimulation were detected by qRT-PCR. After interference with miR-184 in Th17 cells, the concentration of IL-17A in cell culture supernatants was detected by ELISA and the proportion of Th17 cells was analyzed by flow cytometry. The relationship between miR-184 and FOXO1 was verified by online software and dual-luciferase reporter system. After HGTLF treatment of Th17 cells overexpressing miR-184, the protein level of FOXO1 was detected by Western blot. RESULTS: HGTLF could significantly improve liver fibrosis in mice. By qRT-PCR, miR-184 was most significantly expressed after HGTLF drug stimulation, and miR-184 was considered to be the major RNA involved in Th17 cell differentiation. Interference with miR-184 in Th17 cells inhibited the differentiation of Th17 cells. By online software and dual-luciferase reporter system assay, the direct interaction of miR-184 with FOXO1 was confirmed. After HGTLF treatment of Th17 cells overexpressing miR-184, FOXO1 protein levels were significantly up-regulated and inhibited the differentiation of Th17 cells, which was reversed by miR-184 inhibitors. The Vivo experiments also confirmed the improvement effect of HGTLF on liver fibrosis in mice. CONCLUSION: Our results indicated that HGTLF could improve liver fibrosis via down-regulating miR-184 and up-regulating of FOXO1 to inhibit Th17 cell differentiation.

Antiviral efficacy of entecavir for hepatitis B virus rtA181V/T mutants.

BACKGROUND: The amino acid substitution at position 181 of the Hepatitis B virus (HBV) polymerase is a multi-drug resistance affecting both the L-nucleoside and acyclic phosphonate nucleotide groups. Data is limited on the efficacy of entecavir (ETV) rescuing chronic hepatitis B (CHB) patients with rtA181T/V mutation. METHODS: Thirty-one patients with rtA181T/V mutation and 25 patients with rtA181T/V and rtN236T mutation were enrolled. Virological, serological and biochemical outcomes of ETV rescue therapy over 12 months in CHB patients with rtA181T/V mutation strains were investigated. All patients were treated with ETV 0.5 mg/day for 12 months and scheduled follow-up every 3 months. Patients' characteristics, laboratory tests results and clinical outcomes were collected and compared. RESULTS: After emergence of rtA181T/V mutant, serum HBV DNA levels increased over 4 log10 IU/mL, but the total bilirubin, alanine aminotransferase (ALT) levels raised moderately. No significant difference in baseline characteristics was observed between the rtA181T/V group and rtA181T/V + rtN236T group. After 12 months rescue therapy, total 85.7% (48/56) patients achieved HBV DNA undetectable. No significant difference in the mean reduction of serum HBV DNA and biochemical response was observed between both groups (3.59 ± 1.85 vs. 3.76 ± 2.15 log10 IU/ml; P = 0.756 and 90.3 vs. 80.0%; P = 0.272, respectively). The mean HBV DNA reduction, HBsAg and ALT levels were also similar between different rtA181T/sW172 mutations (P > 0.05). HBV DNA level is the only predictor of 12 months antiviral outcomes (odds ratio 6.723, P = 0.022). CONCLUSIONS: The results of the present study suggested that ETV is efficient in rescuing rtA181T/V mutation CHB patients. HBV DNA level could predict viral clearance at the 12th month.

Swine and rabbits are the main reservoirs of hepatitis E virus in China: detection of HEV RNA in feces of farmed and wild animals.

Hepatitis E virus (HEV) infection is recognized as a zoonosis. The prevalence of HEV RNA and anti-HEV antibodies in many animal species has been reported, but the host range of HEV is unclear. The aims of this study were to investigate HEV infection in various animal species and to determine the reservoirs of HEV. Eight hundred twenty-two fecal samples from 17 mammal species and 67 fecal samples from 24 avian species were collected in China and tested for HEV RNA by RT-nPCR. The products of PCR were sequenced and analyzed phylogenetically. The positive rates of HEV RNA isolated from pigs in Beijing, Shandong, and Henan were 33%, 30%, and 92%, respectively, and that from rabbits in Beijing was 5%. HEV RNA was not detectable in farmed foxes, sheep or sika deer, or in wild animals in zoos, including wild boars, yaks, camels, Asiatic black bears, African lions, red pandas, civets, wolves, jackals and primates. Sequence analysis revealed that swine isolates had 97.8%-98.4% nucleotide sequence identity to genotype 4d isolates from patients in Shandong and Jiangsu of China. Phylogenetic analysis showed that swine HEV isolates belong to genotype 4, including subgenotype 4h in Henan and 4d in Beijing and Shandong. The rabbit HEV strains shared 93%-99% nucleotide sequence identity with rabbit strains isolated from Inner Mongolia. In conclusion, swine and rabbits have been confirmed to be the main reservoirs of HEV in China.

Hepatitis E virus genotype 4 isolated from a patient with liver failure: full-length sequence analysis showing potential determinants of virus pathogenesis.

The full-length genome sequence of a genotype 4 strain of Hepatitis E virus (HEV) (CHN-NJ-H2011) from a patient (in Nanjing, China) with liver failure has been determined. Phylogenetic analysis showed that CHN-NJ-H2011 belongs to genotype 4, subtype 4h. Comparative sequence analysis carried out on a 301-bp fragment of ORF2 showed that CHN-NJ-H2011 shares high nucleotide sequence identity (94.3-94.7 %) with porcine viruses (ch-shsw1 and Ch-estw2) isolated in the same geographical region, pointing to the strong possibility of zoonotic transmission of HEV genotype 4. A broader comparison with other genotype 4 isolates revealed 12 unique amino acid substitutions in ORF1 and three in ORF2 that might serve as signatures of disease severity for genotype 4 HEV infection.

The biomarker N-terminal pro-brain natriuretic peptide and liver diseases.

PURPOSE: NT-proBNP has emerged as a powerful diagnostic and prognostic biomarker in heart disease. Studies showed that NT-proBNP is a sensitive biomarker for identifying patients with heart failure caused by hepatitis C virus (HCV) related myocarditis. The purpose of this study was to evaluate the correlation between the serum concentration of NT-proBNP and hepatitis virus infection/liver disease. METHODS: 223 serum samples from blood donors (aged 19~50 years old) were collected as a control group, and 644 samples were obtained from patients infected by hepatitis viruses including 493 HBV: 364 chronic hepatitis (CH), 86 hepatocellular carcinoma (HCC) and 43 liver cirrhosis (LC) and 151 HCV (85 CH, 14 HCC, 52 LC). All samples were assayed with an Elecsys immunoassay analyzer for NT-proBNP concentration. RESULTS: The mean concentration of NT-proBNP in the control group was 21.77 pg/ml and showed no significant variation with either age or gender. Both the mean value and the rate of abnormality of NT-proBNP were significantly higher for the HBV- and HCV-infected groups in comparison with the control group. The mean NT-proBNP value (380.24 pg/ml) and abnormality rate (38.41%) in the HCV group were higher than that of the HBV group. For samples from patients with HBV/HCV-related hepatic disease/pathology, the mean NT-proBNP value (517.19 pg/ml/597.18 pg/ml) were the highest in the liver cirrhosis group. CONCLUSIONS: Hepatic pathologic lesions, particularly cirrhosis, may contribute to the elevation of NT-proBNP in subjects with HBV/HCV infection.

Effect of entecavir plus Ganshuang granule on fibrosis and cirrhosis in patients with chronic hepatitis B.

OBJECTIVE: To investigate the effect of entecavir plus Ganshuang granule (, GSG) on advanced fibrosis and cirrhosis in patients with chronic hepatitis B virus infection. METHODS: One hundred thirty-five patients were randomly assigned to one of two cohorts: GSG cohort (n = 69) or placebo cohort (n = 66). The GSG cohort received entecavir plus GSG and the placebo cohort received entecavir plus placebo for 48 weeks. Liver biopsy was performed at baseline and between weeks 44 and 48 during this placebo-controlled trial. We assessed histological improvement (greater than a two-point decrease using the Knodell in fl ammatory score and no worsening of the Ishak fibrosis score) and fibrosis regression (a decrease of at least one point in the Ishak fibrosis score). RESULTS: There were 95.7% of patients (66/69) in the GSG cohort and 66.7% (44/66) of patients in the placebo cohort who showed necroin fl ammation improvement. The mean reduction in the Knodell necroinflammatory score was 5.1 and 2.6, respectively. There were 89.9% (62/69) of patients in the GSG cohort and 31.8% (21/66) of patients in the placebo cohort who showed at least a one-point improvement in the Ishak fibrosis score. The mean reduction in the Ishak fibrosis score was 1.7 and 0.4, respectively. CONCLUSION: Patients with advanced fibrosis and cirrhosis caused by chronic hepatitis B showed more improvement in liver histology in a shorter time after treatment with entecavir plus GSG compared with entecavir plus placebo.

Genetic characteristics and pathogenicity of human hepatitis E virus in Nanjing, China.

AIM: To investigate the genetic characteristics and pathogenicity of hepatitis E virus (HEV) and assess the potential risk factors for sporadic hepatitis E. METHODS: Sixty-two serum samples from the patients with acute hepatitis E were collected, including 23 cases coinfected with hepatitis B virus. Anti-HEV detection and partial HEV RNA amplification were performed by enzyme immunoassays and reverse transcription-nested polymerase chain reaction (RT-nPCR) method, respectively, and PCR products were sequenced. The isolated human HEV sequences were analyzed phylogenetically. RESULTS: The positive rate of serum HEV RNA were 21.0% (13/62), including 5 cases of liver failure. All the 13 isolates shared a 82.1%-98.0% nucleotide homology with each other and had identities of 74.7%-81.0%, 75.3%-78.6%, 75.3%-80.0% and 82.1%-96.1% with the corresponding regions of HEV genotypes 1-4, respectively. The human HEV strain GS-NJ-12 shared a 100% nucleotide identity with the swine HEV strain swIM6-43 isolated from Inner Mongolia, China. CONCLUSION: Swine may be a principal risk factor for occurrence of sporadic hepatitis E in eastern China, and genotype 4 HEV can induce acute liver failure.

Phylogenetic analysis of the full genome of rabbit hepatitis E virus (rbHEV) and molecular biologic study on the possibility of cross species transmission of rbHEV.

Rabbit HEV strains were isolated in China from farmed rabbits, which may be a reservoir of a novel genotype of HEV. However, there is little information regarding host range and zoonotic potential of rabbit HEV. A previous study from this group identified 25 specific nucleotide positions as possibly being involved in specifying the host range of HEV and/or in determining the severity of hepatitis E disease, whereas this previous analysis did not extend to the new rabbit HEVs. Consequently the complete genome of the rabbit HEV strain ch-bj-n1 was amplified as overlapping fragments using a combination of reverse-transcription-nested polymerase chain reaction and rapid amplification of cDNA ends. The resulting sequence together with two other rabbit HEVs were compared with 91 other HEV isolates representative of genotypes 1-4. Phylogenetic analysis showed that the novel rabbit HEV strain should be classified into a new HEV genotype which has a closer evolutionary relationship with genotype 3 than genotypes 1, 2 and 4, with 20 of the 25 specific nucleotide positions identified as being present in rabbit HEV genome. Alpha-helix, hydrophilicity, and surface probability plots together with antigenic index analysis of the ORF2 protein were all undertaken to investigate the antigenicity of rabbit HEV. The alpha-helix plot and antigenicity index of the rabbit ORF2 protein was nearly identical to that of genotype 1 HEV isolates from human. Furthermore, 20 possible host range determinants in rabbit HEV were identical to those isolates from zoonotic groups, which may suggest a potential possibility of cross species transmission of rabbit HEV.

Analysing complete genome sequence of swine hepatitis E virus (HEV), strain CHN-XJ-SW13 isolated from Xinjiang, China: putative host range, and disease severity determinants in HEV.

Hepatitis E is a worldwide public health problem, particular in areas where hygiene conditions are poor. Hepatitis E virus (HEV) has at least four genotypes: genotypes 1 and 2 exclusively infect human; while genotypes 3 and 4, are considered to be a zoonotic agent, infecting both humans and animals. This study was aimed at determining why genotype 3 and 4 HEV strains isolated from swine are able to cross species borders, whereas genotype1 and 2 strains isolated from humans are not. The full length genome of the swine HEV isolate CHN-XJ-SW13 was amplified as overlapping fragments using reverse-transcription-nested polymerase chain reaction (RT-nPCR) and rapid amplification of cDNA ends (RACE). The sequence of CHN-XJ-SW13 was compared with those of 90 HEV strains covering genotype 1-4 retrieved from GenBank. Possible regions of the viral genome, specifying the host range of HEV or associated with the severity of hepatitis E disease, were then screened for with the aid of the ALIGNX sequences alignment software package. The CHN-XJ-SW13 swine HEV isolate was determined to be a novel subtype of genotype 4, whose sequence provided several valuable clues for tracing the sources of human HEV infection. 25 specific nucleotide positions were identified to possibly being involved specifying the host range of HEV or determining the severity of hepatitis E disease.

Hepatitis E virus infection among animals and humans in Xinjiang, China: possibility of swine to human transmission of sporadic hepatitis E in an endemic area.

Hepatitis E is a worldwide public health problem, especially in areas with poor sanitation. This study examines the potential hepatitis E virus (HEV) animal reservoirs and the current status of HEV infection among animals and humans in an endemic area of Xinjiang, China. One thousand five hundred twenty-one serum samples from 12 different animal species and 296 sera from humans were detected for anti-HEV with an in-house enzyme immunoassay, and partial HEV RNA was amplified with a reverse transcription-nested polymerase chain reaction (RT-nPCR). All these distinct animal species, except jerboa and hoptoad, were positive for anti-HEV. However, HEV RNA was only amplified from pigs and a sporadic hepatitis E case in humans. The human HEV strain (CHN-XJ-HE29) shared 100% nucleotide identity with the swine HEV strain (CHN-XJ-SW50), both of which were collected from the same district; this indicates the possibility of HEV transmission from swine to humans in an endemic area.

[Hepatitis E virus (HEV) genotype and the prevalence of anti-HEV in 8 species of animals in the suburbs of Beijing].

OBJECTIVE: To investigate the prevalence of anti-hepatitis E virus (HEV) and genotypes of hepatitis E virus in 8 species of animals including swine, cattle, sheep, horse, donkey, dog, chicken and duck in the suburb of Beijing. METHODS: Serum samples were collected from the 8 species of animals, and fecal samples of younger swine were collected from 2 stock farms. Anti-HEV was detected by Double Antigen Sandwich Assay. HEV RNA from fecal samples was detected by a reverse transcription nested polymerase chain reaction (RT-nPCR). Parts of the PCR products were cloned and sequenced. The swine HEV sequences were analyzed genetically. RESULTS: The positive rates of anti-HEV in serum specimens of swine, cattle, horse, donkey, sheep, dog, duck and chicken were 80.43% (481/598), 15.02% (52/346), 14.29% (40/280), 0 (0/26), 9.88% (33/334), 0 (0/21), 3.03% (7/231) and 2.53% (8/316), respectively. The anti-HEV prevalence of adult swine (>/= 6 months) and younger swine ( < / = 3 months) were 87.86% (369/420) and 62.92% (112/178) respectively. 74 of 111 (66.67%) pig faces were positive for HEV RNA. Sequence analysis on these positive samples showed that there were 6 groups of HEV designated as bjsw1, bjsw2, bjsw3, bjsw4, bjsw5 and bjsw6. The 6 strains of HEV shared 94.5% - 99.6% sequence identity of partial HEV ORF2 nucleotide with each other. The identities of HEV ORF2 nucleotide sequences between the 6 strains and genotype 1, 2, 3 and 4 were 75.6% - 78.6%, 75.6% - 76.2%, 77.1% - 80.7% and 83.7% - 94.5%, respectively. The sequence identity between the 6 strains and human HEV genotype 4d was 90.0% - 94.5%. CONCLUSION: HEV infection was seen in swine, cattle, horse, sheep, duck and chicken in the suburbs of Beijing. The anti-HEV positive rate appeared the highest in swine and the lowest in dog and donkey. The six strains of HEV isolated from younger swine belonged to genotype 4d.

Zoonotic risk of hepatitis E virus (HEV): A study of HEV infection in animals and humans in suburbs of Beijing.

AIM: To investigate hepatitis E virus (HEV) infection among different animals and workers in pig farms and slaughterhouses, and analyze the genotype of HEV isolated in this study. METHODS: Serum samples were collected from adult swine, cows, sheep, younger swine (< 3 months), and workers in pig farms and slaughterhouses (professional group). Fecal samples were collected from younger swine in the south suburbs of Beijing. Anti-HEV antibody was evaluated by direct sandwich enzyme immunoassay. HEV RNA was extracted from fecal samples and amplified by nested reverse transcription polymerase chain reaction (RT-nPCR). The PCR products were sequenced, and the sequence homology and phylogenetics of the HEV strains isolated from swine were analyzed. RESULTS: The anti-HEV positivity rates in adult swine, cows, sheep, younger swine, professional group and general population were 98.23% (222/226), 29.35% (54/184), 9.80% (20/207), 60.73% (99/164), 42.51% (105/247) and 20.29% (522/2572), respectively. The HEV RNA positivity rate of fecal samples was 22.89% (19/83) and 16/19 samples were positive for HEV RNA amplified with both primers, HEV open reading frame (ORF)1 and HEV ORF2. Sequence analysis of these 16 samples showed that there were two groups, designated BJ-1 and BJ-2. The nucleotide homology of BJ-1 and BJ-2 was 99%. Phylogenetic analysis indicated that both of these groups belonged to genotype 4d. CONCLUSION: Workers in pig farms and slaughterhouses were more likely to contract HEV infection than the general population because of close contact with swine with a high prevalence of anti-HEV.