Jing-Xia Zhang

Increased Th17 cells contribute to disease progression in patients with HBV‐associated liver cirrhosis

Summary.  T helper (Th) 17 cells have been demonstrated to participate in the pathogenesis of HBV‐associated liver damage. However, little is known regarding the immunopathogenic role of liver fibrosis in patients with HBV‐associated liver cirrhosis. The aims of this study were to evaluate whether Th17 cells are related to disease progression in patients and to explore the possible mechanisms. The frequencies of circulating Th17 cells were analysed in 78 patients with hepatitis B and cirrhosis (Child A: 34; Child B: 22; Child C 22) and matched controls. Liver samples were collected from 13 patients with HBV‐associated cirrhosis, 23 patients with chronic hepatitis B and 12 healthy controls for immunohistochemical analysis. IL‐17 receptor expression was studied on liver biopsies and in human hepatic stellate cells as well as their response to recombinant IL‐17 by flow cytometry. Patients with hepatitis B‐associated cirrhosis with more severe disease displayed significant increases in peripheral numbers of Th17 cells as well as in IL‐17 plasma levels. The increased intrahepatic IL‐17+ cells correlated positively with fibrotic staging scores and clinical progression from CHB to cirrhosis. Moreover, many IL‐17+ cells were located in fibrotic areas in the liver of patients with cirrhosis. In vitro, IL‐17 together with IL‐17‐activated monocytes, could promote the activation of stellate cells, which, in turn, aggravated liver fibrosis and the inflammatory response. In summary, increased peripheral and intrahepatic Th17 cells are enriched in patients with hepatitis B and cirrhosis and contribute further to the severity of disease progression through induction of stellate cell activation.

Occult Hepatitis B Virus Infection in Anti-HBs-Positive Infants Born to HBsAg-Positive Mothers in China

Objective To investigate the prevalence of occult HBV infection (OBI) among children and to characterize virology of occult HBV, we conducted an epidemiological survey. Methods 186 HB-vaccinated infants born to HBsAg-positive mothers were included in the study. Serological tests for HBV markers were performed using commercial ELISA kits. Real-time quantitative PCR and nested PCR were used to detect HBV DNA. PCR products of the C and pre-S/S regions were sequenced and analyzed. Results 1.61% (3/186) infants were HBsAg positive, and 4.92% (9/183) infants were considered as occult infection. The viral load of mothers was associated with occult infection (P = 0.020). Incomplete three-dose injections of HB vaccine was associated with HBV infection (P = 0.022). Six OBI infants were positive for anti-HBs, but their titers were not greater than 100 mIU/mL. Seven isolated HBV pre-S/S sequences were obtained from nine OBI infants. Three of the sequences were genotype C, and four of the sequences were genotype C/D. Escape mutation S143L was found in the four sequences of genotype C/D. All seven sequences lacked G145R and other escape mutation in S region. Conclusions Occult HBV infection was detected in anti-HBs positive infants born to HBsAg-positive mothers in China. Occult infection was associated with absent anti-HBs or with low anti-HBs level, high maternal viral loads and escape mutations in the S gene.

Association between genomic heterogeneity of hepatitis B virus and intrauterine infection.

Hepatitis B virus (HBV) intrauterine infection remains to be an important cause for a large number of persistent hepatitis B surface antigen (HBsAg) positive carriers in areas with a high HBV prevalence, particularly in China and Southeast Asia. In this study, the possible association between the HBV genomic heterogeneity and intrauterine infection was investigated by comparing the quasi species isolated from eight pairs of HBsAg-positive mothers and their neonates, who were infected intrauterinely with HBV, with clones from eight HBsAg-positive mothers whose neonates were not infected with HBV. The proportion of clones with specific mutations was compared among different subject groups, and phylogenetic analysis was performed to evaluate the significance of specific mutations. It was observed that the core promoter with conserved major functional regions and conserved hepatitis B e antigen (HBeAg) might be beneficial to HBV maternal-fetal transmission. Particularly, A1762T/G1764A mutations seemed to be disadvantageous for fetal infection. It was also shown that amino acid substitutions located in the immune epitopes of HBsAg were strongly associated with intrauterine HBV transmission. The clones with mutations such as amino acid P110S in preS1 region, P36L in preS2 region and C107R in S region might infect fetuses more readily. In addition, positively selected site analysis confirmed the above results.

Epidemiology of Hepatitis C Virus Infection in Highly Endemic HBV Areas in China

Background Wuwei City has the highest prevalence of hepatitis B virus (HBV) in China. From 2007 to 2011, the average reported incidence rate of hepatitis B was 634.56/100,000 people. However, studies assessing the epidemic features and risk factors of HCV in the general population of Wuwei City are limited. Methods A total of 7189 people were interviewed and screened for HCV antibodies. HCV RNA and HCV genotypes were analyzed by PCR. Relevant information was obtained from the general population using a standardized questionnaire, and association and logistic regression analyses were conducted. Results The anti-HCV prevalence was 1.64% (118/7189), and HCV-RNA was detected in 37.29% (44/118) of the anti-HCV positive samples. The current HCV infection rate was 0.61% (44/7189) in the Wuwei general population. Hepatitis C infection rate was generally higher in the plains regions (χ2 = 27.54,P<0.05), and the most predominant HCV genotypes were 2a (59.1%) and 1b (34.1%). The concurrent HCV and HBV infection rate was 1.37%, and a history of blood transfusion (OR = 17.9, 95% CI: 6.1 to 52.6, p<0.001) was an independent risk factor for HCV positivity. Conclusions Although Wuwei is a highly endemic area for HBV, the anti-HCV positive rate in the general population is low. More than one-third of HCV-infected people were unaware of their infection; this may become an important risk factor for hepatitis C prevalence in the general population. Maintaining blood safety is important in order to help reduce the burden of HCV infection in developing regions of China.

Establishment and characterization of a spontaneously immortalized trophoblast cell line (HPT-8) and its hepatitis B virus-expressing clone

BACKGROUND Most trophoblast cell lines currently available to study vertical transmission of hepatitis B virus (HBV) are immortalized by viral transformation. Our goal was to establish and characterize a spontaneously immortalized human first-trimester trophoblast cell line and its HBV-expressing clone. METHODS Chorionic villi of Asian human first-trimester placentae were digested with trypsin and collagenase I to obtain the primary trophoblast cell culture. A spontaneously immortalized trophoblast cell line (HPT-8) was analyzed by scanning and transmission electron microscopy, cell cycle analysis, immunohistochemistry and immunofluorescence. HPT-8 cells were stably transfected with the adr subtype of HBV (HPT-8-HBV) and characterized by PCR and enzyme-linked immunosorbent assay. RESULTS We obtained a clonal derivative of a spontaneously immortalized primary cell clone (HPT-8). HPT-8 cells were epithelioid and polygonal, and formed multinucleate, giant cells. They exhibited microvilli, distinct desmosomes between adjacent cells, abundant endoplasm, lipid inclusions and glycogen granules, which are all characteristic of cytotrophoblasts. HPT-8 cells expressed cytokeratin 7, cytokeratin 18, vimentin, cluster of differentiation antigen 9, epidermal growth factor receptor, stromal cell-derived factor 1 and placental alkaline phosphatase. They secreted prolactin, estradiol, progesterone and hCG, and were positive for HLA-G, a marker of extravillous trophoblasts. HPT-8-HBV cells were positive for HBV relaxed-circular, covalently closed circular DNA and pre-S sequence. HPT-8-HBV cells also produced and secreted HBV surface antigen and HBV e antigen. CONCLUSIONS We established a trophoblast cell line, HPT-8 and its HBV-expressing clone which could be valuable in exploring the mechanism of HBV viral integration in human trophoblasts during intrauterine infection.

Recombination and Natural Selection in Hepatitis E Virus Genotypes

To gain new insights into the evolutionary processes that created the genetic diversity of the hepatitis E virus (HEV), the Recombination Detection Program (RDP) and SimPlot program were employed to detect recombination events in the genome, then the fixed‐effects likelihood (FEL) method was used to detect natural selection effects on viral proteins. Recombination analysis provided strong evidence for both intergenotype and intragenotype recombination events in the sequences analyzed. Recombination events were found to be distributed non‐randomly, with the highest frequency in the X domain and the helicase. Strain DQ450072 was identified as intergenotype‐recombinant. Natural selection analysis revealed that codons under both negative selection and positive selection were distributed non‐randomly. ORF1 and ORF2 have experienced strong purifying selection across genotypes. Furthermore, potentially important sites were also found under positive selection in the N‐terminal end of ORF2 and the C‐terminal end of ORF3. No significant difference was found among the selective pressures on different genotypes. J. Med. Virol. 84:1396–1407, 2012.

Epidemiological Screening for Hepatitis E Virus in Bile Specimens from Livestock in Northwest China

Hepatitis E has been hypothesized as a zoonosis. However, there is no definite conclusion about which animal species contribute to hepatitis E virus (HEV) infection in humans. In this study, HEV RNA was detected only in swine bile specimens and not in bile specimens collected from cattle, goats, or dogs. We postulate that swine are the main animal reservoir for HEV.

High conservation of hepatitis B virus surface genes during maternal vertical transmission despite active and passive vaccination.

Objective: Our purpose was to explore the relationship between hepatitis B virus (HBV) gene heterogeneity and maternal vertical transmission. Methods: HBsAg-positive mothers and their neonates were selected and classified into a vertical infection neonate group (group N), a vertical infection mother group (group M) and a control group (group C). Serum HBsAg and HBeAg were examined. HBV gene fragments, including the pre-S1, and pre-S2 and S coding regions, were amplified and sequenced, and the genotype and serotype of the sequences were identified. Mutation sites and frequency of mutations were then compared between group N and group C. Results: A total of 104 HBV clone sequences were obtained. All obtained sequences belonged to genotype C and serotype adr. Upon comparing sequences between group N and group C, 4 nonsynonymous mutations were found with significant difference in mutation frequency (p < 0.05). When the mothers were both HBsAg and HBeAg positive, 10 nonsynonymous mutations were found. The frequencies of these mutations were significantly lower in group N than in group C (p < 0.05). Conclusion: The 10 HBV mutations were negatively associated with vertical transmission when maternal HBeAg was positive. Furthermore, the species that were vertically transmitted to the fetus were mainly wild-type.

The risk factors of transmission after the implementation of the routine immunization among children exposed to HBV infected mothers in a developing area in northwest China.

We aimed to evaluate the present situation and possible risk factors of HBV transmission after the implementation of the routine immunization among children exposed to HBV infected mothers in a developing area in northwest China. Two hundred and twenty one HBsAg carrier mothers and 247 children born to them were finally recruited in Wuwei city, Gangsu province, China in 2010. Serum samples were taken from those HBsAg carrier mothers and their children. Children who had detectable HBsAg or HBV DNA were considered to be HBV infection. Conditional logistic regression model was used to identify potential risk factors of HBV mother-to-child transmission. Of the 247 children born to HBsAg carrier mothers, 8 (3.24%) were HBsAg positive, 15 (6.07%) were HBV DNA positive. The rate of HBV mother-to-child transmission was 7.29% (18/247). The univariate analysis and multivariate analysis showed that maternal HBV DNA positive (OR=4.83, 95% CI: 1.38-16.98, p=0.0140), the delayed injection of the first dose of HBV vaccine after premature birth (OR=9.73, 95% CI: 1.78-53.21, p=0.0087) and the missing use of HBV vaccine (OR=8.29, 95% CI: 1.42-48.23, p=0.0186) were significantly associated with an increased risk for HBV mother-to-child transmission. The rate of HBV infection of the children received HBV vaccine and HBIG together after birth (2.56%, 4/156) was lower than those children received HBV vaccine alone (11.39%, 9/79) (χ(2)=7.83, p=0.0052). In conclusion, the rate of mother-to-child transmission of HBV was still high in the northwest of China. Besides the positivity of maternal HBV DNA and the missing of HBV vaccination after birth, the delayed injection of the first dose of HBV vaccine after premature birth was also a possible independent risk factor for HBV mother-to-child transmission. The HBV prevention and treatment guidelines should make it clear that all of the new born infants need to receive HBV vaccine injection after birth in 24 h, including the premature infants.

Epidemiological characteristics of the carriers with coexistence of HBsAg and anti-HBs based on a community cohort study.

The coexistence of HBsAg and anti‐HBs is an atypical serological pattern in HBV infection. There is no epidemiological characteristics of this serological pattern in the community and there is controversy over the molecular mechanisms underlying this pattern. We investigated the epidemiological characteristics of the carriers with HBsAg and anti‐HBs in a longitudinal community cohort study. The prevalence of this atypical serological pattern was 2.93% (122/4169) in HBsAg‐positive populations. The prevalence progressively increased with age from 40 to 70 years old. The rate of HBeAg positive and detectable HBV DNA were both significantly higher in carriers with this pattern than in carriers who were HBsAg positive but anti‐HBs negative (26/122 verse 598/4047, P = 0.046; 86/122 verse 275/529,P < 0.001). After 1 year of follow‐up, 85.19% of the carriers still had coexistence HBsAg and anti‐HBs, 14.81% of the carriers lost their anti‐HBs. Viral sequencing showed that carriers with coexistence of HBsAg and anti‐HBs had higher numbers of residue changes within the S gene than carriers who were HBsAg positive but anti‐HBs negative (2.42 verse 1.33 changes per 100 residues, P < 0.05). Hence, the coexistence of HBsAg and anti‐HBs is a unique serological pattern which may be associated with an increased risk of adverse clinical outcome and may be related to HBsAg immune variants which have genotypic heterogeneity.