Wei-Zhen Xu

Prevalence of occult hepatitis B virus infection among hepatopathy patients and healthy people in China

OBJECTIVE To investigate the prevalence of occult hepatitis B virus (HBV) infection among hepatopathy patients and healthy people in China. METHODS The HBV DNA in 653 sera samples collected from cryptogenic chronic liver disease patients (159), hepatitis B surface antigen (HBsAg) negative hepatocellular carcinoma (HCC) patients (135) and HBsAg-negative healthy people (359) were tested by nested PCR using specific primers of the X region of the HBV genome. We performed real-time PCR to determine the levels of serum HBV-DNA. RESULTS Prevalence of occult HBV infection was 28.3% (45/159), 70.4% (95/135) and 10.6% (38/359) in cryptogenic chronic liver disease patients, HBsAg-negative HCC patients and HBsAg-negative healthy people, respectively. The prevalence of occult HBV infection in IgG anti-HBc-positive subjects was 100% (45/45), 86.7% (85/98) and 33.3% (14/42) in cryptogenic chronic liver disease patients, HBsAg-negative HCC patients and HBsAg-negative healthy people, respectively. In all cases, viral loads were low (<10(4)viral copies/mL). CONCLUSION The prevalence of occult HBV infection was significantly high among hepatopathy patients and healthy people in China. Thus, more meticulous attention should be given to prevent HBV transmission by blood transfusion or organ transplantation in endemic areas, and further studies on clinical implication and mechanism of occult HBV infection are required.

Molecular characterization and functional analysis of occult hepatitis B virus infection in Chinese patients infected with genotype C

Occult HBV infection is defined as the persistence of HBV DNA in individuals negative for HBV surface antigen (HBsAg), and many different mechanisms have been reported in different countries. However, in China, one of the endemic areas for HBV infection, no reports have been published on occult HBV infection. The present study investigated the virological features and the mechanism of occult HBV infection in China. Full‐length HBV DNA from eight patients with occult HBV infection (S1–S8) and three HBsAg‐positive cases (SWT1–SWT3) was cloned and sequenced. Additionally, four entire linear HBV genomes from occult cases were transfected transiently into HepG2 cells. The sequencing results showed two major mutations in patients with occult HBV infection as follows: deletions in the pre‐S1 (S3, S4, and S7) and X (S1, S2, and S5) regions. Such deletions covered the S promoter and the basal core promoter (BCP), and function analysis of these variants also showed a decrease in DNA replication and antigen expression. Two patients with occult infection (S6 and S8) had no mutations capable of interfering with viral replication and gene expression in the major viral population. Thus, the deletions in the S promoter and the BCP regions that disable the regulatory elements may be the reason for the absence of HBsAg, and multiple mechanisms may be responsible for occult HBV infection. J. Med. Virol. 81:826–835, 2009.

VIRAL DELETIONS AMONG HEALTHY YOUNG CHINESE ADULTS WITH OCCULT HEPATITIS B VIRUS INFECTION

To investigate the mechanism and prognosis of occult hepatitis B virus (HBV) infection (OBI) at a molecular level among healthy young adults, the presence of HBV DNA in 1176 sera samples collected from healthy young people after neonatal vaccination was assessed by nested polymerase chain reaction (PCR) using specific primers designed for the X and S regions of the HBV genome. Full-length HBV DNA from 9 patients with OBI (OB1-OB9) was cloned and sequenced. Deletions in the pre-S, basal core promoter (BCP), core (C) and polymerase (P) regions were observed. The data indicate that there is still a substantial risk of OBI in China despite neonatal vaccination. All deletions that were observed in the pre-S, BCP, C and P regions play a direct or indirect role in OBI. The presence of a deletion mutation in the pre-S1 region was considered to play a pivotal role in hepatocarcinogenesis and was found to increase the risk of hepatocellular carcinoma in the cohorts studied.

Altered mRNA levels of MOV10, A3G, and IFN-α in patients with chronic hepatitis B.

To explore the relationship of the MOV10, A3G, and IFN-α mRNA levels with chronic hepatitis B virus (HBV) infection, Blood samples from 96 patients with chronic hepatitis B (CHB) and 21 healthy individuals as control were collected. HBV DNA load and aminotransferase in the serum were tested using real time PCR and velocity methods, respectively. The MOV10, A3G, and IFN-α mRNA levels in the peripheral blood mononuclear cells (PBMC) were examined through qRT-PCR. The MOV10, A3G, and IFN-α mRNA levels in CHB group was significantly lower than those in the control group (P<0.01, P<0.05, P<0.01, respectively). The A3G mRNA level in the high-HBV DNA load group was lower than that in the low-HBV DNA load group (P<0.05). However, no statistical difference was found in the MOV10 and IFN-α mRNA levels between the two HBV DNA load groups. Furthermore, the MOV10 mRNA level showed positive correlation with IFN-α in the control group. These results indicated that the expression of the innate immune factors MOV10, A3G, and IFN-α is affected by chronic HBV infection.

Differential inhibition of lamivudine-resistant hepatitis B virus by allele-specific RNAi.

Drug-resistant hepatitis B virus (HBV) is a serious problem affecting antiviral therapy. In this study, two long-term eukaryotic cell lines with full-length HBV were constructed and contained either lamivudine-resistant mutants (HBV-YIDD) or wild-type virus (HBV-wt). High levels of intracellular viral DNA replication were observed continuously after transfecting the plasmids into HepG2 cells, and HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) were secreted into the cell culture supernatant. A series of experiments showed differential inhibition of HBV gene expression and replication by four specific siRNAs, according to the principles of allele-specific RNAi technology. The results showed that the designed siRNAs with a mismatch in the sixteenth nucleotide of the guide strands could effectively discriminate the HBV-YIDD mutants from the wild-type alleles, thus providing a new insight into the development of antiviral therapy with differing or complementary patterns characteristic of lamivudine-resistant HBV.

The role of Moloney leukemia virus 10 in hepatitis B virus expression in hepatoma cells.

Recent studies have shown that the Moloney leukemia virus 10 (Mov10), a putative RNA helicase, has very broad and potent antiretroviral activities. Hepatitis B virus (HBV) has a reverse transcription process, but the potential role of Mov10 in HBV replication remains unknown. In this study, Mov10 was demonstrated to affect HBV expression in HepG2 and HepG2.2.15 cell lines. The data showed that the over-expression of exogenous Mov10 resulted in an increase of the HBsAg and HBeAg levels in the culture supernatant and HBV mRNA level in transfected cells at a low dose and resulted in a decrease at a high dose, but HBV DNA in culture supernatant was not affected. The knockdown of endogenous Mov10 expression through siRNA treatment could suppress levels of HBsAg, HBeAg and HBV mRNA, but had no effect on HBV DNA. Above results indicate that an appropriate level of exogenous Mov10 is responsible for HBV replication, that any perturbation in the level of Mov10 could affect HBV replication, while the endogenous Mov10 could promote HBV replication in vitro. The precise mechanisms that underlie the action of Mov10 on HBV still need further investigation.

Characterization of two new monoclonal antibodies against human papillomavirus type 16 L1 protein

BackgroundHuman papillomavirus type 16 (HPV16) infection is implicated in cervical carcinogenesis. This study aimed to characterize two new monoclonal antibodies (mAbs) against HPV L1 protein.MethodsThe immunocompetence of AE3 and AG7 mAbs for HPV L1 protein was evaluated by Western blot analysis, immunostaining, hemagglutination inhibition assay, and ELISA. The heavy chain variable region (VH) and light chain variable region (VL) of AE3 and AG7 mAbs were sequenced and analyzed.ResultsBoth mAbs specifically recognized HPV16 L1 and virus-like particles (VLPs). Both the affinity and the titer of AE3 mAb were higher than that of AG7. There were differences in sequences in the complementary determining regions (CDR) 2 and 3 of VL, as well as in the CDR1 and CDR3 of VH. The two mAbs have distinct predicted three-dimensional structures.ConclusionsWe characterized two mAbs neutralizing antibodies for HPV L1 protein, which would help develop genetic-engineered neutralizing antibodies against HPV16 for diagnostic and therapeutic purposes.

Application of allele-specific RNAi in hepatitis B virus lamivudine resistance

Summary.  Understanding the consequences of mutation in the tyrosine–methionine–aspartate–aspartate (YMDD) motif of hepatitis B virus (HBV) genome on replication is critical for treating chronic hepatitis B with lamivudine. Allele‐specific gene silencing by RNAi (allele‐specific RNAi: ASP‐RNAi) is an advanced application of RNAi techniques. Use of this strategy as a means for specifically inhibiting an allele expression of interest suggested that it can specifically suppress the expression of alleles causing disease without inhibiting the expression of corresponding wild‐type alleles. However, no studies have used ASP‐RNAi to address the issue of HBV lamivudine resistance. In this study, we applied ASP‐RNAi into two long‐term eukaryotic cell lines of full‐length HBV containing either lamivudine‐resistant mutants (HBV‐YIDD) or wild type (HBV–WT) which we generated in previously. The designed siRNAs were also used in this eukaryotic expression system together with lamivudine. ELISA and real‐time PCR were performed to monitor virus‐specific protein synthesis and viral DNA replication. The results showed that the base substitutions conferring marked ASP‐RNAi appeared to be largely present in positions 1, 3, 6, 11, 12, 15 and 19 of the sense strand of siRNAs which were different from the most sensitive positions of this application in eukaryotes. In addition, siRNA–lamivudine combinations did not possess the prominent anti‐HBV activity we expected because of some unknown mechanisms. These findings recapitulated many of the features of ASP‐RNAi in hepadnaviruses which provided a new insight into the development of a potent strategy against HBV drug resistance.

Successful establishment and evaluation of a new animal model for studying the hepatitis B virus YVDD mutant

The treatment of infection with lamivudine-resistant mutants of hepatitis B virus (HBV) with mutations in the YMDD motif has become a crucial issue in the clinic. In this work, the plasmids pcDNA3.1 (+)-HBV/C-YVDD and pcDNA3.1 (+)-HBV/C-YMDD were constructed and injected into BALB/c mice using a hydrodynamics-based procedure to investigate viral replication and expression of HBV lamivudine-resistant YVDD mutants in vivo. Compared with the YMDD group, HBsAg levels were higher in sera of mice in the YVDD group, but HBeAg levels were lower on day 1 after injection. Levels of HBcAg in hepatocytes were higher in the YVDD group on day 1, whereas the HBsAg levels were lower. The levels of HBV mRNA in the liver were higher in mice in the YVDD group on day 1 after injection. The results showed that injection with these plasmids resulted in efficient initiation of replication of HBV in mice and also suggested that the combined mutations in YVDD mutants could affect the replication process.