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.

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.

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.

Construction and expression of eukaryotic plasmids containing lamivudine-resistant or wild-type strains of Hepatitis B Virus genotype C.

AIM: To construct eukaryotic expression plasmids of full-length Hepatitis B Virus (HBV) genotype C genome, which contain lamivudine-resistant mutants (YIDD, YVDD) or wild-type strain (YMDD), and to observe the expression of HBV DNA and antigens [hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg)] of the recombinant plasmids in HepG2 cells. METHODS: Three HBV full-length genomes were amplified from the plasmids pMD18T-HBV/YIDD, pMD18T-HBV/YVDD and pMD18T-HBV/YMDD, using PCR. Three recombinant plasmids were generated by inserting each of the PCR products into the eukaryotic expression vector pcDNA3.1 (+), between the EcoRI and HindIII sites. After being characterized by restriction endonuclease digestion, and DNA sequence analysis, the recombinant plasmids were transfected into HepG2 cells. At 48 and 72 h post-transfection, the levels of intracellular viral DNA replication were detected by real-time PCR, and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA. RESULTS: Restriction endonuclease digestion and DNA sequence analysis confirmed that the three recombinant plasmids were correctly constructed. After transfecting the plasmids into HepG2 cells, high levels of intracellular viral DNA replication were observed, and HBsAg and HBeAg were secreted into the cell culture supernatant. CONCLUSION: Eukaryotic expression plasmids pcDNA3.1 (+)-HBV/YIDD, pcDNA3.1 (+)-HBV/YVDD or pcDNA3.1 (+)-HBV/YMDD, which contained HBV genotype C full-length genome, were successfully constructed. After transfection into HepG2 cells, the recombinant plasmids efficiently expressed HBV DNA, HBsAg and HBeAg. Our results provide an experimental basis for the further study of HBV lamivudine-resistant mutants.

Combination therapy of siRNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cell.

BACKGROUND/AIMS: Hepatitis B virus (HBV) infection is a world-wide health problem. The major obstacles for current anti-HBV therapy are the low efficacy and the occurrence of drug resistant HBV mutations. Recent studies have demonstrated that combination therapy can enhance antiviral efficacy and overcome the shortcomings. Here, the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of HBV nuclear localization signal (NLS) was monitored in HepG2.2.15 cells. METHODOLOGY: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. At 48, 72 and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. Intracellular viral DNA and covalently closed circular DNA (cccDNA) was quantified by real-time PCR. HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR. RESULTS: Our data demonstrated that three used siRNAs showed marked anti-HBV effects. The expression of HBsAg and the replication of HBV DNA could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication, even though the final concentration of siRNA in the therapy was the same. More importantly, we showed that combination therapy significantly suppressed HBV cccDNA amplification. CONCLUSION: Our results revealed that combination of siRNAs mediated a stronger inhibition on viral replication and antigen expression in HepG2.2.15 cells, especially, the amplification of cccDNA.

Combination of small interfering RNA and lamivudine on inhibition of human B virus replication in HepG2.2.15 cells.

AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression by combination of siRNA and lamivudine in HepG2.2.15 cells. METHODS: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. The transfected cells were cultured in lamivudine-containing medium (0.05 micromol/L) and harvested at 48, 72 and 96 h. The concentration of HBeAg and HBsAg was determined using ELISA. HBV DNA replication was examined by real-time PCR and the level of HBV mRNA was measured by RT-PCR. RESULTS: In HepG2.2.15 cells treated with combination of siRNA and lamivudine, the secretion of HBeAg and HBsAg into the supernatant was found to be inhibited by 91.80% and 82.40% (2.89+/-0.48 vs 11.73+/-0.38, P<0.05; 4.59+/-0.57 vs 16.25+/-0.48, P<0.05) at 96 h, respectively; the number of HBV DNA copies within culture medium was also significantly decreased at 96 h (1.04+/-0.26 vs 8.35+/-0.33, P<0.05). Moreover, mRNA concentration in HepG2.2.15 cells treated with combination of siRNA and lamivudine was obviously lower compared to those treated either with siRNA or lamivudine (19.44+/-0.17 vs 33.27+/-0.21 or 79.9+/-0.13, P<0.05). CONCLUSION: Combination of siRNA and lamivudine is more effective in inhibiting HBV replication as compared to the single use of siRNA or lamivudine in HepG2.2.15 cells.

Effects of tacrolimus on infection of Friend murine leukemia virus to Fv-4 gene heterozygous mice.

AIM: To investigate the effect of tacrolimus (FK506) on the infection of Friend murine leukemia virus (Friend MuLV) in vivo. METHODS: Three kinds of mice were used including Friend MuLV-sensitive BALB/c mice, Friend MuLV-resistant Fv-4 gene-homozygous mice (Fv-4 mice), and Friend MuLV-resistant Fv-4 gene-heterozygous mice (F1 mice). Tacrolimus was administrated i.p. to those mice in every 2 d. Those treated mice were inoculated i.p. with Friend MuLV once on d 3. The symptoms and viral proliferations in those mice were observed to recognize the Friend MuLV infection. The expression and genotype of Fv-4 gene that resistant against the infection of Friend MuLV were analyzed to confirm the genomic background and related mechanism of the resistance. RESULTS: BALB/c mice and F1 mice, but not Fv-4 mice, appeared obvious early death, spleenomegaly, and viral proliferation after both treatments of viral inoculation and tacrolimus administration, whereas the expression and genotype of Fv-4 gene was not changed in F1 mice and Fv-4 mice with treatment of tacrolimus. Compared to the virus-inoculated control, the Friend MuLV-sensitivity of tacrolimus-treated BALB/c mice and the Friend MuLV-resistance of tacrolimus-treated Fv-4 mice were the same as the controls, but only F1 mice became the symptoms and viral proliferation after both treatments. It suggested the Friend MuLV-resistant F1 mice could be converted to be Friend MuLV-sensitive by treatment of tacrolimus, and this conversion was not depended on the expression and genotype of Fv-4 gene. CONCLUSION: Tacrolimus could not inhibit the infection of Friend MuLV in all mice, furthermore, it could enhance the infection of Friend MuLV in F1 mice. The enhancement may be related to the immunosuppressive effect of tacrolimus.

[Expressions of TNF-alpha and IL-1beta in human ischemic brain tissues].

AIM: To detect the expression of TNF-alpha and IL-1beta in human cerebral ischemic tissues. METHODS: 13 cerebral specimens from patients died of cerebral infarction were divided into three groups, <2 d, 3-5 d, and >5d, according to the lasting time of infarctions. The expression of TNF-alpha and IL-1beta in the cerebral ischemic tissues were examined by immnohistochemical staining. The contralateral tissues were employed as controls. RESULTS: The expression of TNF-alpha and IL-1beta in the cerebral ischemic tissues were significant higher than those in the contralateral tissues. The focal distribution of the TNF-alpha(+) and IL-1beta(+) cells was identical with the ischemic area. The expression of IL-1beta and TNF-alpha peaked at the 3rd to 5th and 2nd day after ichemia, respectively. There were no significant difference between the ischemic and contralateral brains at the 5th day after ischemia for the expression of TNF-alpha and IL-1beta. CONCLUSION: Our results showed the expression of TNF-alpha and IL-1beta in human strok of infarction were similar to those in animal experiments. It is suggested that TNF-alpha and IL-1beta are involved in cerebral ischemic injury, which will be helpful for developing clinically a novel therapy aiming at cerebral ischemic injury.