Assessing hepatitis B virus infectivity in blood components following pathogen reduction using a human hepatocyte model.

Eliminating cccDNA to cure hepatitis B virus infection

A new role for an old marker, HBsAg

The incidence of hepatitis B virus (HBV) infection in dialysis populations has declined over recent decades, largely because of improvements in infection control and widespread implementation of HBV vaccination. Regardless, outbreaks of infection continue to occur in dialysis units, and prevalence rates remain unacceptably high. For a variety of reasons, dialysis patients are at increased risk of acquiring HBV. They also demonstrate different disease manifestations compared with healthy individuals and are more likely to progress to chronic carriage. This paper will review the epidemiology, modes of transmission and diagnosis of HBV in this population. Prevention and treatment will be discussed, with a specific focus on strategies to improve vaccination response, new therapeutic options and selection of patients for therapy.

Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Despite the prevalence of infection, gaining a complete understanding of the molecular mechanisms of HBV infection has been difficult because HBV cannot infect common immortalized cell lines. HepG2.2.15, however, is a well established version of the HepG2 cell line that constitutively expresses HBV. Therefore, comparative proteomics analysis of HepG2.2.15 and HepG2 may provide valuable clues for understanding the HBV virus life cycle. In this study, two-dimensional blue native/SDS-PAGE was utilized to characterize different multiprotein complexes from whole cell lysates between HepG2.2.15 and HepG2. These results demonstrate that two unique protein complexes existed in HepG2.2.15 cells. When these complexes were excised from the gel and subjected to the second dimension separation and the proteins were sequenced by mass spectrometry, 20 non-redundant proteins were identified. Of these proteins, almost 20% corresponded to heat shock proteins, including HSP60, HSP70, and HSP90. Antibody-based supershift assays were used to verify the validity of the distinct protein complexes. Co-immunoprecipitation assays confirmed that HSP60, HSP70, and HSP90 proteins physically interacted in HepG2.2.15 but not HepG2 cells. We further demonstrated that down-regulation of HSP70 or HSP90 by small interfering RNA significantly inhibited HBV viral production but did not influence cellular proliferation or apoptosis. Consistent with these results, a significant reduction in HepG2.2.15 HBV secretion was observed when the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin was used to treat HepG2.2.15 cells. Collectively these results suggest that the interaction of HSP90 with HSP70/HSP60 contributes to the HBV life cycle by forming a multichaperone machine that may constitute therapeutic targets for HBV-associated diseases.

BackgroundThe aim of this study was to investigate the correlation between the expression of hepatitis B surface antigen (HBsAg) in human ovary and placenta and the vertical transmission of hepatitis B virus (HBV).Methodology/Principal FidningsOvarian and placental tissue specimens of pregnant women infected with HBV were collected during cesarean section and immunostained for HBsAg. The sera of the corresponding newborns were tested for HBV markers and HBV DNA. HBsAg was detected in 15 out of 33 (45%) placental tissues and was further detected in capillary endothelial cells in 4 specimens (26%), of which 3 (75%) corresponding infants were infected with HBV in utero. Out of the 33 ovarian tissues, 7 (21%) were positive for HBsAg, of which 2 (28%) showed HBsAg in ovarian follicles and the 2 corresponding infants (100%) had intrauterine HBV infection.Conclusions/SignificanceHBsAg expression in cells of the ovarian follicle or placental capillary endothelium signal a higher risk for intrauterine HBV infection.

Impact of Occult Hepatitis B Virus Infection or Hepatitis B Virus DNA Integration on Efficacy of Chronic Hepatitis C Treatment With Peginterferon and Ribavirin

Pathogen inactivation

Chronic hepatitis B (CHB) puts 250 million people or more at a greatly increased risk to develop terminal liver disease.1 The causative agent, hepatitis B virus (HBV), is a small...

Genetic variability of hepatitis B virus and response to antiviral treatments: Searching for a bigger picture

Bringing to an end mother-to-child transmission of hepatitis B: A role for quantitative hepatitis B surface antigen?

Optimal design of antiviral short-interfering RNA (siRNA) targeting highly divergent hepatitis B virus (HBV) was validated by quantitative structure activity relationship (QSAR) analysis. The potency of 23 synthetic siRNAs targeting 23 sites throughout HBV pregenomic RNA were evaluated at 10 nmol/L by determining the inhibition on the expression of S/P/pregenomic mRNA and hepatitis B surface antigen (HBsAg) quantitatively in HepG2.2.15 cells. Genotype homology within HBV genomes was identified through plentiful computational analysis and the multiple linear regression analysis was made to validate the relationship between the functional siRNAs and primary characteristics. Based on the preliminary results, relationships between different determined endpoints [S/P mRNA, HBsAg, C/P mRNA, hepatitis B e antigen (HBeAg) and viral DNA load] and siRNA efficacy evaluation were investigated. Genotype homology, open reading frame (ORF) S/P, X and C had tight correlation with the ability of siRNAs on inhibiting the expression of S/P/Pregenomic mRNA and HBsAg (P<0.01), of which, ORF C was negatively correlated with the siRNA potency (P<0.05). Further study showed that siRNA potency evaluation was influenced by different determined endpoints. P-target siRNAs showed significant inhibition on the S mRNA and HBsAg expression. S-target siRNAs inhibited the expression of S mRNA and HBsAg strongly. X-target siRNAs played active roles in inhibiting all 5 determined endpoints. C-target siRNAs blocked the expression of C mRNA, HBeAg and viral DNA load significantly. The antiviral potency of siRNA was relevant to its primary characteristics and determined endpoints were important for siRNA efficacy evaluation for complex genome with overlapping ORF, which was helpful for siRNA optimal design.

While an effective vaccine against hepatitis B virus (HBV) has long been available, chronic HBV infection remains a severe global public health concern. Current treatment options have limited effectiveness, and long-term therapy is required to suppress HBV replication; however, complete elimination of the virus is rare. The lack of suitable animal models and infection systems has hindered efforts to unravel the HBV life cycle, particularly the early events in HBV entry, which appear to be highly species- and tissue-specific. Human primary hepatocytes remain the gold standard for HBV replication studies but are limited by availability and variability. While the HepaRG cell line is permissive for HBV replication, other hepatoma cell lines such as HepG2 do not support HBV replication. The recent discovery of sodium taurocholate transporting peptide (NTCP) as a primary receptor for HBV binding has led to the development of replication-competent cell lines such as HepG2-NTCP. Human hepatocytes grown in chimeric mice have provided another approach that allows primary human hepatocytes to be used while overcoming many of their limitations. Although the difficulty in developing HBV infection systems has hindered development of effective treatments, the variability and limited replication efficiency among cell lines point to additional liver-specific factors involved in HBV infection. It is hoped that HBV infection studies will lead to novel drug targets and therapeutic options for the treatment of chronic HBV infection.

Covalently closed circular (ccc) DNA of hepatitis B virus (HBV) existed in the nuclei of HBV infected hepatocytes with a half-life time of 14.3 years in a mathematic model. Viral protein feedback regulation in HBV life cycle to maintain vital viral replication is an important mechanism. Interleukin-6, epithelial growth factor, heme oxygenase-1, histones, and hepatocyte nuclear factors are demonstrated as the key regulators for HBV life cycle. CpG island structure and methylation status are involved in the regulation of HBV DNA replication. Nucleos(t)ide analogues are widely used in the clinical practice for the treatment of chronic hepatitis B patients, although no evidence indicating a direct inhibiton of HBV cccDNA. In the future, along with the study of HBV life cycle, new drugs including RNA interference technique, will pave the way to eliminate the HBV cccDNA from infected hepatocytes resulting final cure of chronic hepatitis B.

Background and Aims Haemodialysis (HD) patients are highly susceptible to blood borne virus infections and prevention strategies include surveillance, segregation, vaccination and strict implementation of standard precautions. As per new guidelines it is mandatory for all HD patients to be tested for hepatitis B core antibody at the beginning of renal replacement therapy. In the present study we sought to determine the prevalence of core antibody positivity and occult HBV infection as evidenced by viraemia in hepatitis B surface antigen (HBsAg) negative patients on maintenance haemodialysis. Method All HD patients (aged≥ 18 years) who were dialyzing in 2019 were included in this cross-sectional study. Retrospective data based on one-off screening for hepatitis B core antibody total (anti-HBc) for all HBsAg negative patients (study population) carried out between May and Oct 2019 was used for analysis. Information was gathered on HBsAg, anti-HBc antibody, surface antibody (anti-HBs) and Hepatitis B virus (HBV) DNA. All anti-HBc antibody positive patients were tested for HBV DNA using quantitative PCR assay. Patients were classified as immune (anti-HBs &amp;gt;10 IU/L) and non-immune (anti-HBs ≤10 IU/L) based on surface antibody levels. Demographic data were compared using Chi square test for categorical variables and Independent T test/Mann Whitney U test for continuous variables between the patients with and without HBV core antibody. Baseline characteristics were compared between the immune and non-immune groups using Chi square test and Independent T test/Mann Whitney U test as appropriate. Results A total of 3704 HBsAg negative HD patients (mean age 63.5±11.3 years, 56.1% male, 57.7% Chinese, 65.4% diabetic and mean vintage of 5.8±5.4 years) were included in the study after exclusion of 147 patients (3.8%) who had chronic HBV infection from the entire HD cohort of 3851 patients. 895 (24.2%) showed evidence of past HBV infection (anti-HBc positive but HBsAg negative) with only 3 (0.1%) patients having detectable HBV DNA. The HBV core antibody positive patients were predominantly elderly, Chinese, males and with high medical dependency compared to the patients without HBV core antibodies (p&amp;lt;0.001). There were no statistically significant differences in diabetic status and dialysis vintage between the patients with and without HBV core antibody. 3202 (86.4%) patients were immune following vaccination (anti-HBs &amp;gt;10 IU/L). The non-immune patients (13.5%) were more likely to be diabetic, with high medical dependency and lower vintage compared to immune patients (p&amp;lt;0.001). Age, race and gender did not differ significantly between the immune and non-immune patients. Conclusion The prevalence of hepatitis B core antibody positivity was 24.2% in our large racially diverse haemodialysis cohort but the presence of occult HBV was low (0.1%). Elderly Chinese males with high comorbidity and poor functional status were more likely to have evidence of past HBV infection. Occult HBV infection has implications in policy development for the prevention of HBV infection in dialysis centres.

Rescue therapy for drug resistant hepatitis B: Another argument for combination chemotherapy?