No abstract

BackgroundHepatitis B virus (HBV) is one of the most common human pathogens that cause aggressive hepatitis and advanced liver disease (AdLD), including liver cirrhosis and Hepatocellular Carcinoma. The persistence of active HBV replication and liver damage after the loss of hepatitis B e antigen (HBeAg) has been frequently associated with mutations in the pre-core (pre-C) and core promoter (CP) regions of HBV genome that abolish or reduce HBeAg expression. The purpose of this study was to assess the prevalence of pre-C and CP mutations and their impact on the subsequent course of liver disease in Morocco.Methods/Principal FindingsA cohort of 186 patients with HBeAg-negative chronic HBV infection was studied (81 inactive carriers, 69 with active chronic hepatitis, 36 with AdLD). Pre-C and CP mutations were analyzed by PCR-direct sequencing method. The pre-C stop codon G1896A mutation was the most frequent (83.9%) and was associated with a lower risk of AdLD development (OR, 0.4; 95% CI, 0.15–1.04; p = 0.04). HBV-DNA levels in patients with G1896A were not significantly different from the other patients carrying wild-type strains (p = 0.84). CP mutations C1653T, T1753V, A1762T/G1764A, and C1766T/T1768A were associated with higher HBV-DNA level and increased liver disease severity. Multiple logistic regression analysis showed that older age (≥40 years), male sex, high viral load (>4.3 log10 IU/mL) and CP mutations C1653T, T1753V, A1762T/G1764A, and C1766T/T1768A were independent risk factors for AdLD development. Combination of these mutations was significantly associated with AdLD (OR, 7.52; 95% CI, 4.8–8; p<0.0001).ConclusionsThis study shows for the first time the association of HBV viral load and CP mutations with the severity of liver disease in Moroccan HBV chronic carriers. The examination of CP mutations alone or in combination could be helpful for prediction of the clinical outcome.

To date, the chimpanzee has been used as the natural infection model for hepatitis B virus (HBV). However, as this model is very costly and difficult to use because of ethical and animal welfare issues, we aimed to establish the tupaia (Tupaia belangeri) as a new model for HBV infection and characterized its intrahepatic innate immune response upon HBV infection. First, we compared the propagation of HBV genotypes A2 and C in vivo in tupaia hepatocytes. At 8-10days post infection (dpi), the level of HBV-A2 propagation in the tupaia liver was found to be higher than that of HBV-C. Abnormal architecture of liver cell cords and mitotic figures were also observed at 8 dpi with HBV-A2. Moreover, we found that HBV-A2 established chronic infection in some tupaias. We then aimed to characterize the intrahepatic innate immune response in this model. First, we infected six tupaias with HBV-A2 (strains JP1 and JP4). At 28 dpi, intrahepatic HBV-DNA and serum hepatitis B surface antigens (HBsAg) were detected in all tupaias. The levels of interferon (IFN)-β were found to be significantly suppressed in the three tupaias infected with HBV A2_JP4, while no significant change was observed in the three infected with HBV A2_JP1. Expression of toll-like receptor (TLR) 1 was suppressed, while that of TLR3 and TLR9 were induced, in HBV A2_JP1-infected tupaias. Expression of TLR8 was induced in all tupaias. Next, we infected nine tupaias with HBV-A2 (JP1, JP2, and JP4), and characterized the infected animals after 31 weeks. Serum HBsAg levels were detected at 31 weeks post-infection (wpi) and IFN-β was found to be significantly suppressed in all tupaias. TLR3 was not induced, except in tupaia #93 and #96. Suppression of TLR9 was observed in all tupaias, except tupaia #93. Also, we investigated the expression levels of cyclic GMP-AMP synthase, which was found to be induced in all tupaias at 28 dpi and in four tupaias at 31 wpi. Additionally, we evaluated the expression levels of sodium-taurocholate cotransporting polypeptide, which was found to be suppressed during chronic HBV infection. Thus, the tupaia infection model of HBV clearly indicated the suppression of IFN-β at 31 wpi, which might have contributed to the establishment of chronic HBV infection.

The cytidine deaminase apolipoprotein B mRNA editing catalytic subunit-3 (APOBEC3) induces G-to-A hypermutation in hepatitis B virus (HBV) genomes and operates as part of the innate antiviral immune system. We investigated the associations between the presence of APOBEC3 variants and HBV carriage in a case-control study in the Moroccan population. A polymorphic deletion affecting the APOBEC3B gene and the H186R variant of APOBEC3G were genotyped in 179 HBV chronic carriers and 216 healthy control subjects. In addition, to assess the overall impact of APOBEC3 deaminases on circulating HBV, we looked for hyperedited forms of the viral genome using the 3DPCR technique and analysed editing context. Data analysis showed that there was no significant difference in the frequencies of deleted APOBEC3B alleles (P = 0.261) or genotypes (P = 0.333) between patients with chronic hepatitis B and control subjects. By contrast, subjects bearing deleted genotype had a faster progression of liver disease than those with the insertion genotype (adjusted OR, 3.72; 95% CI, 0.38-36.12). The analysis of the APOBEC3G H186R polymorphism revealed that R/R genotype frequencies were not significantly different in HBV infected patients and in healthy subjects. 3DPCR was positive in 26 samples (14%) among 179. Amplified viral segments displayed monomorphic G>A transitions highly reminiscent of APOBEC3G activity. Most intriguingly, hemi/homozygous carriers of the APOBEC3B deletion had significantly lower virus loads than patients with the wild type (median 539 vs. 2213 IU/mL, P = 0.0023). This result suggests that genetic variations in APOBEC3 cytidine deaminases do not predispose to chronicity but may modulate the course of persistent HBV infection.