rt269I Type of Hepatitis B Virus (HBV) Polymerase versus rt269L Is More Prone to Mutations within HBV Genome in Chronic Patients Infected with Genotype C2: Evidence from Analysis of Full HBV Genotype C2 Genome

Recently, it has been reported that the rt269I type of hepatitis B virus (HBV) polymerase (Pol) versus the rt269L type is more significantly related to lower viral replication and HBeAg negative infections in chronic hepatitis B (CHB) patients of genotype C2. In this study, we compared mutation rates within HBV genomes between rt269L and rt269I using a total of 234 HBV genotype C2 full genome sequences randomly selected from the HBV database (115 of rt269L and 119 of rt269I type). When we applied the Benjamini and Hochberg procedure for multiple comparisons, two parameters, dN and d, at the amino acids level in the Pol region were significantly higher in the rt269I type than in the rt269L type. Although it could not reach statistical significance from the Benjamini and Hochberg procedure, nonsynonymous (NS) mutations in the major hydrophilic region (MHR) or “a” determinant in the surface antigens (HBsAg ORF) related to host immune escape or vaccine escape are more frequently generated in rt269I strains than in rt269L. We also found that there are a total of 19 signature single nucleotide polymorphisms (SNPs), of which 2 and 17 nonsynonymous mutation types were specific to rt269L and rt269I, respectively: Of these, most are HBeAg negative infections (preC-W28*, X-V5M and V131I), lowered HBV DNA or virion production (C-I97F/L, rtM204I/V) or preexisting nucleot(s)ide analog resistance (NAr) (rtN139K/H, rtM204I/V and rtI224V) or disease severity (preC-W28*, C-I97F/L, C-Q182K/*, preS2-F141L, S-L213I/S, V/L5M, T36P/S/A, V131I, rtN139K/H, rtM204I/V and rtI224V). In conclusion, our data showed that rt269I types versus rt269L types are more prone to overall genome mutations, particularly in the Pol region and in the MHR or “a” determinant in genotype C2 infections and are more prevalent in signature NS mutations related to lowered HBV DNA replication, HBsAg and HBeAg secretion and potential NAr variants and hepatocellular carcinoma (HCC), possibly via type I interferon (IFN-I)-mediated enhanced inflammation. Our data suggest that rt269L types could contribute to liver disease progression via the generation of immune escape or enhanced persistent infection in chronic patients of genotype C2.

Seroprevalence and genotypic characterization of HBV among low risk voluntary blood donors in Nairobi, Kenya

Background Hepatitis B virus (HBV) causes significant morbidity and mortality globally primarily due to its ability to cause hepatitis, liver cirrhosis and hepatocellular carcinoma. The Kenya National Blood Transfusion Services screens for Hepatitis B antibodies using the chemiluminescent microparticle immunoassay method. This test does not inform on the genotypic characteristics of the virus or the actual presence of the virus in blood. This study therefore sought to determine the serologic and genotypic profiles of HBV circulating among the voluntary blood donors in Nairobi. Methods Blood samples were collected in plain and EDTA vacutainers and tested for the Hepatitis B surface antigen (HBsAg). HBV DNA was then extracted from plasma, its overlapping P/S gene amplified and sequenced. The resulting sequences were used to analyze for the circulating genotypes and mutations within the P and S genes. Bivariate statistical analysis was used to determine the association between demographic factors and HBV infection. Results A seroprevalence of 2.3% (n = 7) was reported. The age group 19–28 years was significantly associated with HBV infection. Nine samples were positive for HBV DNA; these included 2 HBsAg positive samples and 7 HBsAg negative samples. Genotype A, sub genotype A1 was found to be exclusively prevalent while a number of mutations were reported in the “a” determinant segment of the major hydrophilic region of the S gene associated with antibody escape. RT mutations including mutation rt181T in the P gene conferring resistance against Lamivudine and other ʟ-nucleoside drugs were detected. Conclusion There is a high prevalence of occult HBV infections among these blood donors and therefore the testing platform currently in use requires revision.

A Hyper-Glycosylation of HBV Surface Antigen Correlates with HBsAg-Negativity at Immunosuppression-Driven HBV Reactivation in Vivo and Hinders HBsAg Recognition In Vitro

Immune-suppression driven Hepatitis B Virus (HBV)-reactivation poses serious concerns since it occurs in several clinical settings and can result in severe forms of hepatitis. Previous studies showed that HBV strains, circulating in patients with HBV-reactivation, are characterized by an enrichment of immune-escape mutations in HBV surface antigen (HBsAg). Here, we focused on specific immune-escape mutations associated with the acquisition of N-linked glycosylation sites in HBsAg (NLGSs). In particular, we investigated profiles of NLGSs in 47 patients with immunosuppression-driven HBV-reactivation and we evaluated their impact on HBsAg-antigenicity and HBV-replication in vitro. At HBV-reactivation, despite a median serum HBV-DNA of 6.7 [5.3–8.0] logIU/mL, 23.4% of patients remained HBsAg-negative. HBsAg-negativity at HBV-reactivation correlated with the presence of >1 additional NLGSs (p < 0.001). These NLGSs are located in the major hydrophilic region of HBsAg (known to be the target of antibodies) and resulted from the single mutation T115N, T117N, T123N, N114ins, and from the triple mutant S113N+T131N+M133T. In vitro, NLGSs strongly alter HBsAg antigenic properties and recognition by antibodies used in assays for HBsAg-quantification without affecting HBsAg-secretion and other parameters of HBV-replication. In conclusion, additional NLGSs correlate with HBsAg-negativity despite HBV-reactivation, and hamper HBsAg-antigenicity in vitro, supporting the role of NGSs in immune-escape and the importance of HBV-DNA for a proper diagnosis of HBV-reactivation.

OCCULT HEPATITIS B VIRUS INFECTION AND ASSOCIATED GENOTYPES AMONG HBSAG-NEGATIVE SUBJECTS IN BURKINA FASO

Background: Occult Hepatitis B virus infection (OBI), is characterized by the absence of detectable HBsAg in the blood of a person assumed to be healthy. It remains a potential transmission threat and risk to HBV chronic infection. The purpose of this study was to determine the OBI prevalence among HBsAg negative subjects and to characterize associated genotypes.Methods: Blood samples of 219 HBsAg-negative subjects tested by ELISA were collected. HBV DNA was investigated in all samples. Viral loads were determined using quantitative real-time PCR. All samples were screened for HBV markers (anti-HBc, anti-HBe, HBsAg). The Pre-S/S region of the HBV genome was sequenced. The database was analyzed using the SPSS and Epi info softwares. Phylogenetic analysis was performed using the BioEdit and MEGA softwares.Results: Of the 219 samples, 20.1 % were anti-HBc positive, 1.8 % HBeAg and 22.8 % were anti-HBe positive. Fifty-six 56 (25.6 %) of the samples had a detectable HBV DNA and viral loads ranging from 4 IU/mL to 13.6 106 IU/mL. Sixteen of them (16/56) had a viral load < 200 IU/mL, resulting in an OBI prevalence of 7.3 % (16/219) in our study. The remaining 40 subjects had viral loads ˃ 200 IU/mL, resulting in a “false OBI” prevalence of 18.3% (40/219). HBV genotype E was predominant followed by the quasi-sub-genotype A3. A single "false OBI" strain had the characteristic mutation G145R. Other mutations were observed and all located in the major hydrophilic region (MHR) of the S gene.Conclusion: The study reported a prevalence of 7.3% of occult hepatitis B infection. It confirms the predominance of genotype E and the existence of a subgroup of quasi-sub-genotype A3 of HBV in Burkina Faso. It further provides information on the existence of “false OBI “. This study has found mutations in the major hydrophilic region (MHR) of the pre-S/S gene of HBV. Key words: HBV, OBI, Genotypes, Real-time PCR, Sequencing