Molecular characterization of hepatitis B virus in blood donors in Botswana.

Hepatitis B virus (HBV) poses a significant threat to blood transfusion safety in sub-Saharan Africa (SSA) where allogeneic blood donations are screened serologically, and more sensitive nucleic acid tests (NATs) are utilized infrequently. HBV strains circulating among blood donors in Botswana are not yet characterized. We designed a cross-sectional study to determine the HBV sub-genotypes and prevalence of hepatitis B surface antigen (HBsAg) among blood donors between November 2014 and October 2015. A total of 12,575 blood donations were screened for HBsAg and 50 consecutive plasma samples were selected for genotyping from confirmed HBsAg+ donations. Overlapping Pol and complete S (Pol/S) open reading frames (ORFs) were sequenced from extracted HBV DNA. To identify any signature amino acids, mutations were compared to sequences from a cohort of chronic HBV patients co-infected with HIV and were treatment naïve. The prevalence of HBsAg+ blood donors was 1.02% (95% CI 0.9-1.2%), and the circulating sub-genotypes were A1 serotype adw2 (36.1%), D2 serotype ayw2 (2.9%), and D3 serotypes ayw 1/2 (58.3%). Prevalence of escape mutations was 14% from HBV isolates of blood donors and 15% from isolates of HBV/HIV co-infected patients (p = 0.6926). The escape mutations sP120L, sG130R, sY134H, and sD144A were identified predominantly among HBV isolates from blood donors. These escape mutations have been associated with accelerated HBV sequelae [e.g., liver cirrhosis (LC) and hepatocellular carcinoma (HCC)], failure to detect HBsAg, inability to respond to immunoglobulin (Ig) therapy, and HBV vaccine escape. Characterizing the HBV burden, circulating sub-genotypes, and clinically relevant mutations among blood donors in Botswana is important to elucidate the efficacy of currently available vaccines, predicting HBV-transmission patterns, understanding the cohort's risk to HBV-related complications, and to developing prevention strategies and effective genotype-based antiretroviral therapies.

Molecular characterization of hepatitis C virus in liver disease patients in Botswana: a retrospective cross-sectional study.

BACKGROUND: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease globally. Direct acting antivirals (DAAs) have proven effective in curing HCV. However, the current standard of care (SOC) in Botswana remains PEGylated interferon-α (IFN-α) with ribavirin. Several mutations have been reported to confer resistance to interferon-based treatments. Therefore, there is a need to determine HCV genotypes in Botswana, as these data will guide new treatment guidelines and understanding of HCV epidemiology in Botswana. METHODS: This was a retrospective cross-sectional pilot study utilizing plasma obtained from 55 participants from Princess Marina Hospital in Gaborone, Botswana. The partial core region of HCV was amplified, and genotypes were determined using phylogenetic analysis. RESULTS: Four genotype 5a and two genotype 4v sequences were identified. Two significant mutations - K10Q and R70Q - were observed in genotype 5a sequences and have been associated with increased risk of hepatocellular carcinoma (HCC), while R70Q confers resistance to interferon-based treatments. CONCLUSION: Genotypes 5a and 4v are circulating in Botswana. The presence of mutations in genotype 5 suggests that some patients may not respond to IFN-based regimens. The information obtained in this study, in addition to the World health organization (WHO) recommendations, can be utilized by policy makers to implement DAAs as the new SOC for HCV treatment in Botswana.

In Silico Prediction of Human Leukocytes Antigen (HLA) Class II Binding Hepatitis B Virus (HBV) Peptides in Botswana.

Hepatitis B virus (HBV) is the primary cause of liver-related malignancies worldwide, and there is no effective cure for chronic HBV infection (CHB) currently. Strong immunological responses induced by T cells are associated with HBV clearance during acute infection; however, the repertoire of epitopes (epi) presented by major histocompatibility complexes (MHCs) to elicit these responses in various African populations is not well understood. In silico approaches were used to map and investigate 15-mers HBV peptides restricted to 9 HLA class II alleles with high population coverage in Botswana. Sequences from 44 HBV genotype A and 48 genotype D surface genes (PreS/S) from Botswana were used. Of the 1819 epi bindings predicted, 20.2% were strong binders (SB), and none of the putative epi bind to all the 9 alleles suggesting that multi-epitope, genotype-based, population-based vaccines will be more effective against HBV infections as opposed to previously proposed broad potency epitope-vaccines which were assumed to work for all alleles. In total, there were 297 unique epi predicted from the 3 proteins and amongst, S regions had the highest number of epi (n = 186). Epitope-densities (Depi) between genotypes A and D were similar. A number of mutations that hindered HLA-peptide binding were observed. We also identified antigenic and genotype-specific peptides with characteristics that are well suited for the development of sensitive diagnostic kits. This study identified candidate peptides that can be used for developing multi-epitope vaccines and highly sensitive diagnostic kits against HBV infection in an African population. Our results suggest that viral variability may hinder HBV peptide-MHC binding, required to initiate a cascade of immunological responses against infection.

HBV genotype characterization and distribution in patients with HBV-related liver diseases in Zhejiang Province, P.R. China: possible association of co-infection with disease prevalence and severity.

BACKGROUND: There are 8 well-documented genotypes of hepatitis B virus (HBV) at this time point. Genotyping can be accomplished based on a partial sequence of hepatitis B virus (HBV) genome such as the pre-S or S gene. Several methods have been developed and used for HBV genotyping including direct sequencing, restriction fragment length polymorphism, line probe assay and enzyme-linked immunoassay. Recently, a novel, rapid and cost-effective genotyping method based on PCR amplification assay using type-specific primers that can identify all six major genotypes has been developed. This study was undertaken to characterise HBV genotypes and investigate the association between the prevalence of different genotypes and the severity of HBV-induced liver diseases. METHODS: Serum samples from carriers of HBV and patients with HBV-related liver diseases from Zhejiang Province were screened for viral serological markers using commercially available radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) kits. Serum HBV DNA load was determined by real-time detection PCR. A type-specific primer based the nested-PCR method was employed in the HBV genotyping. The genotype results obtained were confirmed by direct sequencing of nested PCR amplicons of the pre-S region. Ten samples of each genotype (B and C) were sequenced. RESULTS: The survey on a cohort of 125 HBV carriers in and around Hangzhou City, Zhejiang Province showed the existence of HBV genotypes A (0.8%), B (48%), C (40.8%), D (0.8%), mixed B and C (9.6%) and an absence of E and F genotypes. Distribution of HBV genotypes in patients with liver diseases revealed a statistically insignificant higher prevalence of genotype B in mild chronic hepatitis (CH). Among the three genotypes B, C and mixed B/C infections 11 (73.3%), 3 (20%) and 1 (6.7%), (P<0.05), respectively in subjects with moderate CH, genotype B was significantly predominant. The infection patterns for genotypes B, C and B/C mixed in (i) liver cirrhosis (LC) 4 (23.5%), 10 (58.8%) and 3 (17.7%) and (ii) hepatocellular carcinoma (HCC) 2 (28.6%), 5 (71.4%) and 0 (0.0%) respectively revealed a marked association of C genotype with liver disease; however, the association was statistically insignificant (P>0.05). Differences in positive rate of HBeAg for the three genotypes B, 16 (30.8%), C, 27 (51.9%), and mixed B/C, 9 (17.3%) were significant (P<0.05), with genotype C showing predominance. CONCLUSIONS: These findings show an interesting distribution of HBV A-D genotypes in Zhejiang Province. Furthermore, our results indicate a novel and markedly high prevalence of mixed B/C genotype infections in subjects with severe CH and LC, and a possible association of mixed B/C infections with the severity of liver diseases in this region of Mainland China.

Clinical features of chronic hepatitis B patients with YMDD mutation after lamivudine therapy.

OBJECTIVE: To study the clinical features of chronic hepatitis B (CHB) patients with tyrosine-methionine-aspartate-aspartate (YMDD) mutation after lamivudine therapy. METHODS: This investigation was a retrospective study of 63 CHB patients with YMDD mutation during lamivudine therapy. Clinical data, including period and types of YMDD mutation; hepatitis B virus (HBV) DNA levels and alanine aminotransferase (ALT) levels before and after YMDD mutation were measured. YMDD mutation in the HBV DNA polymerase gene was determined using polymerase chain reaction (PCR) and direct sequencing. HBV DNA quantification was determined using real-time PCR. Relevant serum markers of HBV were measured. The follow-up period was 12 months after YMDD mutation. RESULTS: YMDD mutation occurred 7-44 months (median, 21.5 months) after the start of lamivudine therapy. The majority of the cases (42/63, 66.6%) had YMDD mutants detected between 12 and 24 months. Four types of YMDD mutation were observed in this study, rtL180M/M204V mutation was the predominant type (26/63, 41.3%). A proportion of patients (16/63, 25.4%; 12/63, 19.1%) had higher HBV DNA levels and ALT levels (after mutation vs before mutation), respectively. CONCLUSION: The majority of patients with YMDD mutants had similar or lower HBV DNA levels and ALT levels compared with baseline values. This subset of patients might have benefited from the continued lamivudine therapy. The patients with increased ALT and HBV DNA levels (breakthrough hepatitis) should benefit from the addition of a newer nucleotide analogue (e.g. adefovir).

Down-modulation of heat shock protein 70 and up-modulation of Caspase-3 during schisandrin B-induced apoptosis in human hepatoma SMMC-7721 cells.

AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC-7721 cells in vitro and regulation of Hsp70 and Caspases-3, 7, 9 expression by Sch B. METHODS: Human hepatoma cell line SMMC-7721 was cultured and treated with Sch B at various concentrations. Growth suppression was detected with MTT colorimetric assay. Cell apoptosis was confirmed by DNA ladder detection and flow cytometric analysis. The expression of Hsp70, Caspases-3, 7, 9 were analyzed by Western blot analysis. RESULTS: Sch B inhibited the growth of hepatoma SMMC-7721 cells in a dose-dependent manner, leading to a 50% decrease in cell number (LC50) value of 23.50 mg/L. Treatment with Sch B resulted in degradation of chromosomal DNA into small internucleosomal fragments, evidenced by the formation of a 180-200 bp DNA ladder on agarose gels. FCM analysis showed the peak areas of subdiploid at the increased concentration of Sch B. The results of Western bolt analysis showed that Hsp70 was down-regulated and Caspase-3 was up-regulated, while the activity of Caspases-7, -9 had no significant change. CONCLUSION: Sch B is able to inhibit the proliferation of human hepatoma SMMC-7721 cells and induce apoptosis, which goes through Caspase-3-dependent and Caspase-9-independent pathway accompanied with the down-regulation of Hsp70 protein expression at an early event.

Cloning and expression of cDNAs from hepatitis E virus structural gene.

OBJECTIVE: To obtain recombinant antigen for development of anti-HEV ELISA kit and vaccine against hepatitis E virus infection. METHODS: A 492 base cDNA was collected from 5-terminus of open reading frame 2 (ORF2) among epidemic hepatitis E virus (HEV) isolated from Xinjiang, Western China. The fragment was digested with BamH I and EcoR I, and inserted into vector pGEX-4T-3 which was also digested by the same enzyme. The recombinant plasmid was transformed into E.coli TG-1 and the fusion protein expressed was confirmed by Western blot analysis using serum of a patient with hepatitis E. RESULTS: The recombinant plasmid was identified and confirmed with enzyme digestion, polymerase chain reaction (PCR) and sequencing, respectively. A protein band of about 46 kDa was demonstrated by SDS-PAGE and designated GST-pORF2. The result of Western blot analysis suggested that the fusion protein reacted with anti-HEV positive sera at a dilution of 1:100. CONCLUSION: The recombinant protein GST-pORF2 may be useful in developing anti-HEV ELISA kit and vaccine against hepatitis E virus infection.