Maemu P. Gededzha

Near full-length genome analysis of HCV genotype 5 strains from South Africa.

Hepatitis C virus (HCV) genotype 5 is the predominant genotype in South Africa. However, to date, only 2 full-length genotype 5 genomes have been sequenced and only one is from South Africa. This study characterized HCV genotype 5 sequences from South Africa, including six near full-length genomes, as well as the E1 region from an additional 12 genotype 5 samples. Phylogenetic analysis of these near full-length genome sequences revealed that all genotype 5 sequences formed a close cluster with high bootstrap support. Bayesian analysis of the E1 region was used to estimate the time of the most recent common ancestor (tMRCA). The tMRCA for HCV genotype 5a was estimated at 114-134 years before the last sampling date. In conclusion, this study provides six near full-length genotype 5 nucleotide sequences for use as references to design efficient vaccines and for the development of new antiviral agents, and provides further insight into the diversity of HCV genotypes circulating in South Africa.

Introduction of new subtypes and variants of hepatitis C virus genotype 4 in South Africa.

Hepatitis C virus (HCV) genotype is an important predictor of disease progression and treatment response. This descriptive study investigated the sequence diversity and genotypes of HCV in South Africa based on comparative analysis of the 5′ untranslated region (UTR), C/E1, and NS5B regions of 60 sequences from 52 patients. Genotype distribution in the studied population was as follows: 54% (28/52) were genotype 5, 19% (10/52) were genotype 1, 19% (10/52) were genotype 4, and 2% (1/52) were genotype 3. Three of 52 (6%) individuals were infected with multiple genotypes based on the 5′UTR. Phylogenetic analysis of the 5′UTR was accurate in determining genotypes, while the C/E1 and NS5B coding region was able to differentiate both genotypes and subtypes, including an outlier group. Furthermore, this study observed the existence of distinct variants of HCV which were divergent from confirmed genotype 4 subtypes. For the first time in South Africa, this analysis has shown the presence of HCV subtypes 4k, 4q, and 4r, as well as evidence of intragenotypic recombinant 4l/4q within NS5B. In conclusion, while genotype 5a remains the predominant genotype in South Africa, the current study indicates the introduction of new subtypes and existence of variants of genotype 4 in South Africa. J. Med. Virol. 84:601–607, 2012.

Hepatitis B virus infection in post-vaccination South Africa : occult HBV infection and circulating surface gene variants

BACKGROUND AND OBJECTIVE The aim of this study was to investigate the prevalence of occult hepatitis B virus (HBV) infection and the HBV surface (S) gene variants circulating in the South African population after nearly two decades of universal hepatitis B vaccination. STUDY DESIGN From a previous serosurvey, 201 serum samples with serological evidence of exposure to HBV were identified and these were stratified into post- and pre-vaccine introduction populations. For all samples, HBV DNA was screened and quantified using a real-time PCR assay and results analysed together with HBV serological markers. Where HIV results were available, subset analysis was performed. The HBV S gene was PCR-amplified and sequences analysed for a total of 37 isolates. RESULTS The prevalence of occult HBV infection reduced from 70.4% in the pre-vaccine introduction era to 66.0% post-vaccine introduction. There was an association between HIV infection and an increase in prevalence of occult HBV infection within the post-vaccine introduction population, although this was not statistically significant. Furthermore, sequence analysis revealed the following HBV subgenotypes; A1 (n=34), A2 (n=2) and a rare D4 isolate. HBV S gene variants, including diagnostic escape mutants were isolated. CONCLUSION There was a decline in the prevalence of occult HBV infection in post-vaccination South Africa, although the disease burden remains significant in the HIV co-infected population. After nearly two decades of a universal hepatitis B vaccination programme, no positive selection of vaccine escape mutants were observed.

Prediction of T-cell epitopes of hepatitis C virus genotype 5a

BackgroundHepatitis C virus (HCV) is a public health problem with almost 185 million people estimated to be infected worldwide and is one of the leading causes of hepatocellular carcinoma. Currently, there is no vaccine for HCV infection and the current treatment does not clear the infection in all patients. Because of the high diversity of HCV, protective vaccines will have to overcome significant viral antigenic diversities. The objective of this study was to predict T-cell epitopes from HCV genotype 5a sequences.MethodsHCV near full-length protein sequences were analyzed to predict T-cell epitopes that bind human leukocyte antigen (HLA) class I and HLA class II in HCV genotype 5a using Propred I and Propred, respectively. The Antigenicity score of all the predicted epitopes were analysed using VaxiJen v2.0. All antigenic predicted epitopes were analysed for conservation using the IEDB database in comparison with 406, 221, 98, 33, 45, 45 randomly selected sequences from each of the HCV genotypes 1a, 1b, 2, 3, 4 and 6 respectively, downloaded from the GenBank. For epitope prediction binding to common HLA alleles found in South Africa, the IEDB epitope analysis tool was used.ResultsA total of 24 and 77 antigenic epitopes that bind HLA class I and HLA class II respectively were predicted. The highest number of HLA class I binding epitopes were predicted within the NS3 (63%), followed by NS5B (21%). For the HLA class II, the highest number of epitopes were predicted in the NS3 (30%) followed by the NS4B (23%) proteins. For conservation analysis, 8 and 31 predicted epitopes were conserved in different genotypes for HLA class I and HLA class II alleles respectively. Several epitopes bind with high affinity for both HLA class I alleles and HLA class II common in South Africa.ConclusionThe predicted conserved T-cell epitopes analysed in this study will contribute towards the future design of HCV vaccine candidates which will avoid variation in genotypes, which in turn will be capable of inducing broad HCV specific immune responses.

Should routine serological screening for HCV be mandatory in HIV/AIDS patients enrolling for HAART in South Africa?

To the Editor: Globally, an estimated 170 million people (about 3% of the worlds population) have been infected with hepatitis C virus (HCV). HCV, a member of the Hepacivirus genus in the Flaviviridae family, possesses a single stranded, positive-sense RNA genome of approximately 9.6 kilobase (kb), and is the major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. In Africa, HCV prevalence ranges from <3% in South Africa to <20% in Egypt. In South Africa, there is a low prevalence from 0.16% to 1.8%, but this is higher in high-risk individuals, e.g. 39.4% in haemophiliacs and 4.8% in chronic dialysis patients.

Complete genome analysis of hepatitis B virus in human immunodeficiency virus infected and uninfected South Africans.

Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections are highly endemic in South Africa. Data on the complete genome sequences of HBV in HIV‐positive patients in South Africa are scanty. This study characterized the complete HBV genome isolated from both HIV‐positive and negative patients at the Dr George Mukhari Academic Hospital (DGMAH), Pretoria. Serum samples from nine (five HIV‐positive and four HIV‐negative) patients attending the DGMAH from 2007 to 2011 were serologically tested, amplified, and sequenced for complete genome. Phylogenetic tree was constructed using MEGA6.0. Mutations were analyzed by comparing the sequences with genotype‐matched GenBank references. Eight patients were HBsAg positive, with only one from the HIV positive group being negative. Phylogenetic analysis of the complete genome sequences classified them into five genotypes; A1 (n = 4), A2 (n = 1), C1 (n = 2), D1 (n = 1), and D3 (n = 1). Deletions up to 35 nucleotides in length were identified in this study. No drug resistance mutations were identified in the P ORF, while the L217R mutation was identified in one subgenotype A2 sequence. The double (A1762T/G1764A) and triple (T1753C/A1762T/G1764A) mutations in the Basal core promoter were identified in four and two sequences, respectively. In the core region, mutation G1888A was identified in four of the subgenotype A1 sequences. In conclusion, this study has added to the limited South African data on HBV genotypes and mutations in HBV/HIV co‐infected and HBV mono‐infected patients, based on complete HBV genome analysis. Subgenotype A1 was predominant, and no drug‐resistant mutants were detected in the study. J. Med. Virol. 88:1560–1566, 2016.

Evidence of susceptibility to lamivudine-based HAART and genetic stability of hepatitis B virus (HBV) in HIV co-infected patients: A South African longitudinal HBV whole genome study.

BACKGROUND Reports on the concomitant impact of HIV co-infection and long term highly active anti-retroviral therapy (HAART) on the genetic stability and molecular evolution of HBV are limited in sub-Saharan Africa. MATERIALS AND METHODS This retrospective study investigated the molecular evolution of chronic HBV in HIV co-infected patients on lamivudine (3TC)-based HAART over a 5year period. Four HIV co-infected patients, consecutively recruited and followed-up, were screened for hepatitis B serological markers, and their viral loads determined. The HBV genome was amplified from longitudinal samples and characterized by Bayesian inference, mutational analysis, and identification of immune selection pressure. RESULTS All patients exhibited persistent chronic HBV infection at baseline, as well as over the course of follow-up despite exposure to 3TC-based HAART. The polymerase gene in all isolates was relatively variable prior to HAART initiation at baseline and during the course of follow-up, although primary drug resistance mutations were not detected. All but one patient were infected with HBV subgenotype A1. The divergence rates between baseline and the last follow-up sequences ranged from 0 to 2.0×10(-3) substitutions per site per year (s/s/y). Positive selection pressure was evident within the surface and core genes. CONCLUSION Despite persistent HBV infection in the HIV co-infected patients exposed to long term 3TC-based HAART, the molecular evolution of HBV over a 5year period was unremarkable. In addition, HBV exhibited minimal genetic variability overtime.

Characterization of HCV Genotype 5a Envelope Proteins: Implications for Vaccine Development and Therapeutic Entry Target

Background: Hepatitis C virus (HCV) is one of the major causes of cirrhosis and hepatocellular carcinoma with an estimation of 185 million people with infection. The E2 is the main target for neutralizing antibody responses and the variation of this region is related to maintenance of persistent infection by emerging escape variants and subsequent development of chronic infection. While both E1 and E2 are hypervariable in nature, it is difficult to design vaccines or therapeutic drugs against them. Objectives: The objective of this study was to characterize genotype 5a E1 and E2 sequences to determine possible glycosylation sites, conserved B-cell epitopes and peptides in HCV that could be useful targets in design of vaccine and entry inhibitors. Patients and Methods: This study was conducted through PCR amplification of E1 and E2 regions, sequencing, prediction of B-cell epitopes, analysis of N-linked glycosylation and peptide design in 18 samples of HCV genotype 5a from South African. Results: Differences in the probability of glycosylation in E1 and E2 regions were observed in this study. Three conserved antigenic B-cell epitopes were predicted in the E2 regions and also 11 short peptides were designed from the highly conserved residues. Conclusions: This study provided conserved B-cell epitopes and peptides that can be useful for designing entry inhibitors and vaccines able to cover a global population, especially where genotype 5a is common.