Genetic characterization of G12P[6] and G12P[8] rotavirus strains collected in six African countries between 2010 and 2014.

BACKGROUND: G12 rotaviruses were first observed in sub-Saharan Africa in 2004 and since then have continued to emerge and spread across the continent and are reported as a significant human rotavirus genotype in several African countries, both prior to and after rotavirus vaccine introduction. This study investigated the genetic variability of 15 G12 rotavirus strains associated with either P[6] or P[8] identified between 2010 and 2014 from Ethiopia, Kenya, Rwanda, Tanzania, Togo and Zambia. METHODS: The investigation was carried out by comparing partial VP7 and partial VP4 sequences of the African G12P[6] and G12P[8] strains with the available GenBank sequences and exploring the recognized neutralization epitopes of these strains. Additionally, Bayesian evolutionary analysis was carried out using Markov Chain Monte Carlo (MCMC) implemented in BEAST to estimate the time to the most recent ancestor and evolutionary rate for these G12 rotavirus strains. RESULTS: The findings suggested that the VP7 and VP4 nucleotide and amino acid sequences of the G12 strains circulating in African countries are closely related, irrespective of country of origin and year of detection, with the exception of the Ethiopian strains that clustered distinctly. Neutralization epitope analysis revealed that rotavirus VP4 P[8] genes associated with G12 had amino acid sequences similar to those reported globally including the vaccine strains in RotaTeq and Rotarix. The estimated evolutionary rate of the G12 strains was 1.016 × 10- 3 substitutions/site/year and was comparable to what has been previously reported. Three sub-clusters formed within the current circulating lineage III shows the diversification of G12 from three independent ancestries within a similar time frame in the late 1990s. CONCLUSIONS: At present it appears to be unlikely that widespread vaccine use has driven the molecular evolution and sustainability of G12 strains in Africa. Continuous monitoring of rotavirus genotypes is recommended to assess the long-term impact of rotavirus vaccination on the dynamic nature of rotavirus evolution on the continent.

Molecular characterization of hepatitis B virus X gene in HIV-positive South Africans.

Hepatitis B virus (HBV) infection is a major public health problem worldwide and the major cause of hepatocellular carcinoma (HCC) in South Africa. The role of HBV in HCC is not well understood, although the HBV X gene has been implicated as a critical factor. Data on the HBV X gene in HIV-positive South Africans are limited; thus, we investigated X gene variability in 24 HIV-infected treatment-naïve patients at Dr George Mukhari Academic Hospital. Quantitative and qualitative HBV DNA tests were conducted using real-time and in-house polymerase chain reaction (PCR) assays, respectively, targeting the complete HBV X gene. In-house PCR-positive samples were cloned using the P-Gem T-easy vector System II and sequenced. By phylogenetic analysis, X gene sequences were classified as subgenotype A1 (n = 15), A2 (n = 4), and D1 (n = 4), and one dual infection with subgenotypes as A1 and C. The basal core promoter mutations T1753C, A1762T, and G1764A were identified in the majority of sequences. Genotype D sequences had a 6-nucleotide insertion. In conclusion, subgenotype A1 was predominant, and a rare dual infection of HBV genotype A and C was detected. The 6-nucleotide insertion could represent a unique variant in the region and highlights the need for functional studies of HBV X gene variants, particularly from resource-limited settings.

Functional analysis of 'a' determinant mutations associated with occult HBV in HIV-positive South Africans.

Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in the absence of hepatitis B surface antigen (HBsAg). Occult HBV is associated with the development of hepatocellular carcinoma, reactivation during immune suppression, and virus transmission. Viral mutations contribute significantly to the occult HBV phenotype. Mutations in the 'a' determinant of HBsAg are of particular interest, as these mutations are associated with immune escape, vaccine escape and diagnostic failure. We examined the effects of selected occult HBV-associated mutations identified in a population of HIV-positive South Africans on HBsAg production in vitro. Mutations were inserted into two different chronic HBV backbones and transfected into a hepatocyte-derived cell line. HBsAg levels were quantified by enzyme-linked immunosorbent assay (ELISA), while the detectability of mutant HBsAg was determined using an HA-tagged HBsAg expression system. Of the seven mutations analysed, four (S132P, C138Y, N146D and C147Y) resulted in decreased HBsAg expression in one viral background but not in the second viral background. One mutation (N146D) led to a decrease in HBsAg detected as compared to HA-tag, indicating that this mutation compromises the ability of the ELISA to detect HBsAg. The contribution of occult-associated mutations to the HBsAg-negative phenotype of occult HBV cannot be determined adequately by testing the effect of the mutation in a single viral background, and rigorous analysis of these mutations is required.

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. © 2016 Wiley Periodicals, Inc.

Sustained favourable HIV viral load response in South African patients during concomitant HAART and cancer therapy.

HIV infection has led to an increase of both AIDS defining and non-AIDS defining cancers. Immunosuppressive cancer therapy had been noted for increased HIV viral load in cancer patients infected with HIV before the advent of highly active antiretroviral therapy (HAART). Assessing the outcome of concomitant HAART and cancer treatment in regions endemic for HIV is thus important. From a cohort of 34 cancer patients infected with HIV, 10 (six underwent radiotherapy and four chemotherapy) had at least three serial samples collected before, during and after treatment. From each sample, HIV viral load, CD4 + and CD8 + cell count was investigated. HIV genotypic drug resistance was assessed for six patients with a detectable HIV viral load at baseline. Of the 10 patients; one was HIV positive only, three were HIV and HBV co-infected and six were HIV, HBV and HCV triple infected. Four patients were HAART experienced at recruitment, while six were HAART naive with detectable HIV viral loads. A significant HIV viral load decrease (P = 0.0128) was observed in all six patients with detectable baseline HIV viral loads. The four patients on HAART at recruitment maintained lower than detectable HIV viral load status throughout cancer therapy. A positive correlation between the rise in CD4 + cell count and attaining a lower than detectable HIV viral load was also observed (P = 0.0439). This pilot study supports concomitant treatment of HIV/AIDS and cancer in HIV endemic regions irrespective of the type of cancer diagnosed, prescribed cancer therapy or other viral (HBV, HCV or both) co-infections.

Active co-infection with HBV and/or HCV in South African HIV positive patients due for cancer therapy.

Human immunodeficiency virus (HIV), Hepatitis B virus (HBV) and Hepatitis C virus (HCV) share routes of transmission. There is limited data on the incidence of active co-infection with HBV and/or HCV in cancer patients infected with HIV in Africa. This was a prospective study based on 34 patients with varied cancer diagnosis, infected with HIV and awaiting cancer therapy in South Africa. HIV viral load, CD4+ cell counts, Alanine-aminotransferase and aspartate aminotransferase levels were tested. Exposure to HBV and HCV was assessed serologically using commercial kits. Active HBV and/or HCV co-infection was detected using viral specific nested PCR assays. HCV 5'-UTR PCR products were sequenced to confirm active HCV infection. Active viral infection was detected in 64.7% of patients for HBV, 38.2% for HCV, and 29.4% for both HBV and HCV. Occult HBV infection was observed in 63.6% of the patients, while seronegative HCV infection was found in 30.8% of patients. In addition, CD4+ cell count < 350 cells/µl was not a risk factor for increased active HBV, HCV or both HBV and HCV co-infections. A total of 72.7%, 18.2% and 9.1% of the HCV sequences were assigned genotype 5, 1 and 4 respectively.The study revealed for the first time a high active HBV and/or HCV co-infection rate in cancer patients infected with HIV. The findings call for HBV and HCV testing in such patients, and where feasible, appropriate antiviral treatment be indicated, as chemotherapy or radiotherapy has been associated with reactivation of viral hepatitis and termination of cancer therapy.

Detection, genotyping and quantitation of multiple hpv infections in south African women with cervical squamous cell carcinoma.

In Africa, data is limited on quantitation of human papillomavirus (HPV) types in women with multiple infections. This study applied a real time PCR (qPCR) assay for detection, genotyping and quantitation of multiple HPV infections in 90 tissue blocks of South African women with cervical squamous cell carcinoma. One sample with multiple HPV types was subjected to laser micro-dissection and qPCR. Four samples were negative for ß-globin and these were excluded from the analysis. The HPV DNA positivity rate was 93.0% (80/86). All 80 positives showed the presence of HR HPV types; HPV 68 was the only type negative in all the samples. Overall, HPV 16 was positive in most of the samples (88.8%), followed by HPV 56 (28.7%), HPV 18 (20.0%) and HPV 39 (18.7%). More than half of the samples (65.0%) had multiple infections. HPV 16 was present in majority of single (85.7%) and multiple infections (90.4%). HPV 16 showed higher viral loads in 70.3% of the HPV 16 co-infected samples. In one multiple infected sample laser micro-dissection and qPCR identified HPV 18 with higher viral load as the most likely cause of the invasive lesion. There is large number of multiple HPV infections in South African women with cervical squamous cell carcinoma. HPV 16 is the most frequently detected type and often presents with higher viral load, suggesting it could be responsible for pathogenesis of the lesions in the majority of cases.

Laser micro-dissection and qPCR for identifying specific HPV types responsible for malignancy in penile lesions.

The aim of the study was to identify specific human papillomavirus (HPV) type responsible for malignancy in penile tissue samples using laser micro-dissection and TaqMan quantitative real-time PCR (qPCR). The study was based on two pre-malignant and seven malignant penile tissue samples and laser micro-dissection was performed on all. Genotyping was performed on whole tissue sections and laser micro-dissection samples using qPCR. Two whole tissue section samples were HPV negative while seven were HPV positive. In four samples that were single HPV infections with whole tissue section PCR, identical HPV types were confirmed with laser micro-dissection PCR. Clearly confirming that the single HPV type detected is responsible for malignancy. In two samples that had multiple HPV infections with whole tissue section PCR, only one HPV type with the highest viral load was detected with laser micro-dissection PCR, suggesting that the HPV type with the highest viral load is most likely the cause of that particular lesion. HPV 11 and/or HPV 16 were the only types detected with laser micro-dissection PCR in these cases, compared to multiple HPV types (HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, and HPV 39) initially detected with whole tissue section PCR. HPV 11 was associated with verrucous lesions while HPV 16 was associated with squamous cell carcinoma and PIN 3 lesions. This study confirms that laser micro-dissection and qPCR are essential tools in identifying the HPV types responsible for malignancy in penile lesions, particularly in samples with multiple infections.

Mutations associated with occult hepatitis B in HIV-positive South Africans.

Occult hepatitis B is characterized by the absence of hepatitis B surface antigen (HBsAg) but the presence of HBV DNA. Because diagnosis of hepatitis B virus (HBV) typically includes HBsAg detection, occult HBV remains largely undiagnosed. Occult HBV is associated with increased risk of hepatocellular carcinoma, reactivation to chronic HBV during immune suppression, and transmission during blood transfusion and liver transplant. The mechanisms leading to occult HBV infection are unclear, although viral mutations are likely a significant factor. In this study, sera from 394 HIV-positive South Africans were tested for HBV DNA and HBsAg. For patients with detectable HBV DNA, the overlapping surface and polymerase open reading frames (ORFs) were sequenced. Occult-associated mutations-those mutations found exclusively in individuals with occult HBV infection but not in individuals with chronic HBV infection from the same cohort or GenBank references-were identified. Ninety patients (22.8%) had detectable HBV DNA. Of these, 37 had detectable HBsAg, while 53 lacked detectable surface antigen. The surface and polymerase ORFs were cloned successfully for 19 patients with chronic HBV and 30 patients with occult HBV. In total, 235 occult-associated mutations were identified. Ten occult-associated mutations were identified in more than one patient. Additionally, 15 amino acid positions had two distinct occult-associated mutations at the same residue. Occult-associated mutations were common and present in all regions of the surface and polymerase ORFs. Further study is underway to determine the effects of these mutations on viral replication and surface antigen expression in vitro.

Whole genome analyses of G1P[8] rotavirus strains from vaccinated and non-vaccinated South African children presenting with diarrhea.

Group A rotaviruses (RVAs) are the leading cause of severe gastroenteritis and eventually death among infants and young children worldwide, and disease prevention and management through vaccination is a public health priority. In August 2009, Rotarix™ was introduced in the South African Expanded Programme on Immunisation. As a result, substantial reductions in RVA disease burden have been reported among children younger than 5 years old. Rotavirus strain surveillance post-vaccination is crucial to, inter alia, monitor and study the evolution of vaccine escape strains. Here, full-genome sequence data for the 11 gene segments from 11 South African G1P[8] rotavirus strains were generated, including 5 strains collected from non-vaccinated children during the 2004-2009 rotavirus seasons and 6 strains collected from vaccinated children during the 2010 rotavirus season. These data were analyzed to gain insights into the overall genetic makeup and evolution of South African G1P[8] rotavirus strains and to compare their genetic backbones with those of common human Wa-like RVAs from other countries, as well as with the Rotarix™ and RotaTeq™ G1P[8] vaccine components. All 11 South African G1P[8] strains revealed a complete Wa-like genotype constellation of G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. On the basis of sequence similarities, the South African G1P[8] strains (with the exception of strain RVA/Human-wt/ZAF/1262/2004/G1P[8]) were closely related to each other (96-100% identity in all gene segments). Comparison to the Rotarix™ and RotaTeq™ G1P[8] vaccine components revealed a moderate nucleotide identity of 89-96% and 93-95%, respectively. The results indicated that none of the gene segments of these 11 South African G1P[8] strains were vaccine-derived. This study illustrates that large-scale next generation sequencing will provide crucial information on the influence of the vaccination program on evolution of rotavirus strains. This is the first report to describe full genomic analyses of G1P[8] RVA strains collected from both non-vaccinated and vaccinated children in South Africa.

Diversity of HPV types in cancerous and pre-cancerous penile lesions of South African men: implications for future HPV vaccination strategies.

This study reports the detection of HPV types from cancerous and pre-cancerous penile lesions that were diagnosed histologically. Sixty-six (22 pre-cancerous and 44 cancerous lesions) tissue biopsies, received between 2004 and 2011 by the Anatomical Pathology Department at Dr. George Mukhari Hospital were selected for this study. Total DNA was extracted and genotyped using type specific real-time quantitative polymerase chain reaction (qPCR) for 18 HPV types. Of 66 samples, only 51 were included in the analysis. Overall, HPV 11 (50.9%) and HPV 16 (49.1%) showed almost similar incidence in the study patients. In pre-cancerous lesions, HPV 11 was more frequent (80.0%), followed by HPV 31 and HPV 16 at 25.0% each and other HPV types included 35 (15.0%), 59 (15.0%), 53 (10.0%), 33 (10.0%), 18 (5.0%), 51 (5.0%), 52 (5.0%), 56 (5.0%), and 67 (5.0%). For cancerous lesions, HPV 16 was the most detected (62.9%), followed by HPV 11 (34.3%), and other HPV types included 18 (11.4%), 33 (5.7%), 39 (5.7%), 45 (5.7%), 66 (5.7%), 52 (2.9%), 58 (2.9%), 6 (2.9%), and 67 (2.9%). Several lesions demonstrated multiple HPV infections, ranging from two to six different types in one lesion. The study showed high diversity of HPV types in cancerous and pre-cancerous lesions of South African males with the most frequent being HPV types 11 and 16. The data suggest that boys could directly benefit from vaccination as they are exposed to variety of HPV types as early as 10 years of age in Africa.

Evidence for a change in the epidemiology of hepatitis B virus infection after nearly two decades of universal hepatitis B vaccination in South Africa.

The hepatitis B vaccine has been part of the South African Expanded Program on Immunization since April 1995 but its long-term impact remains unknown. This study tested 1,206 sera collected from patients aged 1-25 years from various health facilities across the country for HBV serological markers and HBV DNA. Based on the year the vaccine was introduced, samples were stratified by age into pre- and post-vaccine introduction populations, which were then compared for evidence of immunity and chronic carriage using the Chi-square test. Where HIV status was known, subset analyses were performed. Immunity to HBV infection increased from 13.0% in the pre- to 57.0% in the post-vaccine introduction population (P < 0.001). This decreased with increasing age within the post-vaccine introduction population (76.1% for 1-5 years, 50.0% for 6-10 years, and 46.3% for 11-16 years). In addition, HBV chronic carriage was significantly (P = 0.003) reduced in the post- (1.4%) compared to the pre-vaccine introduction population (4.2%). The difference in prevalence of active HBV infection in the serologically exposed pre- and post-vaccine introduction populations was not statistically significant. Subset analyses showed that evidence of immunity was significantly (P < 0.001) higher in the HIV negative compared to the HIV positive subset in both populations. Universal hepatitis B vaccination has been a remarkable success, with a significant increase in immunity to HBV infection. The observation that HBV chronic carriage increases as immunity wanes over time calls into question whether the time has come to consider a pre-adolescence vaccine booster dose policy.

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

BACKGROUND: Hepatitis 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. METHODS: HCV 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. RESULTS: A 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. CONCLUSION: The 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.

Whole-genome analyses of DS-1-like human G2P[4] and G8P[4] rotavirus strains from Eastern, Western and Southern Africa.

Group A rotaviruses (RVAs) with distinct G and P genotype combinations have been reported globally. We report the genome composition and possible origin of seven G8P[4] and five G2P[4] human RVA strains based on the genetic evolution of all 11 genome segments at the nucleotide level. Twelve RVA ELISA positive stool samples collected in the representative countries of Eastern, Southern and West Africa during the 2007-2012 surveillance seasons were subjected to sequencing using the Ion Torrent PGM and Illumina MiSeq platforms. A reference-based assembly was performed using CLC Bio's clc_ref_assemble_long program, and full-genome consensus sequences were obtained. With the exception of the neutralising antigen, VP7, all study strains exhibited the DS-1-like genome constellation (P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2) and clustered phylogenetically with reference strains having a DS-1-like genetic backbone. Comparison of the nucleotide and amino acid sequences with selected global cognate genome segments revealed nucleotide and amino acid sequence identities of 81.7-100 % and 90.6-100 %, respectively, with NSP4 gene segment showing the most diversity among the strains. Bayesian analyses of all gene sequences to estimate the time of divergence of the lineage indicated that divergence times ranged from 16 to 44 years, except for the NSP4 gene where the lineage seemed to arise in the more distant past at an estimated 203 years ago. However, the long-term effects of changes found within the NSP4 genome segment should be further explored, and thus we recommend continued whole-genome analyses from larger sample sets to determine the evolutionary mechanisms of the DS-1-like strains collected in Africa.

Variability of the preC/C region of hepatitis B virus genotype A from a South African cohort predominantly infected with HIV.

Hepatitis B virus (HBV) is a serious global health problem, and HBV genotype is an important determinant of disease progression and treatment outcome. The aim of this study was to assess variations of the precore/core (preC/C) region in HBV genotype A. Sequencing of the preC/C and surface (S) genes of HBV was performed on plasma samples from 20 HBV/HIV co-infected and 5 HBV mono-infected individuals. All preC/C study sequences clustered with subgenotype A1, except for two which clustered with subgenotype D4 reference strains. The nucleotide and amino acid variability was far higher in the preC/C region than in the S region. Mutations associated with reduction or failure of HBV e-antigen (HBeAg) production were observed, with a preC start codon mutation being common (24%). Other mutations (e.g., P5H/L and I97L) associated with severe liver disease were also noticed, some of which were located in the major histocompatibility restricted sites. PreC/C intergenotype nucleotide divergence was >7%, while subgenotypes differed by 2.5-7%. Several study sequences were highly divergent from other African subgenotype A1 strains. This study showed that HBeAg-negative chronic hepatitis B is underestimated in subgenotype A1, and also highlighted the variant South African A1 strains. The major advantage of preC/C sequencing is that it informs patient management as HBeAg-negative chronic hepatitis B responds poorly to conventional interferon-α therapy, and some guidelines treat HBeAg-negative chronic hepatitis B differently from HBeAg-positive chronic hepatitis B. These data suggest that subgenotype A1 may be more involved in severe HBV-related diseases.

Maintaining Momentum for Rotavirus Immunization in Africa during the COVID-19 Era: Report of the 13th African Rotavirus Symposium.

The 13th African Rotavirus Symposium was held as a virtual event hosted by the University of Nairobi, Kenya and The Kenya Paediatric Association on 3rd and 4th November 2021. This biennial event organized under the auspices of the African Rotavirus Network shapes the agenda for rotavirus research and prevention on the continent, attracting key international and regional opinion leaders, researchers, and public health scientists. The African Rotavirus Network is a regional network of institutions initially established in 1999, and now encompassing much of the diarrheal disease and rotavirus related research in Africa, in collaboration with the World Health Organization African Regional Office (WHO-AFRO), Ministries of Health, and other partners. Surges in SARS-CoV2 variants and concomitant travel restrictions limited the meeting to a webinar platform with invited scientific presentations and scientific presentations from selected abstracts. The scientific program covered updates on burden of diarrheal diseases including rotavirus, the genomic characterization of rotavirus strains pre- and post-rotavirus vaccine introduction, and data from clinical evaluation of new rotavirus vaccines in Africa. Finally, 42 of the 54 African countries have fully introduced rotavirus vaccination at the time of the meeting, including the two recently WHO pre-qualified vaccines from India. Nonetheless, the full benefit of rotavirus vaccination is yet to be realized in Africa where approximately 80% of the global burden of rotavirus mortality exists.

Low seroprevalence of hepatitis E virus in pregnant women in an urban area near Pretoria, South Africa.

Objectives: Hepatitis E virus (HEV) infection is a globally neglected health problem with a high burden in resource-poor communities. Pregnant women are at increased risk of complications. This pilot study sought to assess the seroprevalence of HEV infection in pregnant women at Dr George Mukhari Academic Hospital, South Africa. Methods: Stored serum samples from 384 HIV-uninfected pregnant women attending the antenatal clinic were initially screened for HEV total antibody. Positive samples were further evaluated for the presence of IgG and IgM antibody isotypes, using commercial ELISA assays. HEV RNA was assessed in antibody-positive samples utilizing qRT-PCR assay. Results: The sample consisted of women with a median age of 31 years (interquartile range: 28-35 years). Total HEV antibody was detected in 12/384 (3.13%, 95% CI: 1.80-5.38) of these pregnant women. All 12 samples were IgG HEV antibody positive, but none tested positive for IgM antibody or for HEV RNA, demonstrating a lack of current or recent exposure. Conclusions: Our study revealed a low seroprevalence of HEV among pregnant women from an urban area north of Pretoria. This observation warrants further attention to the circulation of HEV in this population, and a greater understanding of the epidemiology of the infection in South Africa.

Evolutionary changes between pre- and post-vaccine South African group A G2P[4] rotavirus strains, 2003-2017.

The transient upsurge of G2P[4] group A rotavirus (RVA) after Rotarix vaccine introduction in several countries has been a matter of concern. To gain insight into the diversity and evolution of G2P[4] strains in South Africa pre- and post-RVA vaccination introduction, whole-genome sequencing was performed for RVA positive faecal specimens collected between 2003 and 2017 and samples previously sequenced were obtained from GenBank (n=103; 56 pre- and 47 post-vaccine). Pre-vaccine G2 sequences predominantly clustered within sub-lineage IVa-1. In contrast, post-vaccine G2 sequences clustered mainly within sub-lineage IVa-3, whereby a radical amino acid (AA) substitution, S15F, was observed between the two sub-lineages. Pre-vaccine P[4] sequences predominantly segregated within sub-lineage IVa while post-vaccine sequences clustered mostly within sub-lineage IVb, with a radical AA substitution R162G. Both S15F and R162G occurred outside recognised antigenic sites. The AA residue at position 15 is found within the signal sequence domain of Viral Protein 7 (VP7) involved in translocation of VP7 into endoplasmic reticulum during infection process. The 162 AA residue lies within the hemagglutination domain of Viral Protein 4 (VP4) engaged in interaction with sialic acid-containing structure during attachment to the target cell. Free energy change analysis on VP7 indicated accumulation of stable point mutations in both antigenic and non-antigenic regions. The segregation of South African G2P[4] strains into pre- and post-vaccination sub-lineages is likely due to erstwhile hypothesized stepwise lineage/sub-lineage evolution of G2P[4] strains rather than RVA vaccine introduction. Our findings reinforce the need for continuous whole-genome RVA surveillance and investigation of contribution of AA substitutions in understanding the dynamic G2P[4] epidemiology.

A decade of rotavirus vaccination in Africa - Saving lives and changing the face of diarrhoeal diseases: Report of the 12th African Rotavirus Symposium.

The African Rotavirus Network organised the 12th African Rotavirus Symposium (ARS) from 30 July to 1 August 2019 in Johannesburg, South Africa. The symposium theme "A decade of rotavirus vaccination in Africa - Saving lives and changing the face of diarrhoeal diseases", included sessions aimed at sharing ideas and expertise on prevention and control of diarrhoeal disease in Africa. Inter alia, the delegates reviewed global and regional epidemiological trends on rotavirus diarrhoea, progress and experiences on rotavirus vaccine introduction, including vaccine safety monitoring and impact in Africa, scientific advances in developing newer rotavirus vaccines, surveillance and research on other diarrhoeal pathogens, and providing an enabling environment for networking. Importantly, the 12th ARS served to commemorate the 20th anniversary of the African Rotavirus Network (AfrRN) coinciding with the 50th anniversary of the South African Medical Research Council. Four oral, live-attenuated rotavirus vaccines are currently prequalified by the WHO (Rotarix, RotaTeq, Rotavac and RotaSiil). African countries utilising rotavirus vaccines in routine national immunisation programmes are realising their effectiveness and impact on diarrhoeal disease morbidity. An ~40% reduction in hospitalisations of <5-year-olds with acute gastroenteritis following rotavirus vaccine introduction, was reported between 2006 and 2018 in 92,000 children from the WHO-coordinated African Rotavirus Surveillance Network (AfrRSN) comprising 33 Member States. This was corroborated by a meta-analysis of published data, sourced from January 2000 to August 2018 that reported substantial reductions in rotavirus hospitalisations in countries using rotavirus vaccines. However, it was highlighted that the transition of some countries from Gavi-eligibility and vaccine supply shortfalls present significant challenges to achieving the full impact of rotavirus immunization in Africa. The wide diversity of rotavirus genotypes continues in Africa, with variation observed both geographically and temporally. There is currently no evidence to suggest that the emergence of rotavirus strains not included in the current vaccines do escape vaccine-induced immunity.