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.

Whole Genome Analysis of Human Rotaviruses Reveals Single Gene Reassortant Rotavirus Strains in Zambia.

Rotarix® vaccine was implemented nationwide in Zambia in 2013. In this study, four unusual strains collected in the post-vaccine period were subjected to whole genome sequencing and analysis. The four strains possessed atypical genotype constellations, with at least one reassortant genome segment within the constellation. One of the strains (UFS-NGS-MRC-DPRU4749) was genetically and phylogenetically distinct in the VP4 and VP1 gene segments. Pairwise analyses demonstrated several amino acid disparities in the VP4 antigenic sites of this strain compared to that of Rotarix®. Although the impact of these amino acid disparities remains to be determined, this study adds to our understanding of the whole genomes of reassortant strains circulating in Zambia following Rotarix® vaccine introduction.

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.

Whole-Genome Analyses Identifies Multiple Reassortant Rotavirus Strains in Rwanda Post-Vaccine Introduction.

Children in low-and middle-income countries, including Rwanda, experience a greater burden of rotavirus disease relative to developed countries. Evolutionary mechanisms leading to multiple reassortant rotavirus strains have been documented over time which influence the diversity and evolutionary dynamics of novel rotaviruses. Comprehensive rotavirus whole-genome analysis was conducted on 158 rotavirus group A (RVA) samples collected pre- and post-vaccine introduction in children less than five years in Rwanda. Of these RVA positive samples, five strains with the genotype constellations G4P[4]-I1-R2-C2-M2-A2-N2-T1-E1-H2 (n = 1), G9P[4]-I1-R2-C2-M2-A1-N1-T1-E1-H1 (n = 1), G12P[8]-I1-R2-C2-M1-A1-N2-T1-E2-H3 (n = 2) and G12P[8]-I1-R1-C1-M1-A2-N2-T2-E1-H1 (n = 1), with double and triple gene reassortant rotavirus strains were identified. Phylogenetic analysis revealed a close relationship between the Rwandan strains and cognate human RVA strains as well as the RotaTeq® vaccine strains in the VP1, VP2, NSP2, NSP4 and NSP5 gene segments. Pairwise analyses revealed multiple differences in amino acid residues of the VP7 and VP4 antigenic regions of the RotaTeq® vaccine strain and representative Rwandan study strains. Although the impact of such amino acid changes on the effectiveness of rotavirus vaccines has not been fully explored, this analysis underlines the potential of rotavirus whole-genome analysis by enhancing knowledge and understanding of intergenogroup reassortant strains circulating in Rwanda post vaccine introduction.

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.

Metagenomic Analysis of the Enteric RNA Virome of Infants from the Oukasie Clinic, North West Province, South Africa, Reveals Diverse Eukaryotic Viruses.

Establishing a diverse gut microbiota after birth is essential for preventing illnesses later in life. However, little knowledge exists about the total viral population (virome) present in the gut of infants during the early developmental stage, with RNA viruses being generally overlooked. Therefore, this small pilot longitudinal study investigated the diversity and changes in the enteric RNA virome in healthy infants from South Africa. Faecal samples (n = 12) were collected from four infants at three time points (on average at 8, 13, and 25 weeks), and then sequenced on an Illumina MiSeq platform. The genomic analysis revealed a diverse population of human enteric viruses from the infants' stools, and changes in the enteric virome composition were observed over time. The Reoviridae family, more specifically the Rotavirus genus, was the most common and could be linked to viral shedding due to the administration of live-attenuated oral vaccines in South Africa, followed by the Picornaviridae family including parechoviruses, echoviruses, coxsackieviruses, enteroviruses, and polioviruses. Polioviruses were also linked to vaccine-related shedding. Astroviridae (astroviruses) and Caliciviridae (noroviruses) were present at low abundance. It is evident that an infant's gut is colonized by distinct viral populations irrespective of their health state. Further characterization of the human virome (with a larger participant pool) is imperative to provide more conclusive insights into the viral community structure and diversity that has been shown in the current study, despite the smaller sample size.

Whole Genome In-Silico Analysis of South African G1P[8] Rotavirus Strains Before and After Vaccine Introduction Over A Period of 14 Years.

Rotavirus G1P[8] strains account for more than half of the group A rotavirus (RVA) infections in children under five years of age, globally. A total of 103 stool samples previously characterized as G1P[8] and collected seven years before and seven years after introducing the Rotarix® vaccine in South Africa were processed for whole-genome sequencing. All the strains analyzed had a Wa-like constellation (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1). South African pre- and post-vaccine G1 strains were clustered in G1 lineage-I and II while the majority (84.2%) of the P[8] strains were grouped in P[8] lineage-III. Several amino acid sites across ten gene segments with the exception of VP7 were under positive selective pressure. Except for the N147D substitution in the antigenic site of eight post-vaccine G1 strains when compared to both Rotarix® and pre-vaccine strains, most of the amino acid substitutions in the antigenic regions of post-vaccine G1P[8] strains were already present during the pre-vaccine period. Therefore, Rotarix® did not appear to have an impact on the amino acid differences in the antigenic regions of South African post-vaccine G1P[8] strains. However, continued whole-genome surveillance of RVA strains to decipher genetic changes in the post-vaccine period remains imperative.

Molecular Characterisation of a Rare Reassortant Porcine-Like G5P[6] Rotavirus Strain Detected in an Unvaccinated Child in Kasama, Zambia.

A human-porcine reassortant strain, RVA/Human-wt/ZMB/UFS-NGS-MRC-DPRU4723/2014/G5P[6], was identified in a sample collected in 2014 from an unvaccinated 12 month old male hospitalised for gastroenteritis in Zambia. We sequenced and characterised the complete genome of this strain which presented the constellation: G5-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The genotype A8 is often observed in porcine strains. Phylogenetic analyses showed that VP6, VP7, NSP2, NSP4, and NSP5 genes were closely related to cognate gene sequences of porcine strains (e.g., RVA/Pig-wt/CHN/DZ-2/2013/G5P[X] for VP7) from the NCBI database, while VP1, VP3, VP4, and NSP3 were closely related to porcine-like human strains (e.g., RVA/Human-wt/CHN/E931/2008/G4P[6] for VP1, and VP3). On the other hand, the origin of the VP2 was not clear from our analyses, as it was not only close to both porcine (e.g., RVA/Pig-tc/CHN/SWU-1C/2018/G9P[13]) and porcine-like human strains (e.g., RVA/Human-wt/LKA/R1207/2009/G4P[6]) but also to three human strains (e.g., RVA/Human-wt/USA/1476/1974/G1P[8]). The VP7 gene was located in lineage II that comprised only porcine strains, which suggests the occurrence of independent porcine-to-human reassortment events. The study strain may have collectively been derived through interspecies transmission, or through reassortment event(s) involving strains of porcine and porcine-like human origin. The results of this study underline the importance of whole-genome characterisation of rotavirus strains and provide insights into interspecies transmissions from porcine to humans.

Whole genome and in-silico analyses of G1P[8] rotavirus strains from pre- and post-vaccination periods in Rwanda.

Rwanda was the first low-income African country to introduce RotaTeq vaccine into its Expanded Programme on Immunization in May 2012. To gain insights into the overall genetic make-up and evolution of Rwandan G1P[8] strains pre- and post-vaccine introduction, rotavirus positive fecal samples collected between 2011 and 2016 from children under the age of 5 years as part of ongoing surveillance were genotyped with conventional RT-PCR based methods and whole genome sequenced using the Illumina MiSeq platform. From a pool of samples sequenced (n = 158), 36 were identified as G1P[8] strains (10 pre-vaccine and 26 post-vaccine), of which 35 exhibited a typical Wa-like genome constellation. However, one post vaccine strain, RVA/Human-wt/RWA/UFS-NGS:MRC-DPRU442/2012/G1P[8], exhibited a RotaTeq vaccine strain constellation of G1-P[8]-I2-R2-C2-M2-A3-N2-T6-E2-H3, with most of the gene segments having a close relationship with a vaccine derived reassortant strain, previously reported in USA in 2010 and Australia in 2012. The study strains segregated into two lineages, each containing a paraphyletic pre- and post-vaccine introduction sub-lineages. In addition, the study strains demonstrated close relationship amongst each other when compared with globally selected group A rotavirus (RVA) G1P[8] reference strains. For VP7 neutralization epitopes, amino acid substitutions observed at positions T91A/V, S195D and M217T in relation to the RotaTeq vaccine were radical in nature and resulted in a change in polarity from a polar to non-polar molecule, while for the VP4, amino acid differences at position D195G was radical in nature and resulted in a change in polarity from a polar to non-polar molecule. The polarity change at position T91A/V of the neutralizing antigens might play a role in generating vaccine-escape mutants, while substitutions at positions S195D and M217T may be due to natural fluctuation of the RVA. Surveillance of RVA at whole genome level will enhance further assessment of vaccine impact on circulating strains, the frequency of reassortment events under natural conditions and epidemiological fitness generated by such events.

Impact of Lamivudine-Based Antiretroviral Treatment on Hepatitis B Viremia in HIV-Coinfected South Africans.

This prospective study investigated the impact of lamivudine-containing antiretroviral therapy (ART) on HIV-positive patients in South Africa with baseline hepatitis B virus (HBV) infection. Follow-up samples from 56 HBV/HIV co-infected patients, 25 with occult HBV infection (OBI) and 31 with chronic HBV infection (CHB), were available for analysis. HBV viral loads were quantified at 6, 12, 18, and 24 months post-ART initiation by the COBAS TaqMan HBV Test 48 assay, and the HBV polymerase gene was amplified with an in-house nested polymerase chain reaction assay. During 24 months of lamivudine-based ART, 6 of 8 (75%) OBI and 4 of 6 (67%) CHB patients achieved undetectable levels of HBV DNA, while 2 patients had persistent HBV DNA levels ≥ 2 × 105 despite lamivudine-based ART for 24 months. HIV viremia was undetectable in all patients at 12 months, suggesting high adherence to ART. Several lamivudine-associated HBV resistance mutations, including L180M, A181T, M204I, and M204V, were observed. Sequence analysis also revealed a rare genotype G infection. While resource-limited settings may use lamivudine-based ART because of availability and low cost, antivirals with dual therapy against HBV and HIV (e.g., lamivudine and tenofovir) should always be recommended with the regular monitoring of HBV viremia levels.

Uncovering the First Atypical DS-1-like G1P[8] Rotavirus Strains That Circulated during Pre-Rotavirus Vaccine Introduction Era in South Africa.

Emergence of DS-1-like G1P[8] group A rotavirus (RVA) strains during post-rotavirus vaccination period has recently been reported in several countries. This study demonstrates, for the first time, rare atypical DS-1-like G1P[8] RVA strains that circulated in 2008 during pre-vaccine era in South Africa. Rotavirus positive samples were subjected to whole-genome sequencing. Two G1P[8] strains (RVA/Human-wt/ZAF/UFS-NGS-MRC-DPRU1971/2008/G1P[8] and RVA/Human-wt/ZAF/UFS-NGS-MRC-DPRU1973/2008/G1P[8]) possessed a DS-1-like genome constellation background (I2-R2-C2-M2-A2-N2-T2-E2-H2). The outer VP4 and VP7 capsid genes of the two South African G1P[8] strains had the highest nucleotide (amino acid) nt (aa) identities of 99.6-99.9% (99.1-100%) with the VP4 and the VP7 genes of a locally circulating South African strain, RVA/Human-wt/ZAF/MRC-DPRU1039/2008/G1P[8]. All the internal backbone genes (VP1-VP3, VP6, and NSP1-NSP5) had the highest nt (aa) identities with cognate internal genes of another locally circulating South African strain, RVA/Human-wt/ZAF/MRC-DPRU2344/2008/G2P[6]. The two study strains emerged through reassortment mechanism involving locally circulating South African strains, as they were distinctly unrelated to other reported atypical G1P[8] strains. The identification of these G1P[8] double-gene reassortants during the pre-vaccination period strongly supports natural RVA evolutionary mechanisms of the RVA genome. There is a need to maintain long-term whole-genome surveillance to monitor such atypical strains.

Datasets on the measurement of mechanical properties of ferrite and austenite constitutive phases using nanoindentation and micro hardness techniques.

The major objective of this work is to study the hardness data at the domain of ferrite and Austenite phases. Nanoindentation and microhardness study has been conducted on austenite and ferrite present in the microstructure of hot rolled and heat treated duplex stainless steel (2205 DSS). Furthermore, Optical microscopy and field emission scanning electron microscope (FE-SEM) were used to identify the microstructural distribution and phases present. Austenite reveals higher nanohardness data value than ferrite, as oppose to ferrite average elastic modulus which is higher than that of austenite. Also, higher value of microhardness data was observed for austenite in comparison with the ferrite at different load application.

Impact of rotavirus vaccine introduction and genotypic characteristics of rotavirus strains in children less than 5 years of age with gastroenteritis in Ethiopia: 2011-2016.

INTRODUCTION: A monovalent rotavirus vaccine was introduced in the Ethiopian Expanded Program on Immunization from November 2013. We compared impact of rotavirus vaccine introduction on rotavirus associated acute diarrhea hospitalizations and genotypic characteristics of rotavirus strains pre-and post-vaccine introduction. METHODS: Sentinel surveillance for diarrhea among children <5 years of age was conducted at 3 hospitals in Addis Ababa, Ethiopia from 2011 to 2017. Stool specimens were collected from enrolled children and tested using an antigen capture enzyme immunoassay. Rotavirus positive samples (156 from pre- and 141 from post-vaccination periods) were further characterized by rotavirus genotyping methods to identify the predominant G and P types circulating during the surveillance era. RESULTS: A total of 788 children were enrolled during the pre- (July 2011-June 2013) and 815 children during the post-vaccination (July 2014-June 2017) periods. The proportion of diarrhea hospitalizations due to rotavirus among children <5 years of age declined by 17% from 24% (188/788) in the pre-vaccine period and to 20% (161/185) in post-vaccine introduction era. Similarly, a reduction of 18% in proportion of diarrhea hospitalizations due to rotavirus in children <12 months of age in the post (27%) vs pre-vaccine (33%) periods was observed. Seasonal peaks of rotavirus declined following rotavirus vaccine introduction. The most prevalent circulating strains were G12P[8] in 2011 (36%) and in 2012 (27%), G2P[4] (35%) in 2013, G9P[8] (19%) in 2014, G3P[6] and G2P[4] (19% each) in 2015, and G3P[8] (29%) in 2016. DISCUSSION: Following rotavirus vaccine introduction in Ethiopia, a reduction in rotavirus associated hospitalizations was seen in all age groups with the greatest burden in children <12 months of age. A wide variety of rotavirus strains circulated in the pre- and post-vaccine introduction periods.

Impact of vaccine stock-outs on infant vaccination coverage: a hospital-based survey from South Africa.

Introduction: National population-based immunization coverage surveys provide data for validating official administrative coverage figures. However, these costly and logistically challenging surveys are conducted infrequently. This hospital-based records review determined coverage of birth-dose vaccines, fully immunized under 1-y-old coverage (FIC) of 12- to 59-mo-old children; and the reasons for missed vaccinations. Methods: Rotavirus surveillance in South Africa is based on under-5-y-old children being treated for diarrhoea, and includes photocopying the official vaccination document and collecting data on reasons for missed vaccinations. These data were captured from all 508 records collected from 2011 to 2014, and subjected to descriptive statistical analysis. Results: Bacille Calmette Guérin coverage was 99%; oral polio vaccine birth dose (OPV(0)) coverage was 99%. Coverage for 12- to 59-mo-olds ranged from 75% for the pneumococcal conjugate vaccine third dose to 99% for OPV(0). Several instances of subsequent doses being recorded without prior doses being received resulted in a FIC of 55%. In total, 207 vaccinations were missed by 88 children. Vaccine stock-outs were responsible for 62% of missed vaccinations. Conclusions: Efforts to improve vaccine stock management at facility and district levels should be implemented, and should include vaccinator training and supervision to eliminate vaccine stock-outs and missed vaccination opportunities.

Whole-genome sequencing and analyses identify high genetic heterogeneity, diversity and endemicity of rotavirus genotype P[6] strains circulating in Africa.

Rotavirus A (RVA) exhibits a wide genotype diversity globally. Little is known about the genetic composition of genotype P[6] from Africa. This study investigated possible evolutionary mechanisms leading to genetic diversity of genotype P[6] VP4 sequences. Phylogenetic analyses on 167 P[6] VP4 full-length sequences were conducted, which included six porcine-origin sequences. Of the 167 sequences, 57 were newly acquired through whole genome sequencing as part of this study. The other 110 sequences were all publicly-available global P[6] VP4 full-length sequences downloaded from GenBank. The strength of association between the phenotypic features and the phylogeny was also determined. A number of reassortment and mixed infections of RVA genotype P[6] strains were observed in this study. Phylogenetic analyses demostrated the extensive genetic diversity that exists among human P[6] strains, porcine-like strains, their concomitant clades/subclades and estimated that P[6] VP4 gene has a higher substitution rate with the mean of 1.05E-3 substitutions/site/year. Further, the phylogenetic analyses indicated that genotype P[6] strains were endemic in Africa, characterised by an extensive genetic diversity and long-time local evolution of the viruses. This was also supported by phylogeographic clustering and G-genotype clustering of the P[6] strains when Bayesian Tip-association Significance testing (BaTS) was applied, clearly supporting that the viruses evolved locally in Africa instead of spatial mixing among different regions. Overall, the results demonstrated that multiple mechanisms such as reassortment events, various mutations and possibly interspecies transmission account for the enormous diversity of genotype P[6] strains in Africa. These findings highlight the need for continued global surveillance of rotavirus diversity.

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.

Impact of rotavirus vaccine on all-cause diarrhea and rotavirus hospitalizations in Madagascar.

BACKGROUND: Rotavirus vaccine was introduced into the Extended Program on Immunization in Madagascar in May 2014. We analyzed trends in prevalence of all cause diarrhea and rotavirus hospitalization in children <5years of age before and after vaccine introduction and assessed trend of circulating rotavirus genotypes at Centre Hospitalier Universitaire Mère Enfant Tsaralalàna (CHU MET). METHODS: From January 2010 to December 2016, we reviewed the admission logbook to observe the rate of hospitalization caused by gastroenteritis among 19619 children <5years of age admitted at the hospital. In June 2013-December 2016, active rotavirus surveillance was also conducted at CHUMET with support from WHO. Rotavirus antigen was detected by EIA from stool specimen of children who are eligible for rotavirus gastroenteritis surveillance at sentinel site laboratory and rotavirus positive specimens were further genotyped at Regional Reference Laboratory by RT-PCR. RESULTS: Diarrhea hospitalizations decreased after rotavirus vaccine introduction. The median proportion of annual hospitalizations due to diarrhea was 26% (range: 31-22%) before vaccine introduction; the proportion was 25% the year of vaccine introduction, 17% in 2015 and 16% in 2016. Rotavirus positivity paralleled patterns observed in diarrhea. Before vaccine introduction, 56% of stool specimens tested positive for rotavirus; the percent positive was 13% in 2015, 12% in 2016. Diverse genotypes were detected in the pre-vaccine period; the most common were G3P[8] (n=53; 66%), G2P[4] (n=12; 15%), and G1P[8] (n=11; 14%). 6 distinct genotypes were found in 2015; the most common genotype was G2P[4] (n=10; 67%), the remaining, 5, G12[P8], G3[P8], G1G3[P4], G3G12[P4][P8] and G1G3[NT] had one positive specimen each. CONCLUSIONS: Following rotavirus vaccine introduction all-cause diarrhea and rotavirus-specific hospitalizations declined dramatically. The most common genotypes detected in the pre-vaccine period were G3P[8] and G2P[4] in 2015, the post vaccine period.