ABSTRACT
Prompt detection of viral respiratory pathogens is crucial in managing respiratory infection including severe acute respiratory infection (SARI). Metagenomics next-generation sequencing (mNGS) and bioinformatics analyses remain reliable strategies for diagnostic and surveillance purposes. This study evaluated the diagnostic utility of mNGS using multiple analysis tools compared with multiplex real-time PCR for the detection of viral respiratory pathogens in children under 5 years with SARI. Nasopharyngeal swabs collected in viral transport media from 84 children admitted with SARI as per the World Health Organization definition between December 2020 and August 2021 in the Free State Province, South Africa, were used in this study. The obtained specimens were subjected to mNGS using the Illumina MiSeq system, and bioinformatics analysis was performed using three web-based analysis tools; Genome Detective, One Codex and Twist Respiratory Viral Research Panel. With average reads of 211323, mNGS detected viral pathogens in 82 (97.6%) of the 84 patients. Viral aetiologies were established in nine previously undetected/missed cases with an additional bacterial aetiology (Neisseria meningitidis) detected in one patient. Furthermore, mNGS enabled the much needed viral genotypic and subtype differentiation and provided significant information on bacterial co-infection despite enrichment for RNA viruses. Sequences of nonhuman viruses, bacteriophages, and endogenous retrovirus K113 (constituting the respiratory virome) were also uncovered. Notably, mNGS had lower detectability rate for severe acute respiratory syndrome coronavirus 2 (missing 18/32 cases). This study suggests that mNGS, combined with multiple/improved bioinformatics tools, is practically feasible for increased viral and bacterial pathogen detection in SARI, especially in cases where no aetiological agent could be identified by available traditional methods.
Subject(s)
Bacterial Infections , COVID-19 , RNA Viruses , Viruses , Humans , Child , Child, Preschool , RNA, Viral/genetics , South Africa , Viruses/genetics , RNA Viruses/genetics , Bacteria/genetics , Metagenomics/methods , High-Throughput Nucleotide Sequencing/methods , Sensitivity and SpecificityABSTRACT
Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) amplicon contamination was discovered due to next-generation sequencing (NGS) reads mapping in the negative controls. Environmental screening was undertaken to determine the source of contamination, which was suspected to be evaporation during polymerase chain reaction (PCR) assays while using the coronavirus disease 2019 (COVID-19) ARTIC protocol. A decontamination strategy is hereby documented to assist laboratories that may experience similar amplicon contamination. Routine molecular laboratory environmental screening as a quality control is highly recommended.
Subject(s)
COVID-19 , Laboratories , COVID-19/prevention & control , Decontamination , High-Throughput Nucleotide Sequencing , Humans , SARS-CoV-2/geneticsABSTRACT
The gut of healthy neonates is devoid of viruses at birth, but rapidly becomes colonised by normal viral commensals that aid in important physiological functions like metabolism but can, in some instances, result in gastrointestinal illnesses. However, little is known about how this colonisation begins, its variability and factors shaping the gut virome composition. Thus, understanding the development, assembly, and progression of enteric viral communities over time is key. To explore early-life virome development, metagenomic sequencing was employed in faecal samples collected longitudinally from a cohort of 17 infants during their first six months of life. The gut virome analysis revealed a diverse and dynamic viral community, formed by a richness of different viruses infecting humans, non-human mammals, bacteria, and plants. Eukaryotic viruses were detected as early as one week of life, increasing in abundance and diversity over time. Most of the viruses detected are commonly associated with gastroenteritis and include members of the Caliciviridae, Picornaviridae, Astroviridae, Adenoviridae, and Sedoreoviridae families. The most common co-occurrences involved asymptomatic norovirus-parechovirus, norovirus-sapovirus, sapovirus-parechovirus, observed in at least 40 % of the samples. Majority of the plant-derived viruses detected in the infants' gut were from the Virgaviridae family. This study demonstrates the first longitudinal characterisation of the gastrointestinal virome in infants, from birth up to 6 months of age, in sub-Saharan Africa. Overall, the findings from this study delineate the composition and variability of the healthy infants' gut virome over time, which is a significant step towards understanding the dynamics and biogeography of viral communities in the infant gut.
Subject(s)
Feces , Virome , Humans , South Africa , Infant , Longitudinal Studies , Feces/virology , Infant, Newborn , Gastrointestinal Microbiome , Male , Female , Viruses/classification , Viruses/isolation & purification , Viruses/genetics , Metagenomics , Gastrointestinal Tract/virology , Gastroenteritis/virology , Sapovirus/genetics , Sapovirus/isolation & purification , Sapovirus/classification , Norovirus/genetics , Norovirus/isolation & purification , Norovirus/classification , Picornaviridae/genetics , Picornaviridae/classification , Picornaviridae/isolation & purification , Caliciviridae/genetics , Caliciviridae/isolation & purification , Caliciviridae/classification , MetagenomeABSTRACT
Enterovirus (EV) infections are widespread and associated with a range of clinical conditions, from encephalitis to meningitis, gastroenteritis, and acute flaccid paralysis. Knowledge about the circulation of EVs in neonatal age and early infancy is scarce, especially in Africa. This study aimed to unveil the frequency and diversity of EVs circulating in apparently healthy newborns from the Free State Province, South Africa (SA). For this purpose, longitudinally collected faecal specimens (May 2021-February 2022) from a cohort of 17 asymptomatic infants were analysed using metagenomic next-generation sequencing. Overall, seven different non-polio EV (NPEV) subtypes belonging to EV-B and EV-C species were identified, while viruses classified under EV-A and EV-D species could not be characterised at the sub-species level. Additionally, under EV-C species, two vaccine-related poliovirus subtypes (PV1 and PV3) were identified. The most prevalent NPEV species was EV-B (16/17, 94.1%), followed by EV-A (3/17, 17.6%), and EV-D (4/17, 23.5%). Within EV-B, the commonly identified NPEV types included echoviruses 6, 13, 15, and 19 (E6, E13, E15, and E19), and coxsackievirus B2 (CVB2), whereas enterovirus C99 (EV-C99) and coxsackievirus A19 (CVA19) were the only two NPEVs identified under EV-C species. Sabin PV1 and PV3 strains were predominantly detected during the first week of birth and 6-8 week time points, respectively, corresponding with the OPV vaccination schedule in South Africa. A total of 11 complete/near-complete genomes were identified from seven NPEV subtypes, and phylogenetic analysis of the three EV-C99 identified revealed that our strains were closely related to other strains from Cameroon and Brazil, suggesting global distribution of these strains. This study provides an insight into the frequency and diversity of EVs circulating in asymptomatic infants from the Free State Province, with the predominance of subtypes from EV-B and EV-C species. This data will be helpful to researchers looking into strategies for the control and treatment of EV infection.
ABSTRACT
Respiratory syncytial virus (RSV) is the most predominant viral pathogen worldwide in children with lower respiratory tract infections. The Coronavirus disease 2019 (COVID-19) pandemic and resulting nonpharmaceutical interventions perturbed the transmission pattern of respiratory pathogens in South Africa. A seasonality shift and RSV resurgence was observed in 2020 and 2021, with several infected children observed. Conventional RSV-positive nasopharyngeal swabs were collected from various hospitals in the Free State province, Bloemfontein, South Africa, from children suffering from respiratory distress and severe acute respiratory infection between 2020 to 2021. Overlapping genome fragments were amplified and complete genomes were sequenced using the Illumina MiSeq platform. Maximum likelihood phylogenetic and evolutionary analysis were performed on both RSV-A/-B G-genes with published reference sequences from GISAID and GenBank. Our study strains belonged to the RSV-A GA2.3.2 and RSV-B GB5.0.5a clades. The upsurge of RSV was due to pre-existing strains that predominated in South Africa and circulating globally also driving these off-season RSV outbreaks during the COVID-19 pandemic. The variants responsible for the resurgence were phylogenetically related to pre-pandemic strains and could have contributed to the immune debt resulting from pandemic imposed restrictions. The deviation of the RSV season from the usual pattern affected by the COVID-19 pandemic highlights the need for ongoing genomic surveillance and the identification of genetic variants to prevent unforeseen outbreaks in the future.
Subject(s)
COVID-19 , Genome, Viral , Phylogeny , Respiratory Syncytial Virus Infections , Respiratory Syncytial Virus, Human , SARS-CoV-2 , Whole Genome Sequencing , South Africa/epidemiology , Humans , COVID-19/epidemiology , COVID-19/virology , Respiratory Syncytial Virus Infections/epidemiology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/genetics , Respiratory Syncytial Virus, Human/classification , Respiratory Syncytial Virus, Human/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/classification , SARS-CoV-2/isolation & purification , Child, Preschool , Infant , Child , Pandemics , Seasons , Male , Female , Nasopharynx/virologyABSTRACT
The G2P[4] genotype is among the rotavirus strains that circulate commonly in humans. Several countries have reported its immediate upsurge after the introduction of rotavirus vaccination, raising concern about sub-optimal vaccine effectiveness against this genotype in the long term. This study aimed to gain insight into the evolution of post-vaccine Zambian G2P[4] group A rotavirus (RVA) strains and their overall genetic make-up by analysis of sequence alignments at the amino acid (AA) level. Twenty-nine Zambian G2P[4] rotavirus strains were subjected to whole-genome sequencing using the Illumina MiSeq® platform. All the strains exhibited the typical DS-1-like genotype constellation, and the nucleotide sequences of the 11 genome segments showed high nucleotide similarities (>97%). Phylogenetic analyses together with representative global G2P[4] RVA showed that Zambian strains clustered into human lineages IV (for VP2, VP4, VP7, NSP1, and NSP5), V (for VP1, VP3, VP6, NSP2, and NSP3), and XXIII (for NSP4). The AA differences between the lineages where the study strains clustered and lineages of global reference strains were identified and analyzed. Selection pressure analysis revealed that AA site seven in the Viral Protein 3 (VP3) genome segment was under positive selection. This site occurs in the region of intrinsic disorder in the VP3 protein, and Zambian G2P[4] strains could potentially be utilizing this intrinsically disordered region to survive immune pressure. The Zambian G2P[4] strains from 2012 to 2016 comprised the G2P[4] strains that have been circulating globally since the early 2000s, highlighting the epidemiological fitness of these contemporary G2P[4] strains. Continuous whole-genome surveillance of G2P[4] strains remains imperative to understand their evolution during the post-vaccination period.
Subject(s)
Rotavirus , Humans , Amino Acids , Genomics , Phylogeny , Rotavirus/genetics , Zambia/epidemiology , Viral Proteins/geneticsABSTRACT
Africa has a high level of genetic diversity of rotavirus strains, which is suggested to be a possible reason contributing to the suboptimal effectiveness of rotavirus vaccines in this region. One strain that contributes to this rotavirus diversity in Africa is the G8P[4]. This study aimed to elucidate the entire genome and evolution of Rwandan G8P[4] strains. Illumina sequencing was performed for twenty-one Rwandan G8P[4] rotavirus strains. Twenty of the Rwandan G8P[4] strains had a pure DS-1-like genotype constellation, and one strain had a reassortant genotype constellation. Notable radical amino acid differences were observed at the neutralization sites when compared with cognate regions in vaccine strains potentially playing a role in neutralization escape. Phylogenetic analysis revealed that the closest relationship was with East African human group A rotavirus (RVA) strains for five of the genome segments. Two genome sequences of the NSP4 genome segment were closely related to bovine members of the DS-1-like family. Fourteen VP1 and eleven VP3 sequences had the closest relationships with the RotaTeq™ vaccine WC3 bovine genes. These findings suggest that the evolution of VP1 and VP3 might have resulted from reassortment events with RotaTeq™ vaccine WC3 bovine genes. The close phylogenetic relationship with East African G8P[4] strains from Kenya and Uganda suggests co-circulation in these countries. These findings highlight the need for continued whole-genomic surveillance to elucidate the evolution of G8P[4] strains, especially after the introduction of rotavirus vaccination.
ABSTRACT
Although the introduction of rotavirus vaccines has substantially contributed to the reduction in rotavirus morbidity and mortality, concerns persist about the re-emergence of variant strains that might alter vaccine effectiveness in the long term. The G9 strains re-emerged in Africa during the mid-1990s and have more recently become predominant in some countries, such as Ghana and Zambia. In Rwanda, during the 2011 to 2015 routine surveillance period, G9P[8] persisted during both the pre- and post-vaccine periods. The pre-vaccination cohort was based on the surveillance period of 2011 to 2012, and the post-vaccination cohort was based on the period of 2013 to 2015, excluding 2014. The RotaTeq® vaccine that was first introduced in Rwanda in 2012 is genotypically heterologous to Viral Protein 7 (VP7) G9. This study elucidated the whole genome of Rwandan G9P[8] rotavirus strains pre- and post-RotaTeq® vaccine introduction. Fecal samples from Rwandan children under the age of five years (pre-vaccine n = 23; post-vaccine n = 7), conventionally genotyped and identified as G9P[8], were included. Whole-genome sequencing was then performed using the Illumina® MiSeq platform. Phylogenetic analysis and pair-wise sequence analysis were performed using MEGA6 software. Distinct clustering of three post-vaccination study strains was observed in all 11 gene segments, compared to the other Rwandan G9P[8] study strains. Specific amino acid differences were identified across the gene segments of these three 2015 post-vaccine strains. Important amino acid differences were identified at position N242S in the VP7 genome segment of the three post-vaccine G9 strains compared to the other G9 strains. This substitution occurs at a neutralization epitope site and may slightly affect protein interaction at that position. These findings indicate that the Rwandan G9P[8] strains revealed a distinct sub-clustering pattern among post-vaccination study strains circulating in Rwanda, with changes at neutralization epitopes, which may play a role in neutralization escape from vaccine candidates. This emphasizes the need for continuous whole-genome surveillance to better understand the evolution and epidemiology of the G9P[8] strains post-vaccination.
Subject(s)
Rotavirus Infections , Rotavirus Vaccines , Rotavirus , Child , Humans , Child, Preschool , Rotavirus Infections/epidemiology , Rotavirus Infections/prevention & control , Rwanda/epidemiology , Phylogeny , Vaccination , Genotype , Ghana/epidemiology , Genomics , Cluster Analysis , Amino Acids/genetics , Antigens, Viral/genetics , Capsid Proteins/geneticsABSTRACT
Mozambique introduced the rotavirus vaccine (Rotarix®; GlaxoSmithKline Biologicals, Rixensart, Belgium) in 2015, and since then, the Centro de Investigação em Saúde de Manhiça has been monitoring its impact on rotavirus-associated diarrhea and the trend of circulating strains, where G3P[8] was reported as the predominant strain after the vaccine introduction. Genotype G3 is among the most commonly detected Rotavirus strains in humans and animals, and herein, we report on the whole genome constellation of G3P[8] detected in two children (aged 18 months old) hospitalized with moderate-to-severe diarrhea at the Manhiça District Hospital. The two strains had a typical Wa-like genome constellation (I1-R1-C1-M1-A1-N1-T1-E1-H1) and shared 100% nucleotide (nt) and amino acid (aa) identities in 10 gene segments, except for VP6. Phylogenetic analysis demonstrated that genome segments encoding VP7, VP6, VP1, NSP3, and NSP4 of the two strains clustered most closely with porcine, bovine, and equine strains with identities ranging from 86.9-99.9% nt and 97.2-100% aa. Moreover, they consistently formed distinct clusters with some G1P[8], G3P[8], G9P[8], G12P[6], and G12P[8] strains circulating from 2012 to 2019 in Africa (Mozambique, Kenya, Rwanda, and Malawi) and Asia (Japan, China, and India) in genome segments encoding six proteins (VP2, VP3, NSP1-NSP2, NSP5/6). The identification of segments exhibiting the closest relationships with animal strains shows significant diversity of rotavirus and suggests the possible occurrence of reassortment events between human and animal strains. This demonstrates the importance of applying next-generation sequencing to monitor and understand the evolutionary changes of strains and evaluate the impact of vaccines on strain diversity.
ABSTRACT
Severe acute respiratory infections (SARI) contribute to mortality in children ≤5 years. Their microbiological aetiologies are often unknown and may be exacerbated in light of coronavirus disease 19 (COVID-19). This study reports on respiratory pathogens in children ≤5 years (n = 84) admitted with SARI during and between the second and third waves of COVID-19 infection in South Africa. Nasopharyngeal/oropharyngeal swabs collected were subjected to viral detection using QIAstat-Dx® Respiratory SARS-CoV-2 Panel. The results revealed viral positivity and negativity detection rates of 88% (74/84) and 12% (10/84), respectively. Of the 21 targeted pathogens, human rhinovirus/enterovirus (30%), respiratory syncytial virus (RSV; 26%), and severe acute respiratory syndrome coronavirus 2 (24%) were mostly detected, with other viruses being 20% and a co-infection rate of 64.2% (54/84). Generally, RSV-positive samples had lower Ct values, and fewer viruses were detected during the third wave. Changes in the circulation patterns of respiratory viruses with total absence of influenza virus could be attributed to measures against COVID-19 transmission, which may result in waned immunity, thereby increasing susceptibility to severe infections in the following season. High viral co-infection rate, as detected, may complicate diagnosis. Nonetheless, accurate identification of the pathogens may guide treatment decisions and infection control.
Subject(s)
COVID-19 , Coinfection , Respiratory Tract Infections , Virus Diseases , Viruses , COVID-19/epidemiology , Child , Coinfection/epidemiology , Humans , Pandemics , Respiratory Tract Infections/microbiology , SARS-CoV-2 , South Africa/epidemiologyABSTRACT
Viral respiratory infections contribute to significant morbidity and mortality in children. Currently, there are limited reports on the composition and abundance of the normal commensal respiratory virome in comparison to those in severe acute respiratory infections (SARIs) state. This study characterised the respiratory RNA virome in children ≤ 5 years with (n = 149) and without (n = 139) SARI during the summer and winter of 2020/2021 seasons in South Africa. Nasopharyngeal swabs were, collected, pooled, enriched for viral RNA detection, sequenced using Illumina MiSeq, and analysed using the Genome Detective bioinformatic tool. Overall, Picornaviridae, Paramoxyviridae, Pneumoviridae, Picobirnaviridae, Totiviridae, and Retroviridae families were the most abundant viral population in both groups across both seasons. Human rhinovirus and endogenous retrovirus K113 were detected in most pools, with exclusive detection of Pneumoviridae in SARI pools. Generally, higher viral diversity/abundance was seen in children with SARI and in the summer pools. Several plant/animal viruses, eukaryotic viruses with unclear pathogenicity including a distinct rhinovirus A type, were detected. This study provides remarkable data on the respiratory RNA virome in children with and without SARI with a degree of heterogeneity of known viruses colonizing their respiratory tract. The implication of the detected viruses in the dynamics/progression of SARI requires further investigations.
Subject(s)
COVID-19 , Pneumonia , Respiratory Tract Infections , Viruses , Child , Animals , Humans , Virome , South Africa/epidemiology , Seasons , RNA , Pandemics , Viruses/genetics , Respiratory SystemABSTRACT
This study presents whole genomes of seven bovine rotavirus strains from South Africa and Mozambique. Double-stranded RNA, extracted from stool samples without prior adaptation to cell culture, was used to synthesise cDNA using a self-annealing anchor primer ligated to dsRNA and random hexamers. The cDNA was subsequently sequenced using an Illumina MiSeq platform without prior genome amplification. All strains exhibited bovine-like artiodactyl genome constellations (G10/G6-P[11]/P[5]-I2-R2-C2-M2-A3/A11/A13-N2-T6-E2-H3). Phylogenetic analysis revealed relatively homogenous strains, which were mostly related to other South African animal strains or to each other. It appears that these study strains represent a specific bovine rotavirus population endemic to Southern Africa that was derived through multiple reassortment events. While one Mozambican strain, MPT307, was similar to the South African strains, the second strain, MPT93, was divergent from the other study strains, exhibiting evidence of interspecies transmission of the VP1 and NSP2 genes. The data presented in this study not only contribute to the knowledge of circulating African bovine rotavirus strains, but also emphasise the need for expanded surveillance of animal rotaviruses in African countries in order to improve our understanding of rotavirus strain diversity.
ABSTRACT
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.
Subject(s)
Genome, Viral , Genomics , Reassortant Viruses/genetics , Rotavirus Infections/epidemiology , Rotavirus Infections/virology , Rotavirus/classification , Rotavirus/genetics , Amino Acid Sequence , Capsid Proteins/chemistry , Capsid Proteins/genetics , Capsid Proteins/immunology , Databases, Nucleic Acid , Genomics/methods , Humans , Models, Molecular , Phylogeny , Protein Conformation , Rotavirus Infections/prevention & control , Rwanda/epidemiology , Sequence Analysis, DNA , Vaccination , Viral Vaccines/immunology , Whole Genome SequencingABSTRACT
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.
Subject(s)
Gastrointestinal Tract/virology , Metagenome , RNA, Viral/genetics , Virome , Cohort Studies , Feces/virology , High-Throughput Nucleotide Sequencing , Humans , Infant , Longitudinal Studies , Phylogeny , RNA Viruses/classification , RNA Viruses/genetics , South AfricaABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Computational Biology/methods , Rotavirus Infections/genetics , Rotavirus/genetics , Capsid Proteins/genetics , Child, Preschool , Computer Simulation , Diarrhea/epidemiology , Feces/microbiology , Female , Genetic Variation/genetics , Genome, Viral/genetics , Genotype , High-Throughput Nucleotide Sequencing , Humans , Infant , Male , Phylogeny , RNA, Viral/genetics , Rotavirus Infections/virology , Rwanda/epidemiology , Sequence Analysis, DNA/methods , Vaccination/methods , Whole Genome Sequencing/methodsABSTRACT
This study presents whole genomes of seven bovine rotavirus strains from South Africa and Mozambique. Double-stranded RNA, extracted from stool samples without prior adaptation to cell culture, was used to synthesise cDNA using a self-annealing anchor primer ligated to dsRNA and random hexamers. The cDNA was subsequently sequenced using an Illumina MiSeq platform without prior genome amplification. All strains exhibited bovine-like artiodactyl genome constellations (G10/G6-P[11]/P[5]-I2-R2-C2-M2-A3/A11/A13-N2-T6-E2-H3). Phylogenetic analysis revealed relatively homogenous strains, which were mostly related to other South African animal strains or to each other. It appears that these study strains represent a specific bo-vine rotavirus population endemic to Southern Africa that was derived through multiple reassortment events. While one Mozambican strain, MPT307, was similar to the South African strains, the second strain, MPT93, was divergent from the other study strains, exhibiting evi-dence of interspecies transmission of the VP1 and NSP2 genes. The data presented in this study not only contribute to the knowledge of circulating African bovine rotavirus strains, but also em-phasise the need for expanded surveillance of animal rotaviruses in African countries in order to improve our understanding of rotavirus strain diversity