Longitudinal analysis of the enteric virome in paediatric subjects from the Free State Province, South Africa, reveals early gut colonisation and temporal dynamics.

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.

Evaluation of extraction and enrichment methods for recovery of respiratory RNA viruses in a metagenomics approach.

Viral metagenomics is increasingly applied in viral detection and virome characterization. Different extraction and enrichment techniques may be adopted, however, reports on their effective influence on viral recovery is often conflicting. Using a three step enrichment steps, the effect of three extraction kits and the influence of DNase treatment with or without rRNA removal for respiratory RNA virus recovery from nasopharyngeal swab samples was evaluated. The viral cocktail containing six different RNA viruses pooled in equal volume were subjected to the different extraction and enrichment methods, sequenced using the Illumina MiSeq, and analysed using Genome Detective. The PureLink® Viral RNA/DNA Mini Kit (PureLink) was highly efficient with better recovery of all the viral agents in the cocktail. The use of rRNA treatment resulted in increased viral recovery with PureLink and QIAamp® Viral RNA Mini kit, while having comparable recovery rate as DNase only with the QIAamp® MinElute Virus Spin Kit. The observed low reads and genome coverage of some of the viruses could be attributed to their low abundance. Depending on sample matrix, extraction choice and enrichment strategy may influence recovery of respiratory RNA virus in metagenomics studies, therefore individual evaluation and adoption may be necessary for a robust result.

Whole Genome Analysis of African G12P[6] and G12P[8] Rotaviruses Provides Evidence of Porcine-Human Reassortment at NSP2, NSP3, and NSP4.

Group A rotaviruses (RVA) represent the most common cause of pediatric gastroenteritis in children <5 years, worldwide. There has been an increase in global detection and reported cases of acute gastroenteritis caused by RVA genotype G12 strains, particularly in Africa. This study sought to characterize the genomic relationship between African G12 strains and determine the possible origin of these strains. Whole genome sequencing of 34 RVA G12P[6] and G12P[8] strains detected from the continent including southern (South Africa, Zambia, Zimbabwe), eastern (Ethiopia, Uganda), central (Cameroon), and western (Togo) African regions, were sequenced using the Ion Torrent PGM method. The majority of the strains possessed a Wa-like backbone with consensus genotype constellation of G12-P[6]/P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, while a single strain from Ethiopia displayed a DS-1-like genetic constellation of G12-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2. In addition, three Ethiopian and one South African strains exhibited a genotype 2 reassortment of the NSP3 gene, with genetic constellation of G12-P[8]-I1-R1-C1-M1-A1-N1-T2-E1-H1. Overall, 10 gene segments (VP1-VP4, VP6, and NSP1-NSP5) of African G12 strains were determined to be genetically related to cognate gene sequences from globally circulating human Wa-like G12, G9, and G1 strains with nucleotide (amino acid) identities in the range of 94.1-99.9% (96.5-100%), 88.5-98.5% (93-99.1%), and 89.8-99.0% (88.7-100%), respectively. Phylogenetic analysis showed that the Ethiopian G12P[6] possessing a DS-1-like backbone consistently clustered with G2P[4] strains from Senegal and G3P[6] from Ethiopia with the VP1, VP2, VP6, and NSP1-NSP4 genes. Notably, the NSP2, NSP3, and NSP4 of most of the study strains exhibited the closest relationship with porcine strains suggesting the occurrence of reassortment between human and porcine strains. Our results add to the understanding of potential roles that interspecies transmission play in generating human rotavirus diversity through reassortment events and provide insights into the evolutionary dynamics of G12 strains spreading across selected sub-Saharan Africa regions.