Genomic investigation of the faecal RNA virome in children from Oukasie clinic, North West Province, South Africa

Abstract

The advancements in high-throughput sequencing (HTS) and improvements in bioinformatics tools have enabled partial description of the human gut microbiome and continue to receive increasing attention. Novel enteric eukaryotic viruses have been associated with severe childhood diarrhoea in low-income areas worldwide. New virome data has shown that childhood diarrhoea contains higher abundance of viruses most of which were previously non-pathogenic such as those within the families Adenoviridae, Picornaviridae and Reoviridae. Nevertheless, a huge knowledge gap exists about the composition and diversity of the viruses colonizing the gastrointestinal tract of asymptomatic humans, which may be of clinical importance, especially in low-income countries. A major drawback for this poor characterization of the human gut virome has been attributed to lack of optimised methods to conduct such studies. However, an effective virome enrichment method called NetoVIR, developed by Conceiação-Neto and co-workers in 2015 for preparation of viral metagenomics samples has bridged the gap. In this study, viral metagenomics was employed to characterize the gut RNA virome of children under one-year old from the Oukasie clinic in the North West Province of South Africa. Faecal samples (n=12) were collected from four healthy infants at three time intervals (on average 7, 13 and 25 weeks old), to enable comparison of the changes in virome composition from baseline throughout the collection period. The samples were enriched for viral particles, followed by RNA extraction and RT-PCR. Library construction was done using a Nextera XT library preparation kit. The prepared libraries were sequenced on a MiSeq instrument to generate 251 bp paired-end reads. Using an in-house analysis pipeline, quality control of the generated reads was performed with FASTQc and Prinseq programmes. Quality-filtered reads were de novo assembled using metaSPAdes. Contigs were analysed by BLASTX searched against the NCBI database using DIAMOND, by aligning protein sequences against the NCBI protein database. Lastly, contigs that mapped to viruses were extracted for further statistical analysis. Numerous human enteric viruses were detected in all faecal samples. Reoviridae and in particular rotaviruses were detected in all 12 samples (100 %). However, majority of the viral contigs belonged to Picornaviridae family including viruses such parechoviruses, echoviruses, coxsackieviruses, enteroviruses and polioviruses, making it the most abundant. Astroviridae such as astroviruses and Caliciviridae such as noroviruses were detected at low abundance. Additionally, few sequences matched to plant viruses (pepper mild mottle virus), which was likely introduced through diet. Several viruses of animal origin were also present in gut of two of the participants. This study has proved that viral metagenomics can be an appropriate method in characterization of the human virome, providing insight into viral community structure and diversity of human enteric viruses. Although the faecal samples used in this study were negative by rotavirus screening using ELISA, it is interesting to observe that such high abundance of rotavirus sequences were still detected in the gut of asymptomatic individuals. The detection of polioviruses in one of the participants is a matter of public health concern since polioviruses have been eradicated by vaccination from many countries with only a handful still reporting sporadic cases. However, further analysis revealed that these were oral poliovirus vaccine sequences. It is evident that the infant’s gastrointestinal tract is colonized by different viral populations, irrespective of their health status. Despite the small sample size, this metagenomic study has provided some insight into the composition and diversity of viruses present in the gut of children. Lastly, the obtained data could be useful in the development of prevention strategies, as it provides information on virus species circulating in particular geographic areas, and to some extent can also suggest potential zoonotic transmissions.