Metagenomics detection and characterisation of viruses in faecal samples from Australian wild birds

Jessy Vibin,Anthony Chamings,Marcel Klaassen,Tiffanie M Nelson,Soren Alexandersen,Fiona Collier

doi:10.1038/s41598-018-26851-1

Jessy Vibin, Anthony Chamings + Show 4 more

Open Access

https://doi.org/10.1038/s41598-018-26851-1

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Journal: Scientific Reports	Publication Date: Jun 6, 2018
Citations: 70	License type: open-access

Affiliation: Deakin University, Barwon Health, Geelong Hospital

Abstract

We present an optimised metagenomics method for detection and characterisation of all virus types including single and double stranded DNA/RNA and enveloped and non-enveloped viruses. Initial evaluation included both spiked and non-spiked bird faecal samples as well as non-spiked human faecal samples. From the non-spiked bird samples (Australian Muscovy duck and Pacific black ducks) we detected 21 viruses, and we also present a summary of a few viruses detected in human faecal samples. We then present a detailed analysis of selected virus sequences in the avian samples that were somewhat similar to known viruses, and had good quality (Q20 or higher) and quantity of next-generation sequencing reads, and was of interest from a virological point of view, for example, avian coronavirus and avian paramyxovirus 6. Some of these viruses were closely related to known viruses while others were more distantly related with 70% or less identity to currently known/sequenced viruses. Besides detecting viruses, the technique also allowed the characterisation of host mitochondrial DNA present and thus identifying host species, while ribosomal RNA sequences provided insight into the “ribosomal activity microbiome”; of gut parasites; and of food eaten such as plants or insects, which we correlated to non-avian host associated viruses.

Highlights

Detection and characterisation of viruses by metagenomics (VM) is a relatively new technique that takes advantage of the sensitivity of next-generation sequencing (NGS) while being broadly non-specific for any particular virus present in a given sample
We identified viruses present in selected faecal samples from a Muscovy duck and a group of Pacific black ducks and from selected human faecal samples to show the broad applicability of the method
The spiked Muscovy duck (MUD) faecal sample was subjected to all the enrichment variations, and nucleic acids extracted, and real-time polymerase chain reaction (PCR) assays carried out to determine which method retained the maximum number and amount of the marker viruses (Table S3 of Supplementary material 1)

Summary

Introduction

Detection and characterisation of viruses by metagenomics (VM) is a relatively new technique that takes advantage of the sensitivity of next-generation sequencing (NGS) while being broadly non-specific for any particular virus present in a given sample. Different approaches have been applied based on, for example, the sample type and the sequencing platforms used[1,6,7,8,9,10] Among such proposed protocols, one designated NetoVIR by Conceição-Neto, N et al.[6] was optimised on mock communities and later tested on actual and complex biological samples by the same research group[11,12,13]. One designated NetoVIR by Conceição-Neto, N et al.[6] was optimised on mock communities and later tested on actual and complex biological samples by the same research group[11,12,13] The latter testing was performed without spiking the biological samples with known viruses, which would have served as a useful positive control to validate the protocol. Detergent treatment, ultracentrifugation and filtration using 0.45 μm filter were tried in different combinations to identify the optimal method for virus detection from faecal samples

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