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

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.

The avian gut microbiota: community, physiology and function in wild birds

“…A powerful tool yet to be employed in the study of avian microbiomes is metagenomic sequencing (Riesenfeld et al 2004), which interrogates all gene content in the microbiome. A first use of metagenomics for identifying viruses in wild birds was recently published (Vibin et al 2018), but the bacterial microbiome has not been investigated. Metagenomic sequencing relies on shotgun sequencing and assembly of DNA fragments from the sample, and subsequently using database matching the relevant sequences to known microbial functional groups.…”

Meta-transcriptomic analysis of virus diversity in urban wild birds with paretic disease

“…Suspected intoxication was a common history in those birds presenting en masse. Although seven birds were found dead or died prior to veterinary examination, clinical signs among the remaining birds included paresis (Figure 2a), recumbency or profound weakness (27), loss of righting reflex or difficulty righting (12), and less commonly dyspnea (4), watery or bloody diarrhea (4), clenched claws (1) and a moribund state (1). Although most birds were recumbent, they were not paralyzed as they had good cloacal tone, withdrawal reflexes, and could stand and shuffle, flap or walk across the room when stimulated.…”

“…This method relies on the unbiased sequencing of non-ribosomal RNA, and has been used to identify novel viral species in seemingly healthy animals. In Australia, for example, meta-transcriptomic approaches have been used with invasive cane toads ( Rhinella marina ) (11), waterfowl (12, 13), fish (14) and Tasmanian devils ( Sarcophilus harrisii ) (15). These approaches have also been applied diagnostically in a disease setting, including in domestic animals such as cats ( Felis sylvestris ) (16), dogs ( Canis lupus familiaris ) (17), cattle ( Bos taurus ) with respiratory diseases (18–20), and pythons ( Pythonidae ) with neurological signs (21).…”

Pathogenic viruses: Molecular detection and characterization

“…Monitoring studies in wild birds can be used as an early warning system for the incursion of a range of zoonotic viruses (Michel et al, 2018). Different molecular methods, including metagenomics approaches, have been developed and applied for detection and characterization of viruses in birds (Vibin et al, 2018).…”