Meta-transcriptomic analysis of the virome and microbiome of the invasive Indian myna (Acridotheres tristis) in Australia

Abstract

Invasive species exert a serious impact on native fauna and flora and have become the target of eradication and management efforts worldwide. Invasive avian species can also be important pathogen reservoirs, although their viromes and microbiomes have rarely been studied. As one of the top 100 invasive pest species globally, the expansion of Indian mynas (Acridotheres tristis) into peri-urban and rural environments, in conjunction with increasing free-ranging avian agricultural practices, may increase the risk of microbial pathogens jumping species boundaries. Herein, we used a meta-transcriptomic approach to explore the microbes present in brain, liver and large intestine of 16 invasive Indian myna birds in Sydney, Australia. From this, we discovered seven novel viruses from the families Adenoviridae, Caliciviridae, Flaviviridae, Parvoviridae and Picornaviridae. Interestingly, each of the novel viruses identified shared less than 80% genomic similarity with their closest relatives from other avian species, indicative of a lack of detectable virus transmission between invasive mynas to native or domestic species. Of note, we also identified two coccidian protozoa, Isospora superbusi and Isospora greineri, from the liver and gut tissues of mynas. Overall, these data demonstrate that invasive mynas can harbor a diversity of viruses and other microorganisms such that ongoing pathogen surveillance in this species is warranted.

Keywords: Calicivirus; Chaphamaparvovirus; Evolution; Isospora; Meta-transcriptomics; Pegivirus; Phylogeny; myna.

Fig. 1

Genome characterization and phylogenetic analysis of representative pegiviruses. (A) Genome structure of Pin virus. (B) Structure of the 5’UTR region of Pin virus predicted by RNAfold. (C) Phylogenetic trees of the NS3 gene (shaded pink) and NS5B genes including the RdRp (shaded green) of selected pegiviruses. Branches are scaled according to the number of amino acid substitutions per site and bootstrap support values are shown. Pin virus is shown in red font. Trees are midpoint-rooted for clarity only. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Phylogenetic analysis of myna hepatovirus. Phylogenetic tree of the polyprotein, containing the RdRp region, of relevant picornaviruses focusing on the novel myna hepatovirus identified here (bold red font). The tree was midpoint rooted, corresponding to the division between mammalian and avian viruses. The blue box indicates members of the genus Hepatovirus. Bootstrap values >70% are shown for key nodes. The scale bar indicates the number of amino acid substitutions per site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Phylogenetic tree of the polyprotein, including the RdRp, of myna caliciviruses. The tree was midpoint rooted, corresponding to the division between mammalian calicivirus, sapovirus, norovirus, fish caliciviruses and avian viruses (shaded purple). Some monophyletic groups have been collapsed to improve visualization (shaded green). Bootstrap values >70% are shown for key nodes. The myna caliciviruses obtained here are shown in bold red font. The scale bar indicates the number of amino acid substitutions per site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Phylogenetic relationships of the myna adenoviruses. (A) Amino acid phylogenetic tree of the hexon protein of myna siadenovirus and myna aviadenovirus (bold red font). (B) Phylogenetic tree of DNA polymerase (~232 nt) of the myna aviadenovirus and myna siadenovirus (bold red font). The yellow box shading indicates the avian siadenoviruses while the purple box denotes the avian aviadenoviruses. The scale bar represents the number of substitutions per site and bootstrap values are shown. Both trees are mid-point rooted for clarity only. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Phylogenetic tree of the non-structural gene (NS) of parvoviruses (Parvoviridae). The genome structure and sequence coverage of myna chaphamaparvovirus. The yellow shading denotes the subfamily Hamaparvovirinae that contains the novel myna chaphamaparvovirus (bold red font). The scale bar represents the number of amino acid substitutions per site. The tree is mid-point rooted for clarity only. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Phylogenetic tree of the cytochrome C oxidase subunit 1 (COX1) gene of Isospora sp. Two abundant isosporan transcripts (bold red font) from the Indian myna showed high sequence similarity to Isospora greineri and Isospora superbusi. Yellow shading indicates the avian-associated isosporan coccidial parasites. The scale bar denotes the number of nucleotide substitutions per site and bootstrap values are shown. The tree is mid-point rooted for clarity only. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Supplementary Fig. S1

Comparative analysis of the genomes of Pin virus and goose pegiviruses. (A) Genome characterization of Pin virus (pink) in comparison with Goose pegivirus 1 (blue) and Goose pegivirus 2 (light gray). The pairwise identity (amino acid) of each gene between Pin virus and two goose pegivruses were denoted, respectively. (B) Conserved functional motifs that likely impact virus packaging and replication, including ATP-binding sites (GSGKS helicase motifs, green rectangle), and a nucleotidase domain (QRRGRXGR, red rectangle), two D-X4-D motifs (motif A:yellow), a GX2TTX3N (GVLTTSSSN) motif (motif B: red), and a highly conserved GDD motif (motif C: purple).

Supplementary Fig. S2

Microbial composition of the liver and gut samples of myna birds. Stacked columns indicate the read abundance of each microbial taxon at the family level from the liver and gut tissues of mynas. Only the abundance of top 30 families of microbial taxon is shown. Abundance was calculated based on the sum RPM of each maker per metagenome. The most abundant family across four libraries was the Eimeriidae (shaded dark purple) that includes members of the genus Isospora.