From islands to infectomes: host-specific viral diversity among birds across remote islands

doi:10.21203/rs.3.rs-3995126/v1

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Research Article

From islands to infectomes: host-specific viral diversity among birds across remote islands

https://doi.org/10.21203/rs.3.rs-3995126/v1

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Background

Accelerating biodiversity loss necessitates monitoring the potential pathogens of vulnerable species. With a third of New Zealand's avifauna considered at risk of extinction, a greater understanding of the factors that influence viral transmission in this island ecosystem is needed. We used metatranscriptomics to determine the viruses, as well as other microbial organisms (i.e. the infectomes), of seven bird species, including the once critically endangered black robin (Petroica traversi), on two islands in the remote Chatham Islands archipelago, New Zealand.

Results

We identified 19 likely novel avian viruses across nine viral families. Black robins harboured viruses from the Flaviviridae, Herpesviridae, and Picornaviridae, while introduced starlings (Sturnus vulgaris) and migratory seabirds (Procellariiformes) carried viruses from six additional viral families. Potential cross-species virus transmission of a novel passerivirus (family: Picornaviridae) between native (black robins and grey-backed storm petrels) and introduced (starlings) birds was also observed. Additionally, we identified bacterial genera, apicomplexan parasites, as well as a novel megrivirus linked to disease outbreaks in other native New Zealand birds. Notably, island effects were outweighed by host taxonomy as a significant driver of viral composition, even among sedentary birds.

Conclusions

These findings underscore the value of surveillance of avian populations to identity and minimise escalating threats of disease emergence and spread in these island ecosystems. Importantly, they contribute to our understanding of the potential role of introduced and migratory birds in the transmission of microbes and associated diseases, which could impact vulnerable island-endemic species.

birds

Chatham Islands

New Zealand

infectome

microbiome

cross-species transmission

virus

In an era where an ever-increasing number of species teeter on the brink of extinction, monitoring the emergence of pathogens is an essential tool to inform their conservation mangement. The avian world boasts an array of charismatic species, but few have stories rivaling that of the remarkable population recovery of the Chatham Island black robin (Petroica traversi/black robin/kākāruia/karure)). First identified in 1872 as an insectivorous, forest-dewelling member of the Australasian robins (family: Petroicidae) endemic to the Chatham Islands (Rēkohu/Wharekauri), Aotearoa New Zealand (1), this small passerine holds a unique place in conservation history. By the early 1980s, their population had dwindled to just five individuals, bestowing them the title of “the rarest bird in the world” (2). The survival of a solitary breeding pair, including the only successful breeding female, Old Blue, formed the foundation of ongoing conservation efforts to restore this species (3). Today’s population has rebounded to over 300 birds, all descendants of Old Blue.

Despite their resurgence, the future of the black robin is still faced with uncertainty. New Zealand and its unique flora and fauna have evolved in near geographic isolation since splitting from the Gondwanan supercontinent some 85 million years ago (mya) (4). The Chatham Islands, located around 800 km east of New Zealand’s South Island, have been an epicenter for the evolution of distinctive species since their emergence from the Chatham Rise roughly three mya (5), hosting at least 52 avian species, 18 of which occur nowhere else in the world (6). It is here that, like most New Zealand endemics, black robins existed in geographic isolation devoid of natural predators for an extended period. Human colonisation of the Chatham Islands, followed by the arrival of introduced species, played a pivotal role in the black robin's decline (6, 7). Now, due to their low genetic diversity, these robins are highly suceptible to the looming threats of infectious diseases such as highly pathogenic avian influenza virus (8, 9).

Sedentary black robins co-exist and frequently interact with other avian species. Notably, seabirds (Procellariiformes) that breed in large numbers across the Chatham Islands serve as potential vectors for infectious agents (6). While black robins remain isolated within their now limited home ranges on just two of five major forested islands in the archipelago, seabirds, on the other hand, act as biological bridges, potentially moving pathogens from one island to another through natal dispersal and roosting on different islands. In addition, common starlings (Sturnus vulgaris), an introduced passerine which has been implicated in the predation of black robin nests (3, Reyes et al. In prep), pose another significant potential threat as vectors of disease transmission to black robins. Starlings forage on farmland on nearby Pitt Island and fly over to roost on the predator-free islands at night (3). Determing the infectomes of these avian hosts, in conjunction with black robins, may provide a holisitic view of potential transmission dynamics among these birds and deepens our understanding of future disease risk.

Avian viruses and wider infectomes remain vastly underexplored in New Zealand. While introduced pathogens such as avipoxvirus and avian malaria (Plasmodium spp.) are known to be circulating (10–12), only a few studies have integrated metagenomic approaches to identify all novel infectious agents in avian hosts (13–16). Moreover, many of New Zealand's avian species grapple with limited genetic diversity, rendering them potentially susceptible to severe diseases and mortality from otherwise innocuous infections (17, 18).

Herein, we present a metatranscriptomic analysis from swab samples collected across seven bird species from Mangere and Rangatira Islands. By comparing the infectomes of sedentary black robins with those of other cohabiting and migratory species, we sought to gain a deeper understanding of the transmission dynamics and ecological factors influencing avian viromes and microbiomes in New Zealand. In particular, we aimed to determine whether birds with inter-island movements act as vectors, facilitating the tranmission of microbes to and between species and islands. We emphasise the importance of understanding isolation and interconnectivty in these unqiue ecosystems, particularly in the context of cross-species transmission of pathogens to threatened species.

Bird sampling and swab storage.

Petroica traversi (black robin) and Thinornis novaeseelandiae (shore plover/tchuriwat’/tūturuatu) had oral and cloacal swabs taken during other work scheduled for management purposes, such as banding, by the Department of Conservation. Black robins were sampled on Mangere Island throughout the breeding season from December 2021 to February 2022 and on Rangatira Island during a post-breeding census in March to April 2022. Birds were primarily caught with pull activated drop traps baited with meal worms and, where this was not possible due to a trap shy individual, with a mist net. Shore plovers were sampled on Rangatira Island during a colour band maintenance trip in May 2022 and were caught with noose mats. Starling (Sturnus vulgaris) and seabirds (Procellariiformes), including fairy prions (Pachyptila turtur), broad-billed prions (Pachyptila vittata), sooty shearwater (Ardenna grisea), and grey-backed storm petrels (Garrodia nereis), were caught opportunistically for sampling during the 2021-22 seasons field work on both islands. Oral and cloacal swabs were taken from individuals and stored in sterile tubes filled with 1 mL DNA/RNA shield (Zymo Research). In some cases, a fresh faecal sample was collected instead of obtaining a cloacal swab. After collection, all samples were refrigerated in a solar powered fridge on return to the field hut at the end of the day and were frozen in a chest freezer on return to the main Chatham Island Department of Conservation base, typically within three weeks. Samples were then sent to the Department of Microbiology and Immunology, University of Otago and stored at -80°C until processing.

Total RNA extraction and sequencing.

Table 1

Overview of sample libraries.
Library	Species name	Common name	Location	Number of birds	Age	Collection date
BM1	Petroica traversi	Black robin	Mangere	16	Adult	Dec 2021 – Feb 2022
BM2	Petroica traversi	Black robin	Mangere	11	Pullus	Dec 2021 – Feb 2022
OM1	Pachyptila turtur	Fairy prion	Mangere	5	Adult	Dec 2021 – Jan 2022
OM2	Pachyptila turtur	Fairy prion	Mangere	1	Chick	Dec 2021 – Jan 2022
OM3	Sturnus vulgaris	Common starling	Mangere	1	Adult	Dec 2021 – Jan 2022
OM4	Sturnus vulgaris	Common starling	Mangere	4	Chick	Dec 2021 – Jan 2022
OM5	Ardenna grisea	Sooty shearwater	Mangere	12	Adult	Dec 2021 – Jan 2022
OM6	Ardenna grisea	Sooty shearwater	Mangere	11	Adult	Dec 2021 – Jan 2022
OM7	Pachyptila vittata	Broad-billed prion	Mangere	3	Adult	Dec 2021 – Jan 2022
OM8	Pachyptila vittata	Broad-billed prion	Mangere	12	Chick	Dec 2021 – Jan 2022
OM9	Garrodia nereis	Grey-backed storm petrel	Mangere	6	Adult	Dec 2021 – Jan 2022
BR1	Petroica traversi	Black robin	Rangatira	16	Adult	Mar – Apr 2022
BR2	Petroica traversi	Black robin	Rangatira	16	Adult	Mar – Apr 2022
BR3	Petroica traversi	Black robin	Rangatira	16	Juvenile	Mar – Apr 2022
BR4	Petroica traversi	Black robin	Rangatira	17	Juvenile	Mar – Apr 2022
OR1	Ardenna grisea	Sooty shearwater	Rangatira	10	Adult	Apr – May 2022
OR2	Pachyptila vittata	Broad-billed prion	Rangatira	9	Adult	Apr – May 2022
OR3	Pachyptila vittata	Broad-billed prion	Rangatira	9	Adult	Apr – May 2022
SP1	Thinornis novaeseelandiae	Shore plover	Rangatira	16	Adult	May 2022

Frozen oral and cloacal swabs from individual birds were defrosted and placed together in ZR BashingBead Lysis Tubes (0.1 and 0.5 mm) (Zymo Research) filled with 1 mL of fresh DNA/RNA shield (Zymo Research) using sterile forceps. Lysis tubes were homogenised for five minutes in a mini-beadbeater 24 disruptor (Biospec Products Inc.). Total RNA was then extracted following the ZymoBIOMICS MagBead RNA kit (Zymo Research). RNA concentrations were quantified using a NanoDrop Spectrophotometer (ThermoFisher). Equal quantities (10–15 µL) of RNA from individuals were pooled into 19 groups by species, sampling location (Mangere Island or Rangatira Island), and age group (adults or juveniles) ranging from one to 17 birds per pool (Table 1). The Illumina Stranded Total RNA Prep with Ribo-Zero plus kit (Illumina) was used for library preparation of the pooled samples. Libraries were sequenced on the Illumina NovaSeq 6000 platform and 150 bp paired-end reads were generated.

Virus discovery and abundance estimation.

Raw sequence reads were quality trimmed and then assembled de novo using Trinity (v2.11) (19) with the “trimmomatic” flag. Assembled contigs were screened against the NCBI non-redundant nucleotide (nt) and protein (nr) databases using BLASTn (20) and Diamond BLASTx (v2.02.2) (21) searches, respectively. A sequence similarity e-value cut-off of 1×10^− 10 was set for all searches to reduce false positive hits. Putative virus sequences were then manually screened with additional BLASTn and BLASTx searches using the online BLAST sever (22). Viruses were considered where top hits included other viruses with avian or vertebrate host assignments and excluded non-vertebrate-associated viruses or non-viral (e.g. bird genomic DNA) hits. For putative DNA viruses, nucleotide sequences of the viruses were screened against host references genomes (where available) and the NCBI nt database to exclude endogenous viral elements (EVEs) or misassigned host sequences.

Viral transcript abundances were estimated using the “align and estimate” module within Trinity with the “prep reference” flag set. RNA-seq by Expectation-Maximization (RSEM) (23) was used as the abundance estimation method and Bowtie 2 (24) as the alignment method. Transcript abundances were standardised for inter-library comparisons by dividing RSEM counts by their respective sequencing library depths. To reduce incorrect assignment of viruses to libraries due to index hopping, shorter viral contigs sharing more than 99% nucleotide identity with a longer contig in another library or host species and a read count < 0.1% of the highest count for that virus across the other libraries were considered contamination due to index-hopping and excluded.

Phylogenetic analysis and genome annotation.

Evolutionary relationships of the identified viruses were determined to infer host assignments. We assumed that viruses that clustered with other avian or vertebrate viruses in their respective phylogenies were likely to be infecting the birds sampled here (avian host-associated viruses), while those associated with other host types (e.g. invertebrates, bacteria, fungi, plants or environmental metagenomes) were unlikely to be replicating in their associated bird host and instead represented viruses from sources such as dietary, environmental, or sample processing contamination.

Phylogenies were estimated using protein sequences containing the highly conserved RNA-dependent RNA polymerase (RdRp) or DNA-dependent DNA polymerase (DdDp), or where viral genomes did not encode polymerases, such as those from the Papillomaviridae, major capsid proteins were used. Viruses that could be assigned down to at least order level based on their polymerase or capsid were aligned with their closest genetic relatives identified by BLAST as well as a representative range of viruses from their respective taxonomic orders or families collected from NCBI Taxonomy (25) using the L-INS-i algorithm in MAFFT (v7.450) (26). Alignments were visualised in Geneious Prime (v2020.2.4) (27) and ambiguously aligned regions were trimmed using trimAL (v1.2) (28) with the “automated1” flag set. IQ-TREE (v.1.6.12) (29) with the LG amino acid substitution model and 1000 ultra-fast bootstrapping replicates (30) was used to estimate maximum likelihood trees for each taxon. The “alrt” flag was also added to perform 1000 bootstrap replicates for the SH-like approximate likelihood ratio test (31). Phylogenetic trees were annotated in FigTree (v1.4.4) (32) and rooted at their midpoints.

Alignment and annotation of the Passerivirus GPS genome was done using an annotated genome of its closest relative, Passerivirus A1 (NC_014411), as a reference and was performed in Geneious Prime using the L-INS-i in MAFFT.

Virus nomenclature.

Virus species were considered novel if they shared < 90% RdRp or DdDp protein identity with their closest known relative, or < 80% genome identity with previously described species. Novel viruses were provisionally named with either the common name or genus of the proposed host species. In the case of Passerivirus GPS, “GPS” denotes the initials of the genera of the three host species it was identified in (Garrodia, Petroica, and Sturnus).

Non-viral microbe classification.

Transcripts (i.e. active gene expression) of archaeal, bacterial, and eukaryotic microbes were determined using CCMetagen (33). Reads were first mapped to the NCBI nucleotide database (34), excluding unclassified environmental microbes and cloning vectors, using KMA (35), ensuring a match to a reference was only counted if both paired-end reads mapped to the reference. The KMA output was then processed with CCMetagen to produce taxonomic classifications which were merged and filtered at genus level.

CCMetagen outputs were imported into R and filtered to exclude viruses and microbial genera with abundances < 10 reads pger million (RPM) across all libraries. A subset of the full filtered list, denoted “genera-of-interest” and comprising potentially pathogenic avian microbes or those that had been linked to disease in other native birds in New Zealand (36), were also screened. De novo assembled contigs were also manually screened for the genera-of-interest identified with CCMetagen with additional BLASTn searches and alignment to 16S and 18S ribosomal RNA (rRNA) nucleotide sequences of their closest hits to validate their presence in a sample.

Ecological and microbial diversity analysis.

The effects of host taxonomy (Passeriformes, Procellariiformes, or Charadriiformes) and sampling location (Mangere Island or Rangatira Island) on full family-level virome (exogenous viruses only) and non-viral genus-level microbiome compositions was assessed using non-metric multidimensional scaling (NMDS). Briefly, standardised virome and filtered non-viral microbiome abundances were normalised by library and distance matrices were created using the vdist function available in the vegan package (37) with Bray-Curtis dissimilarity as the distance measure. NMDS was then performed on the distance matrices using the metaMDS function from vegan. Permutational multivariate analysis of variance (PERMANOVA) with the adonis2 function from vegan was used to test for statistical significance of the effect of host taxonomy and location on the virome and microbiome distances. Ecological analysis of avian viromes and non-viral microbiomes was performed in R (v4.1.1) and graphs were plotted using ggplot2 (38). The effects of host taxonomy and location on virome compositions was repeated on non-vertebrate-associated family-level viromes in each library as a control and tested using PERMANOVA in the same manner as vertebrate-associated viromes.

Alpha diversity of non-viral microbiomes for each library was measured as both richness and Shannon diversity, which considers both the number of genera (richness) and their abundances (evenness). Differences in alpha diversity between Passeriformes and Procellariiformes and between black robins from Mangere Island and Rangatira Island were assessed using Welch two sample t-tests. Due to shore plover only being represented by a single sample, they were excluded from comparisons of alpha diversity.

We used metatranscriptomics to reveal the combined oral and cloacal infectomes of seven bird species from Mangere and Rangatira Islands – both part of the Chatham Islands, New Zealand (Table 1) – to characterise their associated viruses and other microbes, and exposure to potential pathogens. We also investigated the factors that influence infectome composition and transmission dynamic patterns between hosts and islands.

Metatranscriptomic sequencing.

High-throughput RNA sequencing of the 19 sequencing libraries (Table 1) generated approximately 49 to 89 million paired-end (150bp) reads per library (Fig. 1). No quality issues were noted for any of the libraries. Raw reads were assembled de novo, generating 0.2 to 1.1 million contigs per library and viral reads accounted for around 0.003–12% of total sequence reads following abundance estimations.

Avian virome compositions and abundance.

We identified sequences belonging to nine viral families across the seven hosts sampled (Fig. 2), with sequences from at least one avian virus identified per host species (range: one to five). Black robin viromes comprised vertebrate-specific viruses from the Flaviviridae, Herpesviridae, and the Picornaviridae, while starling and seabird viromes contained viruses from an additional six viral families. Only herpesvirus-like and picornavirus-like transcripts were identified in shore plover from Rangatira Island (Figs. 2a and 2b). Viral transcripts across the Picornaviridae and Herpesviridae were among the most prevalent and abundant viral transcripts found across the Chatham Island birds sampled (Fig. 2b).

Amino acid sequences containing the viral capsid or polymerase, either the RdRp or DdDp, from 27 viruses sharing sequence similarity with known vertebrate host-associated viruses were recovered, allowing their phylogenetic relationships to be inferred and novelty to be determined. These sequences belonged to 19 distinct and likely novel avian virus species (Figs. 3 and 4 and Table 2). We also identified two viruses from the Arenaviridae and Hantaviridae that were related to viruses from fish hosts in sooty shearwater from Mangere Island, and a hepevirus and picobirnavirus associated with black robins from Rangatira Island, all likely of environmental or dietary origin (Figures S1 and S2). These four viruses were excluded from further discussion and analysis.

Table 2

Avian virus polymerase sequences* described in this study.
Virus taxonomy	Host	Library	GenBank accession	Blastx top hit	Percentage identity (%)	Length (nt)	Novel?
Astroviridae;Pachyptila astrovirus	Pachyptila vittata	OM8	OR645486	AXF38643.1 Avastrovirus 2	56	608	Yes
Reoviridae; Pachyptila rotavirus D	Pachyptila turtur	OM1	OR645487	APR73519.1 Rotavirus D	79.7	240	Yes*
Reoviridae; Sturnus rotavirus A	Sturnus vulgaris	OM4	OR645488	UNY48222.1 Rotavirus sp.	80.7	1887	Yes*
Reoviridae; Pachyptila rotavirus D	Pachyptila vittata	OM7	OR645489	AXF38701.1 Rotavirus D	67.1	2089	Yes*
Caliciviridae; Broad-billed prion calicivirus	Pachyptila vittata	OM8	OR645492	QUS52514.1 Goose calicivirus	53.6	303	Yes
Herpesviridae; Petroica herpesvirus	Petroica traversi	BR4	OR645493	AVQ93817.1 Human betaherpesvirus 6	41.3	272	Yes
Herpesviridae; Ardenna herpesvirus	Ardenna grisea	OM6	OR645494	NP_944402.1 Psittacid alphaherpesvirus 1	89.9	3252	Yes*
Papillomaviridae; Garrodia papillomavirus	Garrodia nereis	OM9	OR645495	NP_647590.1 Psittacus erithacus papillomavirus 1	60.7	873	Yes
Papillomaviridae; Sturnus papillomavirus	Sturnus vulgaris	OM3	OR900097	AYN76738.1 Etapapillomavirus 1	74.0	301	Yes
Flaviviridae; Petroica flavivirus 1	Petroica traversi	BR1	OR645496	QUE43478.1 Goose pegivirus	82.9	226	Yes
Flaviviridae; Petroica flavivirus 2	Petroica traversi	BM2	OR645498	UCJ00138.1 Pin virus	90	214	No*
Flaviviridae; Garrodia flavivirus	Garrodia nereis	OM9	OR645499	UCJ00138.1 Pin virus	52.4	920	Yes
Picornaviridae; Passerivirus GPS	Sturnus vulgaris	OM4	OR645505	YP_003853285.1 Passerivirus A1	80.7	7494	Yes
Picornaviridae; Petroica picornavirus 1	Petroica traversi	BR1	OR645502	UCJ00139.1 Myna hepatovirus	34.5	711	Yes
Picornaviridae; Petroica picornavirus 2	Petroica traversi	BR3	OR645503	UCJ00139.1 Myna hepatovirus	33.7	933	Yes
Picornaviridae; Sturnus picornavirus	Sturnus vulgaris	OM4	OR645506	QNQ73364.1 Soft-shelled turtle systemic septicemia spherical virus	49.4	286	Yes
Picornaviridae; Pachyptila picornavirus	Pachyptila vittata	OM7	OR645507	CBY02484.1 Pigeon picornavirus A	61.3	356	Yes
Picornaviridae; Pachyptila hepato-like virus 1	Pachyptila vittata	OM8	OR645508	ART66868.1 Hepatovirus A	44.8	846	Yes
Picornaviridae; Pachyptila hepato-like virus 2	Pachyptila vittata	OM8	OR645509	YP_009164030.1 Phopivirus	48	643	Yes
Picornaviridae; Pachyptila megrivirus	Pachyptila vittata	OM8	OR645510	QDY92330.1 Red-capped plover megrivirus	64.1	577	Yes
Picornaviridae; Thinornis picornavirus	Thinornis novaeseelandiae	SP1	OR900098	YP_164335.1 anativirus A1	38.6	795
Hepadnaviridae; Garrodia hepatitis B-like virus	Garrodia nereis	OM9	OR645514	AJE59611.1 Duck hepatitis B virus	85.4	537	Yes
*If the virus was found in multiple libraries or species, the library with the longest sequence is detailed here. For a full breakdown of identified viruses see Additional file 5.

Evolutionary relationships of avian DNA viruses.

Four novel virus transcripts were found across three double-stranded DNA (dsDNA) virus families: the Hepadnaviridae (n = 1), Herpesviridae (n = 2), and Papillomaviridae (n = 2) (Fig. 3). A putative exogenous hepatitis B-like virus polymerase segments was found in grey-backed storm petrels from Mangere Island. The virus fell within the genus of avian hepatitis B viruses, Avihepadnavirus, in the Hepadnaviridae. The viruses, named Garrodia hepatitis B-like virus, shared 85% amino acid identity with Duck hepatitis B virus (DHBV) (AJE59611.1) (Table 2) and accounted for 0.014% the total viral reads in the petrel library. Hepatitis B viruses, such as DHBV, infect hepatocytes in the liver of various bird species and can lead to persistent, lifelong infections if infected congenitally (39).

A partial herpesvirus DdDp from black robins on Rangatira Island and a full-length DdDp from sooty shearwater from Mangere Island were also identified. The divergent virus infecting black robins, Petroica herpesvirus, shared only 41% amino acid identity with Human betaherpesvirus 6 (AVQ93817.1), but was phylogenetically placed outside of the Gammaherpesvirinae subfamily. No other avian-associated gamma or betaherpesviruses have been identified to date (40) and the virus was screened against the Petroica traversi genome assembly (GCA_025920805.1) to exclude it as an EVE. In contrast, the Ardenna herpesvirus DdDp in sooty shearwater shared almost 90% amino acid identity with that from Psittacid alphaherpesvirus 1 (NP_944402.1) and fell within the Alphaherpesvirinae subfamily with other avian-associated herpesviruses. Psittacid alphaherpesvirus 1 is the causative agent of Pacheco’s disease, a potentially lethal respiratory disease of parrots (41). A full major capsid protein, amongst other viral transcripts, were also identified from the Ardenna herpesvirus (see Additional file 5). The divergence of the capsid and polymerase proteins from those of Psittacid alphaherpesvirus 1, in additional to the seabird host, suggests this new virus to be a distinct species. The black robin herpesvirus made up 0.017% of the viral abundance for that library, while the sooty shearwater viruses made up around 62%.

Papillomaviruses are small non-enveloped dsDNA viruses that can produce a variety of benign and malignant epithelial lesions in animal hosts (42), primarily mammalian and bird species (43). We identified a complete L1 segment of a papillomavirus, denoted Garrodia papillomavirus, in the grey-backed storm petrel library and a partial L1 segment from a starling library, named Sturnus papillomavirus, both from Mangere Island. Around 0.34% of the total viral reads in the petrel were attributed to the novel papillomavirus, and it fell into an avian host-associated viral clade, sharing 61% amino acid identity with Psttacus erithacus papillomavirus 1 (NP_647590.1), cloned from a papilloma of a grey parrot (Psittacus erithacus) (44). The starling papillomavirus, on the other hand, made up 0.003% of the total viral reads in the starling library and shared 74% amino acid identity with Etapapillomavirus 1 (AYN76738.1) associated with skin lesions in wild British finches (45).

Evolutionary relationships of avian RNA viruses.

Several RdRps from positive-sense single-stranded RNA (+ ssRNA) viruses from the Astroviridae (n = 1), Caliciviridae (n = 1), and Flaviviridae (n = 3) families were identified in black robins and seabirds. Members of the Astroviridae from mammalian and avian hosts can be clearly divided into two distinct genera: Mammastrovirus (mammalian) and Avastrovirus (avian). Pachyptilla astrovirus from broad-billed prions fell into the genus Avastrovirus, exhibiting 56% amino acid identity with Avastrovirus 2 (AXF38643.1) and comprised 0.048% of the total viral reads in the prion library. Avastroviruses are most well studied in poultry, with some species, like Avastrovirus 2, leading to avian nephritis, stunted growth, renal damage, gout and, rarely, mortality (46). A segment of the structural polyprotein from the novel broad-billed prion astrovirus was also recovered (see Additional file 5).

Calicivirus sequences made up approximately 0.01% of the viral abundance in a Mangere Island broad-billed prion library. Sequences of an RdRp sharing around 49–54% amino acid identity with avian-associated calicivirus species such as Duck calicivirus (AXF38657.1) and Goose calicivirus (QUS52514.1), respectively, were identified in the prions. Caliciviruses are not well described in wild birds (47) but can cause gastroenteritis or systematic disease in avian hosts (48).

Several pegi-like viruses (genus: Pegivirus) from the Flavivirdae were found in black robins from both islands and grey-backed storm petrel from Mangere Island, making up around 0.003 to 0.17% of the birds’ total viral abundances. The Rangatira black robin pegiviruses shared 63–89% identity with Goose pegivirus (QUE43478.), while the grey-backed storm petrel and Mangere black robin pegiviruses shared 53–90% amino acid identity with Pin virus identified in Acridotheres tristis (UCJ00138). Some flaviviruses, such as goose pegivirus, have shown lymphotropic pathogenicity and high rates of co-infection with astroviruses, parvoviruses, and circoviruses (49). Other pegiviruses, such as the Montifringilla taczanowskii pegivirus, have been found in respiratory tracts of passerines (50).

Rotaviruses are a group of double-stranded RNA (dsRNA) viruses from the Reoviridae. We identified structural and non-structural sequences from Rotavirus D in broad-billed and fairy prions, as well as Rotavirus A in starlings, with polymerase-containing non-structural segments sharing 67–81% amino acid identity with other members of the genus Rotavirus (Fig. 3). Rotavirus D can cause enteric infections and stunted growth in birds (51) and to date has only been detected in non-human animal hosts. Rotavirus A is also a cause of major gastrointestinal disease in young birds and some genotypes may be able to infect mammalian hosts, including humans (52). The rotavirus D viruses made up 0.021 to 0.18% of total viral reads for their respective libraries and fell above the threshold for exclusion due to index-hopping.

The Picornavirdae and cross-species transmission of a novel Passerivirus.

The Picornaviridae family of single-stranded RNA (ssRNA) viruses within the Picornavirales is the largest family of animal-infecting viruses, with several species causing significant disease in humans, livestock, and wild animals (53). We identified nine likely novel virus species across the Picornaviridae across the subfamilies Enasvirinae, Heptrevirinae (hepatoviruses) and Kodimesavirinae (Fig. 4). Four of these were represented by novel virus transcripts related to bird-associated anativiruses, megriviruses and hepatoviruses found in broad-billed prions from Mangere Island. Of particular interest was Pachyptila megrivirus, which shared ~ 85% amino acid identity with a megrivirus recently discovered in diseased hoiho (yellow-eyed penguin) chicks from the Otago region of New Zealand (OR713086-95) (54) and made up 0.05% of total viral reads in its respective library.

Another notable observation was a novel passeri-like virus, Passerivirus GPS, in both passerines – black robins (endemic) and starlings (introduced) – and grey-backed storm petrels from Mangere Island. Passeriviruses have previously been reported to infect passerines and have been linked to deaths in wild birds and gastroenteric outbreaks in home-reared finches (55, 56). Passerivirus GPS was highly abundant in a sample of four starling chicks (Fig. 2), accounting for around 88% of the total virus reads in the chicks. The virus was less abundant in the larger pools of black robin chicks and grey-backed storm petrel adults, comprising between 0.13 and 0.29% of the total viral reads for the libraries respectively. Consequently, a likely full genome was recovered from starling, including the full-length polyprotein (Fig. 5). The genome was aligned with its closest relative, Passervirus A1 (NC_014411), from finches in Hungary for comparison. The two viruses shared approximately 78% genome (nt) identity and around 82% amino acid identity across the translated polyproteins, supporting Passerivirus GPS to be a novel species within the genus Passerivirus. Futhermore, the polyprotein of Passerivirus GPS (7,296 nt) was nine nucleotides longer than Passerivirus A1 (7,287 nt). The longer novel polyprotein is explained by four amino acid insertions in the L, VPO, VP1, and 2B peptides, and a deletion in the 3A peptide compared to its reference. Note that while the complete 3’ untranslated region (UTR) of the novel virus appeared to be recovered, including the poly-A tail, it is possible that the 5’ UTR is incomplete due to poor alignment and conservation of this region compared to its closest relative. The recovered viral segments in the two non-starling species shared 97.1 to 97.7% nucleotide identity with the starling polyprotein, suggesting recent virus transmission among these hosts. However, the petrel and robin segments did not overlap and therefore could not be compared directly.

Presence and diversity of non-viral microbes.

Non-viral microbes, including archaea, bacteria, and eukaryotes, were characterised using CCMetagen across the seven species of birds (Fig. 6). Various bacterial and eukaryotic genera were of particular interest due to links to disease outbreaks and mortalities in other avian species in New Zealand due to environmental contaminations, or because they contain notable avian pathogens (36, 57). We identified microbes from nine genera of interest (Fig. 6a). Both adult and chick starlings carried the largest number (eight out of nine) of these genera, while other species, such as the fairy prion, shore plover and grey-backed storm petrels had none or only one of these genera present in their microbiomes. Enterococcus, Escherichia, and Mycobacterium were the most widespread of the genera across the birds sampled. Of further note was the high abundance (152 to 58,178 RPM) of parasites from the phylum Apicomplexa (including RNA sequencing reads from the genera Eimeria, Cryptosporidium and Atoxoplasma) in samples of both species in the Passeriformes (black robins and starlings) (Fig. 6a).

Differences in diversity and richness of the full microbiomes of the two most widely sampled groups (passerines and seabirds) was also considered. Of the two groups, Passeriformes had significantly higher microbial richness (p = 0.003) than Procellariiformes with 377 microbial genera unique to Passeriformes and only 87 unique to Procellariiformes (Fig. 6b). However, there was no statistically significant difference in Shannon diversity between the two groups.

Effect of host taxonomy and location on microbiome composition.

We conducted a multivariate analysis examining the potential differences in both avian family-level viromes and full genus-level non-viral microbiomes between host taxonomy (order) and sampling location (island) as key factors (Fig. 7). Bray-Curtis distances was used as the measure of sample microbial beta diversity. Host order was found to be significantly associated with both virome composition (p = 0.043) and non-viral microbiome composition (p = 0.001) when controlling for sampling location. Sampling location was also found to have a significant impact on non-viral microbiomes (p = 0.006), with distinct clustering of samples by island, particularly samples from Rangatira Island, but not on virome compositions (p = 0.121).

Birds are hosts to a plethora of viruses and the migratory and dispersive lifecycles of some species make them important disease vectors, facilitating their transmission across large geographic distances (58, 59). Birds are also a highly diverse group, represented by more than 10,000 species (60). Despite this, studies of bird viruses and non-viral microbiomes are outnumbered ten-to-one by those of mammailan species (61). With over a third of avian populations in Aotearoa New Zealand considered at risk or threatened (62), understanding the dynamics of microbial transmission is necessary to limit the potential impact of future disease outbreaks on these avifauna. We investigated the infectomes of a snapshot of the avian life on two isolated New Zealand islands to better understand transmission and connectivity of microbes between species with differing behavioural ecology and taxonomy. Our findings detail the presence of known and novel microbes associated with the avifauna on the Chatham Islands, including factors that shape their infectomes. In addition, we present evidence for the transmission of a novel passerivirus between native and introduced sedentary and migratory birds.

We identified likely novel avian viruses in all seven hosts sampled, spanning nine viral families. Members of the Flaviviridae, Herpesviridae, and Picornaviridae were the dominant viruses in black robins. Other passerines in New Zealand have recently been shown to carry similar viral families with no evidence of transmission between native and introduced species (13). However, another related robin species, the South Island robin (Petroica australis), has a large diversity of single-stranded DNA viruses which were absent in black robins sampled here (16). Additional viruses from the Astroviridae, Caliciviridae, Hepadnaviridae, Papillomaviridae and Reoviridae families were identified in seabirds and starlings. While surveys of wild and invasive birds have also revealed similar viral families (49, 63), we have broadened the known host range and diversity of these virus families.

Some of the viruses identified here are related to those that cause disease in other avian hosts. For example, the causative agent of Pacheco’s disease (family: Herpesviridae) has been reported in imported and captive parrots in New Zealand (64), and herein we identified a closely related alpha herpesvirus in sooty shearwater from Mangere Island. In contrast, the evolutionary history of the divergent herpesvirus in black robins from Rangatira Island is uncertain as no beta or gamma herpesviruses have been reported in Aves to date (40) and the possibility of contamination from mammalian species such as fur seals that haul-out along coastal margins in the Chathams (65) close to black robin territories or from human interactions such as during routine population surveys over the years should not be ruled out. Similarly, rotaviruses are a common cause of enteric disease in animals, including humans (51, 52), and herein we identified these viruses in migratory seabirds and starlings.

The emergence and introduction of infectious disease from migratory species threatens the maintenance of vulnerable populations. In this context, a major finding from this study was a probable instance of cross-species transmission of a novel passerivirus (family: Picornaviridae), Passerivirus GPS, on Mangere Island found among both passerine species (the black robin and introduced starling), as well as the native grey-backed storm petrel. Passeriviruses have previously only been reported in dead passerines, including wild birds from the Turdidae family and home-reared estrildid finches in association with gastroenteritis outbreaks (55, 56). Hence, the presence of this virus in grey-backed storm petrels represents the first case of passerivirus identified outside the Passeridae hosts. Passerivirus A1 has previously been associated with enteritis causing high mortality in estrildid finches (55). Here, samples were collected from seemingly healthy birds, although the high abundance of Passerivirus GPS in a library of mixed oral and cloacal swabs from four starling chicks is consistent with an enterically transmitted virus. It has been suggested that passeriviruses may be sustained in bird populations and only cause sporadic cases of disease (55), although prevalence data on asymptomatic birds is limited in wild populations. Increasingly, birds have been identified as vectors of viral pathogens without showing clinical signs (9, 59).

There are also gaps in the possible chain of transmission of Passerivirus GPS on the Chatham Islands that will provide important information on the natural ecology of these viruses. Transmission between starlings and black robins is likely due to excreta from starlings contaminating soil under the roosts where robins forage for invertebrates, interspecific interactions due to nest cavity competition, or potentially from the predation of robin nests by starlings (3), making a clear case for a viral host-jumping event between these taxonomically related birds. Grey-backed storm petrels, in contrast, have very different behavioural ecology. These small seabirds feed at sea and tend to nest in dense vegetation, while black robins and starlings dwell in forested areas (7). While possible, the transmission of viruses between these species is less likely to be direct, instead involving other intermediate hosts not yet sampled. The high viral sequence similarity among these hosts also suggests relatively recent cross-species viral transmission. Sampling of the wider avifauna across the archipelago may help to identify missing links within these viral dynamics and confirm suspected hosts.

Among viruses that fell into the Picornaviridae, we identified viral transcripts related to anativiruses, hepatoviruses and megriviruses, all of which have been previously documented in avian hosts (66) and are frequently pathogenic (67, 68). Of particular note was the observation of a megrivirus in broad-billed prions from Mangere Island that was closely related to a megrivirus found in yellow-eyed penguins (Megadyptes antipodes) with diphtheritic disease from the Otago region, New Zealand (54). Indeed, prions have relatively broad habitat distributions along southern New Zealand and the South Atlantic Ocean (69). Hoiho and seabirds such as broad-billed prions breed on the same islands in the Foveaux Strait (70) which may have provided an environment for a historic cross-species viral transmission event to occur without the need for intermediary host jumps. Therefore, the genetic proximity of the broad-billed prion megrivirus to other avian-associated viruses across wider New Zealand suggests that migratory birds play an important role in the transmission of these viruses, the interconnectedness of avian populations, and disease dynamics.

Few studies have directly compared the microbial diversity of seabirds and passerines, particularly where they co-exist in these isolated island ecosystems. Comparisons of the microbiomes of the most widely sampled host orders in this study - Passeriformes (passerines or perching birds) and Procellariiformes (seabirds) - revealed that passeriformes had a larger number of distinct microbes than seabirds. Passerines have previously been shown to carry a number of unique microbial genera compared to non-passerine groups (71). The presence of apicomplexan parasites in both passerine species was of particular interest. Parasites within this phylum, such as Plasmodium relictum, responsible for avian malaria, are relatively widespread in New Zealand birds, infecting around 34 species to-date (72). While no evidence of P. relictum was identified, evidence of other coccidian parasites, such as Atoxoplasma, were observed and have been found to be very prevalent in other passerines, including other native robins (14, 73). While high parasitic loads may be harmful to their host, they are rarely found in high enough abundance in wild populations to cause symptomatic disease. However, in the case of coinfections or in young or immunocompromised inbred native birds, these parasites pose a more urgent threat (74), making identifying and monitoring their prevalence in these vulnerable populations, despite seemingly healthy, a potentially important part of ongoing species management (75).

Microbiomes are influenced by a multitude of intrinsic and extrinsic factors (76). The foraging, breeding and other behavioural ecology differed between taxonomic groups studied here. For example, sea and shore birds often roost on rocky coastlines or burrow under forest cover (6, 77) while passerines reside in forested areas and nest in tree cavities (6). Furthermore, some Passeriformes, like black robins, are entirely insectivorous (1, 2), while Charadriiformes are opportunistic feeders, sourcing small fish and invertebrates (78) and members of the order Procellariiformes hunt at sea for fish and krill (79). Groups such as seabirds, that frequently travel large distances (7) and may come into contact with a larger number of different species than sedentary birds, such as black robins and shore plovers, also presents a key difference in behaviour that may impact differences in their microbiomes. Predictably, we found a significant host effect influencing both virome and non-viral microbiome beta diversity, while location also significantly influenced the diversity of non-viral microbiomes. Host taxonomy is frequently found to be the most significant impactor of virome composition (80), while non-viral microbiomes are well documented as being more strongly influenced by non-host factors such as seasonality and geography (61, 81, 82), diet (83, 84), age (85) and sex (81, 86).

We note a lack of viruses in the Rangatira Island bird viromes, although this could represent disparities in processing, storage, and handling of swab samples between teams and islands (87), rather than a relative lack of viruses. For example, during sampling in March to April when there are fewer daylight hours, fridges used for temporary storage may be turned off during days with poor weather conditions to conserve power. Some viruses, such as avipoxviruses, which are known to infect shore plovers and black robins on the Chatham Islands (12, 17, 64) were similarly not identified, likely due to the types of samples collected. Hence, body site bias will influence what microbes may be identified, and therefore the findings presented here cannot rule out the presence or absence of other microbes that may have different tissue trophism.

In summary, through investigating the infectomes of seven Chatham Island bird species, we have shed light on the role that birds play as reservoirs for various microbes and their potential to act as vectors for disease transmission to threatened native species. The avian populations sampled here hosted a multitude of known and novel microbes. The apparent cross-species transmission of a novel passerivirus, Passerivirus GPS, between passerines and grey-backed storm petrels highlights the potential risks posed by introduced and migratory birds to the maintenance of vulnerable populations. Further research is needed to understand the prevalence and transmission dynamics of this virus, particularly given the migratory status of starlings and its apparent absence in black robins on Rangatira Island. Additionally, various other seabirds carried viruses related to known avian pathogens. Our comparative analysis of microbial diversity indicates that passerines carry a larger number of distinct viral species and microbial genera than seabirds and that host taxonomy outweighs location as a driver of virome composition. Importantly, we did not find evidence of viruses being transmitted between islands. Since the Chatham Islands house a diverse number of avifauna, moving forward, future research should focus on expanding species sampled to validate these trends. Overall, this knowledge not only enhances our understanding of avian infectomes in New Zealand but also the role of introduced and migratory birds as vectors for future disease outbreaks that may impact threatened species, informing the broader field of conservation aimed at preserving the rich avian biodiversity of New Zealand.

Ethics approval.

All sampling of avian species was undertaken by the New Zealand Department of Conservation as part of routine species management.

Consent for publication.

Not applicable.

Availability of data and materials.

Raw sequence reads are available on the NCBI Short Read Archive (SRA) under the BioProject accession PRJNA1021626 while viral sequences identified are available under GenBank accession numbers OR645486 to OR645543 and OR900097 to OR900098. Please see GitHub for detailed R code and accompanying input data used to generate the results described in this study (88).

Competing interests.

The authors declare that they have no competing interests.

Funding.

R.M.G. is supported by a University of Otago Doctoral Scholarship, J.L.G. is funded by a New Zealand Royal Society Rutherford Discovery Fellowship (RDF-20-UOO-007). E.C.H is funded by a National Health and Medical Research Council (Australia) Investigator Grant (GNT2017197).

Authors’ contributions.

Project was conceptualised and edited by R.M.G., J.L.G, K.I., E.C.H., J.W., G.T., add T.M. Manuscript was prepared by R.M.G. and edited E.M.R.R. collected samples from Rangatira Island and J. C. collected samples from Mangere Island. R.M.G, L.L. and J.D. extracted RNA from samples.

Acknowledgments.

We wish to thank the New Zealand Department of Conservation. To Erin Patterson, Jenna Hoverd, Jilleen Chandler, from the Chatham Island district office for the logistic support. To Leon Billows, Matu Booth, Tom Hitchon and Tim Foley for assisting during the seabirds sampling. To Owenga Charters for the trip logistics to the island. Thanks to Hamish Thompson for the native bird illustrations used throughout the manuscript.

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No competing interests reported.

Additionalfile1.pdf
File name: Additional file 1.pdf Title of data: Supplementary Figure 1. Description of data: Supplementary Figure 1. Presence-absence plot of avian and avian-associated viral taxa by host species, location, and age group. Dark and light blue blocks indicate adult and chick viromes from Mangere Island (left), respectively; dark and light orange blocks indicate adult and chick viromes from Rangatira Island (right). Green blocks indicate the presence of avian viruses, while lighter green-yellow blocks and viral taxa preceded by an asterisk (*) indicate avian-associated viruses, likely from dietary or environmental sources. Darker green blocks indicate the presences of likely endogenous viral elements.
Additionalfile2.pdf
File name: Additional file 2.pdf Title of data: Supplementary Figure 2. Description of data: Supplementary Figure 2. Maximum like likelihood trees of avian-associated viruses. Viruses identified as being associated with birds on Mangere Island are highlighted in blue and viruses identified on Rangatira Island are highlighted in orange. Host species associated with the viruses are indicated by bird illustrations. Substitutions per site indicated by key on left-hand side of trees. Nodes with ≥ 95 UFbootstrap support values are denoted by an asterisk (*).
Additionalfile3.pdf
File name: Additional file 3.pdf Title of data: Supplementary Figure 3. Description of data: Supplementary Figure 3. Comparisons of non-viral microbial genera richness and diversity per host species and sampling location. Comparison of full genus-level microbiome microbial richness (left, t-test p-value 0.110) and Shannon indices (right, t-test p-value 0.117) of Mangere (blue) and Rangatira (orange) black robin (top). Venn diagrams of the unique microbial genera of black robin (left), broad-billed prions (middle), and sooty shearwater (right) based on their sampling location – Mangere (blue) or Rangatira (orange) (bottom).
Additionalfile4.pdf
File name: Additional file 4.pdf Title of data: Supplementary Figure 4. Description of data: Supplementary Figure 4. Non-metric multidimensional scaling plots of non-vertebrate family-level virome compositions in Chatham birds. Non-vertebrate family-level virome compositions of sampling libraries (points) were plotted by Bray-Curtis distances and coloured by host order. Non-vertebrate family-level virome compositions were not significantly influenced by host taxonomy or location (PERMANOVA p-value = 0.08 and 0.65, respectively).
Additionalfile5.xlsx
File name: Additional file 5.xlsx Title of data: Extended summary of microbes and virus segments identified. Description of data: An excel spreadsheet containing extended information on the viruses and microbes identified in this study, including polymerase and non-polymerase segments and presence/absence of microbes in each library.

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From islands to infectomes: host-specific viral diversity among birds across remote islands

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