key: cord-0310950-1kq0npyk
authors: Wille, Michelle; Harvey, Erin; Shi, Mang; Gonzalez-Acuña, Daniel; Holmes, Edward C.; Hurt, Aeron C.
title: Sustained virome diversity in Antarctic penguins and their ticks: geographical connectedness and no evidence for low pathogen pressure
date: 2019-12-12
journal: bioRxiv
DOI: 10.1101/2019.12.11.873513
sha: 551314700170030ade3f0a7eb7a3880a928bcbd7
doc_id: 310950
cord_uid: 1kq0npyk

Despite its isolation and extreme climate, Antarctica is home to diverse fauna and associated microorganisms. It has been proposed that the most iconic Antarctic animal, the penguin, experiences low pathogen pressure, accounting for their disease susceptibility in foreign environments. However, there is a limited understanding of virome diversity in Antarctic species, the extent of in situ virus evolution, or how it relates to that in other geographic regions. To test the idea that penguins have limited microbial diversity we determined the viromes of three species of penguins and their ticks sampled on the Antarctic peninsula. Using total RNA-Sequencing we identified 107 viral species, comprising likely penguin associated viruses (n = 13), penguin diet and microbiome associated viruses (n = 82) and tick viruses (n = 8), two of which may have the potential to infect penguins. Notably, the level of virome diversity revealed in penguins is comparable to that seen in Australian waterbirds, including many of the same viral families. These data therefore reject the theory that penguins are subject to lower pathogen pressure. The repeated detection of specific viruses in Antarctic penguins also suggests that rather than being simply spill-over hosts, these animals may act as key virus reservoirs.

experiences low pathogen pressure, accounting for their disease susceptibility in foreign 28 environments. However, there is a limited understanding of virome diversity in Antarctic 29 species, the extent of in situ virus evolution, or how it relates to that in other geographic 30 regions. To test the idea that penguins have limited microbial diversity we determined the 31 viromes of three species of penguins and their ticks sampled on the Antarctic peninsula. 32 Using total RNA-Sequencing we identified 107 viral species, comprising likely penguin 33 associated viruses (n = 13), penguin diet and microbiome associated viruses (n = 82) and tick 34 viruses (n = 8), two of which may have the potential to infect penguins. Notably, the level of 35 virome diversity revealed in penguins is comparable to that seen in Australian waterbirds, 36 including many of the same viral families. These data therefore reject the theory that 37 penguins are subject to lower pathogen pressure. The repeated detection of specific viruses in 38 Introduction 43 Geographical separation and extreme climates have resulted in the long isolation of 44 Antarctica and the subantarctic islands. The result is a unique assemblage of animals, some 45 relying entirely on the frozen continent, with others utilizing the fringes. Such geographic 46 isolation has been proposed to explain why Antarctic fauna supposedly harbour a paucity of 47 viruses, and supported by the observation that captive Antarctic penguins are highly 48 susceptible to infectious diseases (1). It has therefore been hypothesized that Antarctic fauna 49 have evolved in a setting of low "pathogen pressure", reflected in limited microbial diversity 50 and abundance (1, 2). As a consequence, the potential for climate driven and human mediated 51 movement of microorganisms makes the expansion of infectious diseases to the Antarctic a 52 matter of considerable concern (1, 3-5). 53 To date, a small number of viral species have been described in Antarctic fauna (6). 54 Serological studies have revealed that Antarctic penguins are reservoirs for influenza A virus 55 (IAV), avian avulaviruses (formerly avian paramyxoviruses), birnaviruses, herpesviruses, and 56 flaviviruses (7-13). Despite improvements in the molecular tools for virus detection, it is only 57 in recent years that full viral genomes have been characterized (6). For example, 58 adenoviruses, astroviruses, paramyxoviruses, orthomyxoviruses, polyomaviruses and 59 papillomavirus have been identified in Adélie penguins (Pygoscelis adeliae), Chinstrap 60 penguins (Pygoscelis antarctica) and Gentoo penguins (Pygoscelis papua) (6, 14-22). 61 However, sampling is limited and genomic data sparse, such that we have a fragmented 62 understanding of virus diversity in penguins and in Antarctica in general. 63 Antarctic penguins may also be infected by viruses spread by ectoparasites, particularly the 64 seabird tick Ixodes uriae (White) (23). For example, seven different arthropod-borne viruses 65 (arboviruses) were identified in I. uriae ticks collected from King nymphs) were collected from Paradise Bay (Fig 1) . Ticks were collected under rocks within 106 and directly adjacent to penguin colonies and placed in RNAlater (Ambion) and stored at -107 80°C within 4-8 hours of collection. 108 RNA library construction and sequencing 109 RNA library construction, sequencing and RNA virus discovery was carried out as described (Table S1 ). Sequence reads were demultiplexed and trimmed with Trimmomatic followed by de novo 131 assembly using Trinity (34). No filtering of host/bacterial reads was performed before 132 assembly. All assembled contigs were compared to the entire non-redundant nucleotide (nt) 133 and protein (nr) database using blastn and diamond blast (35), respectively, setting an e-value 134 threshold of 1x10 -10 to remove false-positives. Abundance estimates for all contigs were determined using the RSEM algorithm (34). All 136 contigs that returned blast hits with paired abundance estimates were filtered to remove plant, 137 invertebrate fungal, bacterial and host sequences. Viruses detected in the penguin libraries 138 were divided into those likely to infect birds and those likely associated other hosts (36)(37). 139 This division was performed using a combination of phylogenetic analysis and information (Table S1 ). There was a large range in the total viral 185 abundance in both the penguin (0.07-0.7 % total viral reads; 0-0.15 % avian viral reads) and 186 tick libraries (0.03-2.4%) (Table S1, Fig 2) . In addition to likely avian viruses, the penguin 187 libraries contained numerous reads matching insect, plant, or bacterial viruses and 188 retroviruses (Fig 2A, Fig 3) . Retroviruses were excluded from later analyses due to the 189 challenges associated with differentiating exogenous from endogenous sequences using meta- sampling locations that are 130 km apart (Fig 3, Fig S3) . 213 Within the tick libraries, the greatest virus abundance was seen in the adult female ticks, Paramyxoviridae, Picorbirnaviridae, Picornaviridae and Reoviridae (Fig 2B, Fig 3) (see 229 below). Ten of the 13 avian associated viruses identified in the penguins likely represent 230 9 novel avian viral species (Table S2, Fig 4) Table S3 , Fig 3) . The largest diversity was found in the "Narna-Levi", "Noda-Tombus" and 245 "Picorbirna-Pariti" viral groups (36). A number of viruses were highly divergent, including 246 clusters of novel viruses that fell within the Narnaviridae and Leviviridae (Table S3, Fig 3) . (Fig 6A, Fig S9) We also identified a deltacoronavirus and an avastrovirus (Fig 6B, Fig S10-S11 ). The 294 deltacoronavirus was similar to those reported in birds in the United Arab Emirates, 295 Australia, Niger, and Finland, with ~95% identity. A lack of sampling makes it challenging to 296 determine how deltacoronaviruses in Antarctica and other continents may be shared (Fig   297   S10 ). The astrovirus detected was similar (88.3% identity) to a short fragment (1000 bp) 298 previously reported in Adelie penguins on the Ross ice shelf of Antarctica (22) (Table S2) , a 299 pattern confirmed by phylogenetic analysis (Fig 6B) . Phylogenetic analysis also reveals that 300 this virus falls in an outgroup to Group 2 viruses, including Avian Nephritis virus (Fig 6B, 301 Fig S11) . Although we were unable to determine the epidemiology of these viruses in 302 Antarctica, repeated detection on opposite ends of the Antarctic continent makes it possible 303 that this is a penguin specific virus. (Fig 4) . 320 The six other novel virus species identified in the tick libraries comprised five viral families: RdRp sequences currently available, exhibiting just 35.7% amino acid sequence similarity to 328 the divergent tick-borne tetravirus-like virus (Fig S12) . A novel colti-like virus (Reoviridae), 329 Fennes virus, was identified in the adult male, female and nymph libraries, although we were 330 only able to assemble four segments. Notably, Fennes virus falls basal to the existing 331 coltivirus group, exhibiting just 30% amino acid sequence similarity to Shelly headland virus, 332 recently identified in Ixodes holocyclus ticks from Australia (Fig 7) . The partial genome of a 333 Rhabdovirus, Messner virus, was identified in the adult female library. However, this 334 fragment was of low abundance, and only the RdRp segment (Fig S12) . (Table S2) . Combined, these data strongly suggest that penguins are not merely spill-over hosts, but may 379 be central reservoir hosts for a diverse range of viruses. This is apparent in two observations. 380 The first is the repeated detection of specific virus species, such as avian avulaviruses, and In sum, we reveal substantial viral diversity in Antarctic penguins, their diet and their ticks. 433 We therefore expect that additional viruses will be identified with increased sampling, Table S3 , and phylogenetic 632 trees for each virus family can be found in Figs 5-8, S5-S12). 633 

Infectious 450 diseases of Antarctic penguins: current status and future threats

Emerging infectious pathogens of wildlife

A 454 horizon scan of global conservation issues for 2012

The protection of Antarctic terrestrial ecosystems from inter-456 and intra-continental transfer of non-indigenous species by human activities: A review of 457 current systems and practices

Challenges to the Future Conservation of the Antarctic

Viruses associated with Antarctic wildlife: From 461 serology based detection to identification of genomes using high throughput sequencing

Virological studies of Adelie Penguins (Pygoscelis adelia) 464 in Antarctica

Viral infections of Little Blue Penguins 466 (Eudyptula minor) along the southern coast of Australia

Poultry virus infection in 468 Antarctic penguins

Assessing the role of seabirds in the ecology of influenza A viruses

Mounting evidence for the presence of influenza A virus in the avifauna of the 474 Antarctic region

Evidence of ortho-and paramyxoviruses in fauna from 476

Multi-year serological evaluation of three viral 478 agents in the Adelie Penguin (Pygoscelis adeliae) on Ross Island

Newcastle disease virus in penguins from King George Island on the Antarctic region

Evidence for the 484 introduction, reassortment, and persistence of diverse influenza A viruses in Antarctica

Detection of evolutionarily distinct avian influenza A viruses in Antarctica

Chinstrap Penguins (Pygoscelis antarctica) in Antarctica

Genetic and molecular 492 Epidemiological characterization of a novel Adenovirus in Antarctic penguins collected 493 between

A novel 495 papillomavirus in Adelie penguin (Pygoscelis adeliae) faeces sampled at the Cape Crozier 496 colony

Identification of an avian polyomavirus associated with Adelie penguins (Pygoscelis 499 adeliae)

Antarctic penguins as 501 reservoirs of diversity for avian avulaviruses

First report 503 of a feather loss condition in Adelie penguins

Ticks 506 associated with Macquarie Island penguins carry arboviruses from four genera

Isolation of 509 arboviruses (Kemerovo-Group, Sakhalin-Group) from Ixodes uriae collected at Macquarie 510

The 512 isolation of arboviruses including a new Flavivirus and a new Bunyavirus from Ixodes-513 (Ceratixodes)-uriae (Ixodoidea, Ixodidae) collected at

Seabird ticks 516 (Ixodes uriae) distribution along the Antarctic Peninsula

The tick Ixodes uriae (Acari: Ixodidae): Hosts, 518 geographical distribution, and vector roles

Increase in 520 feeding by the tick, Ixodes uriae, on Adelie penguins during a prolonged summer

Habitat requirements of the seabird tick, Ixodes uriae (Acari : Ixodidae), from the Antarctic 524

Peninsula in relation to water balance characteristics of eggs, nonfed and engorged stages

Life cycle 527 of the tick Ixodes uriae in penguin colonies: relationships with host breeding activity

Mapping Application for Penguin Populations and Projected Dynamics (MAPPPD): 531 Data and tools for dynamic management and decision support

Virus-virus interactions 534 and host ecology are associated with RNA virome structure in wild birds

Extensive diversity of 537 RNA viruses in Australian ticks

Full-539 length transcriptome assembly from RNA-Seq data without a reference genome

Fast and sensitive protein alignment using 542 DIAMOND

Redefining the invertebrate 544 RNA virosphere

The evolutionary history of 546 vertebrate RNA viruses

Contaminating viral sequences in high-throughput sequencing viromics: a linkage study of 549 700 sequencing libraries

Fast gapped-read alignment with Bowtie 2

MAFFT multiple sequence alignment software version 7: 553 improvements in performance and usability

trimAl: a tool for automated 555 alignment trimming in large-scale phylogenetic analyses

IQ-TREE: A Fast and Effective 557

Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies

New 560 algorithms and methods to estimate maximum-likelihood phylogenies: assessing the 561 performance of PhyML 3.0

Rhea: a transparent and modular R 563 pipeline for microbial profiling based on 16S rRNA gene amplicons

The 566 vegan package

An R package for reproducible interactive 568 analysis and graphics of microbiome census data

A panel of 571 stably expressed reference genes for real-time qPCR gene expression studies of Mallards 572 (Anas platyrhynchos)

Krishnamurthy SR, Wang D. Extensive conservation of prokaryotic ribosomal 576 binding sites in known and novel picobirnaviruses

Novel avulaviruses in 578 penguins

Serological evidences of influenza A virus infection in Antarctica migratory birds

A novel Coltivirus-585 related virus isolated from free-tailed bats from Cote d'Ivoire is able to infect human cells in 586 vitro

Isolation and 588 characterization of Tarumizu tick virus: A new coltivirus from Haemaphysalis flava ticks

Colorado tick fever -591 clinical, epidemiologic, and laboratory aspects of 228 cases in Colorado in 1973-1974

Active migration is associated 594 with specific and consistent changes to gut microbiota in Calidris shorebirds

Gut microbiota of a long-distance 597 migrant demonstrates resistance against environmental microbe incursions

The family Narnaviridae: simplest of RNA viruses

RNA 602 Viruses in Blechomonas (Trypanosomatidae) and evolution of Leishmaniavirus

A narnavirus in the 605 trypanosomatid protist plant pathogen Phytomonas serpens

RNA phage 607 biology in a metagenomic era

Figure 1. Map of Antarctic peninsula and locations where Antarctic penguins samples and 613

Abundance of viruses found in penguins and their ticks. (A) Abundance of all viral 616 reads found in penguin libraries. (B) Abundance and diversity of avian viruses in each of the 617 penguin libraries. (C) Abundance of the host reference gene RSP13 in penguin libraries

Abundance of all viral reads found in the tick libraries. (E) Abundance and diversity of 619 viruses in each of the tick libraries. (F) Abundance of the host reference gene COX1 in the

Phylogenies of select novel viruses found in penguins. (A) Phylogenetic tree of 635 the VP1, containing the RdRp, of rotaviruses. The tree is midpoint rooted for clarity only

Phylogeny of the concatenated major capsid gene and glycoprotein B gene of the 637

Two betaherpesviruses were used as outgroup to root the tree. The 638 viruses identified in this study are denoted with a filled circle and in bold

Phylogeny of 643 the F gene of Avian avulavirus-17. Detection location for viruses identified in this study and 644

are denoted by either a green filled circle (King George Island) or blue 645 filled triangle (Kopaitik Island)

Avian avulavirus 18 was used as outgroup to root the tree. The scale 648 bar represents the number of nucleotide substitutions per site

ORF1ab, including the RdRp, of avastroviruses. The tree is mid-point rooted for clarity only

Bootstrap values 651 >70% are shown for key nodes. Viruses identified in this study are denoted in bold

Phylogenies of tick arboviruses. (A) The RdRp segment of select members of the 654

Reoviridae, including the genus Coltivirus. (B). The RdRp of select members of the 655

The novel tick viruses identified in this 656 study are denoted with a filled circle and in bold. The tree has been mid-point rooted for 657 clarity only. Bootstrap values >70% are shown for key nodes. The scale bar represents the 658 number of amino acid substitutions per site