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Meta-transcriptomic analysis of virus diversity in urban wild birds 1
with paretic disease 2
3
Wei-Shan Chang1†, John-Sebastian Eden1,2†, Jane Hall3, Mang Shi1, Karrie Rose3,4, Edward C. 4
Holmes1# 5
6
7
1Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and 8
Environmental Sciences and School of Medical Sciences, University of Sydney, Sydney, NSW, 9
Australia. 10
2Westmead Institute for Medical Research, Centre for Virus Research, Westmead, NSW, 11
Australia. 12
3Australian Registry of Wildlife Health, Taronga Conservation Society Australia, Mosman, 13
NSW, Australia. 14
4James Cook University, College of Public Health, Medical & Veterinary Sciences, Townsville, 15
QLD, Australia. 16
17
†Authors contributed equally 18
19
#Author for correspondence: 20
Professor Edward C. Holmes 21
Email: [email protected] 22
Phone: +61 2 9351 5591 23
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
2
24
Abstract: 248 words 25
Importance: 109 words 26
Text: 6296 words 27
Running title: Virome of urban wild birds with neurological signs. 28
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
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Abstract 29
Wild birds are major natural reservoirs and potential dispersers of a variety of infectious 30
diseases. As such, it is important to determine the diversity of viruses they carry and use this 31
information to help understand the potential risks of spill-over to humans, domestic animals, and 32
other wildlife. We investigated the potential viral causes of paresis in long-standing, but 33
undiagnosed disease syndromes in wild Australian birds. RNA from diseased birds was extracted 34
and pooled based on tissue type, host species and clinical manifestation for metagenomic 35
sequencing. Using a bulk and unbiased meta-transcriptomic approach, combined with careful 36
clinical investigation and histopathology, we identified a number of novel viruses from the 37
families Astroviridae, Picornaviridae, Polyomaviridae, Paramyxoviridae, Parvoviridae, 38
Flaviviridae, and Circoviridae in common urban wild birds including Australian magpies, 39
magpie lark, pied currawongs, Australian ravens, and rainbow lorikeets. In each case the 40
presence of the virus was confirmed by RT-PCR. These data revealed a number of candidate 41
viral pathogens that may contribute to coronary, skeletal muscle, vascular and neuropathology in 42
birds of the Corvidae and Artamidae families, and neuropathology in members of the 43
Psittaculidae. The existence of such a diverse virome in urban avian species highlights the 44
importance and challenges in elucidating the etiology and ecology of wildlife pathogens in urban 45
environments. This information will be increasingly important for managing disease risks and 46
conducting surveillance for potential viral threats to wildlife, livestock and human health. More 47
broadly, our work shows how meta-transcriptomics brings a new utility to pathogen discovery in 48
wildlife diseases. 49
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4
Importance 50
Wildlife naturally harbor a diverse array of infectious microorganisms and can be a source of 51
novel diseases in domestic animals and human populations. Using unbiased RNA sequencing we 52
identified highly diverse viruses in native birds in Australian urban environments presenting with 53
paresis. This investigation included the clinical investigation and description of poorly 54
understood recurring syndromes of unknown etiology: clenched claw syndrome, and black and 55
white bird disease. As well as identifying a range of potentially disease-causing viral pathogens, 56
this study describes methods that can effectively and efficiently characterize emergent disease 57
syndromes in free ranging wildlife, and promotes further surveillance for specific potential 58
pathogens of potential conservation and zoonotic concern. 59
60
Keyword: paresis, neurological syndrome, wildlife, birds, meta-transcriptomics, virus 61
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Introduction 62
Emerging and re-emerging infectious diseases in humans often originate in wildlife, with free-63
living birds representing major natural reservoirs and potential dispersers of a variety of zoonotic 64
pathogens (1). Neurological syndromes, such as paresis, are of particular concern as many 65
zoonotic viral pathogens carried by wild birds with the potential to cause neurological disease are 66
also potentially hazardous to poultry, other livestock and humans. Examples of this phenomenon 67
include Newcastle disease virus (NDV, Avulavirus 1; family Paramyxoviridae), West Nile virus 68
(WNV, family Flaviviridae) and avian influenza viruses (family Orthomyxoviridae) (2). 69
Importantly, with growing urban encroachment, the habitats of humans, domestic animals and 70
wildlife increasingly overlap. A major issue for the prevention and control of wildlife and 71
zoonotic diseases is how rapidly and accurately we can identify a pathogen, determine its origin, 72
and institute biosecurity measures to limit cross-species transmission and onward spread. With 73
these ever changing environments, wildlife are also at risk from a conservation perspective, as a 74
number of emerging viral pathogens (WNV, Usutu virus, avian poxvirus, avian influenza, 75
Bellinger River snapping turtle nidovirus) have had adverse population level impacts (3-8). 76
Therefore, a comprehensive understanding of the diversity of the viral community, and the 77
ecology of microbes associated with urban wildlife mass mortality and emergent disease 78
syndromes, will improve our capacity to detect pathogens of concern and lead to improved 79
conservation and public health intervention (3). 80
81
Meta-transcriptomic approaches (i.e. total RNA-sequencing) have revolutionized the field of 82
virus discovery, transforming our understanding of the natural virome in vertebrates and 83
invertebrates (9, 10). This method relies on the unbiased sequencing of non-ribosomal RNA, and 84
has been used to identify novel viral species in seemingly healthy animals. In Australia, for 85
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example, meta-transcriptomic approaches have been used with invasive cane toads (Rhinella 86
marina) (11), waterfowl (12, 13), fish (14) and Tasmanian devils (Sarcophilus harrisii) (15). 87
These approaches have also been applied diagnostically in a disease setting, including in 88
domestic animals such as cats (Felis sylvestris) (16), dogs (Canis lupus familiaris) (17), cattle 89
(Bos taurus) with respiratory diseases (18-20), and pythons (Pythonidae) with neurological signs 90
(21). Notably, meta-transcriptomics has also been used to identify bacterial diseases, such as 91
tularemia infections in Australian ring-tailed possums (Pseudocheirus peregrinus) (22). Hence, 92
metagenomic approaches provide the capacity to rapidly and comprehensively map microbial 93
and viral communities and examine their interactions, improving our understanding of animal 94
health and zoonoses. 95
96
Investigations into wildlife diseases are often neglected and under-resourced. Consequently, 97
while many outbreaks and syndromes are reported, until recently limited molecular screening has 98
been performed to characterize the etiology where a novel organism is present. In Australia, 99
several neglected and undiagnosed disease outbreaks have been described in wild avian species 100
including those of suspected viral etiology. Notable examples include two syndromes termed 101
"clenched claw disease” (23-28) and “black and white bird disease” (29-31) that affect rainbow 102
lorikeets (Trichoglossus moluccanus) and several species of passerines, respectively. 103
104
Clenched claw syndrome (CCS) has been recognized as a form of paresis in rainbow lorikeets in 105
eastern Australia, in which birds present recumbent with poor withdrawal reflexes and clenched 106
feet. Although the syndrome may be multifactorial, a proportion of the cases are characterized by 107
non-suppurative encephalomyelitis and ganglioneuritis, and these cases are suspected to have a 108
viral etiology. Similarly, a series of morbidity and mortality events termed black and white bird 109
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disease (BWBD) have occurred in Australian magpies (Gymnorhina tibicen), Pied currawongs 110
(Strepera graculina), Australian ravens (Corvus coronoides) and magpie larks (Gallina 111
cyanoleuca) along the Australian east coast (31). Diseased birds present in groups, either dead or 112
paretic. Although these emergent disease syndromes are suspected to be caused by viral 113
infections, they have been poorly described, and to date no viral pathogens have been identified 114
in culture, such that the cause and mechanisms of disease remain elusive. 115
By exploiting meta-transcriptomic approaches, validated with clinical manifestation and 116
histopathological findings of infection, we investigated the potential viral etiology in historically 117
archived outbreaks of birds fitting the syndrome descriptions associated with black and white 118
bird disease and clenched claw syndrome in Australia, along with other sporadic cases where 119
viral infection was suspected. In particular, we characterized CCS and BWBD and identified a 120
number of novel viruses from the families Astroviridae, Picornaviridae, Polyomaviridae, 121
Paramyxoviridae, and Parvoviridae in common urban wild birds. 122
Results 123
124
Clinical and histological description of rainbow lorikeets 125
To formulate a syndrome description for CCS, pathology records from the Australian Registry of 126
Wildlife Health from 451 rainbow lorikeets presenting between 1981 and 2019 were reviewed 127
for unexplained non-suppurative inflammation within the central nervous system. A total of 55 128
birds were found to match these search terms. The signalments, clinical signs and histological 129
lesions from these birds are summarized chronologically in Table S1. The index case was a 130
juvenile female rainbow lorikeet found in Mosman, NSW in November 1984, and the last known 131
case was recorded in an adult, female from the same location in May 2007. The majority of cases 132
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occurred in adult birds (28 adults, 20 juveniles, 7 age unspecified), including 24 males, 21 133
females and 10 gender unspecified. Although cases were distributed throughout the year, twice 134
as many CCS events occurred in October than any other single month. 135
136
Clenched feet was the most prevalent presentation (n = 40), followed by an inability to fly (n = 137
13), unspecified neurological signs (n = 8), paresis (n = 6), leg paralysis (n = 3), wing paralysis 138
(n = 2), head tilt (n = 3), tremors (n = 3), ascending or progressive neurological signs (n = 2), 139
head bob (n = 2), opisthotonus (n = 1), ataxia (n = 1), and rolling (n = 1) (Figure 1a,b). Body 140
condition was noted in 29 records and 11 birds were classified as being in good condition, 17 141
birds were considered thin or very thin, and one bird was emaciated. Less common presentations 142
included flying into a window (n = 1) and predation (n = 2). The most common cause of death 143
was euthanasia (n = 37). 144
145
Common microscopic lesions in CCS affected lorikeets are illustrated in Figure 1c-e and include 146
mild to severe perivascular cellular infiltrates ranging from one to eight cells deep, composed of 147
lymphocytes, plasma cells and smaller numbers of macrophages. Gliosis, spongiotic change of 148
the neuropil, Wallerian degeneration and nerve cell body degeneration to necrosis were 149
uncommon and restricted to those animals with moderate to severe inflammation. Non-150
suppurative inflammation within the central nervous system was more prevalent and more severe 151
within the caudal brainstem (28/54), cerebellum (30/54) and spinal cord (44/49), compared with 152
the cerebrum and anterior brain stem (12/54). Peripheral nervous system changes were also 153
noteworthy, as 5 of 47 birds examined had non-suppurative spinal ganglioneuritis, and 18 of 19 154
birds examined had non-suppurative neuritis, often with Wallerian degeneration (13/19). 155
156
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An adult, male red-collared lorikeet (Trichoglossus rubritorquis) from Queensland was also 157
found with a history of euthanasia following presentation with clenched feet, ataxia, and thin 158
body condition. Histological changes in this animal were consistent with CCS, including non-159
suppurative lesions that were mild in the cerebrum, and more moderate in the brain stem, 160
cerebellum and spinal cord. 161
162
Liver from an adult, male rainbow lorikeet with histological evidence of moderate hepatocellular 163
single cell necrosis, karyomegaly, and unusual amphophilic and variably shaped (stellate, 164
discrete and dense, and ground-glass) intranuclear inclusions (Figure 1a) was included in the 165
meta-transcriptomic investigation based on suspicion of one or more underlying viral infections. 166
This bird presented recumbent, with extensive subcutaneous epithelial lined cysts encapsulating 167
clusters of mites distributed along the wings and cranium, and loss of the distal primary feathers 168
and central tail feathers in a pattern suggestive of psittacine circovirus infection (Registry 169
4771.1). 170
171
Clinical and histological description of black and white bird disease 172
A total of 2781 birds in the order Passeriformes were included in the analysis. This identified 51 173
birds with myocardial degeneration or non-suppurative myocarditis. Two cases were excluded 174
from the analysis, due to incongruity of histological lesions compared with those of all other 175
birds (n = 49) examined. The signalments, clinical signs and histological lesions from these birds 176
are summarized chronologically in Table S2. 177
178
Although BWBD was first recognized during an epizootic extending across Victoria, NSW and 179
into Queensland (QLD) in 2006, records from the Australian Registry of Wildlife Health 180
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identified the index cases as magpies and currawongs found in Sydney and the NSW central 181
coast in August 2003. The majority of birds examined were magpies (n = 26), currawongs (n = 182
15), and ravens (n = 6), with adults, juveniles, males and females evenly represented. A solitary 183
adult female figbird and one adult female magpie lark were also among the affected birds. 184
Concurrent mortality was observed in crested pigeons (Ocyphaps olphotes), common koels 185
(Eudynamys scolopacea) silver gulls (Larus novaehollandiae) and indian mynahs (Acridotheres 186
tristis). Presentation of affected birds varied, occurring as nine mass morbidity and mortality 187
events, and 25 sporadic cases. The birds examined from outbreaks in 2003, 2006, 2013 and 2015 188
represent a small fraction of affected birds, and accumulatively total 105 birds reported formally 189
and more than 250 reported informally. Suspected intoxication was a common history in those 190
birds presenting en masse. Although seven birds were found dead or died prior to veterinary 191
examination, clinical signs among the remaining birds included paresis (Figure 2a), recumbency 192
or profound weakness (27), loss of righting reflex or difficulty righting (12), and less commonly 193
dyspnea (4), watery or bloody diarrhea (4), clenched claws (1) and a moribund state (1). 194
Although most birds were recumbent, they were not paralyzed as they had good cloacal tone, 195
withdrawal reflexes, and could stand and shuffle, flap or walk across the room when stimulated. 196
Most ravens had additional clinical signs indicative of central nervous system dysfunction, 197
including ataxia, head tilt, circling, and unusual head posture (4/6). Other species were alert and 198
could accurately grasp items with their beaks, despite being recumbent. The body condition of 199
affected birds varied with 21 noted to be in good body condition, while 27 were classified as 200
thin, very thin or emaciated. 201
202
Gross lesions in affected birds were rare, but included hydropericardium, epicardial hemorrhage, 203
pulmonary congestion to hemorrhage, hemorrhage into the gastrointestinal lumen and fibrinous 204
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serositis. Microscopic lesions characteristic of BWBD include the unifying feature of 205
degeneration of cardiac myocytes with or without non-suppurative perivascular and interstitial 206
cardiac inflammation (Figure 2 b-e). Myocyte degeneration or non-suppurative inflammation 207
within skeletal muscle was observed in 24 of the 45 birds examined (four animals were 208
unavailable for examination). Non-suppurative inflammation of the central nervous system was 209
observed in 25 birds. Lesions were most severe in the central cerebellar white matter and 210
included one to four cell deep perivascular cuffs, spongiotic change of the neuropil, degeneration 211
to necrosis of nerve cell bodies and multifocal gliosis. Other common histological features of the 212
syndrome included acute hepatic necrosis or inflammation (25/49), necrosis or non-suppurative 213
gastro-intestinal inflammation (21/49), non-suppurative interstitial nephritis (8/49), mild 214
vasculopathy to marked fibrino-necrotising vasculitis (19/49), non-suppurative perivascular 215
infiltrates throughout serosal surfaces (20/49) and non-suppurative interstitial pancreatitis 216
(11/49). Less common lesions included perivascular pulmonary, cardiac and neural hemorrhage, 217
meningitis and peripheral neuritis. Ravens generally had more severe and widespread central 218
nervous system lesions, and lacked skeletal muscle, renal and serosal inflammation that was 219
evident in the other species. In birds other than ravens, paresis was most likely associated with 220
lesions in striated muscle and the peripheral nervous system. Although Leucocytozoon and 221
microfilaria are common hemoparasites of the bird species examined, and were found in our 222
metagenomic data (32), these organisms were not relevant within disease-related lesions in our 223
cases. Ancillary diagnostic testing for known bacterial and viral pathogens, Chlamydophyla 224
species and intoxication revealed no significant findings. 225
226
Meta-transcriptomic virus identification 227
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Archived samples including brain, liver, heart, and kidneys from representative cases of diseased 228
birds were pooled and used to generate nine RNA-sequencing libraries that resulted in 7,725,034 229
to 26,555,569 paired reads per pool, for meta-transcriptomic analysis. From these data we 230
discovered eight viruses from the RNA virus families, Astroviridae, Paramyxoviridae, and 231
Picornaviridae, the DNA virus families Adenoviridae, Circoviridae, Parvoviridae and 232
Polyomaviridae. Two additional viruses - a hepacivirus and a narnavirus - were also identified 233
but unlikely associated with any clinical syndrome. 234
235
Avian avulavirus in brain samples of lorikeets with clenched claw syndrome 236
The Paramyxoviridae are a large group of enveloped linear negative-sense RNA viruses that 237
range from 15.2 to 15.9 kb in length. We identified paramyxovirus-like contigs in the brain 238
libraries of rainbow lorikeets. A RT-PCR designed to amplify the paramyxovirus L protein (301 239
nt) identified matching RNA in 4/5 brain tissues, corresponding to the four birds presenting with 240
neurological symptoms. Ten overlapping RT-PCRs were then performed to confirm the genomic 241
sequence (Table S4), revealing a typical 3’-N(455 aa)-P(446 aa)-M(366 aa)-F(619 aa)-HN(528 242
aa)-L(2263)-5’ organization. Based on the phylogenetic analysis of the L and M protein, the 243
novel paramyxovirus identified here - termed Avian paramyxovirus 5/rainbow 244
lorikeet/Australia/CCS - fell into the clade comprising the genus Avulavirus, exhibiting 86.7% 245
and 89.1% amino acid pairwise identity to its closest relative - APMV-5/budgerigar/Japan/TI/75 246
(GenBank accession number: LC168759). 247
248
To provide a provisional assessment of the virulence of this novel virus, we compared F protein 249
cleavage sites between the lorikeet avulavirus and known pathotypes of NDV (Avulavirus 1) that 250
commonly causes disease in avian species, including the velogenic, lentogenic, mesogenic and 251
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asymptomatic vaccine types. Specifically, the precursor F glycoprotein (F0) is cleaved into F1 252
and F2 subunits for the progeny virus to be infectious and to enable replication. The F protein 253
cleavage position for virulent or mesogenic strains contain a furin recognition site comprising 254
multiple basic amino acids. Notably, the lorikeet avulavirus had an F cleavage ‘RRRKKRF’ 255
motif identical to pathogenic NDV strains, suggesting its potential virulence, although this 256
remains to be confirmed (Figure 3). 257
258
DNA viruses identified in sporadic cases or mortality events rainbow lorikeets 259
In addition to RNA viruses, we identified three DNA viruses in the context of sporadic mortality 260
events of rainbow lorikeets: avian chapparvovirus, beak and feather disease virus and lorikeet 261
adenovirus. In addition to the RNA-sequencing results, we extracted DNA from corresponding 262
tissues for DNA virus validation through PCR and rolling circle amplification (RCA) assays. 263
264
Lorikeet chapparvovirus 265
The Parvoviridae are a family of small, non-enveloped, dsDNA animal viruses with linear 266
genomes of ~5 kb in length. We identified parvovirus-like transcripts in both liver RNA-seq 267
libraries from diseased lorikeets. Using RCA to enrich for circular DNAs, we recovered a 268
complete genome of a novel lorikeet chapparvovirus, comprising 4,271 nt with two distinct 269
ORFs that encoded the non-structural protein NS1 (670 aa) and the structural protein VP (542 270
aa). We further designed specific primers to amplify the targeted VP region (~248 bp) for 271
screening both the RNA (positive in two liver samples) and DNA (all positive) products. In 272
addition, we inferred two separate phylogenetic trees based on the complete NS1 and VP 273
proteins to determine the evolutionary relationships between the lorikeet virus and other 274
parvoviruses. This revealed that the closest relative to the lorikeet chapparvovirus identified here 275
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was an avian-associated red-crown crane parvovirus, yc-9 (GenBank accession number: 276
KY312548.1), although this shares only 48.2% amino acid identity in NS1 and 46.9% identity in 277
VP (Figure 4). 278
279
Beak and feather disease virus 280
Beak and feather disease virus (BFDV), a member of Circoviridae, is highly prevalent in 281
Australian wild birds, particularly psittacine species (33). We identified BFDV-like contigs in all 282
lorikeet RNA libraries, sharing 95% nucleotide identity to known strains of BFDV. Remapping 283
the raw reads to the closest BFDV strain (GenBank accession number: KM887928) gave 284
coverage of the whole virus genome, comprising 2,014 nt, and encoding a replication-associated 285
protein (290 amino acids; aa) and a capsid protein (247 aa) (Figure 5A). We then screened all the 286
RNA samples from rainbow lorikeets using PCR primers targeting the capsid protein (596 nt) 287
(GenBank accession number: KM887928), with the PCR products then Sanger sequenced. The 288
BFDVs identified from individual birds carried distinctive single nucleotide variants, and 289
sequences from each case formed strong phylogenetic clusters suggesting that these results are 290
not due to cross-sample contamination (Figure 5B). 291
292
Lorikeet adenovirus 293
Adenovirus-like contigs were identified from both the brain and liver libraries of rainbow 294
lorikeets. Phylogenetic analyses were performed based on the products of PCR primers designed 295
based on the obtained sequences of polymerase (pol, 843 aa) and hexon (hexon, 580 aa) proteins. 296
The virus identified, termed lorikeet adenovirus (LoAdv), exhibited 76% amino acid identity to 297
the most closely related skua adenovirus (GenBank accession number: YP.0049359313) (Figure 298
6). 299
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300
A novel lorikeet hepatovirus in clenched clawed lorikeets 301
A complete hepatovirus-like (Picornavirales) contig was identified in one of the liver libraries of 302
diseased lorikeets, exhibiting relatively high read abundance (~10,000x coverage depth). The 303
genome of this novel lorikeet hepatovirus, termed lorikeet hepatovirus (LoHepaV), is 7,339 nt in 304
length, and encodes a polyprotein of 2070 amino acids, excluding poly-A tails, that is similar in 305
structure to most Avihepatoviruses (Figure 6). Interestingly, this virus was most closely related 306
to Hepatovirus sp. isolate HepV-bat3206/Hipposideros_armiger/2011 identified from a round-307
leaf bat, although these two viruses only exhibit 46.3% amino acid identity across the 308
polyprotein. Recently, a lorikeet picornavirus-LoPV-1 (GenBank accession number: MK443503) 309
was identified in fecal samples from rainbow lorikeets in China (34), although this only 310
exhibited 17.4% amino acid identity with the lorikeet hepatovirus identified here (Figure 7). 311
312
Novel picornavirus identified in black and white bird disease cases 313
The Picornaviridae are a large family of single-strand positive-sense RNA viruses, with 314
genomes ranging from 6.7-10.1 kb in length. We identified a highly abundant complete 315
picornavirus-like contig in the library of archived black and white diseases cases. Remapping 316
raw reads to the picornavirus-like transcript showed complete genome coverage (~300x coverage 317
depth). We named this novel virus black and white bird picornavirus (BWBDpicoV). A RT-PCR 318
targeting this picornaviral contig (~220 bp) was positive in 2/6 of the brain tissues tested from an 319
Australian magpie (Cracticus tibicen) and an Australian raven (Corvus coronoides): notably, the 320
two positive birds were those presenting with non-suppurative encephalomyelitis. 321
322
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BWBDpicoV exhibits classical picornavirus features, with a genome length of 7,011 nt 323
excluding the poly A tails. This genome encodes a polyprotein of 2070 amino acid residues, as 324
well as a 5’ untranslated region (UTR) of 508 nt that includes the putative internal ribosome 325
entry site (IRES). The genome organisation of BWBDpicoV is similar to that of other 326
picornaviruses, with the characteristic gene order: 5′-VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 327
3Cpro, 3Dpol-3′ (Figure 7). BWBDpicoV also exhibited such typical picornavirus features as 328
rhv-like domains (111-222,307-424 aa), an RNA-helicase (1172-1271 aa), and an RNA-329
dependent RNA polymerase (RdRp; 2903-2154 aa). Phylogenetic analysis revealed that 330
BWBDpicoV was most closely related to seal picornavirus (GenBank accession number: 331
NC.009891.1), a member of the genus Aquamavirus (Figure 7). However, BWBD picornavirus 332
exhibited low amino acid similarity to the polyproteins of any of these viruses: only 29.5% with 333
Kunsagivirus A (GenBank accession number: YP_009505615.1) and 30.8% with Seal 334
picornavirus type 1 (GenBank accession number: YP_001487152.1). According to the 335
International Committee on Taxonomy of Viruses (ICTV), members of a genus within the 336
Picornaviridae should share at least 40% amino acid sequence identity in the polyprotein region. 337
As such, our BWBD may represent a new virus genus. 338
339
A divergent black and white bird astrovirus in a BWBD outbreak from Nowra, NSW 340
Several astrovirus-like reads were found in the RNA-seq library of the suspected BWBD (i.e. 341
non-suppurative encephalitis of undetermined etiology) outbreak from Nowra, NSW (Table 1). 342
This novel virus, termed black and white bird astrovirus (BWBDastV), was highly divergent in 343
sequence, and contained only 25 reads that covered the partial RdRp of Astrovirus 344
Pygoscelis/DT/2012 (GenBank accession number: KM587711.1) (see below). 345
346
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Reads from BWBDastV were used to develop PCR assays to bridge the gap in the RdRp. Using 347
RT-PCR amplification of a 195 nt region targeting the RdRp, we obtained positive hits for 348
BWBDastV in 4 of 5 brain samples. Based on RdRp protein identity and phylogenetic analysis, 349
there was 69.8% amino acid sequence similarity to the closest astrovirus, Astrovirus 350
Pygoscelis/DT/2012 (GenBank accession number: KM587711) sampled from Adelie penguins 351
(Pygoscelis adeliae) in Antarctica (Figure 8). Combining the PCR results with clinical and 352
histopathology patterns of diseased birds from the Nowra outbreak, we suggest that BWBDastV 353
might be the cause of the BWBD outbreak (Figure 10B). 354
355
Novel magpie polyomavirus in the Nowra outbreak 356
In addition to the novel astrovirus, our RNA-seq analysis identified a novel circular avian 357
polyomavirus in brain and heart tissue from one non-diseased Australian magpie from the Nowra 358
outbreak. Of note, the histopathology of BWBD was not consistent with polyomavirus infection 359
which is not known to be associated with myositis or encephalitis in birds. The genome of the 360
novel magpie polyomavirus - termed magpolyV - comprised 5115 bp with an overall GC content 361
of 46.5%, and we were able to recover full length genome of this virus using RCA and PCR 362
assays. Based on sequence similarity with existing reference polyomavirus proteins we predict 363
that the genome of the novel virus encodes capsid proteins VP1, VP2, VP3 and open reading 364
frame-X (ORF-X) from the late region, as well as two alternative transcripts, large T antigen 365
(LT) and small T antigen (ST) (Figure 9). Consistent with most known polyomaviruses, 366
magpolyV retains the typical conserved motifs and splicing sites in LT, including HPDKGG 367
(DnaJ domain), LRELL and LLGLL (LXXLL- CR1 motif), LFCDE (LXCXE,a pRB1-binding 368
motif) and GAVPEVNLE (ATPase motif). However, the consensus sequence CXCXXC for 369
protein phosphatase 2A binding, mostly found in mammalian polyomaviruses, was not present. 370
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Phylogenetic analysis revealed that this magpie polyomavirus consistently clustered within avian 371
lineages, exhibiting 90.3% amino acid its closest relative - butcherbird polyomavirus isolate 372
AWH19840 (accession number: KF360862) - in the LT protein, and 92% amino acid similarity 373
in VP1. 374
375
Discussion 376
We characterize the clinical and histological features of CCS and BWBD and apply a meta-377
transcriptomic approach to identify potential viral etiological agents in wild Australian birds 378
presenting with hepatic inclusion bodies, paresis and non-suppurative myocarditis or 379
encephalomyelitis, elucidating the complex nature of viral disease investigation amid multiple 380
infections. Next-generation metagenomic sequencing, in combination with traditional gross and 381
histological examination, identified several candidate pathogens for disease syndromes that had 382
been reported for decades as undiagnosed, but likely of viral origin. Application of this 383
methodology in the face of emergent disease syndromes or mass mortality events will facilitate 384
the early detection of viruses infecting wildlife, greatly contributing to disease control, 385
conservation of wild populations and the more rapid identification of novel pathogens. 386
387
Clenched claw syndrome 388
A key element of our paper was the investigation of CCS, a paretic disease of unknown etiology 389
in rainbow lorikeets (Trichoglossus haematodus) and a single red-collared lorikeet 390
(Trichoglossus rubritorquis) in Australia. The diseased lorikeets present with paresis and were 391
initially often found recumbent or sitting on their hocks with feet clenched, the passive position 392
for the avian foot. Affected birds kept in care progressively developed head tilt, ataxia, and 393
paralysis. The disease syndrome has primarily occurred along coastal New South Wales and 394
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South-East Queensland since the early 1980s. Although previous studies report 5-10% of free-395
living rainbow lorikeets rescued annually presenting with this syndrome, it seems likely that the 396
clinical syndrome in fact encompasses two or more disease etiologies (35). For example, lead 397
poisoning and plant based intoxication have been proposed as contributing to syndromes 398
described as clenched claw or drunken lorikeet syndrome (36). We focused on the investigation 399
of 55 cases of CCS characterized by non-suppurative encephalomyelitis and often 400
ganglioneuritis in which a viral etiology has been suspected. The meta-transcriptomic results, in 401
conjunction with the clinical signs, histopathologic data and confirmation through PCR assays, 402
suggest that the Avulavirus (APMV-5) played a potential leading role in the pathogenesis of 403
CCS in these diseased birds (Figure 10a). An increasing incidence of clinical AMPV infection in 404
wild birds is reported worldwide (37). APMVs have been categorized into 12 serotypes, and 405
APMV1 (NDV) causes significant economic impact on the poultry industry as well as population 406
level impacts on wild birds (37). Both NDV and APMV5 have been reportedly associated with 407
disease outbreaks where mortality rates approach 100%, although the pathogenicity of APMV5 408
varies substantially among aviary species. APMV5 was first isolated from a fatal outbreak of 409
caged budgerigars in Japan in 1974, followed by epizootic outbreaks in budgerigars in the UK 410
(38) and Australia (39). Interestingly, APMV5 will not replicate in chicken embryonated eggs, it 411
lacks a virion hemagglutinin, and it is not pathogenic to chickens (40). Phylogenetically, the 412
AMPV5 identified here is closely related to the Brisbane and Japanese strains identified from 413
budgerigars (41). Because of the possibility of cross-species transmission, the prevalence and 414
pathogenicity of avian paramyxoviruses circulating among wild birds in Australia clearly merits 415
further investigation. 416
417
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Detection of avian chapparvovirus, avian adenovirus and beak and feather disease viruses in 418
rainbow lorikeets 419
Chapparvoviruses are being increasingly identified in metagenomic studies, and seemingly have 420
a wide host range (42), including bats (43), and the feces of turkeys (44), red-crowned cranes 421
(45), wild rats (46) and pigs (47). Importantly, a murine kidney parvovirus was recently found in 422
immunodeficient laboratory mice strains, causing renal failure and kidney fibrosis (48), 423
indicating that some chapparvoviruses are associated with highly virulent infections. 424
425
Avian polyomavirus, beak and feather disease virus, and avian adenovirus appear to be prevalent 426
in psittacine species in Australia (49), compatible with the data presented here. As multi-viral 427
infection is common, our analysis of viral communities associated with neglected avian diseases 428
enabled a comprehensive understanding beyond the one-host, one-virus system (13). Many 429
known viral infections are relevant clinical issues in psittacine species due to their association 430
with acute death, disease, the capacity to induce immunocompromise, and difficulties in 431
treatment and control (50). These have been associated with a variety of RNA viruses including 432
avian paramyxovirus (37), avian influenza virus (51, 52), and avian bornavirus (53), and DNA 433
viruses such as psittacine beak and feather disease virus (PBFD) (54), avian polyomavirus 434
infection (APV) (55), psittacid herpesvirus (PsHV) (56), psittacine adenovirus (PsAdV) (57), 435
poxvirus infection (58), and papillomavirus infection (59, 60). 436
437
The role of astroviruses, polyomaviruses and picornaviruses in black and white bird disease 438
Viruses of the family Astroviridae comprise genomes of 6.8-7kb, infect multiple mammalian 439
species including humans, and also avian species. Astrovirus infection is most commonly 440
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associated with gastroenteritis, and rarely neurological symptoms. A number of novel divergent 441
novel astroviruses in wild birds have recently been discovered through metagenomic surveillance 442
(61-63). Many avian astroviruses, including avian nephritis virus, duck and turkey astroviruses 443
and duck hepatitis virus 2, have considerable economic impact on the poultry industry and these 444
viruses are widely distributed in wild birds (62). Despite this, the diversity and ecology of these 445
viruses, including their potential interspecies transmission events, are largely unknown. 446
Previously, astrovirus-associated central nervous system impairment had been reported in mink, 447
human, bovine, ovine, and swine (64-68). 448
449
In mammals such as dogs and cats, astroviruses are commonly isolated with enteric bacterial 450
pathogens in sporadic gastroenteritis outbreaks (69), while in birds, astrovirus infection 451
manifests as a broader disease spectrum including enteritis, hepatitis and nephritis, but rarely 452
neurological symptoms. The astrovirus identified here (BWBDastV) was present in each bird 453
with non-suppurative encephalomyelitis, concurrent with myositis, coelomitis and myocarditis 454
from a single outbreak. However, formal classification of the virus will require additional 455
sequence information. In addition, further surveillance and in situ diagnostic modalities are 456
required to reveal the association between BWBDastV, clinical illness of the birds, and the 457
histological lesions observed. 458
459
A novel polyomavirus - magpolyV - was identified from the brain and heart of one of the non-460
diseased Australian magpies (Cracticus tibicen) that died in the same BWBD outbreak. An avian 461
polyomavirus (APV) - budgerigar fledgling disease polyomavirus – has been documented in 462
young budgerigars and other psittacine species, causing feather abnormalities, abdominal 463
distension, head tremors and skin hemorrhages, reaching infection rates of up to 100% in aviaries 464
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worldwide (70). The clinical presentation and degree of susceptibility to APV infection varies 465
from skin diseases to acute death. In the case of non-psittacine species, goose polyomavirus has 466
been characterized as the etiologic agent of fatal hemorrhagic nephritis and enteritis of European 467
geese (HNEG) (71). Other species of polyomaviruses have been identified in finches (72), crows 468
(73) butcherbirds (74), and a novel APV was recently associated with a fatal outbreak in canaries 469
(75). Moreover, a recent study revealed an APV isolated from pigeon feces in China showing 470
almost 99% identity with previously identified psittacine strains, suggesting a much broader host 471
range of APVs and undermined cross-species transmission (76). 472
473
Picornaviruses are commonly identified infecting a variety of avian species in metagenomic 474
studies. In the context of overt disease, notable avian picornaviruses include avian 475
encephalomyelitis viruses in chickens, pheasants, turkeys (77), duck hepatitis A in ducklings 476
(78), and a novel poecivirus that has been identified strongly associated with Avian keratin 477
disorder (AKD) in Alaskan birds (79, 80). We identified two novel picornaviruses in this study - 478
black and white bird picornavirus and a lorikeet hepatovirus - the pathogenic potential of which 479
merits further investigation. Interestingly, a novel lorikeet picornavirus (LoPv-1, GenBank 480
accession number MK443503) was identified in the feces of healthy rainbow lorikeets 481
(Trichoglossus haematodus) in China (34), although the polyprotein only shared 12.6% 482
similarity with the lorikeet hepatovirus identified here. 483
484
The high frequency of microbial co-infections suggests that rather than being the primary cause 485
of the disease outbreak, some novel avian viruses might augment another pathogen, causing 486
immunosuppression in immuno-compromised hosts (81). Although we were successful in 487
elucidating seven candidate viral pathogens that eluded detection through viral culture, it is clear 488
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that additional experimental evidence such as in vivo inoculation experiments and in situ 489
methodologies to identify pathogens within lesions are critical for clarifying the relationship 490
between infection and the emergent disease syndromes we consider. Notably, the metagenomic 491
approach utilized here is not limited to viruses and can be useful in the detection of bacteria, 492
fungi, protozoan and metazoan parasites. For example, the same data set provided a meta-493
transcriptomic signal of Leucocytozoon manifestation in BWBD affected birds (32), although 494
our histological data suggests that this was not a primary or secondary pathogen. 495
496
The utility of meta-transcriptomics in the context of a thorough and multi-disciplinary diagnostic 497
approach is to rapidly identify viruses and other microbial species, including those that are 498
highly divergent, with greater sensitivity and capability than conventional diagnostic tests. The 499
sequencing data and PCR assays developed here enhance diagnostic capabilities for avian 500
disease and enable epidemiological surveillance to better understand the ecology and impacts of 501
the viruses described. More broadly, our study highlights the value of proactive viral discovery 502
in wildlife, and targeted surveillance in response to an emerging infectious disease event that 503
might be associated with veterinary and public health. 504
505
Materials and methods 506
Animal ethics 507
Wild birds were examined under the auspices of NSW Office of Environment and Heritage 508
Licenses to Rehabilitate Injured, Sick or Orphaned Protected Wildlife (#MWL000100542). 509
Diagnostic specimens from recently deceased birds were collected under the approval of Taronga 510
Animal Ethics Committee’s Opportunistic Sample Collection Program, pursuant to NSW Office 511
of Environment and Heritage issued scientific licenses #SL10469 and SL100104. 512
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513
Sample collection 514
Samples were collected between 2002 and 2013 from 18 birds, predominantly from within the 515
Sydney basin and the coastal center of NSW. Fresh portions of brain, liver, heart, and kidney 516
were collected aseptically and frozen at -80oC. Additionally, a range of tissues were fixed in 10% 517
neutral buffered formalin, processed in ethanol, embedded with paraffin, sectioned, stained with 518
hematoxylin and eosin, and mounted with a cover slip prior to examination by light microscopy. 519
Giemsa stains were applied to a subset of tissue samples to determine whether protozoa were 520
present within lesions. 521
522
Historical Case Review 523
The records of the Australian Registry of Wildlife Health dating back to 1981 were interrogated 524
to identify rainbow lorikeets with clinical signs relating to the nervous system, and those with 525
unexplained non-suppurative inflammation in the central nervous system. An additional search 526
was conducted to identify passerine birds with unexplained non-suppurative inflammation within 527
cardiac or skeletal muscle. Retrieved records were investigated to determine the signalment, 528
clinical signs and histological lesions of affected animals. 529
530
The severity of non-suppurative inflammation was graded on a scale of 0-4, where 0 indicated no 531
discernible lesions and 4 represented severe and extensive mononuclear cell inflammation. 532
Degeneration and necrosis were graded on a scale of 0-4 where 1 represented mild, multifocal 533
single cell degeneration or necrosis, and 4 characterized extensive coagulative necrosis or 534
malacia. Wallerian degeneration of the white matter tracts within the brain, spinal cord, central 535
and peripheral nerves was noted when present. In rainbow lorikeets the lesions were graded in 536
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the cerebrum, brain stem, cerebellum, spinal cord, ganglia, and peripheral nerves. Whereas in the 537
passerines, degeneration or necrosis, and non-suppurative inflammation were graded in skeletal 538
and cardiac muscle, brain, liver, gastrointestinal tract, pancreas, blood vessel walls, and renal 539
interstitium. 540
541
Avian influenza and NDV were excluded using real-time PCR on oropharyngeal, conjunctival 542
and cloacal swabs from 22 magpies and 12 other wild birds (including currawongs, magpies and 543
ravens) (Flu DETECT, Zoetis). Serological testing of plasma from the same birds comprised 544
hemagglutination inhibition to identify NDV antibodies, and competitive ELISA tests targeting 545
Avian influenza virus and flavivirus antibodies. Sixty-eight tissue samples from a sub-group of 546
13 magpies, currawongs and ravens identified as having acute histological lesions suggestive of 547
viral infection were subjected to further avian influenza, NDV and WNV specific rt-PCR and 548
virus isolation. Virus isolation was attempted in both mammalian and insect cell lines (Peter 549
Kirland pers comm.). Additional viral culture was attempted on the same samples inoculated into 550
chick embryos while assessing hemagglutinating agents, and in Vero cells assessing cytopathic 551
effect consistent with WNV infection. Clearview antigen capture ELISA (Unipath, Mountain 552
View, Calif.) targeting Chlamydophila species was conducted on splenic tissue from eight birds 553
with BWBD (magpies, currawongs and a raven and magpie lark). Liver and lung tissues from 554
eight magpies and currawongs were subjected to routine aerobic and anaerobic bacterial and 555
fungal culture. Finally, liver tissue from eight magpies and currawongs, representing the 2006 556
and 2015 BWBD epizootics, were subjected to toxicological testing to exclude the presence of a 557
variety of acaricides, fungicides, organophosphates, carbamates, synthetic pyrethroids, and 558
organochlorines and their metabolites. 559
560
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RNA extraction, library construction and sequencing 561
Viral RNA was extracted from the brain and liver, heart and kidney samples of animals using the 562
RNeasy Plus Mini Kit (Qiagen, Germany). RNA concentration and integrity were determined 563
using a NanoDrop spectrophotometer (Thermo) and TapeStation (Agilent). RNA samples were 564
then pooled in equal proportions based on animal tissue type and syndrome. Illumina RNA 565
libraries were prepared on the pooled samples following rRNA depletion using a RiboZero Gold 566
kit (Epidemiology) at the Australian Genome Research Facility (AGRF), Melbourne. The rRNA 567
depleted libraries were then sequenced on an Illumina HiSeq 2500 system (paired 100 nt reads) 568
569
Virome meta-transcriptomics 570
Unbiased sequencing of RNA aliquots extracted from diseased animals in each outbreak were 571
pooled based on host species, clinical syndrome and histological findings as shown in Table 1. 572
The RNA sequencing reads were trimmed of low quality bases and any adapter sequences before 573
de novo assembly using Trinity 2.1.1 (82). The assembled sequence contigs were annotated using 574
both nucleotide and protein BLAST searches against the NCBI non-redundant sequence 575
database. To identify low abundance organisms, the sequence reads were also annotated directly 576
using a BlastX search against the NCBI virus RefSeq viral protein database using Diamond (83) 577
with an e-value cutoff of <10−5. Open reading frames were then predicted from the viral contigs 578
in Geneious v11.1.2 (84) with gene annotation and functional predictions made against the 579
Conserved domain databases (CDD) (85). All sequences were aligned using the E-INS-i 580
algorithm in MAFFT version 7 (86). Virus abundance was assessed using a read mapping 581
approach, in which reads were mapped to the assembled viral contigs using the BBmap program 582
(87). 583
584
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Rolling-circle amplification assays for circular DNA viruses 585
To recover the complete genomes of the polyomaviruses and circoviruses identified through our 586
RNA-Seq analysis, we enriched for circular DNA using rolling-circle amplification (RCA) (88). 587
Briefly, genomic DNA extracted from animal tissue and combined with a reaction buffer 588
containing random primers, 1X phi29 Buffer, DTT, Bovine Serum Albumin, dNTPs, and phi29 589
DNA polymerase (Thermo Scientific, Australia) and then incubated at 30°C for 16h. The RCA 590
products were then purified using the Monarch PCR & DNA Cleanup Kit (NEB), then quantified 591
using the Qubit dsDNA broad range assay. Nextera XT DNA libraries were then prepared and 592
sequenced on an Illumina MiSeq to a depth of ~2M reads (2 X 150nt). 593
594
RT-PCR assays and Sanger sequencing 595
Total liver and brain RNA from individual birds was reverse transcribed with SuperScript IV 596
VILO Mastermix (Invitrogen). The cDNA generated from the sampled tissues was used for viral 597
specific PCRs targeting regions identified by RNA-Seq. RT-PCR primers were designed to 598
bridge any genomic gaps based on detected transcripts of the paramyxoviruses, polyomaviruses, 599
adenoviruses, and astroviruses identified in the RNA-seq data (Tables S3 and S4). Accordingly, 600
all PCRs were performed using Platinum SuperFi DNA polymerase (Invitrogen) with a final 601
concentration of 0.2 µM for both forward and reverse primers. All PCR products were visualized 602
by agarose gel electrophoresis and Sanger sequencing. Long RT-PCR products were also 603
sequenced using Nextera XT and MiSeq, as per RCA assays above. 604
605
Phylogenetic analysis 606
Conserved domains within the viral proteins produced were used in phylogenetic analyses to 607
determine the evolutionary relationships of the viruses identified here. Specifically, in the case of 608
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RNA viruses we used amino acid sequences of the RNA-dependent RNA polymerase (RdRp) 609
that is the most conserved protein among this group. After removing all ambiguously aligned 610
regions using TrimAl (89), phylogenetic trees were inferred using the maximum likelihood 611
method (ML) implemented in PhyML version 3.0 (90), employing a Subtree Pruning and 612
Regrafting topology searching algorithm and the LG model of amino acid substitution. Bootstrap 613
resampling with 1000 replications under the same substitution model was used to assess nodal 614
support. All phylogenetic trees were then visualized using FigTree v1.4.3 615
(http://tree.bio.ed.ac.uk/software/figtree). 616
617
Nucleotide Sequence Accession Numbers 618
The RNA sequencing data in this study has been deposited in the GenBank Sequence Reads 619
Archive under accession numbers PRJNAXXXX. All genome sequences of identified viruses 620
have been uploaded in GenBank under accession numbers XXXXX to XXXXXX. 621
622
Acknowledgements 623
This research was supported by the Taronga Conservation Society Australia, Taronga 624
Conservation Science Initiative, and New South Wales National Parks and Wildlife Service. 625
ECH is supported by an ARC Australian Laureate Fellowship (FL170100022). We thank Drs. 626
Bill Hartley, Rod Reece, Cheryl Sangster, Shannon Donahoe, Richard Montali, and Cathy 627
Shilton for their diagnostic contributions on individual cases. Virology personnel at the NSW 628
Department of Planning, Industry and Environment’s Elizabeth Macarthur Agricultural Institute, 629
and CSIRO’s Australian Animal Health Laboratories are acknowledged for their considerable 630
body of work pursuing viral culture in the BWBD investigation. 631
632
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References 633
1. Kruse H, kirkemo AM, Handeland K. 2004. Wildlife as source of zoonotic infections. 634
Emerg Infect Dis 10:2067-72. 635
2. Reed KD, Meece JK, Henkel JS, Shukla SK. 2003. Birds, migration and emerging 636
zoonoses: West Nile virus, Lyme disease, influenza A and enteropathogens. Clin Med Res 637
1:5-12. 638
3. Daszak P, Cunningham AA, Hyatt AD. 2000. Emerging infectious diseases of wildlife--639
threats to biodiversity and human health. Science 287:443-9. 640
4. Smith KF, Sax DF, Lafferty KD. 2006. Evidence for the role of infectious disease in 641
species extinction and endangerment. Conserv Biol 20:1349-57. 642
5. LaDeau SL, Kilpatrick AM, Marra PP. 2007. West Nile virus emergence and large-scale 643
declines of North American bird populations. Nature 447:710-3. 644
6. Lachish S, Bonsall MB, Lawson B, Cunningham AA, Sheldon BC. 2012. Individual and 645
population-level impacts of an emerging poxvirus disease in a wild population of great tits. 646
PLoS One 7:e48545. 647
7. Tompkins DM, Carver S, Jones ME, Krkosek M, Skerratt LF. 2015. Emerging infectious 648
diseases of wildlife: a critical perspective. Trends Parasitol 31:149-59. 649
8. Zhang J, Finlaison DS, Frost MJ, Gestier S, Gu X, Hall J, Jenkins C, Parrish K, Read AJ, 650
Srivastava M, Rose K, Kirkland PD. 2018. Identification of a novel nidovirus as a potential 651
cause of large scale mortalities in the endangered Bellinger River snapping turtle 652
(Myuchelys georgesi). PLoS One 13:e0205209. 653
9. Shi M, Lin XD, Chen X, Tian JH, Chen LJ, Li K, Wang W, Eden JS, Shen JJ, Liu L, 654
Holmes EC, Zhang YZ. 2018. The evolutionary history of vertebrate RNA viruses. Nature 655
556:197-202. 656
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
30
10. Shi M, Lin XD, Tian JH, Chen LJ, Chen X, Li CX, Qin XC, Li J, Cao JP, Eden JS, 657
Buchmann J, Wang W, Xu J, Holmes EC, Zhang YZ. 2016. Redefining the invertebrate 658
RNA virosphere. Nature 540:539-543. 659
11. Russo AG, Eden JS, Enosi Tuipulotu D, Shi M, Selechnik D, Shine R, Rollins LA, Holmes 660
EC, White PA. 2018. Viral discovery in the invasive Australian cane toad (Rhinella 661
marina) using metatranscriptomic and genomic approaches. J Virol 92:e00768-18. 662
12. Vibin J, Chamings A, Collier F, Klaassen M, Nelson TM, Alexandersen S. 2018. 663
Metagenomics detection and characterisation of viruses in faecal samples from Australian 664
wild birds. Sci Rep 8:8686. 665
13. Wille M, Eden JS, Shi M, Klaassen M, Hurt AC, Holmes EC. 2018. Virus-virus 666
interactions and host ecology are associated with RNA virome structure in wild birds. Mol 667
Ecol 27:5263-5278. 668
14. Geoghegan JL, Di Giallonardo F, Cousins K, Shi M, Williamson JE, Holmes EC. 2018. 669
Hidden diversity and evolution of viruses in market fish. Virus Evol 4:vey031. 670
15. Chong R, Shi M, Grueber CE, Holmes EC, Hogg CJ, Belov K, Barrs VR. 2019. Fecal viral 671
diversity of captive and wild Tasmanian devils characterized using virion-enriched 672
metagenomics and meta-transcriptomics. J Virol 93:e00205-19. 673
16. Ng TF, Mesquita JR, Nascimento MS, Kondov NO, Wong W, Reuter G, Knowles NJ, 674
Vega E, Esona MD, Deng X, Vinje J, Delwart E. 2014. Feline fecal virome reveals novel 675
and prevalent enteric viruses. Vet Microbiol 171:102-11. 676
17. Moreno PS, Wagner J, Mansfield CS, Stevens M, Gilkerson JR, Kirkwood CD. 2017. 677
Characterisation of the canine faecal virome in healthy dogs and dogs with acute diarrhoea 678
using shotgun metagenomics. PLoS One 12:e0178433. 679
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
31
18. Mitra N, Cernicchiaro N, Torres S, Li F, Hause BM. 2016. Metagenomic characterization 680
of the virome associated with bovine respiratory disease in feedlot cattle identified novel 681
viruses and suggests an etiologic role for influenza D virus. J Gen Virol 97:1771-84. 682
19. Holman DB, Klima CL, Ralston BJ, Niu YD, Stanford K, Alexander TW, McAllister TA. 683
2017. Metagenomic sequencing of bronchoalveolar lavage samples from feedlot cattle 684
mortalities associated with Bovine Respiratory Disease. Genome Announc 5: e01045-17. 685
20. Zhang M, Hill JE, Fernando C, Alexander TW, Timsit E, van der Meer F, Huang Y. 2019. 686
Respiratory viruses identified in western Canadian beef cattle by metagenomic sequencing 687
and their association with bovine respiratory disease. Transbound Emerg Dis 66:1379-688
1386. 689
21. Hyndman TH, Shilton CM, Stenglein MD, Wellehan JFX, Jr. 2018. Divergent 690
bornaviruses from Australian carpet pythons with neurological disease date the origin of 691
extant Bornaviridae prior to the end-Cretaceous extinction. PLoS Pathog 14:e1006881. 692
22. Eden JS, Rose K, Ng J, Shi M, Wang Q, Sintchenko V, Holmes EC. 2017. Francisella 693
tularensis ssp. holarctica in ringtail possums, Australia. Emerg Infect Dis 23:1198-1201. 694
23. DA P. 1993. Diseases of free-ranging birds in Australia. W.B. Saunders Company, 695
Philadelphia. 696
24. Mackie J. 2012. Pathology of Australian native wildlife, Philip Ladds. 90:509. CSIRO 697
Publishing. 698
25. McOrist S, Perry RA. 1986. Encephalomyelitis in free-living rainbow lorikeets 699
(Trichoglossus haematodus). Avian Pathology 15:783-789. 700
26. Reece RL. 1994. The pathology registry and some interesting cases. In: Wildlife. 701
Proceedings of the Post Graduate Foundation in Veterinary Science, Sydney. p 217-233. 702
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
32
27. Rose K. Common diseases of urban wildlife. 1990). In Wildlife of Australia, Proceedings 703
327 of the Post Graduate Foundation in Veterinary Science, Sydney, Australia. p 365-427. 704
28. Rosenwax A , Phalen DN. 2010. Update on lorikeet paralysis. In Proceedings of the 705
Association of Avian Veterinarians Australasian Committee Annual Conference, Hobart, 706
Australia. p85-89. 707
29. Wildlife Health Australia Factsheet. 2017. Neurological syndrome in "black and white" 708
birds in Australia May 2017. Available at: 709
https://www.wildlifehealthaustralia.com.au/Portals/0/Documents/FactSheets/Avian/Neurol710
ogical%20Syndrome%20in%20Black%20and%20White%20Birds.pdf. 711
30. Anonymous. 2014. Pied Currawong (Strepera versicolour) with black and white bird 712
disease. Available at http://arwh.org/node/188 [Accessed 16 Nov 2015]. abstr Australian 713
Registry of Wildlife Health, 714
31. Jarratt C, Rose K. 2016. Mass mortality event in Australian magpies (Cracticus tibicen) 715
and Australian ravens (Corvus coronoides) in NSW, 2015. In 'Association of Avian 716
Veterinarians Australasian Committee (AAVAC). 717
32. Charon J, Grigg MJ, Eden JS, Piera KA, Rana H, William T, Rose K, Davenport MP, 718
Anstey NM, Holmes EC. 2019. Novel RNA viruses associated with Plasmodium vivax in 719
human malaria and Leucocytozoon parasites in avian disease. PLoS Pathog 15:e1008216. 720
33. Amery-Gale J, Marenda MS, Owens J, Eden PA, Browning GF, Devlin JM. 2017. A high 721
prevalence of beak and feather disease virus in non-psittacine Australian birds. J Med 722
Microbiol 66:1005-1013. 723
34. Wang H, Yang S, Shan T, Wang X, Deng X, Delwart E, Zhang W. 2019. A novel 724
picornavirus in feces of a rainbow lorikeet (Trichoglossus moluccanus) shows a close 725
relationship to members of the genus Avihepatovirus. Arch Virol 164:1911-1914. 726
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
33
35. Booth RJ, Hartley WJ, McKee JJ. 2001. Polioencephalomyelitis in rainbow lorikeets 727
(Trichoglossus haematodus). Volume 157. In Veterinary Conservation Biology, Wildlife 728
Health and Management in Australia. Proceedings of International Joint Conference. 729
Sydney, Australia. 730
36. McLelland JM, Gartrell BD, Morgan KJ, Roe WD, Johnson CB. 2011. The role of lead in 731
a syndrome of clenched claw paralysis and leg paresis in Swamp Harriers (Circus 732
approximans). J Wildl Dis 47:907-16. 733
37. Gogoi P, Ganar K, Kumar S. 2017. Avian paramyxovirus: a brief review. Transbound 734
Emerg Dis 64:53-67. 735
38. Gough RE, Manvell RJ, Drury SE, Naylor PF, Spackman D, Cooke SW. 1993. Deaths in 736
budgerigars associated with a paramyxovirus-like agent. Vet Rec 133:123. 737
39. Amery-Gale J, Hartley CA, Vaz PK, Marenda MS, Owens J, Eden PA, Devlin JM. 2018. 738
Avian viral surveillance in Victoria, Australia, and detection of two novel avian 739
herpesviruses. PLoS One 13:e0194457. 740
40. Samuel AS, Paldurai A, Kumar S, Collins PL, Samal SK. 2010. Complete genome 741
sequence of avian paramyxovirus (APMV) serotype 5 completes the analysis of nine 742
APMV serotypes and reveals the longest APMV genome. PLoS One 5:e9269. 743
41. Mustaffa-Babjee A, Spradbrow PB, Samuel JL. 1974. A pathogenic paramyxovirus from a 744
budgerigar (Melopsittacus undulatus). Avian Dis 18:226-30. 745
42. Penzes JJ, de Souza WM, Agbandje-McKenna M, Gifford RJ. 2019. An ancient lineage of 746
highly divergent parvoviruses infects both vertebrate and invertebrate hosts. Viruses 747
11:E525. 748
43. Baker KS, Leggett RM, Bexfield NH, Alston M, Daly G, Todd S, Tachedjian M, Holmes 749
CE, Crameri S, Wang LF, Heeney JL, Suu-Ire R, Kellam P, Cunningham AA, Wood JL, 750
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
34
Caccamo M, Murcia PR. 2013. Metagenomic study of the viruses of African straw-751
coloured fruit bats: detection of a chiropteran poxvirus and isolation of a novel adenovirus. 752
Virology 441:95-106. 753
44. Reuter G, Boros A, Delwart E, Pankovics P. 2014. Novel circular single-stranded DNA 754
virus from turkey faeces. Arch Virol 159:2161-4. 755
45. Wang Y, Yang S, Liu D, Zhou C, Li W, Lin Y, Wang X, Shen Q, Wang H, Li C, Zong M, 756
Ding Y, Song Q, Deng X, Qi D, Zhang W, Delwart E. 2019. The fecal virome of red-757
crowned cranes. Arch Virol 164:3-16. 758
46. Yang S, Liu Z, Wang Y, Li W, Fu X, Lin Y, Shen Q, Wang X, Wang H, Zhang W. 2016. 759
A novel rodent Chapparvovirus in feces of wild rats. Virol J 13:133. 760
47. Palinski RM, Mitra N, Hause BM. 2016. Discovery of a novel Parvovirinae virus, porcine 761
parvovirus 7, by metagenomic sequencing of porcine rectal swabs. Virus Genes 52:564-7. 762
48. Roediger B, Lee Q, Tikoo S, Cobbin JCA, Henderson JM, Jormakka M, O'Rourke MB, 763
Padula MP, Pinello N, Henry M, Wynne M, Santagostino SF, Brayton CF, Rasmussen L, 764
Lisowski L, Tay SS, Harris DC, Bertram JF, Dowling JP, Bertolino P, Lai JH, Wu W, 765
Bachovchin WW, Wong JJ, Gorrell MD, Shaban B, Holmes EC, Jolly CJ, Monette S, 766
Weninger W. 2018. An atypical parvovirus drives chronic tubulointerstitial nephropathy 767
and kidney fibrosis. Cell 175:530-543. 768
49. Hulbert CL, Chamings A, Hewson KA, Steer PA, Gosbell M, Noormohammadi AH. 2015. 769
Survey of captive parrot populations around Port Phillip Bay, Victoria, Australia, for 770
psittacine beak and feather disease virus, avian polyomavirus and psittacine adenovirus. 771
Aust Vet J 93:287-92. 772
50. Katoh H, Ogawa H, Ohya K, Fukushi H. 2010. A review of DNA viral infections in 773
psittacine birds. J Vet Med Sci 72:1099-106. 774
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
35
51. Jiao P, Song Y, Yuan R, Wei L, Cao L, Luo K, Liao M. 2012. Complete genomic sequence 775
of an H5N1 influenza virus from a parrot in southern China. J Virol 86:8894-5. 776
52. Hawkins MG, Crossley BM, Osofsky A, Webby RJ, Lee CW, Suarez DL, Hietala SK. 777
2006. Avian influenza A virus subtype H5N2 in a red-lored Amazon parrot. J Am Vet Med 778
Assoc 228:236-41. 779
53. Staeheli P, Rinder M, Kaspers B. 2010. Avian bornavirus associated with fatal disease in 780
psittacine birds. J Virol 84:6269-75. 781
54. Ramis A, Latimer KS, Niagro FD, Campagnoli RP, Ritchie BW, Pesti D. 1994. Diagnosis 782
of psittacine beak and feather disease (PBFD) viral infection, avian polyomavirus 783
infection, adenovirus infection and herpesvirus infection in psittacine tissues using DNA in 784
situ hybridization. Avian Pathol 23:643-57. 785
55. Bernier G, Morin M, Marsolais G. 1981. A generalized inclusion body disease in the 786
budgerigar (Melopsittacus undulatus) caused by a papovavirus-like agent. Avian Dis 787
25:1083-92. 788
56. Simpson CF, Hanley JE, Gaskin JM. 1975. Psittacine herpesvirus infection resembling 789
pacheco's parrot disease. J Infect Dis 131:390-6. 790
57. Raue R, Gerlach H, Muller H. 2005. Phylogenetic analysis of the hexon loop 1 region of an 791
adenovirus from psittacine birds supports the existence of a new psittacine adenovirus 792
(PsAdV). Arch Virol 150:1933-43. 793
58. McDonald SE, Lowenstine LJ, Ardans AA. 1981. Avian pox in blue-fronted Amazon 794
parrots. J Am Vet Med Assoc 179:1218-22. 795
59. Latimer KS, Niagro FD, Rakich PM, Campagnoli RP, Ritchie BW, McGee ED. 1997. 796
Investigation of parrot papillomavirus in cloacal and oral papillomas of psittacine birds. 797
Vet Clin Pathol 26:158-163. 798
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
36
60. Cooper JE, Lawton MP, Greenwood AG. 1986. Papillomas in psittacine birds. Vet Rec 799
119:535. 800
61. Fernandez-Correa I, Truchado DA, Gomez-Lucia E, Domenech A, Perez-Tris J, Schmidt-801
Chanasit J, Cadar D, Benitez L. 2019. A novel group of avian astroviruses from 802
Neotropical passerine birds broaden the diversity and host range of Astroviridae. Sci Rep 803
9:9513. 804
62. Chu DK, Leung CY, Perera HK, Ng EM, Gilbert M, Joyner PH, Grioni A, Ades G, Guan 805
Y, Peiris JS, Poon LL. 2012. A novel group of avian astroviruses in wild aquatic birds. J 806
Virol 86:13772-8. 807
63. Honkavuori KS, Briese T, Krauss S, Sanchez MD, Jain K, Hutchison SK, Webster RG, 808
Lipkin WI. 2014. Novel coronavirus and astrovirus in Delaware Bay shorebirds. PLoS One 809
9:e93395. 810
64. Vu DL, Bosch A, Pinto RM, Guix S. 2017. Epidemiology of classic and novel human 811
astrovirus: gastroenteritis and beyond. Viruses 9: E33. 812
65. Pfaff F, Schlottau K, Scholes S, Courtenay A, Hoffmann B, Hoper D, Beer M. 2017. A 813
novel astrovirus associated with encephalitis and ganglionitis in domestic sheep. 814
Transbound Emerg Dis 64:677-682. 815
66. Boros A, Albert M, Pankovics P, Biro H, Pesavento PA, Phan TG, Delwart E, Reuter G. 816
2017. Outbreaks of neuroinvasive astrovirus associated with encephalomyelitis, weakness, 817
and paralysis among weaned pigs, Hungary. Emerg Infect Dis 23:1982-1993. 818
67. Li L, Diab S, McGraw S, Barr B, Traslavina R, Higgins R, Talbot T, Blanchard P, Rimoldi 819
G, Fahsbender E, Page B, Phan TG, Wang C, Deng X, Pesavento P, Delwart E. 2013. 820
Divergent astrovirus associated with neurologic disease in cattle. Emerg Infect Dis 821
19:1385-92. 822
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
37
68. Blomstrom AL, Widen F, Hammer AS, Belak S, Berg M. 2010. Detection of a novel 823
astrovirus in brain tissue of mink suffering from shaking mink syndrome by use of viral 824
metagenomics. J Clin Microbiol 48:4392-6. 825
69. Toffan A, Jonassen CM, De Battisti C, Schiavon E, Kofstad T, Capua I, Cattoli G. 2009. 826
Genetic characterization of a new astrovirus detected in dogs suffering from diarrhoea. Vet 827
Microbiol 139:147-52. 828
70. Rott O, Kroger M, Muller H, Hobom G. 1988. The genome of budgerigar fledgling disease 829
virus, an avian polyomavirus. Virology 165:74-86. 830
71. Guerin JL, Gelfi J, Dubois L, Vuillaume A, Boucraut-Baralon C, Pingret JL. 2000. A novel 831
polyomavirus (goose hemorrhagic polyomavirus) is the agent of hemorrhagic nephritis 832
enteritis of geese. J Virol 74:4523-9. 833
72. Circella E, Caroli A, Marino M, Legretto M, Pugliese N, Bozzo G, Cocciolo G, Dibari D, 834
Camarda A. 2017. Polyomavirus infection in Gouldian finches (Erythrura gouldiae) and 835
other pet birds of the family Estrildidae. J Comp Pathol 156:436-439. 836
73. Johne R, Wittig W, Fernandez-de-Luco D, Hofle U, Muller H. 2006. Characterization of 837
two novel polyomaviruses of birds by using multiply primed rolling-circle amplification of 838
their genomes. J Virol 80:3523-31. 839
74. Bennett MD, Gillett A. 2014. Butcherbird polyomavirus isolated from a grey butcherbird 840
(Cracticus torquatus) in Queensland, Australia. Vet Microbiol 168:302-11. 841
75. Halami MY, Dorrestein GM, Couteel P, Heckel G, Muller H, Johne R. 2010. Whole-842
genome characterization of a novel polyomavirus detected in fatally diseased canary birds. 843
J Gen Virol 91:3016-22. 844
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
38
76. Li Q, Niu K, Sun H, Xia Y, Sun S, Li J, Wang F, Feng Y, Peng X, Zhu L, Fan X, Qin Y, 845
Ding J, Jiang H, Xu G. 2019. Complete genome sequence of an avian polyomavirus strain 846
first isolated from a pigeon in China. Microbiol Resour Announc 8: e01490-18. 847
77. Goto Y, Yaegashi G, Kumagai Y, Ogasawara F, Goto M, Mase M. 2019. Detection of 848
avian encephalomyelitis virus in chickens in Japan using RT-PCR. J Vet Med Sci 81:103-849
106. 850
78. Yugo DM, Hauck R, Shivaprasad HL, Meng XJ. 2016. Hepatitis virus infections in 851
poultry. Avian Dis 60:576-88. 852
79. Zylberberg M, Van Hemert C, Dumbacher JP, Handel CM, Tihan T, DeRisi JL. 2016. 853
Novel picornavirus associated with avian keratin disorder in Alaskan birds. MBio 7: 854
e00874-16. 855
80. Zylberberg M, Van Hemert C, Handel CM, DeRisi JL. 2018. Avian keratin disorder of 856
Alaska black-capped chickadees is associated with Poecivirus infection. Virol J 15:100. 857
81. Chan JF, To KK, Chen H, Yuen KY. 2015. Cross-species transmission and emergence of 858
novel viruses from birds. Curr Opin Virol 10:63-9. 859
82. Zhao QY, Wang Y, Kong YM, Luo D, Li X, Hao P. 2011. Optimizing de novo 860
transcriptome assembly from short-read RNA-Seq data: a comparative study. BMC 861
Bioinformatics 12 Suppl 14:S2. 862
83. Buchfink B, Xie C, Huson DH. 2015. Fast and sensitive protein alignment using 863
DIAMOND. Nat Methods 12:59-60. 864
84. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper 865
A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A. 2012. Geneious 866
Basic: an integrated and extendable desktop software platform for the organization and 867
analysis of sequence data. Bioinformatics 28:1647-9. 868
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
39
85. Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He 869
J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, 870
Yamashita RA, Zhang D, Zheng C, Bryant SH. 2015. CDD: NCBI's conserved domain 871
database. Nucleic Acids Res 43:D222-6. 872
86. Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: 873
improvements in performance and usability. Mol Biol Evol 30:772-80. 874
87. Bushnell B. 2016. BBMap short-read aligner, and other bioinformatics tools 875
https://sourceforge.net/projects/bbmap/ accessed Jan 2019. 876
88. Rockett R, Barraclough KA, Isbel NM, Dudley KJ, Nissen MD, Sloots TP, Bialasiewicz S. 877
2015. Specific rolling circle amplification of low-copy human polyomaviruses BKV, 878
HPyV6, HPyV7, TSPyV, and STLPyV. J Virol Methods 215-216:17-21. 879
80. Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T. 2009. trimAl: a tool for automated 880
alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25:1972-3. 881
90. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New 882
algorithms and methods to estimate maximum-likelihood phylogenies: assessing the 883
performance of PhyML 3.0. Syst Biol 59:307-21. 884
885
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
40
Table 1. Details of the 9 RNA-seq libraries, the avian species, and associated disease syndrome 886
studied here. 887
Library Host Organ Reads (pair
reads)
Suspected Disease
RBL-L-2 Rainbow lorikeet Liver 26,555,569 Sporadic mortality events
RBL-L-3 Rainbow lorikeet Liver 25,509,669 CCS,sporadic mortality events
RBL-B-9 Rainbow lorikeet Brain 23,994,093 CCS, sporadic mortality events
RBL-B-46 Rainbow lorikeet Brain 24,868,929 CCS
Nowra-B-14 Australian magpie, Magpie
lark, Pied Currawong
Brain 25,598,183 Nowra BWBD outbreak
Nowra-L-15 Australian magpie, Magpie
lark, Pied Currawong
Liver 20,413,140 Nowra BWBD outbreak
BWBD-L-6 Australian magpie, Pied
currawong, Australian raven
Liver 7,725,034 Archived BWBD
BWBD-B-8 Australian magpie, Pied
currawong, Australian raven
Brain 25,779,043 Archived BWBD
BWBD-O-
13
Australian magpie, Pied
currawong, Australian raven
Heart,
Kidney
23,374,876 Archived BWBD
*CCS - Clenched Claw syndrome. BWBD - Black and white bird disease. 888
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
41
Figure Legends 889
890
Figure 1. (a) Photomicrograph of rainbow lorikeet hepatocellular intranuclear inclusion bodies 891
(closed arrowheads) and karyomegaly with a stellate chromatin pattern (open arrowhead). (b) 892
Paretic rainbow lorikeet with clenched claws. (c-e) Photomicrographs of lesions characteristic of 893
clench claw syndrome in rainbow lorikeets. Encephalitis within the central cerebellar white 894
matter (c) with perivascular lymphoplasmacytic infiltrates (black arrow), vascular intramural 895
mononuclear cell infiltrates (black arrowhead), gliosis, dilation of axonal chambers (*) and 896
degenerating nerve cell bodies (open arrowheads). Spinal ganglion (d) with multifocal 897
mononuclear cell infiltrates (*), swollen axons and dilated axonal chambers within a white 898
matter tract (white bar). A peripheral nerve (e) with perivascular mononuclear cell infiltration 899
(black arrow), and Wallerian degeneration illustrated by dilated axonal chambers (black 900
arrowhead) and a macrophage within an axonal chamber signifying a digestion chamber (open 901
arrowhead). 902
903
Figure 2. (a) A paretic, but alert, Australian magpie with black and white bird disease. (b-e) 904
Photomicrographs of lesions characteristic of black and white bird disease. Severe perivascular 905
pulmonary hemorrhage (*). Myocardium (c) with a mononuclear cell infiltrate (*) and myocyte 906
degeneration (black arrowhead) signified by hypereosinophilic myofibrils and a pyknotic and 907
peripheralized nucleus. Proventricular mononuclear cell infiltrates (d) within the lamina propria 908
(white *) and surrounding serosal blood vessels (black *). Acute hepatic necrosis (e*). 909
910
Figure 3. Phylogeny and characterization of a novel avian avulavirus identified from a rainbow 911
lorikeet with clenched claw disease. (a) A maximum likelihood phylogeny of the M gene (366 912
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
42
amino acids) of avian avulavirus plus representative members of the Paramyxoviridae, including 913
viruses from the genera Avulavirus, Rubulavirus, Ferlavirus, Henipavirus, Morbillivirus, 914
Aquaparamyxovirus and Respirovirus. The tree was midpoint rooted for clarity only. The scale 915
bar indicates the number of amino acid substitutions per site. Bootstrap values >70% are shown 916
for key nodes. (b) Characterization of the virulence determination site in the F gene by 917
comparison to representative NDV strains. The typical RRKKR cleavage site motif is 918
highlighted (red rectangle). 919
920
Figure 4. Characterization and phylogeny of the lorikeet chapparvovirus identified here. (b) 921
Schematic representation and read abundance of the genome of lorikeet chapparvovirus. 922
Maximum likelihood phylogenies of the (B) NS gene and (C) VP gene. The tree was midpoint 923
rooted for clarity only. The scale bar indicates the number of amino acid substitutions per site. 924
Bootstrap values >70% are shown for key nodes. 925
926
Figure 5. Genomic organization and phylogeny of the beak and feather disease virus (BFDV) 927
identified in this study. (a) BFDV circular genome, annotated with two genes - the capsid protein 928
(Cap) and the replicase-associated protein (replicase). (b) Sanger sequencing of each case 929
detected positive with BFDV. The ML phylogeny of the cap gene (596 nt) was inferred and the 930
BFDVs identified from the same case fell into the corresponding clade. Phylogenetic trees were 931
estimated based on (c) the Cap gene and (d) the Replicase protein. The tree was midpoint rooted 932
for clarity only. The scale bar indicates the number of amino acid substitutions per site. 933
Bootstrap values >70% are shown for key nodes. 934
935
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
43
Figure 6. Phylogeny of the novel lorikeet adenovirus identified in this study. ML phylogenies of 936
the (a) Hexon and (b) polymerase protein were inferred with representative members of the 937
Adenoviridae. The tree was midpoint rooted for clarity only. The scale bar indicates the number 938
of amino acid substitutions per site. Bootstrap values >70% are shown for key nodes. 939
940
Figure 7. Black and white bird disease picornavirus and lorikeet hepatovirus identified in this 941
study. (a) Schematic representation and read abundance of the black and white picornavirus 942
(upper left) and lorikeet hepatovirus (upper right). (b) ML phylogenetic tree of the polyprotein 943
(containing the RdRp gene) of both viruses. Bootstrap resampling (1,000 replications) was used 944
to assess node support where >70%. GenBank accession numbers are given in the taxa labels. 945
Trees were midpoint rooted for clarify. The scale bars shows the number of amino acid distance 946
in substitution per site. 947
948
Figure 8. Phylogenetic analysis of magpie astrovirus. ML phylogeny of the partial RdRp gene 949
(267 amino acids) of astroviruses was inferred along with representative members of the 950
Avastrovirus and Mamastrovirus genera. GenBank accession numbers are given in the taxa 951
labels. Trees were midpoint rooted for clarity only, and branch support was estimated using 952
1,000 bootstrap replicates, with those >70% shown at the major nodes. 953
954
Figure 9. Genome characterization and phylogenetic analysis of the black and white bird 955
polyomavirus identified in this study. (a) Schematic depiction and read abundance of the 956
butcherbird polyomavirus/magpie. ML phylogenetic trees of the (b) large antigen (blue) and (c) 957
VP1 (yellow) protein of the butcherbird polyomavirus/magpie. Bootstrap support (1,000 958
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
44
replicates) was used to assess node support where >70%. GenBank accession numbers are given 959
in the taxa labels. The scale bars show the numbers of substitutions per site. 960
961
Figure 10. Summary of cases with meta-transcriptomic pathogen identification and confirmation 962
through PCR assays. (a) Clenched claw syndrome. (b) Black and white bird disease. Blue: PCR 963
positive, yellow: negative, dark yellow: weak positive, NS: non-suppurative, CCS: Clenched 964
claw syndrome, BWBD: Black and white bird disease. 965
966
Supplementary Information 967
Table S1. Presentation and pathology of rainbow lorikeets with clench-claw syndrome. 968
Table S2. Presentation and pathology of passerines with myocardial degeneration and 969
myocarditis. 970
Table S3. PCR primers used to amplify viral sequences from bird tissues. 971
Table S4. Primers used to recover full length genome of the viruses identified. 972
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
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Figure 1 973
974
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
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Figure 2 975
976
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
47
Figure 3 977
978
979
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
48
Figure 4 980
981
982
983
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
49
Figure 5 984
985
986
2,014 bp
Replicase
Circovirus
Cyclovirus
A B
Capsid
Beak and feather disease virus
0.3
C D
YP 009506308 Chimp Cyclovirus
YP 610962 Starling circovirus
AKO84210 Fox circovirus
YP 803548 Gull circovirusNP 059530 Columbid circovirus
YP 009051962 Human circovirus
NP 047277 Beak and feather disease virus
YP 004152330 Goat Cyclovirus
NP 150370 Goose circovirus
YP 004152334 Chicken Cyclovirus
YP 007697653 Canine circovirus
Beak and feather disease virus/lorikeet
NP 065679 Porcine circovirus 1NP 937957 Porcine circovirus 2
YP 009389457 Giant panda circovirus
YP 009506324 Cyclovirus PK5034
YP 764456 Raven circovirus
YP 009315911 Porcine circovirus 3
YP 004152332 Bat Cyclovirus
YP 009134741 Zebra finch circovirus
NP 573443 Canary circovirus
YP 009091699 Swan circovirus
YP_009506322 Cyclovirus TN25
YP 803551 Finch circovirus
ACV69956 Duck circovirus
100
97
98
98
100
98
71
92
100
100
83
100
100
70
0.5
Beak and feather disease virus/lorikeet
NP 065678 Porcine circovirus 1
YP 009389456 Giant panda circovirus 1
NP 150368 Goose circovirus
YP 009051960 Human circovirus
YP 004152329 Goat Cyclovirus YP 009506305 Chicken Cyclovirus
YP 009506323 Cyclovirus PK5034
YP 803546 Gull circovirus
YP 803549 Finch circovirus
NP 047275 Beak and feather disease virus
ACV69955 Duck circovirus
YP 009506307 Chimp Cyclovirus
AKO84209 Fox circovirus
YP 610960 Starling circovirus
YP 007697652 Canine circovirus
YP 009091698 Swan circovirus
YP 764455 Raven circovirus
YP 009134739 Zebra finch circovirus
YP 009506321 Cyclovirus TN25
NP 573442 Canary circovirus
NP 059527 Columbid circovirus
NP 937956 Porcine circovirus 2
YP 004152331 Bat Cyclovirus
100
9876
84
90
77
90
93
100
84
88
96
0.02
Lorikeet 5604.1 Liver
Lorikeet 2989.1 Brain
Lorikeet 8856.2 Brain
Lorikeet 2989.1 Liver
Lorikeet 4771.2 Liver
Lorikeet 5789.1 Brain
Lorikeet 5604.1 Brain
78
100
100
96
100
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
50
Figure 6 987
988
989
PolA BHexon
0.3
YP 009047094.1 Pigeon adenovirus
YP 009051654.1 Lizard adenovirus
NP 046314.1 Bovine adenovirus
YP 009505686.1 Bottlenose dolphin adenovirus
YP 009508600.1 Psittacine aviadenovirus
YP 009252208.1 Penguin siadenovirus A
YP 009047155.1 Duck adenovirus
YP 004414800.1 Raptor adenovirus
YP 003933581.1 Turkey adenovirus
AP 000478.1 Turkey siadenovirus A
YP 009310429.1 Pigeon adenovirus
ADP30814.1 Skua adenovirus 1
YP 006383556.1 Goose adenovirus
YP 009389490.1 Squirrel adenovirus
NP 659515.1 Ovine adenovirus
YP 009373238.1 Deer mastadenovirus
NP 108658.1 Porcine adenovirus
YP 009414575.1 Odocoileus adenovirus
AAX51172.1 Avirulent turkey hemorrhagic enteritis virus
AP 000342.1 Murine mastadenovirus
ACW84422.1 Great tit adenovirus
YP 001552247.1 Snake adenovirus
AAL89791.1 Sturgeon chtadenovirus A
AAC64523.1 Turkey adenovirus
YP 009112716.1 Psittacine adenovirus 3
AAF86924.1 Frog adenovirus
92
99
100
100
100
100
88
100
93
100
95
99
100
98
77
100
75
98
100
100
100
82
100
94
81
82
100
98
100
Lorikeet adenovirus
0.3
YP 009505695.1 Bottlenose dolphin adenovirus
YP 009047163.1 Duck adenovirus 2
YP 009310437.1 Pigeon adenovirus
AP 000351.1 Murine mastadenovirus
ADP30822.1 Skua adenovirus
AAC64532.1 Turkey adenovirus 3
YP 004414808.1 Raptor adenovirus
YP 009508608.1 Psittacine aviadenovirus
YP 009389499.1 Squirrel adenovirus
YP 001552255.1 Snake adenovirus
YP 009373247.1 Deer mastadenovirus
AP 000486.1 Turkey siadenovirus
NP 046323.1 Bovine adenovirus
ALB78146.1 Penguin siadenovirus A
NP 659523.1 Ovine adenovirus
YP 009112724.1 Psittacine adenovirus
Lorikeet adenovirus
AAX51180.1 Avirulent turkey hemorrhagic enteritis virus
YP 009414582.1 Odocoileus adenovirus
YP 009051662.1 Lizard adenovirus
AAF86932.1 Frog adenovirus
YP 006383564.1 Goose adenovirus 4
CAD42235.1 Sturgeon ichtadenovirus
100
100
100
100
100
100
100
100
76
83100
100
10095
100
100
82
71
86
100
88
100
Fowl aviadenovirus
Fowl aviadenovirus
Bovine adenovirus
Bovine adenovirusDuck atadenovirus
Duck atadenovirus
Bat adenovirus Bat adenovirus
Primate adenovirus Primate adenovirus
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
51
Figure 7 990
991
992
993
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
52
Figure 8 994
995
996
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
53
Figure 9 997
998
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint
54
Figure 10 999
1000
1001
+ Positive+ weak postive
Negative
A
B
.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint