Identification of an avian polyomavirus associated with Adélie
penguins (Pygoscelis adeliae)
Varsani, A., Porzig, E. L., Jennings, S., Kraberger, S., Farkas, K., Julian, L., ... &
Ainley, D. G. (2015). Identification of an avian polyomavirus associated with Adélie
penguins (Pygoscelis adeliae). Journal of General Virology, 96, 851-857.
doi:10.1099/vir.0.000038
10.1099/vir.0.000038
Society for General Microbiology
Version of Record
http://cdss.library.oregonstate.edu/sa-termsofuse
Journal of General Virology (2015), 96, 851–857
Short
Communication
DOI 10.1099/vir.0.000038
Identification of an avian polyomavirus associated
with Adélie penguins (Pygoscelis adeliae)
Arvind Varsani,1,2,3 Elizabeth L. Porzig,4 Scott Jennings,5
Simona Kraberger,1 Kata Farkas,1 Laurel Julian,1 Melanie Massaro,6
Grant Ballard4 and David G. Ainley7
Correspondence
Arvind Varsani
arvind.varsani@canterbury.ac.nz
1
School of Biological Sciences and Biomolecular Interaction Centre, University of Canterbury,
Private Bag 4800, Christchurch, 8140, New Zealand
2
Department of Plant Pathology and Emerging Pathogens Institute, University of Florida,
Gainesville, FL, USA
3
Electron Microscope Unit, Division of Medical Biochemistry, Department of Clinical Laboratory
Sciences, University of Cape Town, Observatory, 7700, South Africa
4
Point Blue Conservation Science, Petaluma, CA, USA
5
Department of Fisheries and Wildlife, Oregon Cooperative Fish and Wildlife Research Unit,
US Geological Survey, Oregon State University, Corvallis, OR, USA
6
School of Environmental Sciences, Charles Sturt University, Albury, NSW, Australia
7
HT Harvey and Associates, Los Gatos, CA, USA
Received 4 November 2014
Accepted 20 December 2014
Little is known about viruses associated with Antarctic animals, although they are probably
widespread. We recovered a novel polyomavirus from Adélie penguin (Pygoscelis adeliae) faecal
matter sampled in a subcolony at Cape Royds, Ross Island, Antarctica. The 4988 nt Adélie
penguin polyomavirus (AdPyV) has a typical polyomavirus genome organization with three ORFs
that encoded capsid proteins on the one strand and two non-structural protein-coding ORFs on
the complementary strand. The genome of AdPyV shared ~60 % pairwise identity with all
avipolyomaviruses. Maximum-likelihood phylogenetic analysis of the large T-antigen (T-Ag) amino
acid sequences showed that the T-Ag of AdPyV clustered with those of avipolyomaviruses,
sharing between 48 and 52 % identities. Only three viruses associated with Adélie penguins have
been identified at a genomic level, avian influenza virus subtype H11N2 from the Antarctic
Peninsula and, respectively, Pygoscelis adeliae papillomavirus and AdPyV from capes Crozier and
Royds on Ross Island.
There is extremely little information about pathogens and
parasites associated with Antarctic animals (Barbosa &
Palacios, 2009; Kerry & Riddle, 2009). Of all Antarctic
vertebrates, the penguins are possibly the best studied.
Antarctic penguins have been showing signs of disease of
unknown pathology in recent years, in particular unexplained incursions of feather loss in Adélie penguins
(Pygoscelis adeliae) and Emperor penguins (Aptenodytes
forsteri) in the Ross Sea (Ainley/Ballard Antarctic field team
observations at Cape Crozier for Adélie penguins for 2011/
2012 season and personal communication with Gerald L.
Kooyman for Emperor penguins observed in mid-1990s at
Cape Washington) and also as reported by Barbosa et al.
The GenBank/EMBL/DDBJ accession number for the genome
sequence of the Adélie penguin polyomavirus is KP033140.
One supplementary figure and one supplementary table are available
with the online Supplementary Material.
000038 G 2015 The Authors
Printed in Great Britain
(2014) in the Antarctic Peninsula. Similar feather-loss
patterns have been noted previously in African penguins
(Spheniscus demersus) at rehabilitation centres and in
Magellanic penguins (Spheniscus magellanicus) in colonies
in Argentina (Kane et al., 2010).
Despite the wealth of information on penguin biology and
ecology, there is limited information about their viruses.
Most of the early studies have relied on serological assays
for identifying putative paramyxoviruses, orthomyxoviruses, flavirviruses and birnaviruses in wild penguin
populations (Alexander et al., 1989; Austin & Webster,
1993; Gardner et al., 1997; Miller et al., 2010; Morgan &
Westbury, 1981; 1988; Morgan et al., 1981, 1985; Smith
et al., 2008; Thomazelli et al., 2010) and herpesviruses and
togaviruses in captive individuals (Kincaid et al., 1988;
Tuttle et al., 2005). A handful of recent studies have identified some of the viruses (avipoxviruses, Newcastle disease
851
A. Varsani and others
viruses, adenovirus, avian influenza virus and papillomavirus) at a molecular level (Carulei et al., 2009; Hurt et al.,
2014; Kane et al., 2012; Lee et al., 2014; Thomazelli et al.,
2010; Varsani et al., 2014).
Our team recently identified a novel papillomavirus associated with Adélie penguins breeding at Cape Crozier, Ross
Island, Antarctica (Varsani et al., 2014). Here, we report
investigations at Cape Royds, a much smaller colony
(~3000 vs 280 000 pairs) on the opposite coast of the island
(Lyver et al., 2014), where conditions are much more
severe (Dugger et al., 2014). As part of an ongoing diet
study in a subcolony at Cape Royds, we had access to faecal
matter from which we could identify prey hard parts as
well as viral pathogens. We placed a 1 m2 tray on the ground
within an area where Adélie penguins nest. The tray,
constructed of a hardwood frame holding a densely meshed
(mesh ~0.5 mm), stainless steel hardware cloth, was placed
before breeding commenced to passively collect faecal material
over the 2012/2013 breeding season. Adélie penguins build
their nests over the tray and defecate onto it through the
course of the ~3 month breeding season. Furthermore, the
penguins in their subcolonies nest at high density and
thereby protect their nests from predators and competitors.
Thus, faecal samples from the tray may originate from
multiple penguin territories, but they are exclusively from
Adélie penguins. We collected faeces from the tray at the end
of the season (late January) once the nests were unoccupied.
At the end of the 2012/2013 Adélie penguin breeding
season, ~50 ml of the faecal matter was transferred from
the tray into 50 ml sterile tubes using sterile wooden
spatulas. The faecal samples were stored and transferred to
the laboratory in a frozen state. Approximately half of the
faecal matter from the tray was resuspended and homogenized in 50 ml SM buffer [0.1 M NaCl, 50 mM Tris/HCl
(pH 7.4), 10 mM MgSO4] and processed as described
previously (Varsani et al., 2014). In brief, the supernatant
of the homogenate was recovered after centrifugation at
10 000 g for 20 min. This was then sequentially filtered
through 0.45 and 0.2 mm (pore size) syringe filters and 3 g
of PEG 8000 (Sigma) was mixed gently with 20 ml filtrate.
The 15 % (w/v) PEG 8000 filtrate solution was then
centrifuged for 20 min at 10 000 g and the pellet was
resuspended in 2 ml SM buffer at 4 uC overnight. A High
Pure Viral Nucleic Acid kit (Roche Diagnostics) was used
to purify viral DNA from 200 ml of the final resuspended
solution. We used rolling-circle amplification with a TempliPhi
kit (GE Healthcare) to enrich for circular DNA, and this was
sequenced on an Illumina HiSeq 2000 sequencer at the Beijing
Genomics Institute (Hong Kong). ABySS v.1.3.5 (Simpson
et al., 2009) was used to de novo assemble (kmer564) the
paired-end reads. The translation products of the assembled
contigs that were .500 nt were analysed for similarity to
known viral proteins using BLASTX (Altschul et al., 1990).
Translation products of a 4981 nt de novo-assembled contig
were found to have significant similarities to proteins
encoded by avian polyomaviruses.
852
Polyomaviruses are non-enveloped viruses (~40–45 nm in
diameter) with circular dsDNA genomes (~4600–5700 nt).
Their genomes are bidirectionally transcribed and encode
three structural proteins (VP1, VP2 and VP3) from one
strand, which assemble to form the icosahedral viral capsid,
and at least two non-structural proteins on the complementary strand (large and small tumour antigens: T-Ag
and t-Ag, respectively). T-Ag and t-Ag are transcribed early
in infection. In a few mammalian polyomaviruses and some
avian polyomaviruses, an additional ORF is expressed (VP4)
that is thought to play a role in capsid assembly and release
(Gerits & Moens, 2012).
Polyomaviruses are known to infect a wide range of
mammalian and avian hosts. Apart from four human
polyomaviruses that belong to the genus Wukipolyomavirus
(WU polyomavirus, KI polyomavirus, Human polyomavirus
6 and Human polyomavirus 7), all remaining mammalian
polyomaviruses belong to the genus Orthopolyomavirus. All
the avian-infecting polyomaviruses belong to the genus
Avipolyomavirus (Johne et al., 2011). Six avipolyomaviruses
species have been identified to date, namely: Avian polyomavirus (infecting various parrot species) (Müller & Nitschke,
1986), Goose hemorrhagic polyomavirus (infecting geese and
ducks) (Guerin et al., 2000), Finch polyomavirus (infecting
Pyrrhula pyrrhula griseiventris), Crow polyomavirus (infecting Corvus monedula) (Johne et al., 2006), Canary polyomavirus (infecting Serinus canaria) (Halami et al., 2010)
and Butcherbird polyomavirus (infecting Cracticus torquatus)
(Bennett & Gillett, 2014). Avian polyomaviruses are known
to cause inflammatory disease in birds; in some species the
acute clinical disease can result in high mortality (Guerin
et al., 2000; Johne & Müller, 2007; Krautwald et al., 1989)
and in some species chronic disease of skin and feathers
(Krautwald et al., 1989; Wittig et al., 2007).
Based on the 4981 nt de novo-assembled contig sequence
encoding a putative T-Ag, we designed a set of abutting
primers Pry-AP-F: 59-GCATCCAAGGCTGAGGTCCAAGCCG-39 and Pry-AP-F: 59-CCCGATGGGGATTTCCAGCAGC-39 to recover the complete viral genome using
KAPA Hifi Hotstart DNA polymerase (Kapa Biosystems)
and using the following protocol: initial denaturation at
95 uC for 3 min, followed by 25 cycles of 98 uC for 20 s,
60 uC for 15 s and 72 uC for 5 min and a final extension at
72 uC for 5 min. The resulting amplicon was resolved on a
0.7 % agarose gel (stained with SYBR Safe DNA gel stain),
excised, gel purified and cloned into the pJET1.2 plasmid
(ThermoFisher). The recombinant plasmid containing the
amplicon was Sanger-sequenced by primer walking at
Macrogen Inc. (South Korea). The Sanger-sequenced contigs
were assembled using DNAbaser v.4 (Heracle BioSoft
S.R.L.). All nucleotide and amino acid pairwise identities
were calculated using SDT v.1.2 (Muhire et al., 2014).
A simple analysis of the 4988 nt viral sequence revealed
that its genome architecture was similar to polyomaviruses
and that its genome shared ~60 % pairwise identity with
the genomes of other avipolyomaviruses (Fig. 1). We have
Journal of General Virology 96
Identification of avian polyomavirus in Adélie penguin
Full genome
(b)
APyV (AF241168) 61
FPyV (DQ192571) 62 64
CPyV (DQ192570) 61 60 61 60
BuPyV (KF360862) 60 60 61 61 69
(c)
87
GHPyV (AY140894)
CPyV (DQ192570)
BuPyV (KF360862)
CaPyV (GU345044)
APyV (AF241168)
GHPyV (AY140894) 62 60 61 61 65 66
AdPyV (KP033140)
T-Ag
FPyV (DQ192571)
VP1
93
80
73
(d)
67
T-Ag
aa
nt
44 47 50 48 48 52
54 48 48 50 48
52 48 49 50
51 52 49
CaPyV (GU345044) 61 60 61
69 59
CPyV (DQ192570) 61 60 63 60
FPyV (DQ192571) 62 63
61
55
(g)
VP2
71 59 56 56 60
30 37 39 37 40 42
AdPyV (KP033140)
45 37 35 34 35
APyV (AF241168) 60
62 60 61 65
61 62 61
CaPyV (GU345044) 64 64 63
75 70
CPyV (DQ192570) 62 62 65 66
71
BuPyV (KF360862) 63 62 63 61 71
36 38 38 37
35 36 39
CaPyV (GU345044) 60 61 58
57 49
CPyV (DQ192570) 59 59 60 59
47
BuPyV (KF360862) 59 61 62 62 67
GHPyV (AY140894) 63 63 64 67 68 67
GHPyV (AY140894) 61 58 63 63 63 63
aa
33 40 39 37 38 42
AdPyV (KP033140)
42 39 37 37 38
APyV (AF241168) 61
FPyV (DQ192571) 66 62
CaPyV (GU345044) 62 63 59
38 39 40 39
30 36 36
57 45
CPyV (DQ192570) 62 60 62 60
47
BuPyV (KF360862) 62 65 63 62 69
GHPyV (AY140894) 61 58 65 62 63 64
GHPyV (AY140894)
BuPyV (KF360862)
CPyV (DQ192570)
CaPyV (GU345044)
FPyV (DQ192571)
APyV (AF241168)
FPyV (DQ192571) 64 62
AdPyV (KP033140)
GHPyV (AY140894)
BuPyV (KF360862)
CPyV (DQ192570)
CaPyV (GU345044)
FPyV (DQ192571)
APyV (AF241168)
AdPyV (KP033140)
FPyV (DQ192571) 62 68
VP3
nt
FPyV (DQ192571)
55 56 55 55 58 58
aa
APyV (AF241168)
nt
APyV (AF241168) 64
33
AdPyV (KP033140)
aa
40
GHPyV (AY140894)
BuPyV (KF360862)
CPyV (DQ192570)
(f)
VP1
AdPyV (KP033140)
47
64 55
CPyV (DQ192570) 60 61 60 60
BuPyV (KF360862) 59 60 60 59 70
GHPyV (AY140894) 62 60 56 59 64 67
(e)
nt
53
36 43 40 46
46 41 39
CaPyV (GU345044) 54 60 58
FPyV (DQ192571) 63 63
GHPyV (AY140894)
CPyV (DQ192570)
BuPyV (KF360862)
CaPyV (GU345044)
APyV (AF241168)
FPyV (DQ192571)
AdPyV (KP033140)
GHPyV (AY140894) 63 60 60 59 65 67
51 39 47 44 46
APyV (AF241168) 58
CaPyV (GU345044)
BuPyV (KF360862) 60 62 61 60 71
39 42 41 35 36 41
AdPyV (KP033140)
FPyV (DQ192571)
APyV (AF241168) 60
APyV (AF241168)
AdPyV (KP033140)
60
aa
AdPyV (KP033140)
nt
t-Ag
GHPyV (AY140894)
VP3
CaPyV (GU345044) 60 60 60
CPyV (DQ192570)
AdPyV [KP033140]
4988 nt
100
Pairwise identity (%)
AdPyV (KP033140)
VP2
BuPyV (KF360862)
t-Ag
CaPyV (GU345044)
(a)
Fig. 1. (a) Genome organization of Adélie penguin polyomavirus (AdPyV). (b) Percentage pairwise identities of the full genomes
of the avipolyomaviruses. (c–g) Nucleotide (nt) and amino acid (aa) percentage pairwise identities of the avipolyomavirus
sequences of the large T-Antigen (T-Ag) (c), small T-Antigen (t-Ag) (d), VP1 (e), VP2 (f) and VP3 (g). APyV, Avian
polyomavirus; FPyV, Finch polyomavirus; CaPyV, Canary polyomavirus; CPyV, Crow polyomavirus; BuPyV, Butcherbird
polyomavirus; GHPyV, Goose hemorrhagic polyomavirus.
http://vir.sgmjournals.org
853
A. Varsani and others
(a)
T-Ag
(b)
VP1:VP2
65
62
100
98
ChPyV1 (JX520657)
100 PaVePyV2a (HQ385748)
100 PaVePyV2c (HQ385749)
OtsPyV2 (JX520658)
79 GaPyV (HQ385752)
BaPyV-B1130 (JQ958893)
100
BaPyV-A1055 (JQ958886)
MCPyV (EU375803)
100 100 BaPyV-B0454 (JQ958888)
PaVePyV1a (HQ385746)
100 PaVePyV1b (HQ385747)
100 ChPyV (FR692334)
Avipolyomavirus
NJPyV (KF954417)
BaPyV-C1109 (JQ958889)
100
100
VmPyV1 (AB767298)
BaPyV-R104 (JQ958887)
92
100
Wukipolyomavirus
CarPyV (JX520659)
100 PiRuPyV (JX159984)
100
OtPyV1 (JX520664)
98
100 PaVePyV3 (JX159980)
Orthopolyomavirus
EiPyV1 (JX520660)
OraPyV2 (FN356901)
RaPyV (JQ178241)
65 PaVePyV4 (JX159981)
100
RaPyV (JQ178241)
100 ChPyV (FR692334)
BaPyV-R104 (JQ958887)
NJPyV (KF954417)
100
100
62
BaPyV-C1109 (JQ958889)
PiRuPyV (JX159984)
98
85
100 100 VmPyV1 (AB767298)
OtPyV1 (JX520664)
73
80
ChPyV1 (JX520657)
CarPyV (JX520659)
EiPyV1 (JX520660)
OtsPyV2 (JX520658)
100
86
BaPyV-B1130 (JQ958893)
100 PaVePyV1a (HQ385746)
73 100 BaPyV-A1055 (JQ958886)
100 PaVePyV1b (HQ385747)
MCPyV (HM011542)
BaPyV-B0454 (JQ958888)
GaPyV (HQ385752)
HPyV12 (JX308829)
96
97
100 OraPyV2 (FN356901)
99 PaVePyV2a (HQ385748)
100 PaVePyV4 (JX159981)
100 PaVePyV2c (HQ385749)
100
AtPyV1 (JX159987)
PaVePyV3 (JX159980)
100
100
OraPyV1 (FN356900)
HaPyV (X02449)
MPyV (J02288)
100 TSPyV (GU989205)
HPyV12 (JX308829)
OraPyV1 (FN356900)
100
97
MPyV (J02288)
TSPyV (GU989205)
100
HaPyV (X02449)
AtPyV1 (JX159987)
PaScPyV2 (JX159983)
PaScPyV2 (JX159983)
100
100 HPyV9 (HQ696595)
PaVePyV5 (JX159982)
100
100 HPyV-915 (FR823284)
100 HPyV-915 (FR823284)
100
HPyV9 (HQ696595)
PaVePyV5 (JX159982)
100 MafPyV1 (JX159986)
98 MafPyV1 (JX159986)
100 LPyV (K02562)
100 LPyV (K02562)
100 YBPyV-1 (AB767294)
100 YBPyV1 (AB767294)
HPyV6 (HM011560)
100
100 SA12 (AY614708)
100
97
HPyV7 (HM011566)
YBPyV-2 (AB767295)
75
91
STLPyV
(JX463183)
BKPyV (V01108)
100
100
MWPyV
(JQ898291)
JCPyV (J02226)
100
MXPyV (JX259273)
SV40 (J02400)
SeOPyV (KM282376)
88 HPyV10 (JX262162)
88
DPyV (KC594077)
AdPyV (KP033140)
APyV (AF241168)
BPyV (D13942)
100
65
FPyV (DQ192571)
SLPyV (GQ331138)
100 SSPyV (JX159989)
CaPyV (GU345044)
100
SqPyV (AM748741)
GHPyV (AY140894)
CePyV1 (JX159988)
BuPyV (KF360862)
100
100
BatPyV (FJ188392)
CPyV (DQ192570)
98
69
EqPyV (JQ412134)
MasPyV (AB588640)
MPtV (M55904)
AEPyV (KF147833)
78
AEPyV (KF147833)
97 BaPyV-R266 (JQ958891)
100
KIPyV (EF127906)
PtPyV (JX520662)
100
100
WUPyV (EF444549)
BaPyV-A504 (JQ958890)
100
81
99 BaPyV-R266 (JQ958891)
BaPyV-AT7 (JQ958892)
100
PtPyV (JX520662)
SLPyV (GQ331138)
95
100
BaPyV-A504 (JQ958890)
BPyV (D13942)
99
BaPyV-AT7 (JQ958892)
MiPyV (JX520661)
MiPyV (JX520661)
BatPyV (FJ188392)
96
68
EqPyV (JQ412134)
MasPyV (AB588640)
100 97
MPtV (M55904)
SeOPyV (KM282376)
96
SV40 (J02400)
CePyV1 (JX159988)
97 100
100
100 SqPyV (AM748741)
JCPyV (J02226)
SSPyV J(X159989)
100 BKPyV (V01108)
DPyV (KC594077)
100 SA12 (AY614708)
STLPyV (JX463183)
100 YBPyV2 (AB767295)
100
BPCP-1 (EU069819)
MWPyV (JQ898291)
100
100
BPCV-2 (EU277647)
HPyV10 (JX262162)
100
AdPyV (KP033140)
MXPyV (JX259273)
99
APyV
(AF241168)
HPyV6 (HM011560)
98
100
FPyV (DQ192571)
HPyV7 (HM01156)
100
61
CaPyV (GU345044)
KIPyV (EF127906)
100
GHPyV (AY140894)
WUPyV (EF444549)
100 100
CPyV (DQ192570)
BuPyV (KF360862)
JEECV (AB543063)
0.5 amino acid substitutions per site
0.5 amino acid substitutions per site
Fig. 2. ML phylogenetic trees of the large T-Antigen (T-Ag) (a) and VP1 : VP2 concatenated amino acid sequences (b). The T-Ag
ML phylogenetic tree was rooted with papillomavirus E1 sequences and the VP1 : VP2 tree was mid-point rooted. Branches with
,60 % bootstrap support have been collapsed. See Table S1, available in the online Supplementary Material, for full names.
854
Journal of General Virology 96
Identification of avian polyomavirus in Adélie penguin
tentatively named this viral genome Adélie penguin polyomavirus (AdPyV). The T-Ag (1983 nt), t-Ag (537 nt), VP1
(1083 nt), VP2 (1050 nt) and VP3 (705 nt) share ~54–
64 % pairwise nucleotide identity and ~30–58 % amino
acid identity with homologous ORFs encoded by avipolyomaviruses (Fig. 1).
included the T-Ag-like sequences encoded by Bandicoot
papillomatosis carcinomatosis virus 1 and 2 (BPCV-1 and 2, respectively) (Woolford et al., 2007) and Japanese eel
endothelial cells-infecting virus (Mizutani et al., 2011) and
selected avian papillomavirus E1 sequences (to root the
tree). These datasets were aligned using MUSCLE (Edgar,
2004). The alignments were used to infer maximumlikelihood (ML) phylogenetic trees using PhyML 3.0
(Guindon et al., 2010); 1000 bootstrap replicates using
the RtRev+G+I+F model of substitution (for both
aligned datasets) chose the best-fit model using ProTest
(Darriba et al., 2011). The T-Ag ML phylogenetic tree was
rooted with the papillomavirus E1 sequences, whereas the
VP1 : VP2 one was mid-point rooted. All branches with
,60 % bootstrap support were collapsed using Mesquite
v.2.7 (http://mesquiteproject.org/). The ML phylogenetic
analysis of the T-Ag clearly showed that the AdPyV clusters
with other avipolyomaviruses (Fig. 2). As noted previously
(Woolford et al., 2007), the T-Ag-like protein sequence
encoded by BPCV-1 was phylogenetically basal to the
avipolyomaviruses (Fig. 2). Within the protein sequences
of the T-Ag, we identified the polyomavirus conserved
region 1 (CR1; LIRLL) and hexapeptide (HPDKGG),
retinoblastoma protein binding motif (pRB; LYCEE), a
putative nuclear localization signal (NLS; PPKSQP), a
We also undertook posterior mapping of paired-end reads
from Illumina sequencing of the 4988 nt AdPyV genome
using Bowtie 2 v.2.2.3 (Langmead & Salzberg, 2012). The reads
mapped across the whole genome with .250-fold coverage
(Fig. S1). The de novo-assembled contig was 99.9 % similar to
the PCR–recovered and cloned genome that was Sangersequenced.
Given that there is some movement of individual penguins
between colonies on Ross Island (Dugger et al., 2010), we
screened the faecal sample from Cape Crozier reported by
Varsani et al. (2014) and also checked the sequence reads
resulting from this sample for AdPyV but found no
evidence of it in the sample. Similarly, we did not find any
evidence of the P. adeliae papillomavirus (Varsani et al.,
2014) in the Cape Royds sample.
We assembled two datasets of amino acid sequences, the TAgs and concatenated VP1 : VP2 of representative wuki-,
ortho- and avipolyomaviruses. In the T-Ag dataset, we also
(a)
(b)
(c)
CR1
DnaJ
pRB
(d)
(e)
Bits
4
2
0
AdPyV (KP033140)
APyV (AF241168)
FPyV (DQ192571)
CaPyV (GU345044)
GHPyV (AY140894)
CPyV (DQ192570)
BuPyV (KF360862)
BCPV-1 (EU069819)
BCPV-2 (EU277647)
NLS
Zinc finger motif
(f)
Bits
4
2
0
AdPyV (KP033140)
APyV (AF241168)
FPyV (DQ192571)
CaPyV (GU345044)
GHPyV (AY140894)
CPyV (DQ192570)
BuPyV (KF360862)
BCPV-1 (EU069819)
BCPV-2 (EU277647)
ATPase motif
ATPase motif
Fig. 3. Conserved CR1 (a), DnaJ (b), pRB (c), NLS (d), zinc-finger (e) and ATPase (f) motifs identified in the T-Ag of the
avipolyomaviruses. The sequence log illustration is shown above the motifs to highlight the conserved motif sequences. Gaps in
the alignments are denoted by dashes. See Table S1 for abbreviations.
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A. Varsani and others
zinc-finger motif (CQDCKQQKANTPFGKLKSKWMGGHCDDH) and ATPase motifs (GPVNSKT and GSVPVNLE),
which are all relatively conserved among all polyomaviruses
(Ehlers & Moens, 2014) and BCPVs (Woolford et al., 2007)
(Fig. 3). The consensus CXCX2C protein phosphate 2A
binding domain that is found in almost all mammalian tAgs was not found to be encoded by AdPyV. In the
VP1 : VP2 concatenated amino acid sequence ML phylogenetic tree, the avipolyomavirus sequences are nested
within those of the orthopolyomaviruses; VP1 : VP2 of the
wukipolyomaviruses are the most divergent; hence the
proposal by Johne et al. (2011) to establish a new genus for
these viruses. Nonetheless, the VP1 of AdPyV shared the
highest pairwise amino acid identity (58 %) with that of
butcherbird polyomavirus and goose hemorrhagic polyomavirus (GHPyV), whereas the VP2 was most closely
related to GHPyV (42 %) (Fig. 1).
Based on the ML phyologenetic analysis of the T-Ag coupled
with pairwise identities of the ORFs, AdPyV represents a
new species of avipolyomaviruses. As highlighted by Varsani
et al. (2014) for the P. adeliae papillomavirus, we are confident that AdPyV is associated with Adélie penguins and
that the chances of contamination by the South Polar skua
(Stercorarius maccormicki; preys on Adélie penguin eggs and
chicks), the only other bird in the subcolony, are extremely
slim; the skua do not nest within the faecal trays, are chased
away by the penguins, are outnumbered by the penguins by
.200 : 1, and in fact one skua pair defends ~1000 nests, and
the tray, within its territory. This report expands our current
knowledge of the host range of avipolyomaviruses and to
the best of our knowledge this is the first report of a
polyomavirus associated with penguins. The identification of this novel avipolyomavirus will enable us to design
specific PCR probes to determine its prevalence and host
range, given that this information is unknown; furthermore,
complete genomes can be recovered from samples that test
positive to determine viral diversity.
To the best of our knowledge, this is one of the five
genomes of viruses associated with Antarctic animals to be
characterized to date; previously identified viral genomes
include those of a P. adeliae papillomavirus and an avian
influenza virus subtype H11N2 from Adélie penguins
(Hurt et al., 2014; Varsani et al., 2014) and adenoviruses
from South Polar skuas and chinstrap penguins (Pygoscelis
antarctica) (Lee et al., 2014; Park et al., 2012). This highlights
the poor knowledge of viruses associated with Antarctic
animals. It is worth noting that this poor knowledge of
viruses in Antarctica goes beyond just Antarctic animals.
Only a handful of other complete viral genomes have been
characterized from soil sampled in the McMurdo Dry
Valleys (Meiring et al., 2012; Swanson et al., 2012) and fresh
water ponds (López-Bueno et al., 2009; Zawar-Reza et al.,
2014).
The emergence and re-emergence of viruses is a major
problem that is being driven by habitat and climate change,
which can cause changes in the movement and behaviour
856
of animals and can indirectly result in increased contact
with pathogen reservoirs. With this in mind, it is essential
to increase our knowledge of viruses circulating in the
Antarctic in order to identify pathogens that may pose
significant threats to Antarctic animals.
Acknowledgements
The field work for this study was supported by the US National
Science Foundation (NSF) under grant ANT-0944411, with logistics
supplied by the US Antarctic Program. The field work was conducted
under Animal Care and Use Permit #4130 through Oregon State
University, Corvallis, OR, USA, and Antarctic Conservation Act
Permit #2006-010 from NSF.
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