RESEARCH ARTICLE

Scavengers as Prospective Sentinels of Viral Diversity: the

Snowy Sheathbill Virome as a Potential Tool for Monitoring
Virus Circulation, Lessons from Two Antarctic Expeditions
Gabriel Zamora,a Sebastian Aguilar Pierlé,b Johana Loncopan,a Loreto Araos,a Francisco Verdugo,a Cecilia Rojas-Fuentes,c,f
Lucas Krüger,d,e Aldo Gaggero,c Gonzalo P. Barrigaa

a Laboratory of Emerging Viruses, Virology Program, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile
b Inorevia, Pepinière Paris Santé Cochin, Paris, France
c Laboratory of Environmental Virology, Virology Program, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile
d Instituto Antártico Chileno, Punta Arenas, Chile
e Fundación Instituto de Biodiversidad de Ecosistemas Antárticos y Subantárticos, Las Palmeras, Ñuñoa, Santiago, Chile
Facultad de Ciencias de la Salud, Programa Magister en Ciencias Químico Biológicas, Universidad Bernardo O’Higgins, Santiago, Chile
f

ABSTRACT Antarctica is a unique environment due to its extreme meteorological and

geological conditions. In addition to this, its relative isolation from human influences
has kept it undisturbed. This renders our limited understanding of its fauna and its asso-
ciated microbial and viral communities a relevant knowledge gap to fill. This includes
members of the order Charadriiformes such as snowy sheathbills. They are opportunistic
predator/scavenger birds distributed on Antarctic and sub-Antarctic islands that are in
frequent contact with other bird and mammal species. This makes them an interesting
species for surveillance studies due to their high potential for the acquisition and trans-
port of viruses. In this study, we performed whole-virome and targeted viral surveillance
for coronaviruses, paramyxoviruses, and influenza viruses in snowy sheathbills from two
locations, the Antarctic Peninsula and South Shetland. Our results suggest the potential

Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.

role of this species as a sentinel for this region. We highlight the discovery of two human
viruses, a member of the genus Sapovirus GII and a gammaherpesvirus, and a virus previ-
ously described in marine mammals. Here, we provide insight into a complex ecological
picture. These data highlight the surveillance opportunities provided by Antarctic scav-
enger birds.
IMPORTANCE This article describes whole-virome and targeted viral surveillance for
coronaviruses, paramyxoviruses, and influenza viruses in snowy sheathbills from the
Antarctic Peninsula and South Shetland. Our results suggest an important role of
this species as a sentinel for this region. This species’ RNA virome showcased a diver-
sity of viruses likely tied to its interactions with assorted Antarctic fauna. We high-
light the discovery of two viruses of likely human origin, one with an intestinal
impact and another with oncogenic potential. Analysis of this data set detected a va-
riety of viruses tied to various sources (from crustaceans to nonhuman mammals), Editor David T. Pride, University of California,
depicting a complex viral landscape for this scavenger species. San Diego
Copyright © 2023 Zamora et al. This is an
KEYWORDS surveillance, emerging viruses, Antarctica, snowy sheathbill, zoonosis open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.

A ntarctica is populated by a diverse and unique fauna with its associated microor-
ganisms; however, our knowledge of the microbial and viral diversity on this conti-
nent, including pathogens with zoonotic potential, remains limited (1, 2). The region’s
Address correspondence to Gonzalo P. Barriga,
gonzalo.barriga@uchile.cl.
The authors declare no conflict of interest.
Received 19 August 2022
geographic isolation has been associated with viral paucity and probable endemic evo- Accepted 29 March 2023
lution (3). Currently, a small number of viral species have been described in Antarctic Published 25 May 2023
fauna, mostly through studies of penguin populations and a few screens of additional

May/June 2023 Volume 11 Issue 3 10.1128/spectrum.03302-22 1

Snowy Sheathbill as a Sentinel of Viral Diversity Microbiology Spectrum

TABLE 1 Pools positive by pancoronavirus and panparamyxovirus primers per expeditiona

No. of samples

ECA57 ECA58

Snowy Positive Snowy Positive

Primer type Environmental sheathbill (total) Environmental sheathbill (total)
Pan-CoV 6 2 8 (25) 3 3 6 (21)
Viral isolated 1 0 1 (25) 1 5 6 (21)

Pan-PMX 5 1 6 (25) 6 6 12 (21)

AV-18 5 1 6 (25) 5 6 11 (21)
AV-19 0 0 0 (25) 2 0 2 (21)
Viral isolated 0 0 0 (25) 0 3 3 (21)
aNotethat the samples were analyzed by RT-qPCR against influenza virus and by RT-PCR against NDV. Avulavirus
17 was also tested for; however, no positive samples were detected. AV-18, avulavirus 18; CoV, coronavirus; PMX,
paramyxovirus.

bird species (2–5). Little is known about Antarctic fauna members of the order
Charadriiformes and the viruses that they carry. Birds of this order, which includes po-
lar skuas (Catharacta spp.), Antarctic terns (Sterna vittata), and snowy sheathbills
(Chionis albus), breed on the Antarctic Peninsula during the Antarctic summer and
then migrate northward during the nonbreeding season (6). This implies the move-
ment of the microorganisms (and potential pathogens) that they harbor between
Antarctica and South America (7). Such species are an interesting prospect to provide
us with a detailed picture of the viral diversity in Antarctica. Snowy sheathbills are
opportunistic predator/scavenger birds distributed on Antarctic and sub-Antarctic
islands (2). This species breeds near penguin colonies (6). The isolation and extreme cli-
mate of its breeding grounds made snowy sheathbills flexible enough to take advant-
age of various food sources. These include sea algae, crustaceans, and placentas and
carcasses of marine mammals (2). Their frequent contact with different bird and mam-
mal species, in addition to their foraging/scavenging behavior, makes them an appeal-
ing species for surveillance studies due to their high potential for the acquisition and
transport of viruses (8, 9).
In this study, we performed targeted viral surveillance for coronavirus, paramyxovirus

Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.

(PMX), and influenza virus complemented by RNA virome characterization in snowy
sheathbills from the Antarctic Peninsula and South Shetland. Our results suggest a role
of this species as a potential sentinel for this region. This species’ RNA virome showcased
a diversity of viruses likely tied to Antarctic fauna. We highlight the discovery of two
viruses of likely human origin, a member of the genus Sapovirus GII and a gammaherpes-
virus (Epstein-Barr virus), and additional viruses from marine mammalians. The path by
which these viruses were introduced into Antarctica remains to be resolved. These data
highlight the surveillance opportunities provided by Antarctic scavenger birds.

RESULTS
Surveillance for avian viruses previously detected in Antarctica. First, we attempted
to gauge the circulation of economically/ecologically relevant viruses in our sampled
population. For this, we targeted previously reported avian viruses in Antarctica. More
specifically, we targeted viruses with known impacts on poultry or wild fauna, includ-
ing coronaviruses, paramyxoviruses, and influenza viruses (Table 1). Eight samples posi-
tive for coronavirus were identified during Chilean Antarctic Expedition 57 in 2020
(ECA57/2020) on Nelson Island, and 6 positive samples were identified during ECA58/
2022 around Isabel Riquelme Islet. The samples were positive using degenerate pri-
mers. It should be noted that all samples were positive for the Antartic1 replicase gene.
Furthermore, one coronavirus isolate was partially sequenced for each year (Table 1). The
samples clustered with a deltacoronavirus sequence from a gentoo penguin (Fig. 1). Next,
eight samples positive for paramyxovirus were detected during ECA57 (Nelson Island)
(Fig. 2), and all samples were positive for avulavirus 18 by reverse transcription-PCR (RT-PCR)

May/June 2023 Volume 11 Issue 3 10.1128/spectrum.03302-22 2

Snowy Sheathbill as a Sentinel of Viral Diversity Microbiology Spectrum

A KX588673.1

KX588674.1
B JN788791.1_Avian_coronavirus_Anas_acuta_2009
81
KU321641.1
JN788797.1_Avian_coronavirus_Phalacrocorax_carbo_2009
82
LC364342.1 Gammacoronavirus
99
100
LC364344.1e
100 JN788849.1_Avian_coronavirus_Anas_americana_2010
LC364343.1
82

JQ065044.1
JN788809.1_Avian_coronavirus_2009
NC_011549.1 76
NC_016996.1 JN788818.1_Avian_coronavirus_Phalacrocorax_carbo_2009
MG764094.1 88
MG764125.1 JN788814.1_Avian_coronavirus_2009
96
MG764133.1

JN788854.1_Avian_coronavirus_Phalacrocorax_carbo_2010
94
MG764136.1 Deltacoronavirus 93
95 99
MG764139.1
100 JN788855.1_Avian_coronavirus_2010
MG764146.1

MG764156.1
JN788846.1_Avian_coronavirus_Phalacrocorax_carbo_2010
JN788791.1 83

75
JN788797.1
JN788819.1_Avian_coronavirus_Phalacrocorax_carbo_2009
99
JN788849.1
82

JN788809.1 JN788816.1_Avian_coronavirus_Phalacrocorax_carbo_2009
76
JN788818.1
88
JN788814.1 JN788850.1_Avian_coronavirus_Anas_penelope_2010
JN788854.1
93
JN788805.1_Avian_coronavirus_2009
JN788855.1

100
JN788846.1

7 1JN788844.1_Avian_coronavirus_Phalacrocorax_2010
83

JN788819.1
70 100
JN788816.1
JN788845.1_Avian_coronavirus_Phalacrocorax_carbo_2010
JN788850.1
100

JN788805.1
JN788853.1_Avian_coronavirus_isolate_2009
7 1JN788844.1
83
100
JN788845.1
100
Gammacoronavirus JN788803.1_Avian_coronavirus_2009
JN788853.1 99
83
JN788803.1 JN788852.1_Avian_coronavirus_isolate_Phalacrocorax_carbo_2009
99
JN788852.1
70
70
JN788856.1
JN788856.1_Avian_coronavirus_2009
100 100
JN788857.1

87 JN788857.1_Avian_coronavirus_2009
JN788813.1
100 87
JN788815.1
JN788813.1_Avian_coronavirus_2009
94
JN788793.1
100
60 JN788806.1
85
JN788815.1_Avian_coronavirus_iPhalacrocorax_carbo_2009
JN788817.1
94
JN788841.1 JN788793.1_Avian_coronavirus_Phalacrocorax_carbo_2009
99 99
JN788865.1

Antartic_coronavirus_penguin_2022_1 JN788806.1_Avian_coronavirus_Phalacrocorax_carbo_2009
100
Antartic_coronavirus_penguin_2022_2
85
99
MT025058.1_MAG
JN788817.1_Avian_coronavirus_2009
Deltacoronavirus
NC_016994.1
JN788841.1_Avian_coronavirus_J1463_Ardea_cinerea_2009
JN788880.1
99
JN788885.1
JN788865.1_Avian_coronavirus__Ardea_cinerea_2009
JN788883.1
85

JN788881.1
Antartic_coronavirus_penguin_2022_1
JN788882.1 100
93
JN788884.1 Antartic_coronavirus_penguin_2022_2
93
JN788887.1 99
94 MT025058.1_MAG__Gentoo_penguin_Antartic1_2020

Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.

JN788888.1

JN788886.1

MG764117.1
NC_016994.1_coronavirus_Night-heron_2012

MG764121.1
79
JN788880.1_Avian_coronavirus_Ardeola_bacchus_2008
MG764120.1
75
MG764118.1
JN788885.1_Avian_coronavirus_Ardeola_bacchus_2008
MG764119.1 Gammacoronavirus
MG764157.1
JN788883.1_Avian_coronavirus_Ardeola_bacchus_2008
100 85
KT222536.1

72
JN788788.1 JN788881.1_Avian_coronavirus_Ardeola_bacchus_2008
JN788837.1

91 MG764116.1 JN788882.1_Avian_coronavirus_Ardeola_bacchus_2008

95 JN788796.1 93
100
JN788867.1
JN788884.1_Avian_coronavirus_2008
94
100

NC_016995.1
93
JN788887.1_Avian_coronavirus_2008
JN788838.1

JQ065042.2 94
100 JN788888.1_Avian_coronavirus_Ardeola_bacchus_2008
JQ065043.2
100

100 NC_016992.1
Deltacoronavirus JN788886.1_Avian_coonavirus_Ardeola_bacchus_2008
100 NC_011550.1

NC_016993.1

FIG 1 Phylogenetic tree including members of the gamma- and deltacoronavirus genera. Sequence comparison was performed using 78 sequences from
the gamma- and deltacoronavirus genera; the coronavirus isolated in our study (shown in green) appears to be closely related to a deltacoronavirus
sequenced in 2009 and clusters with a sample of a partially sequenced virus from a gentoo penguin (shown in red). Phylogenetic distances were
calculated using the GTR method. A bootstrap analysis with 1,000 replicates was applied, and the numbers indicate bootstrap values. (A) Complete
phylogenetic tree; (B) zoomed-in view of the Antarctic sequence clade (green).

(Table 1). Twelve samples were positive using panparamyxovirus primers during ECA58
(Isabel Riquelme Islet) (Fig. 2). Samples from this expedition were positive for avulaviruses 18
and 19 by RT-PCR (Table 1). In total, we identified 5 environmental samples positive for avu-
lavirus 18 and 6 snowy sheathbill samples positive for avulavirus 19 (total of 11 samples).
Only one sample was negative using all tested avulavirus primer pairs (avulavirus 17, 18,

May/June 2023 Volume 11 Issue 3 10.1128/spectrum.03302-22 3

Snowy Sheathbill as a Sentinel of Viral Diversity Microbiology Spectrum

FIG 2 Locations of sampling sites for snowy sheathbills in the Antarctic Peninsula’s Harmony Point, Nelson Island, and Duroch Islands. Shown are the

Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.

positions of sampling sites at the Southern Hemisphere scale (a) and the Antarctic Peninsula scale (b), the location of Harmony Point on Nelson Island (c),
and the location for Kopaitic Island/Isabel Riquelme Islet on the Duroch Islands (d). Samples from locations in panel c were collected in 2020, and samples
from locations in panel d were collected in 2022.

or 19 or Newcastle disease virus [NDV]), suggesting a probable novel sequence (Table 1).
Two environmental samples from ECA58/2022 were coinfected with avulaviruses 18 and 19
(Table 1).
To show influenza virus circulation, we screened samples from 14 snowy sheathbills
from 2 locations (Kopaitic Island and Isabel Riquelme Islet) (Fig. 2) for the presence of
antibodies. We detected antibodies against the nucleoprotein (NP) in 2 snowy sheath-
bill samples (Fig. 3).
Diversity and composition of the snowy sheathbill virome. In order to obtain a
fuller and more detailed picture of the viral diversity harbored by this endemic bird spe-
cies, we characterized the snowy sheathbill viromes collected from two Antarctic loca-
tions (Fig. 4; see Fig. S1 in the supplemental material). Sequencing yielded 18,259,501
usable reads, of which 1,817,608 were of a viral origin. This allowed us to assemble 971
viral contigs of between 8,500 and 200 nucleotides (nt), with 142 hits associated with
known viruses. The snowy sheathbill’s scavenger behavior is best portrayed by consider-
able viral diversity (Fig. 5 and Fig. S1); the library contained reads matching avian,
human, nonhuman mammalian, shrimp, insect, plant, and bacterial viruses (Fig. 5). Most
matches were plausible considering the snowy sheathbill’s lifestyle, and they are dis-
played at a viral family resolution (Fig. S1) and then up to the species level (Fig. 4), with
further identity information being provided by BLAST analysis (see Table S2 in the sup-
plemental material).

May/June 2023 Volume 11 Issue 3 10.1128/spectrum.03302-22 4

Snowy Sheathbill as a Sentinel of Viral Diversity Microbiology Spectrum

FIG 3 Avian influenza virus (AIV) seroprevalence in snowy sheathbills. Shown are data from competitive nucleoprotein (NP) ELISAs of serum samples
obtained from snowy sheathbills. Rhombus dots display Isabel Riquelme Islet samples, and circles display Kopaitic Island samples. Overall, there was a 14%
AIV seroprevalence. Abs, antibodies.

Totals of 17 viral families and 4 groups of unclassified viruses were identified (Fig. 4
and Fig. S1). The snowy sheathbill library was enriched primarily in Ackermannviridae,
Caliciviridae, Parvoviridae, Myoviridae, Nimaviridae, and Picornaviridae. Fig. 4 also details
the classifications of the different viral categories with further species resolution and con-
firms most of our findings.
Although the identification of multiple viruses was possible by sequencing (Fig. 4
and Fig. S1), we focused on viruses with potential zoonotic risk and human or mamma-

Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.

lian viruses. Furthermore, the identities of the potential hosts for these viruses are fur-
ther clarified in Table S2.
We detected a partial human sapovirus genome with 75.0% identity to a sapovirus
of North American origin (Fig. 6 and Table S1). Both methods of taxonomic classifica-
tion identified this sequence. A likely human gammaherpesvirus with 98.9% identity to
a Chinese herpesvirus variant strongly associated with nasopharyngeal carcinoma was
also detected (Table S1). Similarly, this virus was also identified by both methods, but
the visual resolution of the chart does not display the above-mentioned herpesvirus.
Finally, a partial sequence from a picorna-like virus with 100% identity to a unique
Antarctic fur seal virus described in 2017 was found in the data set (Table S1).
Phylogenic analyses of the identified gammaherpesvirus and picorna-like virus were
not pertinent since the assembled contigs were too well conserved between different
species to resolve any branches. Instead, we used their respective highest-similarity
hits found in the NCBI nt database, and we considered the E value as the first quality
filter for the BLAST search results. Hits with an E value of ,0.0001 are considered good
hits for homology matches (Table S1).

DISCUSSION
An increase in fieldwork efforts/expeditions paired with improvements in methods
dedicated to the screening of microbial diversity through massively parallel sequenc-
ing has accelerated the discovery of novel pathogens in Antarctica, including bacteria
and viruses (4, 5, 7, 10). However, almost all research dedicated to the characterization
of the microbial diversity in Antarctic bird species has focused on penguin species (3,

May/June 2023 Volume 11 Issue 3 10.1128/spectrum.03302-22 5

Snowy Sheathbill as a Sentinel of Viral Diversity Microbiology Spectrum

Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.

FIG 4 Krona plot representing an overview of all viral sequences identified by massively parallel sequencing. Taxa with .0.009% representation among the
analyzed reads are shown in this Krona chart. Their names are included along with their percentages of representation. The relative abundance of viral sequences is
displayed as follows: inner circles represent higher taxonomic ranks, and more detailed taxonomic ranks (up to the species level) are presented in the outer circles.
An interactive version of this chart is available on the Krona website (see the URL listed in Materials and Methods). Navigational controls are at the top left, and
details of the selected node are at the top right. The chart is zoomed in to place the “Viruses” domain at the root.

10, 11). This is probably due to their high level of representation among Antarctic birds
(2). Some studies have sampled scavenger species such as the snowy sheathbill, but
the number of individuals was usually low, and viruses were not always targeted, cer-
tainly not on a metagenomic scale (10, 12). This encouraged us to assess whether a
bird species such as the snowy sheathbill could provide us with a complementary pic-
ture of viral diversity using targeted surveillance and metatranscriptomic methods (11,
13). Furthermore, the opportunistic feeding behavior associated with this particular
bird species provided an opportunity to gain improved insights into Antarctic viral diversity
(2, 6). To our knowledge, this is the most abundant sampling of snowy sheathbills for a viral
screening study so far (Fig. 2). In addition to targeted viral screening, the snowy sheathbill
virome was used to assemble 971 viral contigs of between 8,500 and 200 nt, with 142 hits

May/June 2023 Volume 11 Issue 3 10.1128/spectrum.03302-22 6

Snowy Sheathbill as a Sentinel of Viral Diversity Microbiology Spectrum

on mammal carcasses
Scavenger behavior

or feces
Co pen ces
ns gu
um in
er
in
fe

ng r l
g

ave avio ma
Sc eh am ses
b m s s
a e
on carc fec
or

Normal dietary

Scavenger behavior
on mammal carcasses
or feces
y
ar
et
di
al

Sc eha ird s
rm

av vi
b n b sse
No

en or
o ca

ge
ca

r
Gut microbiota

FIG 5 The snowy sheathbill as a sentinel of Antarctic viral diversity. Here, we display how the snowy sheathbill’s
opportunistic feeding behavior can influence its harbored viral diversity and its likely source.

Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.

associated with known viruses. Interestingly, a high percentage of contigs showed no signifi-
cant identity, suggesting potentially new highly divergent viruses in these assembled con-
tigs. Considerable viral diversity was found, with representatives from 17 viral families and 4
groups of unclassified viruses being identified in the studied snowy sheathbill pool (Fig. 6
and Fig. S1). Moreover, we identified sequences likely related to viruses from pigs, seals, and
humans in the virome of this scavenger bird (Fig. 5), although it should not be neglected
that undersampling may have affected the diversity reported here. These data support the
idea that scavenger birds from Antarctica provide an opportunity to study the diversity of
viruses in this region. Importantly, this is, to our knowledge, the most diverse virome
described from an Antarctic bird species so far (14).
Recently, a study describing the Pygoscelis genus virome suggested that flying bird spe-
cies act as potential vectors for the transfer of pathogens among different penguin colonies
separated by distances of around 200 km (11). A common example of viruses associated
with penguin colonies is avian paramyxoviruses; these viruses were first described in birds
from the Antarctic in 1970 using a serological approach (15). Later on, additional studies
revealed the presence of avulaviruses in penguins using PCR and transcriptomic techniques
(5, 15, 16). We detected paramyxoviruses in 18 samples taken from two locations in different
years. This strongly suggests that flying bird species, particularly scavengers, are potential
vectors and reservoirs for viruses, likely without experiencing disease. This could also entail
that paramyxoviruses are widespread in these populations, which would become evident
through larger screens. It should be noted that all sampled individuals were evaluated by a
veterinarian and showed no clinical signs of disease.

May/June 2023 Volume 11 Issue 3 10.1128/spectrum.03302-22 7

Snowy Sheathbill as a Sentinel of Viral Diversity Microbiology Spectrum

99

99

99
99

81

99
34

99

67

89
19

24
6

33

99

99

99

MG012410.1 |Sapovirus GII.2 strain Hu/US/2014/GII.2/Nashville9521

26

68

99

99

23
77

97
MG012429.1 |Sapovirus GIV strain Hu/PE/2016/GIV.1/Lima1861
92

90

99
90
14
62

11
MG012401.2 |Sapovirus GI.3 strain Hu/KE/2006/GI.3/Siaya1927
18

52

83

99
MZ576278.1 |Sapovirus Sapozj-9
91

65
99

87

23
99 MG012416.1 |Sapovirus GII.3 strain Hu/NI/2010/GII.3/Leon6708
99

95

21

MG012409.1 |Sapovirus GII.1 strain Hu/US/2015/GII.1/Nashville9385

25

99

99

97 90
97
99

MG012422.1 |Sapovirus GIV strain Hu/US/2015/GIV.1/Portland3644

MG012444.1 |Sapovirus GII.1 strain Hu/US/2015/GII.1/Nashville9343

99

99
75
86
81
10

70
68

99

59
MG012451.1 |Sapovirus GII.5 strain Hu/PE/2016/GII.5/Lima1868
94
18
99
99
56
50

MN161591.1 |Sapovirus GII isolate TZ-Lib02-1

99
99

70

97
MN161596.1 |Sapovirus GI isolate TZ-Lib07-1
99

90

0
MG012406.1 |Sapovirus GII.1 strain Hu/US/2014/GII.1/Nashville9510
99
2
24

70
75 99 MG012413.1 |Sapovirus GII.2 strain Hu/US/2013/GII.2/Nashville9795
99

99
47
MG012424.1 |Sapovirus GIV strain Hu/US/2014/GIV.1/Portland3629
0 99
99

19 99 MN161595.1 |Sapovirus GIV isolate TZ-Lib06-1

99
34
0
99

66
99

MG012408.1 |Sapovirus GII.1 strain Hu/US/2013/GII.1/Nashville9691-2

86

78

99

13
Sapovirus GII strain Chionis Albus/ Antarctica/ 2022/GII
98

93

93

88
92

MG012404.1 |Sapovirus GII.1 strain Hu/US/2015/GII.1/Nashville9353

93

72
90

97
89

99
99 MF944258.1 |Sapovirus sp. strain 20151018
76

47
99
62
MG012455.1 |Sapovirus GIV.1 strain Hu/US/2014/GIV.1/Portland3651
1

Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.

19

99 MN102400.1 |Sapovirus GII.3 strain 03-99S-004/2010/TW

1
98

85
91 66
0
1
99
MG012418.1 |Sapovirus GII.3 strain Hu/US/2014/GII.3/Nashville9513
99

0 5
23
59

86 MG012447.1 |Sapovirus GII.5 strain Hu/US/2015/GII.5/Nashville9349

87

44
MG012438.1 |Sapovirus GI.1 strain Hu/US/2014/GI.1/Nashville9478
78
89
0

77

MN161593.1 |Sapovirus GI isolate TZ-Lib04-1 polyprotein

95
1
99
99

11

52

2
MH933795.1 |Sapovirus sp. isolate HumanSaV/CMR/Kumba-HP15/2014
3

FIG 6 Phylogenetic tree including members of the Sapovirus genus. Sequence comparison was performed using 141 sequences from the Sapovirus genus;
the sequence found in snowy sheathbills is highlighted by a red star. Phylogenetic distances were calculated using the Kimura 2-parameter method. A
bootstrap analysis with 1,000 replicates was applied, and numbers indicate bootstrap values. All GenBank accession numbers are included in Tables S1 and
S2 in the supplemental material. (A) Complete phylogenetic tree; (B) zoomed-in view of the clade including the newly identified sapovirus sequence.

Similar to what has been observed previously for other scavenger bird species, there
are few reports describing snowy sheathbill individuals with viral infections. This is likely
due to a potent immune system and/or a lower stomachic pH killing the viruses (17–19).
Here, we present the first isolation of a paramyxovirus in snowy sheathbills (Table 2). We
detected avulaviruses 18 and 19; however, neither sample was positive for avulavirus 17,
and one sample was not positive for avulavirus 17, 18, or 19 or NDV, suggesting a poten-
tially novel Antarctic paramyxovirus. Extensive characterization of these paramyxoviruses
after isolation is under way in our laboratory. Note that snowy sheathbills shared a habi-
tat with Antarctic penguins at the time of avulavirus 17, 18, and 19 detection, suggesting
the probable circulation of the virus.

May/June 2023 Volume 11 Issue 3 10.1128/spectrum.03302-22 8

Snowy Sheathbill as a Sentinel of Viral Diversity Microbiology Spectrum

TABLE 2 Primers used in this study together with their targets, target lengths, cycle numbers, and corresponding referencesa
Primer Length Annealing No. of
set Direction Sequence (bp) Target temp (°C) cycles Reference
IZS FW 59-CDCAYGARTTYTGYTCNCARC-39 180 RdRp 45 50 45
RV 59-RHGGRTANGCRTCWATDGC-39

Gentoo FW 59-CCCACTAACAAAGGTAGATC-39 270 RdRp 54 45 This study

RV 59-CAGAGCAATGGTCGTCTT-39

PMX FW 59-GARGGIYIITGYCARAARNTNTGGAC-39 121 RdRp 41 35 46

RV 59-TIAYIGCWATIRIYTGRTTRTCNCC-39

APMV1 FW 59-AGTGATGTGCTCGGACCTTC-39 121 M 41 40 47

RV 59-CCTGAGGAGAGGCATTTGCTA-39

AV-17 FW 59-TTRTCATCCTACTCTACCTAT-39 281 L 45 45 This study

RV 59-AGGTCATTCATATAGAGTGT-39

AV-18 FW 59-GCATCAAGTAAGCAATATCT-39 310 L 45 45 This study

RV 59-CTCAAGAAGAGTCATTATGTAA-39

AV-19 FW 59-GGTCACAGATTCTACAAC-39 395 L 45 45 This study

RV 59-GCTCAGTCCTACTTATTATTG-39

Inf-A FW 59-GACCRATCCTGTCACCTCTGAC-39 105 M 60 40 48

RV 59-AGGGCATTYTGGACAAAKCGTCTA-39
Probe 59-FAM-TGCAGTCCTCGCTCACTGGGCACG-BHQ1-39
aFW, forward; RV, reverse; RdRp, RNA-dependent RNA polymerase; FAM, 6-carboxyfluorescein; BHQ1, black hole quencher 1.

In contrast to other Antarctic bird screenings by PCR on fecal samples from snowy
sheathbills (3) and massively parallel sequencing studies on penguins that have shown the
presence of influenza A virus (IAV), we did not detect the above-mentioned virus using ei-
ther of these techniques. We believe that this could be due to our sample size or the time
when the samples were taken (December 2020 and January 2022). Nevertheless, we
detected 2 individuals with antibodies against IAV, suggesting IAV infection at a different
period of its cycle (Fig. 3). Antibodies to IAV have been detected previously in a southern

Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.

giant petrel that spent its nonbreeding season over Argentina and the Falkland/Malvinas
Islands, supporting the general idea that migratory animals might be contaminated by
IAVs during their nonbreeding season (20). Note that the test used for IAV detection has
been validated for domestic chickens, turkeys, ducks, and geese according to the manufac-
turer’s instructions. In addition to this, Claes et al. (21) previously tested the same kit on
mallards, mute swans, Canadian geese, and Peking ducks. These data suggest that the kit
is relatively plastic in terms of its application to other species. It should also be noted that
the NP targeted in this assay can vary between species. Considering this, we think that the
most likely outcome is several false-negative results, although this is merely a hypothesis.
It should be noted that this is currently the gold-standard test for Antarctic penguins.
Of relevance was the detection and isolation of avian coronaviruses. Until now, in
Antarctica, avian coronaviruses were detected only through antibodies or by next-gen-
eration sequencing (NGS) and only in penguins (3, 11, 22). We were able to isolate the
virus from snowy sheathbills for molecular characterization and the identification of its
potential zoonotic impact. This work is ongoing in our laboratory.
The habitats and foraging and nesting habits of bird species are likely to impact the risk
of the acquisition of pathogens infectious to them and other species (23). Birds foraging
close to livestock show a higher incidence of the presence of enteric pathogens than birds
foraging away from such sites (24, 25). Bird species foraging in anthropic sites such as rub-
bish dumps have a higher likelihood of exposure to zoonotic pathogens (23). Therefore, the
characteristics of the environment and the habits of a given bird species affect colonization
by different microorganisms (e.g., bacteria and viruses). Obligate scavenger birds such as
black vultures, a bird common in rubbish dumps, can be used to detect pathogens such as

May/June 2023 Volume 11 Issue 3 10.1128/spectrum.03302-22 9

Snowy Sheathbill as a Sentinel of Viral Diversity Microbiology Spectrum

Salmonella enterica and Chlamydia psittaci as they could act as potential dispersers and reser-
voirs of these pathogens (14). Their role as a sentinel of pathogen diversity in rubbish dumps
was therefore proposed (14). Studies on other obligate scavenger species such as the
Andean condor have also supported the idea that understanding the factors involved in the
circulation and emergence of pathogens in these birds is key to preserving ecosystems and
human health, highlighting their value for surveillance (26). Generally, it is considered that
the scavenger lifestyle amplifies the exposure of such bird species to a variety of pathogens
due to potential contact with infected prey or carcasses, hence the interest in them as
potential sentinels. This has also been observed for additional bird species that exhibit scav-
enger behaviors, such as corvids, owls, and falcons, and different studies have confirmed the
detection of diverse viral pathogens in them (6, 27–29). The focus on snowy sheathbills in
this study offers a unique opportunity for the intersection of their scavenger behavior and
their unique habitat, providing unprecedented insight for viral surveillance.
One of the most interesting findings was the identification of several viruses related
to known mammalian sequences in the samples (Fig. 4 and 6; see also Table S1 in the
supplemental material). We highlight one picornavirus similar to one described previ-
ously in Antarctic fur seals (Arctocephalus gazella) (Table S1) (30). The presence of such
mammalian viruses (16, 31, 32) in snowy sheathbills is likely a result of their scavenger
behavior, therefore also supporting the idea that scavengers are appropriate monitors/
sentinels of Antarctic virus circulation/introduction. Moreover, we detected a virus sim-
ilar to human sapovirus GII. Such viruses belong to the Caliciviridae family and are im-
portant causative agents of acute epidemic gastroenteritis in humans (33) (Fig. 5 and 6
and Fig. S1). Human infection occurs via contaminated food or water, and the respec-
tive diseases are therefore designated foodborne diseases (34). Recently, the presence
of human sapoviruses and other gastrointestinal viruses such as noroviruses in farm
animals has attracted increasing attention. This is due to the fact that transmission
from animals to humans and vice versa would have far-reaching consequences for epi-
demiology and food safety (33, 34). Ultimately, this finding suggests the circulation of
human viruses on the Antarctic continent. The impact that this virus could have on
Antarctic fauna is unknown and complex to estimate. The identification of several
human-associated viruses in our study could be considered an indication of contami-
nation. However, we consider that the techniques and precautions applied for sample

Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.

collection and preparation make contamination rather unlikely. The same sample treat-
ment for viral enrichment was applied in a bat virome study, with no biases concerning
human-associated viruses observed (35). It should also be mentioned that at least one
of the sampled populations in this study is in contact with humans (General Bernardo
O’Higgins military base). Humans have shared this particular environment with snowy
sheathbills for 75 years, which likely has an impact. In addition to this, one of the
human-associated viruses identified in this study was a gammaherpesvirus with 98.9%
identity to a Chinese herpesvirus. The level of identity to this geographically distant
isolate makes contamination an unlikely option. We consider that these arguments col-
lectively make contamination an unlikely event.
The gammaherpesvirus found in our samples was closely related to a specific Chinese
herpesvirus that may cause nasopharynx cancer in humans (36) (Table S1). Epstein-Barr virus
infection and environmental exposure to specific substances are considered risk factors,
even though this condition is endemic in some Asian areas where a genetic predisposition
for oncogenesis has been established (36). The identification of such a virus is surprising and
concerning, particularly because the consequences or the probability of transmission to ma-
rine mammals is unknown. Its origin is a puzzling question that is yet to be solved.
We highlight the need for viral surveillance in remote regions, a goal that possibly
can be achieved by monitoring scavenging seabirds. We found considerable viral di-
versity in snowy sheathbills (Fig. 5), likely associated with their opportunistic diet. We
show evidence of several potential human virus introductions in Antarctica and show-
case the snowy sheathbill as a candidate sentinel of viral diversity. We highlight the im-
portance of continuous avian surveillance, including other Antarctic scavenger birds;

May/June 2023 Volume 11 Issue 3 10.1128/spectrum.03302-22 10

Snowy Sheathbill as a Sentinel of Viral Diversity Microbiology Spectrum

this will be critical for elucidating and better understanding the mechanisms of virus
introduction and circulation in Antarctica.

MATERIALS AND METHODS

Sample collection. Samples were collected on two separate trips during Chilean Antarctic Expedition
57 (ECA57) and Chilean Antarctic Expedition 58 (ECA58). ECA57 took place from 15 to 18 December
2020 on Nelson Island, more specifically on the Harmony Point coast (62°18900S, 59°13900W). ECA58 took
place from 13 January to 9 February 2022 in the Antarctic Peninsula Isabel Riquelme Islet and Kopaitic
Island (on the Duroch Islands) (63°19950S, 57°539550W) (Fig. 2). Harmony Point is an island considered to
be devoid of the presence of humans. Sampling during ECA57 was performed around the island’s pen-
guin colony’s nesting grounds. Sampling during ECA58 was performed at the General Bernardo
O’Higgins military base’s penguin colony site. This is in contrast to ECA57 due to the direct interactions
of the sampled subjects with humans. During 2020, a total of 124 direct environmental samples of
Antarctic avifauna were collected, of which 22 samples were identified for the following specific species:
giant petrel (Macronectes giganteus) (n = 8), snowy sheathbill (Chionis albus) (n = 7), Weddell seal
(Leptonychotes weddellii) (n = 6), and Antarctic shag (Leucocarbo bransfieldensis) (n = 1). These samples were col-
lected with sterile swabs and placed into 1.5-mL Eppendorf tubes with viral transport medium (VTM) from VQIR
(catalog number 611901) (7). The samples were kept at ambient temperature in a cooler (near 0°C) before being
stored at 280°C. It should be noted that during this expedition, we were evacuated from Antarctica due to a co-
ronavirus disease (COVID) outbreak, which impacted our sampling capacity/numbers. Serum samples were
taken from the medial metatarsal vein. During ECA58, a total of 105 samples were collected: cloacal swabs and
serum samples (n = 14) and environmental samples (which correspond to fecal dropping samples) (n = 33)
from snowy sheathbills (Chionis albus) and environmental samples from Antarctic terns (Sterna vittata) (n = 33)
and chinstrap penguins (Pygoscelis antarctica) (n = 25) were taken between three islands (Nelson, Isabel
Riquelme Islet, and Kopaitic Island). Serum samples were taken from the medial metatarsal vein. Samples were
stored at 280°C. All sampled individuals were examined by a veterinarian.
Screening for anti-influenza A virus antibodies. An influenza A virus antibody test kit blocking
enzyme-linked immunosorbent assay (ELISA) was used according to the manufacturer’s instructions (AI
MultiS-Screen, catalog number 99-53101; Idexx).
RNA extraction. The samples were homogenized in VTM. After decanting the particles present in
the sample, 60 m L of the supernatant was taken, and pools were prepared for each of 5 samples with
100 m L of VTM. The pools were clarified by subsequent centrifugation at 8,000  g for 10 min at 4°C and
stored at 280°C. RNA was extracted from 150 m L of the supernatant obtained in the previous step, using
TRIzol reagent (Invitrogen) based on the Chomczynski-Sacchi method (37), according to the manufac-
turer’s instructions. The RNA obtained was resuspended in 30 m L of nuclease-free water and stored at
220°C. The RNA concentration was analyzed by absorption using a Synergy HTX multimodal reader
(Agilent Technologies, USA).
Targeted screening. Screening for coronavirus, including the Antartic1 replicase (this study); para-
myxovirus (PMX); and influenza virus was performed using Brilliant III Ultra-Fast RT-PCR master mix
(Agilent Technologies, USA).

Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.

Concerning the sensitivity of our screen for influenza virus detection, degenerate primers were used for RT-
quantitative PCR (RT-qPCR) screening. We followed WHO recommendations, considering a positive result until
cycle 38. Coronavirus and paramyxovirus detection was performed by RT-PCR, and positive identification was
determined by the presence or absence of a band of the expected size in an agarose gel.
Virus isolation. For the amplification and isolation of coronaviruses and paramyxoviruses, passages
were made in 8- to 10-day-old specific-pathogen-free embryonated eggs obtained from the Institute of
Public Health (ISP), Chile. The inoculated sample was prepared from 100 m L of the supernatant obtained
after the homogenization and clarification of the samples (pools and/or individual samples) and 100 m L
of VTM, inoculating 100 m L per egg in duplicate. Finally, the inoculated eggs were incubated in a humid
environment at 37°C, where they were left for 48 h. Once the incubation time was over, the eggs were
left at 4°C for a minimum of 4 h for euthanization. After incubation, the allantoic fluid was collected,
obtaining between 2 and 3 mL per egg. The allantoic fluids obtained were stored at 280°C.
Viral purification and concentration. Ultracentrifugation was performed based on a protocol
described previously by Fumian et al. (38). Six snowy sheathbill fecal samples were taken, homogenized in
1 mL of VTM, and clarified at 7,000  g for 10 min at 4°C. The supernatant obtained was recovered and
centrifuged at 13,000  g for 30 min at 4°C. This new supernatant was transferred to a cryogenic tube,
and the volume was made up to 1 mL. Samples were centrifuged at 30,000  g for 3 h 15 min. Next,
900 m L of the supernatant was removed, resuspended in 150 m L of 0.25 N glycine buffer (pH 9.5), and
incubated on ice for 30 min. The suspension was neutralized by the addition of 150 m L of 2 phosphate-
buffered saline (PBS) (pH 7.2). The supernatant, rich in viral particles, was centrifuged at 12,000  g for
15 min at 4°C, and viruses were recovered by centrifugation at 30,000  g for 3 h 15 min at 4°C. Finally,
the supernatant was discarded, and the pellet was resuspended in 200 m L of 1 PBS (pH 7.2). DNases and
RNases were added to a final concentration of 10 U. The samples were stored at 280°C until use.
Depletion, library preparation, and NGS. After total RNA enrichment for viral particles by centrifu-
gation, total RNA was depleted for rRNA using the Illumina Ribozero Plus kit with a custom protocol in
Magelia (Inorevia, Paris, France). After bioinformatic verification of probe compatibility for bacterial/bird
selection, 2 m L of total RNA was used in the instrument for the fully automated molecular enrichment of
viral sequences. After depletion, total RNA was run in the Bioanalyzer 2100 system using a nano-RNA
chip to determine rRNA contamination. Next, the Illumina total stranded RNA protocol for library

May/June 2023 Volume 11 Issue 3 10.1128/spectrum.03302-22 11

Snowy Sheathbill as a Sentinel of Viral Diversity Microbiology Spectrum

preparation was performed using approximately 10 ng of depleted RNA. Library preparation yielded
20 nM high-quality sequenceable material, which was then run in a NextSeq 500 Mid Output kit v2 (150
cycles) cartridge. This yielded a total of 36,519,002 sequences in pairs.
Bioinformatic analyses. Read quality control was evaluated using FastQC v0.11.9, and read preprocess-
ing was performed with fastx-toolkit v0.0.13 using the fastx_trimmer, fastx_artifacts_filter, and fastx-clipper
tools. Orphaned reads were separated from the total sample using the makepairs package of pairfq v0.17.0.
The Kraken2-bracken protocol was implemented for taxonomic read assignment. The sample assembly was
generated using SPAdes genome assembler v3.13.0 (metaSPAdes mode). To avoid overestimation, only contigs
with a minimum size established at 200 nucleotides were selected. For contig clustering and nonredundancy,
CD-HIT software was used, covering the nonrepresentative sequences at 80%.
Bowtie2 v2.4.2 was used to recapture clean reads mapped to the contig sequence database. Sample
processing was implemented in Samtools v1.13. Read quantification was performed using samtools
view -c -F 260.
A BLAST analysis was also performed on the contigs using the NCBI BLASTN algorithm. A supple-
mental table was generated with the top hits for the generated contigs with an E value cutoff of E208.
This enabled us to clarify the potential host of origin for the identified viruses.
As a complement, the reads were run using the Kaiju Web tool as previously described (35). This enabled
us to produce a summary file with the number of reads assigned per taxon, which was then loaded into Krona
(39) for interactive visualization. The interactive plot can be found at https://kaiju.binf.ku.dk/output/10391
-7724754407/krona.html?dataset=0&node=3062&collapse=true&color=false&depth=13&font=11&key=true.
Oxford Nanopore MinION sequencing. The coronavirus isolate was sequenced using the Oxford
Nanopore MinION platform. Briefly, starting with 250 ng of high-molecular-weight (50-kbp) double-stranded
DNA (dsDNA) as the input, fragments with an average distribution of 11 kbp were generated by the use of a g-
Tube for DNA shearing (Covaris) and centrifugation at 2,000  g for 1 min. Using the NEBNext FFPE (formalin-
fixed, paraffin-embedded) DNA repair mix (New England BioLabs) and NEBNext Ultra II end repair/dA-tailing
module (New England BioLabs) reagents and according to the instructions provided by Oxford Nanopore
Technologies, the ends of the DNA were repaired, to which a poly(A) tail was attached. The repaired DNA was
purified using AMPure XP (Beckman Coulter) according to the manufacturer’s instructions and using a 2 vol-
ume with respect to the sample. For all subsequent washes, this volume ratio of beads to sample was main-
tained in order to maximize the DNA concentrations recovered at the expense of fragment size. According to
the protocol provided by Oxford Nanopore Technologies, the NEBNext quick ligation module reagent (New
England BioLabs) was used for the ligation of the adapter to the nucleotide sequence. For the following steps,
the library preparation kit for MinION SQK-DCS 109 (Oxford Nanopore Technologies) was used, according to
the company’s instructions. The library was loaded onto a MinION flow cell (R9.4.1). For sequencing, the
minKNOW v3.4.5 program was used (40).
Genome assembly. The quality of the reads was assessed using NanoPlot v1.23.1 (41). Adapter
sequences were removed with Porechop v0.2.4. (https://github.com/rrwick/Porechop) using a 10,000-
read alignment. Reads were quality cut using the Nanofilt v2.3.0 tool (41). Assembly was performed
using Unicycler v0.4.8-beta (40) in long-sequence mode. Finally, the quality of the assembly was eval-
uated using the QUAST v5.0.2 tool (42).
Sapovirus phylogenetic analyses. We compiled a sapovirus database with 141 sequences (Fig. 5;

Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.

see also Table S1 in the supplemental material). Alignments were performed using MUSCLE (http://www
.drive5.com/muscle/) (43). Phylogenetic trees were constructed with MEGA6 (http://www.megasoftware
.net) and IQ-TREE on the IQ-TREE Web server (http://www.cibiv.at/software/iqtree/) (44) by using the
maximum likelihood (ML) method. The robustness of the tree topologies was assessed with 1,000 boot-
strap replicates. Phylogenetic trees were constructed using ML inference with the Kimura 2-parameter
nucleotide substitution model.
Deltacoronavirus phylogenetic analyses. We compiled a coronavirus database with 78 sequences
(Table S2). Alignments were performed using MUSCLE (http://www.drive5.com/muscle/) (43). Phylogenetic
trees were constructed with MEGA6 (http://www.megasoftware.net) and IQ-TREE on the IQ-TREE Web
server (http://www.cibiv.at/software/iqtree/) (44) by using the ML method. The robustness of the tree top-
ologies was assessed with 1,000 bootstrap replicates. Phylogenetic trees were constructed using ML infer-
ence with the general time-reversible (GTR) parameter nucleotide substitution model.
Ethics statement. The study described here was approved by the Ethics Committee of the Faculty
of Medicine at the Universidad de Chile (approval number 20349-med.uch).
Data availability. The data supporting the findings of this study can be found in this article and its
supplemental material. All Illumina sequencing data used for this RNA sequencing (RNA-seq) study have
been submitted to the Sequence Read Archive (SRA) under BioProject accession number PRJNA904746.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 0.5 MB.
SUPPLEMENTAL FILE 2, XLS file, 0.1 MB.
SUPPLEMENTAL FILE 3, EPS file, 0.5 MB.

ACKNOWLEDGMENTS
We are supported by Instituto Antártico Chileno (INACH) RT_35-19 (GB-P), ANID
Chile, through Fondecyt grant number 11200228 (G.P.B.). L.K. is supported by INACH’s

May/June 2023 Volume 11 Issue 3 10.1128/spectrum.03302-22 12

Snowy Sheathbill as a Sentinel of Viral Diversity Microbiology Spectrum

Programa Áreas Marinas Protegidas (AMP 24 03 052) and the Instituto Milénio Base
(ICN_2021_002).
We thank the Chilean Army for help on the field trip.
Conceptualization, G.P.B. Data curation, A.G., S.A.P., C.R.-F., and G.P.B. Formal
analysis, G.Z. and G.P.B. Funding acquisition, G.P.B. Investigation, F.V., G.Z., J.L., and
G.P.B. Methodology, S.A.P., A.G., and G.P.B. Project administration, G.P.B. Sample
collection, J.L., L.A., L.K., and G.P.B. Supervision, G.P.B. Validation, G.Z., C.R.-F., and S.A.P.
Visualization, G.P.B. Writing – original draft, G.P.B., S.A.P., A.G., and L.K. Writing – review
and editing, S.A.P., A.G., L.K., and G.P.B. All authors approved the final version of the
manuscript.
We declare that there are no conflicts of interest associated with this work.
G.P.B. had full access to all data in this study and takes complete responsibility for the
integrity of the data and the accuracy of the data analysis. G.P.B. affirms that the
manuscript is an honest, accurate, and transparent account of the study being reported;
that no important aspects of the study have been omitted; and that any discrepancies
from the study as planned have been explained.

REFERENCES
1. Hernandez MM, Berón MP, Zumpano F, Giardino G, Pon JPS. 2021. Time-
activity budget of the snowy sheathbill (Chionis albus) wintering at a sea
lion (Otaria flavescens) haul-out in Argentina. Waterbirds 44:376–381.
https://doi.org/10.1675/063.044.0313.
2. Favero M. 1996. Foraging ecology of pale-faced sheathbills in colonies of
southern elephant seals at King George Island, Antarctica. J Field Ornithol
67:292–299.
3. Hurt AC, Vijaykrishna D, Butler J, Baas C, Maurer-Stroh S, Silva-de-la-
Fuente MC, Medina-Vogel G, Olsen B, Kelso A, Barr IG, González-Acuña D.
2014. Detection of evolutionarily distinct avian influenza A viruses in Ant-
arctica. mBio 5(3):e01098-14. https://doi.org/10.1128/mBio.01098-14.
4. Smeele ZE, Burns JM, Van Doorsaler K, Fontenele RS, Waits K, Stainton D,
Shero MR, Beltran RS, Kirkham AL, Berngartt R, Kraberger S, Varsani A.
2018. Diverse papillomaviruses identified in Weddell seals. J Gen Virol 99:
549–557. https://doi.org/10.1099/jgv.0.001028.
5. Neira V, Tapia R, Verdugo C, Barriga G, Mor S, Ng TFF, García V, Del Río J,
Rodrigues P, Briceño C, Medina RA, González-Acuña D. 2017. Novel avula-
12. Thomazelli LM, Araujo J, Oliveira DB, Sanfilippo L, Ferreira CS, Brentano L,
Pelizari VH, Nakayama C, Duarte R, Hurtado R, Branco JO, Walker D,
Durigon EL. 2010. Newcastle disease virus in penguins from King George
Island on the Antarctic region. Vet Microbiol 146:155–160. https://doi
.org/10.1016/j.vetmic.2010.05.006.
13. Wille M, Eden J-S, Shi M, Klaassen M, Hurt AC, Holmes EC. 2018. Virus-virus
interactions and host ecology are associated with RNA virome structure
in wild birds. Mol Ecol 27:5263–5278. https://doi.org/10.1111/mec.14918.
14. Plaza PI, Blanco G, Madariaga MJ, Boeri E, Teijeiro ML, Bianco G, Lambertucci
SA. 2019. Scavenger birds exploiting rubbish dumps: pathogens at the gates.
Transbound Emerg Dis 66:873–881. https://doi.org/10.1111/tbed.13097.
15. Wille M, Aban M, Wang J, Moore N, Shan S, Marshall J, González-Acuña D,
Vijaykrishna D, Butler J, Wang J, Hall RJ, Williams DT, Hurt AC. 2019. Ant-
arctic penguins as reservoirs of diversity for avian avulaviruses. J Virol 93:
e00271-19. https://doi.org/10.1128/JVI.00271-19.
16. Austin FJ, Webster RG. 1993. Evidence of ortho- and paramyxoviruses in
fauna from Antarctica. J Wildl Dis 29:568–571. https://doi.org/10.7589/

Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.

viruses in penguins, Antarctica. Emerg Infect Dis 23:1212–1214. https://
doi.org/10.3201/eid2307.170054.
6. McClelland GTW, McKechnie AE, Chown SL. 2016. Basal metabolic rate of
the black-faced sheathbill (Chionis minor): intraspecific variation in a phy-
logenetically distinct island endemic. Physiol Biochem Zool 89:141–150.
https://doi.org/10.1086/685411.
7. Barriga GP, Boric-Bargetto D, San Martin MC, Neira V, van Bakel H,
Thompsom M, Tapia R, Toro-Ascuy D, Moreno L, Vasquez Y, Sallaberry M,
Torres-Pérez F, González-Acuña D, Medina RA. 2016. Avian influenza virus
H5 strain with North American and Eurasian lineage genes in an Antarctic
penguin. Emerg Infect Dis 22:2221–2223. https://doi.org/10.3201/eid2212
.161076.
8. Chown SL, Lee JE, Hughes KA, Barnes J, Barrett PJ, Bergstrom DM, Convey
P, Cowan DA, Crosbie K, Dyer G, Frenot Y, Grant SM, Herr D, Kennicutt MC,
II, Lamers M, Murray A, Possingham HP, Reid K, Riddle MJ, Ryan PG,
Sanson L, Shaw JD, Sparrow MD, Summerhayes C, Terauds A, Wall DH.
2012. Conservation. Challenges to the future conservation of the Antarc-
tic. Science 337:158–159. https://doi.org/10.1126/science.1222821.
9. Rands MRW, Adams WM, Bennun L, Butchart SHM, Clements A, Coomes
D, Entwistle A, Hodge I, Kapos V, Scharlemann JPW, Sutherland WJ, Vira B.
2010. Biodiversity conservation: challenges beyond 2010. Science 329:
1298–1303. https://doi.org/10.1126/science.1189138.
10. Hurt AC, Su YCF, Aban M, Peck H, Lau H, Baas C, Deng Y-M, Spirason N,
Ellström P, Hernandez J, Olsen B, Barr IG, Vijaykrishna D, Gonzalez-Acuna
D. 2016. Evidence for the introduction, reassortment, and persistence of
diverse influenza A viruses in Antarctica. J Virol 90:9674–9682. https://doi
.org/10.1128/JVI.01404-16.
11. Wille M, Harvey E, Shi M, Gonzalez-Acuña D, Holmes EC, Hurt AC. 2020.
Sustained RNA virome diversity in Antarctic penguins and their ticks.
ISME J 14:1768–1782. https://doi.org/10.1038/s41396-020-0643-1.
0090-3558-29.4.568.
17. Uhart MM, Quintana F, Karesh WB, Braselton WE. 2003. Hematology,
plasma biochemistry, and serosurvey for selected infectious agents in
southern giant petrels from Patagonia, Argentina. J Wildl Dis 39:359–365.
https://doi.org/10.7589/0090-3558-39.2.359.
18. Globig A, Staubach C, Sauter-Louis C, Dietze K, Homeier-Bachmann T,
Probst C, Gethmann J, Depner KR, Grund C, Harder TC, Starick E, Pohlmann
A, Höper D, Beer M, Mettenleiter TC, Conraths FJ. 2017. Highly pathogenic
avian influenza H5N8 clade 2.3.4.4b in Germany in 2016/2017. Front Vet Sci
4:240. https://doi.org/10.3389/fvets.2017.00240.
19. Osman AM, Oladele OA, Ibrahim AM, Mahamoud MA, Mohamed MA,
Nwachukwu NO. 2021. Seroprevalence of Newcastle disease in backyard
chickens in selected districts of Banadir region, Somalia. Trop Anim Health
Prod 53:192. https://doi.org/10.1007/s11250-021-02622-5.
20. de Souza Petersen E, de Araujo J, Krüger L, Seixas MM, Ometto T,
Thomazelli LM, Walker D, Durigon EL, Petry MV. 2017. First detection of
avian influenza virus (H4N7) in giant petrel monitored by geolocators in
the Antarctic region. Mar Biol 164:62. https://doi.org/10.1007/s00227-017
-3086-0.
21. Claes G, Vangeluwe D, Van der Stede Y, van den Berg T, Lambrecht B,
Marché S. 2012. Evaluation of four enzyme-linked immunosorbent assays
for the serologic survey of avian influenza in wild bird species. Avian Dis
56(4 Suppl):949–954. https://doi.org/10.1637/10165-040912-Reg.1.
22. Morgan IR, Westbury HA. 1981. Virological studies of Adelie penguins
(Pygoscelis adeliae) in Antarctica. Avian Dis 25:1019–1026. https://doi
.org/10.2307/1590077.
23. Benskin CMH, Wilson K, Jones K, Hartley IR. 2009. Bacterial pathogens in
wild birds: a review of the frequency and effects of infection. Biol Rev
Camb Philos Soc 84:349–373. https://doi.org/10.1111/j.1469-185X.2008
.00076.x.

May/June 2023 Volume 11 Issue 3 10.1128/spectrum.03302-22 13

Snowy Sheathbill as a Sentinel of Viral Diversity
24. Hald B, Skov MN, Nielsen EM, Rahbek C, Madsen JJ, Wainø M, Chriél M,
Nordentoft S, Baggesen DL, Madsen M. 2016. Campylobacter jejuni and
Campylobacter coli in wild birds on Danish livestock farms. Acta Vet
Scand 58:11. https://doi.org/10.1186/s13028-016-0192-9.
25. Sulzner K, Kelly T, Smith W, Johnson CK. 2014. Enteric pathogens and anti-
microbial resistance in turkey vultures (Cathartes aura) feeding at the
wildlife-livestock interface. J Zoo Wildl Med 45:931–934. https://doi.org/
10.1638/2012-0217.1.
26. Wiemeyer GM, Plaza PI, Bustos CP, Muñoz AJ, Lambertucci SA. 2021. Ex-
posure to anthropogenic areas may influence colonization by zoonotic
microorganisms in scavenging birds. Int J Environ Res Public Health 18:
5231. https://doi.org/10.3390/ijerph18105231.
27. Shriner SA, Root JJ. 2020. A review of avian influenza A virus associations in
synanthropic birds. Viruses 12:1209. https://doi.org/10.3390/v12111209.
28. Wille M, Holmes EC. 2020. Wild birds as reservoirs for diverse and abun-
dant gamma- and deltacoronaviruses. FEMS Microbiol Rev 44:631–644.
https://doi.org/10.1093/femsre/fuaa026.
29. Chang W-S, Eden J-S, Hall J, Shi M, Rose K, Holmes EC. 2020. Metatran-
scriptomic analysis of virus diversity in urban wild birds with paretic dis-
ease. J Virol 94:e00606-20. https://doi.org/10.1128/JVI.00606-20.
30. Krumbholz A, Groth M, Esefeld J, Peter H-U, Zell R. 2017. Genome
sequence of a novel picorna-like RNA virus from feces of the Antarctic fur
seal (Arctocephalus gazella). Genome Announc 5(36):e01001-17. https://
doi.org/10.1128/genomeA.01001-17.
31. Shin D-L, Siebert U, Haas L, Valentin-Weigand P, Herrler G, Wu N-H. 2022.
Primary harbour seal (Phoca vitulina) airway epithelial cells show high sus-
ceptibility to infection by a seal-derived influenza A virus (H5N8). Trans-
bound Emerg Dis 69:e2378–e2388. https://doi.org/10.1111/tbed.14580.
32. Cortés-Hinojosa G, Adkesson MJ, Cárdenas-Alayza S, Seguel M, Pavés H,
Wellehan JFX. 2021. Adenovirus diversity in fur seal and penguin colonies
of South America. J Wildl Dis 57:964–969. https://doi.org/10.7589/JWD-D
-20-00118.
33. Bank-Wolf BR, König M, Thiel H-J. 2010. Zoonotic aspects of infections
with noroviruses and sapoviruses. Vet Microbiol 140:204–212. https://doi
.org/10.1016/j.vetmic.2009.08.021.
34. Davidson I, Stamelou E, Giantsis IA, Papageorgiou KV, Petridou E, Kritas SK.
2022. The complexity of swine caliciviruses. A mini review on genomic di-
versity, infection diagnostics, world prevalence and pathogenicity. Patho-
gens 11:413. https://doi.org/10.3390/pathogens11040413.
35. Aguilar Pierlé S, Zamora G, Ossa G, Gaggero A, Barriga GP. 2022. The Myo-
tis chiloensis guano virome: viral nucleic acid enrichments for high-reso-
lution virome elucidation and full alphacoronavirus genome assembly.
Viruses 14:202. https://doi.org/10.3390/v14020202.
36. Hui KF, Chan TF, Yang W, Shen JJ, Lam KP, Kwok H, Sham PC, Tsao SW,
Kwong DL, Lung ML, Chiang AKS. 2019. High risk Epstein-Barr virus
May/June 2023 Volume 11 Issue 3
Microbiology Spectrum
variants characterized by distinct polymorphisms in the EBER locus are
strongly associated with nasopharyngeal carcinoma. Int J Cancer 144:
3031–3042. https://doi.org/10.1002/ijc.32049.
37. Chomczynski P, Sacchi N. 1987. Single-step method of RNA isolation by
acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Bio-
chem 162:156–159. https://doi.org/10.1016/0003-2697(87)90021-2.
38. Fumian TM, Leite JPG, Castello AA, Gaggero A, de Caillou MSL, Miagostovich
MP. 2010. Detection of rotavirus A in sewage samples using multiplex qPCR
and an evaluation of the ultracentrifugation and adsorption-elution methods
for virus concentration. J Virol Methods 170:42–46. https://doi.org/10.1016/j
.jviromet.2010.08.017.
39. Ondov BD, Bergman NH, Phillippy AM. 2011. Interactive metagenomic
visualization in a Web browser. BMC Bioinformatics 12:385. https://doi
.org/10.1186/1471-2105-12-385.
40. Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Completing bacterial genome
assemblies with multiplex MinION sequencing. Microb Genom 3:e000132.
https://doi.org/10.1099/mgen.0.000132.
41. De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. 2018.
NanoPack: visualizing and processing long-read sequencing data. Bioin-
formatics 34:2666–2669. https://doi.org/10.1093/bioinformatics/bty149.
42. Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality assess-
ment tool for genome assemblies. Bioinformatics 29:1072–1075. https://
doi.org/10.1093/bioinformatics/btt086.
43. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accu-
racy and high throughput. Nucleic Acids Res 32:1792–1797. https://doi
.org/10.1093/nar/gkh340.
44. Trifinopoulos J, Nguyen L-T, von Haeseler A, Minh BQ. 2016. W-IQ-TREE: a
fast online phylogenetic tool for maximum likelihood analysis. Nucleic
Acids Res 44:W232–W235. https://doi.org/10.1093/nar/gkw256.
45. Lelli D, Papetti A, Sabelli C, Rosti E, Moreno A, Boniotti MB. 2013. Detec-
tion of coronaviruses in bats of various species in Italy. Viruses 31:
2679–2689. https://doi.org/10.3390/v5112679.
46. van Boheemen S, Bestebroer TM, Verhagen JH, Osterhaus ADME, Pas SD,
Herfst S, Fouchier RAM. 2012. A family-wide RT-PCR assay for detection of
paramyxoviruses and application to a large-scale surveillance study. PLoS
One 7:e34961. https://doi.org/10.1371/journal.pone.0034961.
47. Wise MG, Suarez DL, Seal BS, Pedersen JC, Senne DA, King DJ, Kapczynski DR,
Spackman E. 2004. Development of a real-time reverse-transcription PCR for
detection of Newcastle disease virus RNA in clinical samples. J Clin Microbiol
42:329–338. https://doi.org/10.1128/JCM.42.1.329-338.2004.
48. World health Organization (WHO). 2013. Real-time RT-PCR protocol for the
detection of avian influenza A(H7N9) virus. WHO, Geneva, Switzerland. https://
Downloaded from https://journals.asm.org/journal/spectrum on 30 June 2023 by 45.184.86.11.
www.fda.gov/media/85454/download.
10.1128/spectrum.03302-22 14