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Unionid mussels are threatened by multiple environmental stressors and have experienced mass mortality events over the last several decades, but the role of infectious disease in unionid health and population declines remains poorly understood. Although several microbial agents have been found in unionids, to date only one virus has been documented—Lea plague virus (Arenaviridae) in propagated Triangle Shell mussels (Hyriopsis cumingii) in China. We used next-generation DNA sequencing to screen hemolymph of seven individ- uals of five unionid species from the Upper Mississippi River basin, USA for viruses. We identified the complete polyprotein gene of a novel picornalike virus in one individual of the Wabash Pigtoe (Fusconaia flava). The virus is a member of the Nora virus clade of picornalike viruses and is most closely related to viruses from arthropods in China. We did not detect viruses in another Wabash Pigtoe or in animals of the other four species. It is premature to make inferences about the role of this virus in the health of Wabash Pigtoes or other unionid species or the origin or transmission of this virus. Nevertheless, to our knowledge, our results represent the first report of a virus in wild North American unionids. Technologies based on next-generation DNA sequencing should prove useful for identifying new viruses and investigating their role in unionid health and disease.
Phylogenetic tree of picornalike viruses. The major glycoprotein nucleic acid sequences of each virus were aligned using the codon-based Prank algorithm (Loytynoja 2014) implemented in the program TranslatorX (Abascal et al. 2010), with the Gblocks algorithm (Castresana 2000) applied to remove poorly aligned regions. The maximum-likelihood method implemented in the computer program PhyML (Guindon et al. 2010) was then applied to the resulting 1,332-position nucleic acid alignment, with the model of molecular evolution estimated from the data. Taxon names indicate abbreviated virus names (see below), host, country, and year of collection. The novel picornalike virus from the Wabash Pigtoe is indicated with an arrow. Numbers beside branches show statistical confidence of clades based on 1,000 bootstrap replicates of the data. Scale bar indicates nucleotide substitutions per site. Taxon abbreviations and GenBank accession numbers: NoV: Nora virus (NC_007919); HoV-6: Hubei odonate virus 6 (NC_033071); HplV-66: Hubei picornalike virus 66 (NC_033133); HoV-7: Hubei odonate virus 7 (NC_033232); WplV-47: Wenzhou picornalike virus 47 (NC_033150); MRplV-1: Mississippi River picornalike virus 1 (MK301250); CplV-17: Changjiang picornalike virus 17 (KX884555); BplV-116: Beihai picornalike virus 116 (NC_032635); BsV-2: Beihai shrimp virus 2 (NC_032594); WcV-6: Wenling crustacean virus 6 (NC_032810); WcV-5: Wenling crustacean virus 5 (NC_032839); BplV-114: Beihai picornalike virus 114 (NC_032633); BplV-115: Beihai picornalike virus 115 (NC_032618); BssV-2: Beihai sea slater virus 2 (NC_032622); BplV-113: Beihai picornalike virus 113 (NC_032559); BplV-112: Beihai picornalike virus 112 (NC_032571).
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Freshwater Mollusk Biology and Conservation 22:81–84, 2019
ÓFreshwater Mollusk Conservation Society 2019
NOTE
A NOVEL PICORNA-LIKE VIRUS IN A WABASH PIGTOE
(FUSCONAIA FLAVA) FROM THE UPPER MISSISSIPPI
RIVER, USA
Tony L. Goldberg
1
*, Christopher D. Dunn
1
, Eric Leis
2
, and Diane L. Waller
3
1
Department of Pathobiological Sciences, University of Wisconsin-Madison, 1656 Linden Drive,
Madison, WI 53706 USA
2
U.S. Fish and Wildlife Service, La Crosse Fish Health Center-Midwest Fisheries Center, 555 Lester
Avenue, Onalaska, WI 54650 USA
3
U.S. Geological Survey, Upper Midwest Environmental Sciences Center, 2630 Fanta Reed Road, La
Crosse, WI 54603 USA
ABSTRACT
Unionid mussels are threatened by multiple environ-
mental stressors and have experienced mass mortality
events over the last several decades, but the role of
infectious disease in unionid health and population
declines remains poorly understood. Although several
microbial agents have been found in unionids, to date only
one virus has been documented—Lea plague virus
(Arenaviridae) in propagated Triangle Shell mussels
(Hyriopsis cumingii) in China. We used next-generation
DNA sequencing to screen hemolymph of seven individ-
uals of five unionid species from the Upper Mississippi
River basin, USA for viruses. We identified the complete
polyprotein gene of a novel picornalike virus in one
individual of the Wabash Pigtoe (Fusconaia flava). The
virus is a member of the Nora virus clade of picornalike
viruses and is most closely related to viruses from
arthropods in China. We did not detect viruses in another
Wabash Pigtoe or in animals of the other four species. It is
premature to make inferences about the role of this virus
in the health of Wabash Pigtoes or other unionid species or
the origin or transmission of this virus. Nevertheless, to
our knowledge, our results represent the first report of a
virus in wild North American unionids. Technologies
based on next-generation DNA sequencing should prove
useful for identifying new viruses and investigating their
role in unionid health and disease.
KEY WORDS: Unionidae, Fusconaia flava, Wabash Pigtoe,
Mississippi River, virus, next-generation DNA sequencing
INTRODUCTION
Freshwater mussels (order Unionida) face mounting threats
from habitat loss and alteration, invasive species, poor water
quality and pollutants, hydrologic changes, and other stressors
(Strayer et al. 2004; Dudgeon et al. 2006; Downing et al. 2010;
Haag and Williams 2014). Unexplained mortality events have
been documented since at least the 1970s, but their causes
remain poorly understood (Haag and Williams 2014). Union-
ids are susceptible to a variety of metazoan, protozoan, fungal,
and viral infections (Carella et al. 2016), which may contribute
to mussel mortality as primary or secondary factors. Recently,
we described a coordinated effort to investigate potential
pathogens associated with unionid mass mortality events (Leis
et al. 2018).
Viruses are likely culprits in mass die-offs of wildlife
species, accounting for a higher percentage of disease-
associated events across all animal taxa than other classes of
pathogens (Fey et al. 2015). Viruses are also more likely to
emerge (appear in new places, new hosts, and new clinical
contexts) than other classes of pathogens because of their
error-prone replication and ensuing ability to mutate, evolve,
and ‘‘jump’’ to new species (Woolhouse et al. 2005). Viruses
are major causes of mortality in marine bivalves (Zannella et
al. 2017). To our knowledge, the only virus described from
unionids to date is Lea plague virus, an arenavirus (family
Arenaviridae) responsible for mass mortality of Triangle
Shell mussels (Hyriopsis cumingii Lea) in southern China
(Carella et al. 2016); these mussels are cultivated at high
density for freshwater pearl production. We surveyed five
unionid species from the Upper Mississippi River basin, USA
to investigate whether viruses may be present in North
American unionids.
*Corresponding Author: tony.goldberg@wisc.edu
81
METHODS
We sampled a total of seven individuals: one Threeridge
(Amblema plicata) and two Wabash Pigtoes (Fusconaia flava),
collected from the Mississippi River north of Brownsville,
Minnesota (43843.1370N, 91815.373 0W) on September 16,
2016, and one Threeridge, one Giant Floater (Pyganodon
grandis), one Plain Pocketbook (Lampsilis cardium), and one
Fatmucket (Lampsilis siliquoidea), collected from the La-
Crosse River below Neshonoc Dam in Wisconsin (43854.874 0
N, 9184.5860W) on September 30, 2016. We opened the
mussels slightly with reverse pliers and collected a single,
approximately 1-mL hemolymph sample from each animal
using a needle and syringe inserted into the anterior adductor
muscle sinus, which is a nonlethal sampling method
(Gustafson et al. 2005). We then transferred the hemolymph
to a microcentrifuge tube, placed it on ice during transporta-
tion, and stored it at 808C until the samples were processed
for molecular analysis. This sampling was part of a pilot
monitoring effort to characterize microbes in the hemolymph
of mussels across the Upper Mississippi River basin.
To identify viruses in hemolymph, we used a virus
discovery method based on next-generation DNA sequencing
(NGS). NGS methods are ‘‘agnostic’’ —they can detect not
only known viruses but also unknown viruses that are
genomically similar to known viruses, without prior knowl-
edge of which viruses may be present (Munang’andu et al.
2017). These methods have revolutionized the study of
invertebrate viruses, revealing their extraordinary diversity
and deep evolutionary history (Shi et al. 2016; Wolf et al.
2018).
We used published methods optimized for detecting
viruses of all genomic compositions in fluids and tissues,
including those of aquatic organisms (Sibley et al. 2016;
Toohey-Kurth et al. 2017). Briefly, we extracted total nucleic
acids from 200 lL of hemolymph using the QIAamp MinElute
virus spin kit (Qiagen Inc., Valencia, CA, USA) and converted
RNA to double-stranded complementary DNA (dscDNA)
using the Superscript dscDNA synthesis kit (Invitrogen,
Carlsbad, CA, USA) with random hexamer priming. We then
prepared dscDNA for paired-end NGS on an Illumina MiSeq
instrument (MiSeq Reagent Kit v3, 2x150 cycle, Illumina, San
Diego, CA, USA) using the Nextera XT DNA sample prep kit
(Illumina). NGS reads were quality trimmed and analyzed for
similarity to viruses in the GenBank database as described by
Sibley et al. (2016) and Toohey-Kurth et al. (2017).
Figure 1. Phylogenetic tree of picornalike viruses. The major glycoprotein nucleic acid sequences of each virus were aligned using the codon-based Prank
algorithm (Loytynoja 2014) implemented in the program TranslatorX (Abascal et al. 2010), with the Gblocks algorithm (Castresana 2000) applied to remove
poorly aligned regions. The maximum-likelihood method implemented in the computer program PhyML (Guindon et al. 2010) was then applied to the resulting
1,332-position nucleic acid alignment, with the model of molecular evolution estimated from the data. Taxon names indicate abbreviated virus names (see below),
host, country, and year of collection. The novel picornalike virus from the Wabash Pigtoe is indicated with an arrow. Numbers beside branches show statistical
confidence of clades based on 1,000 bootstrap replicates of the data. Scale bar indicates nucleotide substitutions per site. Taxon abbreviations and GenBank
accession numbers: NoV: Nora virus (NC_007919); HoV-6: Hubei odonate virus 6 (NC_033071); HplV-66: Hubei picornalike virus 66 (NC_033133); HoV-7:
Hubei odonate virus 7 (NC_033232); WplV-47: Wenzhou picornalike virus 47 (NC_033150); MRplV-1: Mississippi River picornalike virus 1 (MK301250);
CplV-17: Changjiang picornalike virus 17 (KX884555); BplV-116: Beihai picornalike virus 116 (NC_032635); BsV-2: Beihai shrimp virus 2 (NC_032594);
WcV-6: Wenling crustacean virus 6 (NC_032810); WcV-5: Wenling crustacean virus 5 (NC_032839); BplV-114: Beihai picornalike virus 114 (NC_032633);
BplV-115: Beihai picornalike virus 115 (NC_032618); BssV-2: Beihai sea slater virus 2 (NC_032622); BplV-113: Beihai picornalike virus 113 (NC_032559);
BplV-112: Beihai picornalike virus 112 (NC_032571).
GOLDBERG ET AL.82
RESULTS
We obtained a total of 31,907,949 sequence reads (average
4,558,278 reads per individual mussel) with an average length
of 109 base pairs after quality trimming. We did not detect any
viruses in the animals from the La Crosse River or in the
Threeridge and one Wabash Pigtoe from the Mississippi River.
Sequences from the other Wabash Pigtoe mapped to a
picornalike virus with approximately 12-fold coverage,
yielding a complete open reading frame of 6,990 nucleotides
encoding a putative viral polyprotein gene of 2,329 amino
acids (GenBank accession number MK301250). The virus is a
member of the Nora virus-related clade of picornalike viruses,
named for the Nora virus of Drosophila fruit flies (Habayeb et
al. 2006), which have genomes of approximately 10,000 bases
of single-stranded, positive-sense RNA and infect a diverse
array of aquatic, marine, and terrestrial invertebrates (Shi et al.
2016). The virus is most closely related to the Wenzhou
picornalike virus 47 strain WHCCII11151 (GenBank acces-
sion number NC_033150) found in unspecified insects in
China in 2013 (Shi et al. 2016). It is more distantly related to
the Changjiang picornalike virus 17 strain CJLX30705
(GenBank accession number KX884555) found in unspecified
crayfish in China in 2014 (Shi et al. 2016) (Fig. 1).
DISCUSSION
The presence of a virus in a North American unionid is not
surprising, given the ubiquity of invertebrate viruses world-
wide (Shi et al. 2016; Munang’andu et al. 2017; Wolf et al.
2018). At present, no inferences should be made about the
role, if any, of this virus in the health of Wabash Pigtoes or
any other species it may infect. The phylogenetic similarity of
the Mississippi River picornalike virus 1 to arthropod viruses
from China is interesting as evidence of the global distribution
of the Nora virus clade of picornalike viruses, but because
current data on these viruses are geographically biased,
inferences about transmission or geographic spread also are
premature. However, our detection of this virus in the
hemolymph of only one mussel of seven indicates that such
viruses are not present in all animals, even of the same species
at the same place and time. We have not previously detected a
virus similar to the Mississippi River picornalike virus 1 in any
other sample sequenced in our laboratory despite analyzing
hundreds of samples from diverse sources, supporting the
conclusion that our results do not represent contamination.
Our results suggest that NGS-based methods will be useful
for identifying viruses in unionids and for investigating the
role, if any, of viruses in mortality events. We are currently
applying such methods to investigate unionid mass mortality
events in the Clinch River, Tennessee (Leis et al. 2018).
Applying these methods to carefully selected groups of
mussels of different health and disease states across different
geographic regions should provide useful information for
understanding how viruses may contribute to unionid declines
in general.
ACKNOWLEDGMENTS
We thank Nick Bloomfield, Kyle Mosel, Katie Lieder, and
Sara Erickson (Midwest Fisheries Center, U.S. Fish and
Wildlife Service [USFWS]) for assistance with mussel
collection. The findings and conclusions in this article are
those of the authors and do not necessarily represent the views
of the USFWS. Any use of trade, firm, or product names is for
descriptive purposes only and does not imply endorsement by
the USFWS, the U.S. Geological Survey, or the U.S.
Government.
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GOLDBERG ET AL.84
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