Identification and characterization of Highlands J virus from a Mississippi sandhill crane using unbiased next-generation sequencing

Abstract

Advances in massively parallel DNA sequencing platforms, commonly termed next-generation sequencing (NGS) technologies, have greatly reduced time, labor, and cost associated with DNA sequencing. Thus, NGS has become a routine tool for new viral pathogen discovery and will likely become the standard for routine laboratory diagnostics of infectious diseases in the near future. This study demonstrated the application of NGS for the rapid identification and characterization of a virus isolated from the brain of an endangered Mississippi sandhill crane. This bird was part of a population restoration effort and was found in an emaciated state several days after Hurricane Isaac passed over the refuge in Mississippi in 2012. Post-mortem examination had identified trichostrongyliasis as the possible cause of death, but because a virus with morphology consistent with a togavirus was isolated from the brain of the bird, an arboviral etiology was strongly suspected. Because individual molecular assays for several known arboviruses were negative, unbiased NGS by Illumina MiSeq was used to definitively identify and characterize the causative viral agent. Whole genome sequencing and phylogenetic analysis revealed the viral isolate to be the Highlands J virus, a known avian pathogen. This study demonstrates the use of unbiased NGS for the rapid detection and characterization of an unidentified viral pathogen and the application of this technology to wildlife disease diagnostics and conservation medicine.

Full text

Journal
of
Virological
Methods
206
(2014)
42–45
Contents
lists
available
at
ScienceDirect
Journal
of
Virological
Methods
j
o
ur
nal
ho
me
pag
e:
www.elsevier.com/locate/jviromet
Short
communication
Identification
and
characterization
of
Highlands
J
virus
from
a
Mississippi
sandhill
crane
using
unbiased
next-generation
sequencing
Hon
S.
Ipa,∗,
Michael
R.
Wileyb,
Renee
Longa,
Gustavo
Palaciosb,
Valerie
Shearn-Bochslerb,
Chris
A.
Whitehousea,1
aU.S.
Geological
Survey,
National
Wildlife
Health
Center,
Madison,
WI,
USA
bCenter
for
Genomic
Sciences,
United
States
Army
Medical
Research
Institute
of
Infectious
Diseases,
Frederick,
MD,
USA
Article
history:
Received
21
February
2014
Received
in
revised
form
19
May
2014
Accepted
20
May
2014
Available
online
29
May
2014
Keywords:
Highlands
J
virus
Wildlife
disease
Conservation
Pathogen
discovery
Next-generation
sequencing
Rapid
identification
a
b
s
t
r
a
c
t
Advances
in
massively
parallel
DNA
sequencing
platforms,
commonly
termed
next-generation
sequenc-
ing
(NGS)
technologies,
have
greatly
reduced
time,
labor,
and
cost
associated
with
DNA
sequencing.
Thus,
NGS
has
become
a
routine
tool
for
new
viral
pathogen
discovery
and
will
likely
become
the
standard
for
routine
laboratory
diagnostics
of
infectious
diseases
in
the
near
future.
This
study
demonstrated
the
application
of
NGS
for
the
rapid
identification
and
characterization
of
a
virus
isolated
from
the
brain
of
an
endangered
Mississippi
sandhill
crane.
This
bird
was
part
of
a
population
restoration
effort
and
was
found
in
an
emaciated
state
several
days
after
Hurricane
Isaac
passed
over
the
refuge
in
Mississippi
in
2012.
Post-mortem
examination
had
identified
trichostrongyliasis
as
the
possible
cause
of
death,
but
because
a
virus
with
morphology
consistent
with
a
togavirus
was
isolated
from
the
brain
of
the
bird,
an
arbovi-
ral
etiology
was
strongly
suspected.
Because
individual
molecular
assays
for
several
known
arboviruses
were
negative,
unbiased
NGS
by
Illumina
MiSeq
was
used
to
definitively
identify
and
characterize
the
causative
viral
agent.
Whole
genome
sequencing
and
phylogenetic
analysis
revealed
the
viral
isolate
to
be
the
Highlands
J
virus,
a
known
avian
pathogen.
This
study
demonstrates
the
use
of
unbiased
NGS
for
the
rapid
detection
and
characterization
of
an
unidentified
viral
pathogen
and
the
application
of
this
technology
to
wildlife
disease
diagnostics
and
conservation
medicine.
Published
by
Elsevier
B.V.
This
is
an
open
access
article
under
the
CC
BY-NC-SA
license
(http://creativecommons.org/licenses/by-nc-sa/3.0/).
The
Mississippi
sandhill
crane
(Grus
canadensis
pulla)
is
a
crit-
ically
endangered
subspecies
that
had
decreased
to
about
30–40
birds
in
the
1970s
due
to
unrestricted
hunting
and
the
loss
of
their
preferred
habitat
of
wet
pine
savannas
(Ellis
et
al.,
2000).
An
effort
to
increase
the
size
of
the
population
of
Mississippi
sandhill
cranes
began
with
the
creation
of
the
Mississippi
Sandhill
Crane
National
Wildlife
Refuge
in
Jackson
County,
MS
in
1975
and
beginning
in
1981,
10–15
birds
raised
in
captivity
are
released
each
year
(USFWS,
2011).
This
is
the
largest
crane
restoration
project
in
the
world.
Cur-
rently,
there
are
only
about
110–130
individuals
remaining,
and
a
better
understanding
of
the
factors
that
limit
population
growth
of
the
Mississippi
sandhill
crane
is
needed
to
support
restoration
of
these
birds.
∗Corresponding
author
at:
Diagnostic
Virology
Laboratory,
U.S.
Geological
Survey,
National
Wildlife
Health
Center,
6006
Schroeder
Road,
Madison,
WI
53711,
USA.
Tel.:
+1
608
270
2464;
fax:
+1
608
270
2415.
E-mail
address:
hip@usgs.gov
(H.S.
Ip).
1Current
address:
Molecular
and
Translational
Sciences
Division,
United
States
Army
Medical
Research
Institute
of
Infectious
Diseases,
Frederick,
MD,
USA.
Highlands
J
virus
(HJV)
is
an
arbovirus
in
the
genus
Alphavirus,
family
Togaviridae.
HJV
is
a
member
of
the
western
equine
encephalitis
(WEE)
complex.
Other
members
of
this
complex
found
in
the
United
States
include
WEE
virus
(WEEV)
and
Fort
Mor-
gan
virus
(FMV).
However,
HJV
is
the
only
member
of
the
WEE
complex
found
in
the
eastern
United
States
having
a
distribution
similar
to
that
of
eastern
equine
encephalitis
virus
(EEEV),
and
circulates
under
apparently
identical
transmission
cycles,
sharing
the
same
enzootic
mosquito
vector
(Culiseta
melanura)
and
verte-
brate
amplifying
hosts
such
as
passerine
birds
(Cilnis
et
al.,
1996;
Scott
and
Weaver,
1989).
HJV,
unlike
EEEV,
has
not
been
shown
to
be
pathogenic
to
humans
or
horses,
with
the
exception
of
a
sin-
gle
report
of
the
virus
being
isolated
from
the
brain
of
a
horse
that
died
with
encephalitis
in
Florida
in
1964
(Karabatsos
et
al.,
1988)
and
four
human
encephalitis
cases
that
were
co-infected
with
St.
Louis
Encephalitis
virus
(SLEV)
(Meehan
et
al.,
2000).
HJV
is
an
important
poultry
pathogen
and
has
caused
widespread
infec-
tion
of
turkeys
in
North
Carolina
in
the
past
(Ficken
et
al.,
1993),
mortality
in
chukar
partridges
in
South
Carolina
(Eleazer
and
Hill,
1994),
and
decreased
egg
production
in
domestic
turkeys
(Guy
et
al.,
1994;
Wages
et
al.,
1993).
HJV
isolations
or
antibodies
to
the
http://dx.doi.org/10.1016/j.jviromet.2014.05.018
0166-0934/Published
by
Elsevier
B.V.
This
is
an
open
access
article
under
the
CC
BY-NC-SA
license
(http://creativecommons.org/licenses/by-nc-sa/3.0/).

H.S.
Ip
et
al.
/
Journal
of
Virological
Methods
206
(2014)
42–45
43
Table
1
Known
reported
detections
of
Highlands
J
virus
in
wild
animal
species.
Scientific
nameaCommon
name
Reference
Agelaius
phoeniceus
Red-winged
blackbird
Forrester
and
Spalding
(2003)
Aphelocoma
coerulescens Florida
scrub
jay Forrester
and
Spalding
(2003)
Baeolophus
bicolor
Tufted
titmouse
McLean
et
al.
(1985)
Bombycilla
cedrorum
Cedar
waxwing
Howard
et
al.
(2004)
Bonasa
umbellus
Ruffed
grouse
Howard
et
al.
(2004)
Buteo
jamaicensis
Red-tailed
hawk
Allison
and
Stallknecht
(2009)
Cardinalis
cardinalis
Northern
cardinal
McLean
et
al.
(1985)
Carpodacus
purpureus
Purple
finch
Howard
et
al.
(2004)
Catharus
fuscescens Veery
Howard
et
al.
(2004)
Colaptes
auratus
Northern
(common)
flicker
Main
et
al.
(1988)
Colinus
virginianus
Bobwhite
quail
Forrester
and
Spalding
(2003)
Columbina
passerina
Common
ground
dove
Forrester
and
Spalding
(2003)
Cyanocitta
cristata
Blue
jay
Forrester
and
Spalding
(2003)
Dendroica
pensylvanica
Chestnut-sided
warbler
Howard
et
al.
(2004)
Dumetella
carolinensis
Gray
catbird
Howard
et
al.
(2004)
Egretta
caerulea
Little
blue
heron
Forrester
and
Spalding
(2003)
Geothlypis
trichas
Common
yellowthroat
Main
et
al.
(1988)
Grus
canadensis
pulla Mississippi
sandhill
crane This
report
Hirundo
rustica
Barn
swallow
McLean
et
al.
(1985)
Hylocichla
mustelina
Wood
thrush
Howard
et
al.
(2004)
Icterus
galbula
Northern
oriole
Howard
et
al.
(2004)
Melospiza
georgiana
Swamp
sparrow
Main
et
al.
(1988)
Melospiza
melodia
Song
sparrow
McLean
et
al.
(1985)
Miniotilta
varia
Black
and
white
warbler
Forrester
and
Spalding
(2003)
Myiarchus
crinituss Great
crested
flycatcher McLean
et
al.
(1985)
Pandion
haliaetus
Osprey
Forrester
and
Spalding
(2003)
Passer
domesticus
House
sparrow
Johnson
(1960)
Picoides
pubescens
Downy
woodpecker
Howard
et
al.
(2004)
Pipilo
erythrophthalmus
Eastern
towhee
Forrester
and
Spalding
(2003)
Piranga
olivacea Scarlet
tanager Howard
et
al.
(2004)
Poecile
atricapillus
Black-capped
chickadee
Main
et
al.
(1988)
Quiscalus
quiscula Common
grackle
Forrester
and
Spalding
(2003)
Seiurus
aurocapillus
Ovenbird
Howard
et
al.
(2004)
Setophaga
ruticilla
American
redstart
Howard
et
al.
(2004)
Spinus
tristis
American
goldfinch
Howard
et
al.
(2004)
Sturnella
magna
Eastern
meadowlark
Forrester
and
Spalding
(2003)
Toxostoma
rufum Brown
thrasher
Howard
et
al.
(2004)
Turdus
migratorius
American
robin
Main
et
al.
(1988)
Vireo
flavifrons
Yellow-throated
vireo
Howard
et
al.
(2004)
Vireo
gilvus
Warbling
vireo
Howard
et
al.
(2004)
Vireo
olivaceus
Red-eyed
vireo
Howard
et
al.
(2004)
Zonotrichia
albicollis
White-throated
sparrow
Howard
et
al.
(2004)
Mammals
Peromyscus
gossypinus
Cotton
mouse
Day
et
al.
(1996)
Sigmodon
hispidus
Cotton
rat
Day
et
al.
(1996)
Equus
ferus
caballus
Horse
Karabatsos
et
al.
(1988)
aScientific
names
in
bold
designate
species
in
which
the
virus
has
been
isolated.
virus
have
been
reported
from
at
least
19
species
of
North
Amer-
ican
wild
birds
(see
Table
1).
While
it
has
usually
been
assumed
that
HJV
is
nonpathogenic
to
wild
birds,
HJV
has
been
suspected
to
be
the
cause
of
death
in
a
die-off
of
Florida
scrub
jays
(Aph-
elocoma
coerulescens)
that
took
place
between
1979
and
1980
at
the
Archbold
Biological
Station,
Highlands
County,
Florida
and
in
the
death
of
house
sparrow
(Passer
domesticus)
nestlings
(Forrester
and
Spalding,
2003).
In
addition,
an
epornitic
of
HJV
occurred
in
upstate
New
York
in
1986
(Howard
et
al.,
2004).
To
our
knowl-
edge,
the
virus
has
not
been
isolated
previously
from
any
species
of
cranes.
In
August
2012,
an
attempt
was
made
to
retrieve
a
tagged
Mississippi
sandhill
crane
(U.S.
Fish
and
Wildlife
Service
band#
788-53521)
due
to
lack
of
movement
activity
of
its
radio
trans-
mitter
two
days
after
the
passage
of
Hurricane
Isaac.
This
bird
(Bird#
721)
was
an
adult
female
from
the
class
of
2007.
The
bird
was
in
poor
condition
and
was
euthanized
on
8/31/2012
in
the
field.
The
carcass
was
submitted
to
the
U.S.
Geological
Sur-
vey
National
Wildlife
Health
Center
(NWHC)
for
pathological
and
microbiological
analyses
for
the
possible
cause
of
death.
A
full
necropsy
was
performed.
The
bird
was
found
to
be
emaciated
and
infected
with
intestinal
trematodes
and
nematodes
but
was
otherwise
unremarkable
with
the
exception
of
a
mild
meningoen-
cephalitis.
Samples
from
the
brain
were
submitted
to
the
NWHC
Diagnostic
Virology
Laboratory
for
virus
culture
and
identification.
A
homogenate
of
the
tissue
was
filtered
through
a
0.22
␮m
syringe
filter
and
inoculated
onto
Vero
tissue
culture
cells
as
described
(Docherty
et
al.,
2004).
A
virus
was
recovered
as
evidence
by
the
observation
of
cytopathic
effects.
The
virus
had
a
typical
mor-
phology
of
a
togavirus
by
electron
microscopic
examination
and
real
time
RT-PCR
tests
for
West
Nile
virus
and
EEEV
were
negative
(data
not
shown).
Instead
of
continuing
to
pursue
conventional
diagnosis
by
rul-
ing
out
successive
viral
agents
individually,
it
was
decided
to
apply
the
concept
of
complete
sequence
characterization
using
next
generation
sequencing
(NGS)
technology.
Conventional
methods,
including
serological
assays,
polymerase
chain
reaction
(PCR),
or
microarrays
require
some
prior
knowledge
of
the
virus,
or
at
least
virus
family;
however,
NGS
is
capable
of
randomly
sequencing
the
entire
nucleic
acid
content
of
a
sample
without
the
bias
of
sequenc-
ing
technologies
such
as
Sanger,
which
are
dependent
on
selecting
primers
based
on
a
priori
information.

44
H.S.
Ip
et
al.
/
Journal
of
Virological
Methods
206
(2014)
42–45
Fig.
1.
Whole
genome
molecular
phylogenetic
analysis
by
Maximum
Likelihood
method
of
members
of
the
Alphavirus
genus.
The
evolutionary
history
was
inferred
by
using
the
Maximum
Likelihood
method
based
on
the
JTT
matrix-based
model
(Jones
et
al.,
1992).
The
tree
with
the
highest
log
likelihood
(−70442.5088)
is
shown.
Initial
tree(s)
for
the
heuristic
search
were
obtained
automatically
by
applying
Neighbor-Joining
and
BioNJ
algorithms
to
a
matrix
of
pairwise
distances
estimated
using
a
JTT
model,
and
then
selecting
the
topology
with
superior
log
likelihood
value.
The
tree
is
drawn
to
scale,
with
branch
lengths
measured
in
the
number
of
substitutions
per
site.
The
analysis
involved
24
amino
acid
sequences.
All
positions
containing
gaps
and
missing
data
were
eliminated.
There
were
a
total
of
1402
pos-
itions
in
the
final
dataset.
Evolutionary
analyses
were
conducted
in
MEGA6
(Tamura
et
al.,
2013).
Total
RNA
was
extracted
from
viral
supernatant
with
a
com-
mercial
RNA
kit
according
to
manufacturer’s
instructions.2The
RNA
was
amplified
using
sequence-independent
single
primer
amplification
(SISPA)
as
previously
described
(Djikeng
et
al.,
2008).
Amplicons
were
sheared
to
∼400
bp
and
used
as
start-
ing
material
for
Illumina
TRU-seq
DNA
libraries
construction.3
Sequencing
was
performed
on
an
Illumina
MiSeq
using
a
2
×
250
kit
obtaining
3.9
million
paired-end
reads.
Illumina
and
SISPA
adapter
sequences
were
trimmed
from
the
sequencing
reads
using
Cutadapt-1.2.1
(Martin,
2011),
quality
filtering
was
conducted
with
Prinseq-lite
(-min
len
50-derep
14-lc
method
dust-lc
threshold
3-
trim
ns
left
1-trim
ns
right
1-trim
qual
right
15)
(Schmieder
and
Edwards,
2011)
and
reads
were
assembled
into
contigs
using
Ray
Meta
with
kmer
length
=
25
(Boisvert
et
al.,
2012).
Contigs
were
aligned
to
NCBI
sequence
database
using
BLAST.4A
contig
of
11,365
nucleotide
(nt)
was
generated
and
found
to
be
99%
identical
at
the
nucleotide
level
and
to
have
98.6%
coverage
to
a
HJV
previ-
ously
isolated
from
the
brain
of
a
red-tailed
hawk
in
Georgia
(Fig.
1)
(Allison
and
Stallknecht,
2009).
The
missing
regions
included
80
nt
from
the
beginning
of
the
5end
and
81
nt
from
the
3end
of
2Applied
Biosystems
MagMax
Viral
RNA
Extraction
Kit,
Life
Technologies,
Grand
Island,
NY.
3Illumina,
Inc.,
San
Diego,
CA.
4Available
at:
http://www.ncbi.nlm.nih.gov/BLAST/.
the
genomic
RNA,
and
are
typical
of
the
coverage
drop-off
when
amplifying
using
random
PCR.
Adapter
trimmed
reads
were
aligned
back
to
the
assembled
HJV
sequence
using
Bowtie2
(Langmead
and
Salzberg,
2012)
and
custom
scripts
to
generate
a
final
con-
sensus
sequence.
The
isolate
has
been
named
Mississippi
Sandhill
Crane/Mississippi/186745/2012
(HJV)
and
deposited
in
GenBank
as
accession
KJ409555.
When
compared
to
the
closest
HJV
strain,
56
nt
changes
were
found,
45
synonymous
and
11
nonsynonymous
(Supplemental
Table
1).
See
Table
S1
as
supplementary
file.
Supplementary
data
asso-
ciated
with
this
article
can
be
found,
in
the
online
version,
at
http://dx.doi.org/10.1016/j.jviromet.2014.05.018.
We
were
unable
to
determine
if
the
HJV
is
the
formal
cause
of
the
emaciation
and
ultimately
the
death
of
Bird#
127.
While
the
presence
of
encephalitis
and
the
isolation
of
HJV
from
the
brain
are
highly
suggestive,
we
have
not
demonstrated
the
presence
of
the
virus
in
the
brain
lesions
at
the
present
time.
Whether
cranes
in
general,
or
the
Mississippi
sandhill
crane
subspecies
in
particu-
lar,
are
especially
susceptible
to
HJV
should
be
investigated
in
the
future
by
laboratory
challenge
experiments.
This
study
shows
an
example
of
how
NGS
can
be
used
quickly
to
determine
the
identity
of
a
novel
viral
isolate
and
at
the
same
time
to
derive
a
nearly
complete
genome
sequence
of
the
virus.
While
a
truly
universal
NGS
approach
should
also
accommodate
pathogens
with
a
DNA
genome,
the
ability
of
NGS
technology
to
sequence
the
entire
nucleotide
coding
space
of
a
sample
rapidly
and
without
the
need
for
a
priori
sequence
information
will
greatly
assist
in
the
characterization
of
novel
emerging
pathogens
as
well
as
help
to
eliminate
the
costly
and
time-consuming
sequential
one-by-one
tests
to
rule
out
known
viruses
currently
being
performed
in
most
diagnostic
virology
laboratories.
Conflict
of
interests
The
authors
declare
that
they
had
no
conflict
of
interests
with
respect
to
their
authorship
or
the
publication
of
this
manuscript.
Funding
Work
performed
at
the
USGS
National
Wildlife
Health
Cen-
ter
was
supported,
in
part,
by
funding
from
the
Department
of
the
Interior’s
Ecosystems
Program.
Work
performed
in
the
Genomics
Center
at
USAMRIID
was
supported
by
1881290
CB2851
(TMTI0021
09
RD
T)
Genomics
Center-High
Speed
Sequencing
for
Rapid
Response
and
Countermeasure
Development.
Acknowledgments
We
thank
members
of
the
USGS
National
Wildlife
Health
Cen-
ter
for
their
continued
dedication
to
the
service
on
behalf
of
the
nation’s
wildlife.
In
particular,
we
thank
Craig
Radi
and
Kathy
Kurth
at
the
Wisconsin
Veterinary
Diagnostic
Laboratory
for
electron
microscopy
and
EEEV
RT-PCR
analysis.
Any
use
of
trade,
product,
or
firm
names
is
for
descriptive
purposes
only
and
does
not
imply
endorsement
by
the
U.S.
Government.
Opinions,
interpretations,
conclusions,
and
recom-
mendations
are
those
of
the
author(s)
and
are
not
necessarily
endorsed
by
the
U.S.
Army.
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