Journal of Virological Methods 206 (2014) 42–45

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Journal of Virological Methods

j ournal homepage: www.elsevier.com/locate/jviromet

Short communication

Identification and characterization of Highlands J from a

Mississippi sandhill crane using unbiased next-generation sequencing

a,∗ b a b

Hon S. Ip , Michael R. Wiley , Renee Long , Gustavo Palacios ,

b a,1

Valerie Shearn-Bochsler , Chris A. Whitehouse

a

U.S. Geological Survey, National Wildlife Health Center, Madison, WI, USA

b

Center for Genomic Sciences, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD, USA

a b s t r a c t

Article history: Advances in massively parallel DNA sequencing platforms, commonly termed next-generation sequenc-

Received 21 February 2014

ing (NGS) technologies, have greatly reduced time, labor, and cost associated with DNA sequencing. Thus,

Received in revised form 19 May 2014

NGS has become a routine tool for new viral pathogen discovery and will likely become the standard for

Accepted 20 May 2014

routine laboratory diagnostics of infectious diseases in the near future. This study demonstrated the

Available online 29 May 2014

application of NGS for the rapid identification and characterization of a virus isolated from the brain of an

endangered Mississippi sandhill crane. This was part of a population restoration effort and was found

Keywords:

in an emaciated state several days after Hurricane Isaac passed over the refuge in Mississippi in 2012.

Highlands J virus

Post-mortem examination had identified trichostrongyliasis as the possible cause of death, but because

Wildlife disease

Conservation a virus with morphology consistent with a togavirus was isolated from the brain of the bird, an arbovi-

Pathogen discovery ral etiology was strongly suspected. Because individual molecular assays for several known arboviruses

Next-generation sequencing were negative, unbiased NGS by Illumina MiSeq was used to definitively identify and characterize the

Rapid identification 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- Highlands J virus (HJV) is an arbovirus in the genus ,

ically endangered subspecies that had decreased to about 30–40 family Togaviridae. HJV is a member of the western equine

in the 1970s due to unrestricted hunting and the loss of their encephalitis (WEE) complex. Other members of this complex found

preferred habitat of wet pine savannas (Ellis et al., 2000). An effort in the United States include WEE virus (WEEV) and Fort Mor-

to increase the size of the population of Mississippi sandhill cranes gan virus (FMV). However, HJV is the only member of the WEE

began with the creation of the Mississippi Sandhill Crane National complex found in the eastern United States having a distribution

Wildlife Refuge in Jackson County, MS in 1975 and beginning in similar to that of eastern equine encephalitis virus (EEEV), and

1981, 10–15 birds raised in captivity are released each year (USFWS, circulates under apparently identical transmission cycles, sharing

2011). This is the largest crane restoration project in the world. Cur- the same enzootic vector ( melanura) and verte-

rently, there are only about 110–130 individuals remaining, and a brate amplifying hosts such as passerine birds (Cilnis et al., 1996;

better understanding of the factors that limit population growth of Scott and Weaver, 1989). HJV, unlike EEEV, has not been shown to

the Mississippi sandhill crane is needed to support restoration of be pathogenic to humans or horses, with the exception of a sin-

these birds. 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

∗

Corresponding author at: Diagnostic Virology Laboratory, U.S. Geological Survey, an important poultry pathogen and has caused widespread infec-

National Wildlife Health Center, 6006 Schroeder Road, Madison, WI 53711, USA.

tion of turkeys in North Carolina in the past (Ficken et al., 1993),

Tel.: +1 608 270 2464; fax: +1 608 270 2415.

mortality in chukar partridges in South Carolina (Eleazer and Hill,

E-mail address: [email protected] (H.S. Ip).

1 1994), and decreased egg production in domestic turkeys (Guy

Current address: Molecular and Translational Sciences Division, United States

Army Medical Research Institute of Infectious Diseases, Frederick, MD, USA. et al., 1994; Wages et al., 1993). HJV isolations or 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.

a

Scientific name Common 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 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 Day et al. (1996)

Sigmodon hispidus Day et al. (1996)

Equus ferus caballus Horse Karabatsos et al. (1988)

a

Scientific names in bold designate species in which the virus has been isolated.

virus have been reported from at least 19 species of North Amer- and infected with intestinal trematodes and nematodes but was

ican wild birds (see Table 1). While it has usually been assumed otherwise unremarkable with the exception of a mild meningoen-

that HJV is nonpathogenic to wild birds, HJV has been suspected cephalitis. Samples from the brain were submitted to the NWHC

to be the cause of death in a die-off of Florida scrub jays (Aph- Diagnostic Virology Laboratory for virus culture and identification.

elocoma coerulescens) that took place between 1979 and 1980 at A homogenate of the tissue was filtered through a 0.22 ␮m

the Archbold Biological Station, Highlands County, Florida and in syringe filter and inoculated onto Vero tissue culture cells as

the death of house sparrow (Passer domesticus) nestlings (Forrester described (Docherty et al., 2004). A virus was recovered as evidence

and Spalding, 2003). In addition, an epornitic of HJV occurred in by the observation of cytopathic effects. The virus had a typical mor-

upstate New York in 1986 (Howard et al., 2004). To our knowl- phology of a togavirus by electron microscopic examination and

edge, the virus has not been isolated previously from any species of real time RT-PCR tests for and EEEV were negative

cranes. (data not shown).

In August 2012, an attempt was made to retrieve a tagged Instead of continuing to pursue conventional diagnosis by rul-

Mississippi sandhill crane (U.S. Fish and Wildlife Service band# ing out successive viral agents individually, it was decided to apply

788-53521) due to lack of movement activity of its radio trans- the concept of complete sequence characterization using next

mitter two days after the passage of Hurricane Isaac. This bird generation sequencing (NGS) technology. Conventional methods,

(Bird# 721) was an adult female from the class of 2007. The including serological assays, polymerase chain reaction (PCR), or

bird was in poor condition and was euthanized on 8/31/2012 in microarrays require some prior knowledge of the virus, or at least

the field. The carcass was submitted to the U.S. Geological Sur- virus family; however, NGS is capable of randomly sequencing the

vey National Wildlife Health Center (NWHC) for pathological and entire nucleic acid content of a sample without the bias of sequenc-

microbiological analyses for the possible cause of death. A full ing technologies such as Sanger, which are dependent on selecting

necropsy was performed. The bird was found to be emaciated primers based on a priori information.

44 H.S. Ip et al. / Journal of Virological Methods 206 (2014) 42–45

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 currently being performed in most

Fig. 1. Whole genome molecular phylogenetic analysis by Maximum Likelihood

method of members of the Alphavirus genus. The evolutionary history was inferred diagnostic virology laboratories.

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.

Conflict of interests

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

The authors declare that they had no conflict of interests with

value. The tree is drawn to scale, with branch lengths measured in the number of

respect to their authorship or the publication of this manuscript.

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 Funding

et al., 2013).

Work performed at the USGS National Wildlife Health Cen-

Total RNA was extracted from viral supernatant with a com-

ter was supported, in part, by funding from the Department

2

mercial RNA kit according to manufacturer’s instructions. The

of the Interior’s Ecosystems Program. Work performed in the

RNA was amplified using sequence-independent single primer

Genomics Center at USAMRIID was supported by 1881290 CB2851

amplification (SISPA) as previously described (Djikeng et al.,

(TMTI0021 09 RD T) Genomics Center-High Speed Sequencing for

2008). Amplicons were sheared to ∼400 bp and used as start-

Rapid Response and Countermeasure Development.

3

ing material for Illumina TRU-seq DNA libraries construction.

Sequencing was performed on an Illumina MiSeq using a 2 × 250

Acknowledgments

kit obtaining 3.9 million paired-end reads. Illumina and SISPA

adapter sequences were trimmed from the sequencing reads using

We thank members of the USGS National Wildlife Health Cen-

Cutadapt-1.2.1 (Martin, 2011), quality filtering was conducted with

ter for their continued dedication to the service on behalf of the

Prinseq-lite (-min len 50-derep 14-lc method dust-lc threshold 3-

nation’s wildlife. In particular, we thank Craig Radi and Kathy Kurth

trim ns left 1-trim ns right 1-trim qual right 15) (Schmieder and

at the Wisconsin Veterinary Diagnostic Laboratory for electron

Edwards, 2011) and reads were assembled into contigs using Ray

microscopy and EEEV RT-PCR analysis.

Meta with kmer length = 25 (Boisvert et al., 2012). Contigs were

4 Any use of trade, product, or firm names is for descriptive

aligned to NCBI sequence database using BLAST. A contig of

purposes only and does not imply endorsement by the U.S.

11,365 nucleotide (nt) was generated and found to be 99% identical

Government. Opinions, interpretations, conclusions, and recom-

at the nucleotide level and to have 98.6% coverage to a HJV previ-

mendations are those of the author(s) and are not necessarily

ously isolated from the brain of a red-tailed hawk in Georgia (Fig. 1)

endorsed by the U.S. Army.

(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

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