Application of Broad-Spectrum Resequencing …jvi.asm.org/content/84/18/9557.full.pdf... Lyssavirus...

JOURNAL OF VIROLOGY, Sept. 2010, p. 9557–9574 Vol. 84, No. 180022-538X/10/$12.00 doi:10.1128/JVI.00771-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Application of Broad-Spectrum Resequencing Microarray forGenotyping Rhabdoviruses�

Laurent Dacheux,1‡ Nicolas Berthet,2‡ Gabriel Dissard,3 Edward C. Holmes,4 Olivier Delmas,1Florence Larrous,1 Ghislaine Guigon,3 Philip Dickinson,5 Ousmane Faye,6 Amadou A. Sall,6

Iain G. Old,7 Katherine Kong,5 Giulia C. Kennedy,5 Jean-Claude Manuguerra,8Stewart T. Cole,9 Valerie Caro,3 Antoine Gessain,2 and Herve Bourhy1*

Institut Pasteur, Lyssavirus Dynamics and Host Adaptation Unit, Paris, France1; Institut Pasteur, Epidemiology andPathophysiology Oncogenic Virus Unit, CNRS URA3015, Paris, France2; Institut Pasteur, Genotyping of

Pathogens and Public Health Technological Platform, Paris, France3; Center for Infectious Disease Dynamics,Department of Biology, The Pennsylvania State University, University Park, Pennsylvania4; Affymetrix,

Santa Clara, California5; Institut Pasteur de Dakar, Arbovirology Laboratory, Dakar, Senegal6;Institut Pasteur, European Office, Paris, France7; Institut Pasteur, Laboratory for

Urgent Responses to Biological Threats, Paris, France8; and Institut Pasteur,Bacterial Molecular Genetics Unit, Paris, France9

Received 12 April 2010/Accepted 29 June 2010

The rapid and accurate identification of pathogens is critical in the control of infectious disease. To this end,we analyzed the capacity for viral detection and identification of a newly described high-density resequencingmicroarray (RMA), termed PathogenID, which was designed for multiple pathogen detection using databasesimilarity searching. We focused on one of the largest and most diverse viral families described to date, thefamily Rhabdoviridae. We demonstrate that this approach has the potential to identify both known and relatedviruses for which precise sequence information is unavailable. In particular, we demonstrate that a strategybased on consensus sequence determination for analysis of RMA output data enabled successful detection ofviruses exhibiting up to 26% nucleotide divergence with the closest sequence tiled on the array. Using clinicalspecimens obtained from rabid patients and animals, this method also shows a high species level concordancewith standard reference assays, indicating that it is amenable for the development of diagnostic assays. Finally,12 animal rhabdoviruses which were currently unclassified, unassigned, or assigned as tentative species withinthe family Rhabdoviridae were successfully detected. These new data allowed an unprecedented phylogeneticanalysis of 106 rhabdoviruses and further suggest that the principles and methodology developed here may beused for the broad-spectrum surveillance and the broader-scale investigation of biodiversity in the viral world.

The ability to simultaneously screen for a large panel ofpathogens in clinical samples, especially viruses, will representa major development in the diagnosis of infectious diseasesand in surveillance programs for emerging pathogens. Cur-rently, most diagnostic methods are based on species-specificviral nucleic acid amplification. Although rapid and extremelysensitive, these methods are suboptimal when testing for alarge number of known pathogens, when viral sequence diver-gence is high, when new but related viruses are anticipated, orwhen no clear viral etiologic agent is suspected. To overcomethese technical difficulties, newer technologies have been em-ployed, especially microarrays dedicated to pathogen detec-tion. Indeed, DNA microarrays have been shown to be a pow-erful platform for the highly multiplexed differential diagnosisof infectious diseases. For example, pathogen microarrays canbe simultaneously used to screen various viral or bacterialfamilies and have been successfully used in the detection of

microbial agents from different clinical samples (10–12, 19, 32,35, 41, 42, 48).

The “classical” DNA microarrays developed so far are basedon the use of long-oligonucleotide pathogen-specific probes(�50 nucleotides [nt]). Although powerful in terms of sensi-tivity, these diagnostic tools have the disadvantage of de-creased specificity, making it necessary to target multiplemarkers, and rely on hybridization patterns for pathogen iden-tification, leading to unquantifiable errors (4). Moreover, thesemethods lack comprehensive information about the pathogenat the single-nucleotide level, which could represent a majorproblem when the sequences in question show a high degree ofsimilarity (21). The microarray-based pathogen resequencingassay represents a promising alternative tool with which toovercome these limitations. This method identifies each spe-cific pathogen and is capable of resequencing, or “fingerprint-ing,” multiple pathogens in a single test. Indeed, this technol-ogy uses tiled sets of 105 to 106 probes of 25mers, whichcontain one perfectly matched and three mismatched probesper base for both strands of the target genes (16). This tech-nology also offers the potential for a single test that detects anddiscriminates between a target pathogen and its closest phylo-genetic neighbors, which expands the repertoire of identifiableorganisms far beyond those that are initially included in thearray. Successful results have been obtained using this tech-

* Corresponding author. Mailing address: Unite Dynamique deslyssavirus et adaptation a l’hote, Institut Pasteur, 25 rue du DocteurRoux, 75724 Paris Cedex 15, France. Phone: 33 1 45 68 87 85. Fax: 331 40 61 30 20. E-mail: [email protected].

‡ Contributed equally to this work.� Published ahead of print on 7 July 2010.

9557

on June 1, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

http://jvi.asm.org/

nology, especially for the detection of broad-spectrum respira-tory tract pathogens using respiratory pathogen microarrays (2,25, 26) or the detection of a broad range of biothreat agents (1,23, 36, 45). The amplification step, which is more often limitingfor this technology, has also benefited from recent develop-ments. Phi29 polymerase-based amplification methods provideamplified DNA with minimal changes in sequence and relativeabundance for many biomedical applications (3, 31, 40). Theamplification factor varied from 106 to 109, and it was alsodemonstrated that coamplification occurred when viral RNAwas mixed with bacterial DNA (3). This whole-transcriptomeamplification (WTA) approach can also be successfully appliedto viral genomic RNA of all sizes. Amplifying viral RNA byWTA provides considerably better sensitivity and accuracy ofdetection than random reverse transcription (RT)-PCR in thecontext of resequencing microarrays (RMAs) (3).

The rhabdoviruses are single-stranded, negative-sense RNAgenome viruses classified into six genera, three of which—Vesiculovirus, Lyssavirus, and Ephemerovirus—include arthro-pod-borne agents that infect birds, reptiles, and mammals, aswell as a variety of non-vector-borne mammalian or fish viruses(International Committee on Taxonomy of Viruses database[ICTVdb]) (reviewed in reference 7). These rhabdoviruses arethe etiological agents of human diseases, such as rabies, thatcause serious public health problems. Some rhabdoviruses alsocause important economic losses in livestock. The three othersgenera include Nucleorhabdovirus and Cytorhabdovirus, whichare arthropod-borne viruses infecting plants, and Novirhab-dovirus, which comprises fish viruses. Other than the well-characterized rhabdoviruses that are known to be importantfor agriculture and public health, there is also a constantlygrowing list of rhabdoviruses, isolated from a variety of verte-brate and invertebrate hosts, that are partially characterizedand are still waiting for definitive genus or species assignment.Considering the large spectrum of potential animal reservoirsof these viruses compared to the few identified virus species, itis highly likely that the number of uncharacterized rhabdovi-ruses is immense.

Unclassified or unassigned viruses have been tentativelyidentified as members of the family Rhabdoviridae by electronmicroscopy, based on their bullet-shaped morphology—a char-acteristic trait of members of this family—or using their anti-genic relationships based on serological tests (9, 38). Genesequencing and phylogenetic relationships have then been pro-gressively applied to complete this initial virus taxonomy (6, 22,27). Importantly, a strongly conserved domain in the rhabdovi-rus genome, within the polymerase gene, is a useful target forthe exploration of the distant evolutionary relationships amongthese diverse viruses (6). This region corresponds to block IIIof the viral polymerase, a region predicted to be essential forRNA polymerase function, as it is highly conserved amongmost of the RNA-dependent RNA polymerases (14, 33, 46). Adirect application using this sequence region was recently de-scribed for lyssavirus RNA detection in human rabies diagnosis(13). Taking advantage of these characteristics, this polymer-ase region was also used to design probes for high-densityRMAs, also called PathogenID arrays (Affymetrix), which areoptimized for the detection and sequence determination ofseveral RNA viruses, particularly rhabdoviruses (1).

In the present study, PathogenID microarrays containing

probes for the detection of up to 126 viruses were tested usinga consensus sequence determination strategy for the analysis ofoutput RMA data. We demonstrate that this approach has thepotential to identify, in experimentally infected and clinicalspecimens, known but also phylogenetically related rhabdovi-ruses for which precise sequence information was not avail-able.

MATERIALS AND METHODS

Design of the PathogenID microarray for rhabdovirus detection. Two gener-ations of PathogenID arrays were used in this study: PathogenID v1.0, containingprobes for the detection of 42 viruses (including 3 prototype rhabdoviruses), 50bacteria, and 619 toxin or antibiotic resistance genes (previously described inreference 1), and PathogenID v2.0, which is able to detect 126 viruses (including30 different rhabdoviruses), 124 bacteria, 673 toxin or antibiotic resistance genes,and two human genes as controls. These arrays include prototype sequences ofall of the species (or genotypes) of the genus Lyssavirus, of the other majorgenera defined in the family Rhabdoviridae, such as Ephemerovirus and Vesicu-lovirus, and of 13 rhabdoviruses awaiting classification or tentatively classifiedamong minor groups such as the Le Dantec and Hark Park groups (6). For all ofthe selected probes tiled on the two versions of the PathogenID array, the sameconserved region of the viral polymerase gene was used (block III). However, thesize of the target region tiled on the array was longer in the second version (upto 937 nt in length for some sequences, compared to roughly 500 nt in the firstversion) (Tables 1 and 2).

Virus strains and biological samples analyzed. Detailed descriptions of all ofthe prototype and field virus strains used in this study and their sources are listedin Tables 1 and 2. Briefly, 16 and 31 different viruses were tested using Patho-genID v1.0 (15 lyssaviruses and 1 vesiculovirus) and PathogenID v2.0 (14 lyssa-viruses, 1 vesiculovirus, and 12 unassigned and 4 tentative species of animalrhabdoviruses according to ICTVdb), respectively. Samples tested included invitro-infected cells, a synthetic nucleotide target (when the corresponding virusstrain was not available), brain biopsy specimens obtained from experimentallyinfected mice, and biological specimens from various animals (bat, cat, dog, andfox brains) and humans (brain, saliva, and skin biopsy specimens).

Extraction and amplification of viral RNA. RNA extraction from biologicalsamples was processed with TRI Reagent (Molecular Research Center) accord-ing to the manufacturer’s recommendations. After extraction, viral RNAs werereverse transcribed and then amplified using the whole-transcriptome amplifica-tion (WTA) protocol (QuantiTect Whole Transcriptome kit; Qiagen) as de-scribed previously (3).

Microarrays assay. All of the amplification products obtained from viral RNAwere quantified by Quantit BR (Invitrogen) according to the manufacturer’sinstructions or by the NanoDrop ND-1000 spectrophotometer instrument(Thermo Scientific). A recommended amount of target DNA was fragmentedand labeled according to GeneChip Resequencing Assay manual (Affymetrix).The microarray hybridization process was carried out according to the protocolrecommended by the manufacturer (Affymetrix). All of the details and param-eter settings for the data analysis (essentially conversion of raw image filesobtained from scanning of the microarrays into FASTA files containing thesequences of base calls made for each tiled region of the microarray) have beendescribed previously (1). The base call rate refers to the percentage of base callsgenerated from the full-length tiled sequence.

Data analysis. In the first approach, resequencing data obtained by the Patho-genID v1.0 microarray were manually submitted to the NCBI nr/nt database forBLASTN query. The default BLAST options were modified. The word size wasset to 7 nt. The expected threshold was increased from its default value of 10 to100,000 to reduce the filtering of short sequences and sequences rich in unde-termined calls, which can assist correct taxonomic identification. To avoid false-negative results induced by high numbers of undetermined nucleotides in thesequences, the “low complexity level filter” (�F) was also turned off. BLASTsorts the resulting hits according to their bit scores so that the sequence that isthe most similar to the entry sequence appears first. Identification of the virusstrains tested was considered successful only when the best hit was unique andcorresponded to the expected species or isolate (according to the nucleotidesequences of these viruses already available in the NCBI nr/nt database).

In the second approach, an automatic bioinformatics-based analysis of RMAdata provided by PathogenID v2.0 was developed, including a consensus se-quence determination strategy completed with a systematic BLAST strategy. Thegeneral workflow of this strategy is represented in Fig. 1. A Perl script reads the

9558 DACHEUX ET AL. J. VIROL.


.org/D

ownloaded from

http://jvi.asm.org/

input data, which consist of one FASTA file per sample that contains all of thesequences read by the GSEQ software from the hybridization. A modified ver-sion of the filtering process described by Malanoski et al. (29) is applied to thesequences. The retained sequences contain stretches of nucleotides that areascertained according to the following algorithm. Briefly, sequences that do notcontain subsequences fulfilling specific parameters (minimum nucleotide length[m] and maximum undetermined nucleotide content [N]) defined by the user arediscarded. These parameters differ from those described in the original filteringprocess, where m was fixed to 20 and N was a value depending on m, leading tothe filtering out of all short subsequences, even with a high base call rate. Forsubsequence determination, the program starts from the first base call of thesequence considered and searches for the first m base window area that scoresthe elongation threshold defined by the user, which represents another differencefrom the filtering process described by Malanoski et al., where this elongationthreshold was fixed at 60% (29). The subsequence is extended by one base (m �1) if the percentage of N remains inferior to the elongation threshold. When thisthreshold is exceeded, the elongation is stopped and the subsequence is con-served. This process is reiterated until the end of the sequence is reached togenerate as many informative sequences as possible. All of our analyses wereperformed with the following filtering parameters: m � 12, N � 10, and elon-gation threshold � 10%.

A systematic BLAST strategy to search for sequence homologues was thenperformed with the filtered sequences containing subsequences. These se-

quences individually undergo a BLAST analysis based on a local viral andbacterial database (sequences obtained after filtering from the NCBI nr/nt da-tabase, updated and used for BLAST queries in December 2009), and thetaxonomies of the best BLAST hits are retrieved (Fig. 1A). The default BLASToptions were modified as previously described. When several hits obtain thehighest bit score, the script automatically retrieves the taxonomies of the 10 firstBLAST hits. The final taxonomic identification of each virus strain tested wasdone by the user as follows: (i) identification at the species or isolate level whena unique best hit corresponds to the expected species or isolate, (ii) identificationat the genus level (if available) when multiple best viral hits exist and correspondto different species within the same genus of the family Rhabdoviridae, (iii)identification at the family level when multiple best viral hits exist and corre-spond to different rhabdoviruses genera, or (iv) negative or inaccurate identifi-cation when a BLAST query is not possible or when multiple best hits correspondto other viral families, respectively.

For the consensus sequence determination strategy, resequencing data ob-tained from rhabdoviral tiled sequences are filtered as previously described andthen submitted to a multiple alignment with CLUSTAL W (39), from which aconsensus sequence is determined (Fig. 1B). For each sequence in the alignment,if a called base has undetermined calls on both sides, it is replaced by anundetermined call. If different calls appear in the sequences for a given position,the majority base call is added to the consensus. The positions that contain anundetermined call or a gap are not considered in the majority base call compu-

TABLE 1. Description of virus species belonging to the family Rhabdoviridae used for selection of tiled sequences and for validation of thePathogenID v1.0 microarray

Genus and speciesa

(abbreviation) Strain Host species/vector Origin

Yr offirst

isolation

Tiledregionb (nt)

Length(nt)

Biological sampletested

GenBankaccession no.

Origin of tiled sequencesLyssavirus Rabies virus

(RABV)PV Vaccine 7452–7953 502 NC_001542

Vesiculovirus Vesicularstomatitis Indianavirus (VSIV)

VSVLMS 7453–7953 497 K02378

Ephemerovirus Bovineephemeral fevervirus (BEFV)

BB7721 Bos taurus Australia 1968 7454–7952 498 NC_002526

Rhabdoviruses testedLyssavirus species

Rabies virus (RABV) 8764THA Human Thailand 1983 Human brain EU293111Rabies virus (RABV) 9147FRA Red fox France 1991 Fox brain EU293115Rabies virus (RABV) 93128MAR Fixed strain Morocco ? Mouse brain GU815994Rabies virus (RABV) 9811CHI Dog China 1998 Mouse brain GU815995Rabies virus (RABV) 0435AFG Dog Afghanistan 2004 Mouse brain GU815996Rabies virus (RABV) 9001FRA Dog bitten by

batFrench Guiana 1990 Mouse brain EU293113

Rabies virus (RABV) 9026CI Dog Ivory Coast 1990 Mouse brain GU815997Rabies virus (RABV) 9105USA Fox USA 1991 Fox brain GU815998Rabies virus (RABV) 9233GAB Dog Gabon 1992 Dog brain GU815999Rabies virus (RABV) 93127FRA Fixed strain France ? Mouse brain GU816000Rabies virus (RABV) 9503TCH Fixed strain

(Vnukovo,SAD)

Czechoslovakia ? Mouse brain GU816001

Rabies virus (RABV) 9737POL Raccoon dog Poland 1997 Mouse brain GU816002Rabies virus (RABV) Challenge virus

strain(CVS_IP13)

Fixed strain Mouse brain GU816003

Rabies virus (RABV) ERA Fixed strain Mouse brain GU816005Rabies virus (RABV) LEP Fixed strain Chicken embryo

fibroblastsGU816004

Vesiculovirus Vesicularstomatitis Indianavirus (VSIV)

Orsay(0503FRA)

Fixed strain BSR cellsc GU816006

a Classifications and names of viruses correspond to approved virus taxonomy according to ICTVdb. Names in italics are those of validated virus species.b Position according to the reference Pasteur virus genome (NC_001542) after alignment of all of the tiled sequences with the reference sequence.c Clone of the baby hamster kidney cell line BHK-21.

VOL. 84, 2010 RHABDOVIRUS IDENTIFICATION BY RESEQUENCING MICROARRAY 9559


.org/D

ownloaded from

http://jvi.asm.org/

TA

BL

E2.

Des

crip

tions

ofvi

rus

spec

ies

belo

ngin

gto

the

fam

ilyR

habd

oviri

dae

used

for

sele

ctio

nof

tiled

sequ

ence

san

dfo

rva

lidat

ion

ofth

ePa

thog

enID

v2.0

mic

roar

ray

Gen

usor

grou

paan

dsp

ecie

s(a

bbre

viat

ion)

aU

A/T

S/U

Cb

Stra

inH

ost

spec

ies/

vect

oror

sour

ceT

iled

regi

on(n

t)c

Len

gth

(nt)

Bio

logi

cal

sam

ple

test

edO

rigi

nof

sam

ple

Yr

offir

stis

olat

ion

Gen

Ban

kac

cess

ion

no.

Ori

gin

oftil

edse

quen

ces

Lys

savi

rus

Gen

otyp

e1,

Rab

ies

viru

s(R

AB

V)

PVV

acci

ne70

40–7

977

937

NC

_001

542

Gen

otyp

e2,

Lag

osba

tvi

rus

(LB

V)

8619

NG

AB

at,E

idol

onhe

lvum

7040

–797

793

7N

iger

ia19

56E

U29

3110

Gen

otyp

e3,

Mok

ola

viru

s(M

OK

V)

MO

KV

Cat

7040

–797

793

7Z

imba

bwe

1981

NC

_006

429

Gen

otyp

e4,

Duv

enha

gevi

rus

(DU

VV

)94

286S

AB

at,M

inop

teru

ssp

ecie

s70

40–7

977

937

Sout

hA

fric

a19

81E

U29

3120

Gen

otyp

e5,

Eur

opea

nba

tly

ssav

irus

1(E

BL

V-1

)89

18F

RA

Bat

,Ept

esic

usse

rotin

us70

40–7

977

937

Fra

nce

1989

EU

2931

12G

enot

ype

6,E

urop

ean

bat

lyss

aviru

s2

(EB

LV

-2)

9018

HO

LB

at,M

yotis

dasy

cnem

e70

40–7

977

937

Net

herl

ands

1986

EU

2931

14G

enot

ype

7,A

ustr

alia

nba

tly

ssav

irus

(AB

LV

)A

BL

hH

uman

7040

–797

793

7A

ustr

alia

1986

AF

4180

14

Ves

icul

oviru

sC

hand

ipur

avi

rus

(CH

PV)

I65

3514

Hum

an70

40–7

981

935

Indi

a19

65A

J810

083

Isfa

han

viru

s(I

SFV

)91

026-

167

Phl

ebot

omus

papa

tasi

7040

–798

193

5Ir

an19

75A

J810

084

Ves

icul

arst

omat

itis

New

Jers

eyvi

rus

(VSN

JV)

VSV

NJ-

OB

osta

urus

,equ

ine/

Cul

exni

grip

alpu

s,C

ulic

oide

ssp

ecie

s,M

anso

nia

indu

bita

ns

7040

–798

193

5U

nite

dSt

ates

1949

AY

0748

04

Ves

icul

arst

omat

itis

Indi

ana

viru

s(V

SIV

)V

SVL

MS

?70

40–7

981

935

??

K02

378

Peri

net

viru

s(P

ER

V)

TS

Ar

Mg

802

Cul

exan

tenn

atus

7089

–750

240

5M

adag

asca

r19

78A

Y85

4652

Spri

ngvi

rem

iaof

carp

viru

s(S

VC

V)

TS

VR

-139

0C

yprin

usca

rpio

7040

–798

193

5Y

ugos

lavi

a19

71U

1810

1

Eph

emer

oviru

sA

dela

ide

Riv

ervi

rus

(AR

V)

DPP

61B

osta

urus

7089

–750

240

8A

ustr

alia

1981

AY

8546

35B

ovin

eep

hem

eral

feve

rvi

rus

(BE

FV

)B

B77

21B

osta

urus

7089

–750

240

8A

ustr

alia

1968

AY

8546

42K

imbe

rley

viru

s(K

IMV

)T

SC

S36

8B

osta

urus

7089

–750

240

8A

ustr

alia

1980

AY

8546

37K

oton

kan

viru

sd(K

OT

V)

UA

IbA

r233

80C

ulic

oide

ssp

ecie

s70

89–7

502

408

Nig

eria

1967

AY

8546

38

Oth

erdi

mar

habo

dvir

uses

d

Alm

piw

argr

oup

Alm

piw

arvi

rus

(AL

MV

)U

AM

RM

4059

Abl

epha

rus

bout

onii

virg

atus

7089

–750

241

1A

ustr

alia

1966

AY

8546

45H

umpt

ydo

ovi

rus

(HD

OO

V)

UA

CS

79L

asio

hele

asp

ecie

s70

89–7

502

411

Aus

tral

ia19

75A

Y85

4643

Oak

-Val

evi

rus

(OV

RV

)U

AC

S13

42C

ulex

spec

ies

7089

–750

240

8A

ustr

alia

1981

AY

8546

70H

art

Park

grou

pF

land

ers

viru

s(F

LA

NV

)U

A61

-748

4C

ulis

eta

mel

anur

a70

89–7

502

410

Uni

ted

Stat

es19

61A

F52

3199

Nga

inga

nvi

rus

(NG

AV

)U

AN

RM

1455

6C

ulic

oide

sbr

evita

rsis

7089

–750

240

8A

ustr

alia

1970

AY

8546

49Pa

rry

Cre

ekvi

rus

(PC

RV

)U

AO

R18

9C

ulex

annu

liros

tris

7089

–750

240

8A

ustr

alia

1972

AY

8546

47W

onga

belv

irus

(WO

NV

)U

AC

S26

4C

ulic

oide

sau

stro

palp

alis

7089

–750

240

8A

ustr

alia

1979

AY

8546

48L

eD

ante

can

dK

ern

Can

yon

grou

pF

ukuo

kavi

rus

(FU

KV

)U

AF

UK

-11

Cul

icoi

des

punc

tatu

s70

89–7

502

408

Japa

n19

82A

Y85

4651

Le

Dan

tec

viru

s(L

DV

)U

AD

akH

D76

3H

uman

7089

–750

240

8Se

nega

l19

65A

Y85

4650

Tib

roga

rgan

grou

p,T

ibro

garg

anvi

rus

(TIB

V)

UA

CS

132

Cul

icoi

des

brev

itars

is70

89–7

502

408

Aus

tral

ia19

76A

Y85

4646

Oth

eran

imal

rhab

dovi

ruse

sT

upai

arh

abdo

viru

s(T

UPV

)U

AT

RV

1591

Tup

aia

bela

nger

i70

89–7

502

408

Tha

iland

?N

C_0

0702

0Si

gma

viru

s(S

IGM

AV

)U

A23

4HR

CD

roso

phila

mel

anog

aste

r62

20–6

642

408

??

X91

062

Sea

trou

trh

abdo

viru

s(S

TR

V)

UC

28/9

7Sa

lmo

trut

tatr

utta

7108

–757

641

5Sw

eden

1996

AF

4349

92

Rha

bdov

irus

este

sted

Lys

savi

rus

Gen

otyp

e1

Rab

ies

viru

s(R

AB

V)

9312

7FR

AF

ixed

stra

inM

ouse

brai

nF

ranc

e?

GU

8160

00R

abie

svi

rus

(RA

BV

)87

64T

HA

Hum

anH

uman

brai

nT

haila

nd19

83E

U29

3111

Rab

ies

viru

s(R

AB

V)

0833

9FR

AH

uman

(pro

babl

yco

ntam

inat

edby

bat)

Hum

ansa

liva

Fra

nce

(Fre

nch

Gui

ana)

2008

GU

8160

07

Rab

ies

viru

sh(R

AB

V)

0702

9SE

NH

uman

Skin

biop

sySe

nega

l20

06G

enot

ype

2,L

agos

bat

viru

s(L

BV

)86

19N

GA

Bat

,Eid

olon

helv

umM

ouse

brai

nN

iger

ia19

56E

U29

3110

Gen

otyp

e3,

Mok

ola

viru

s(M

OK

V)

8610

0CA

MSh

rew

Mou

sebr

ain

Cam

eroo

n19

81N

C_0

0642

9G

enot

ype

4,D

uven

hage

viru

s(D

UV

V)

8613

2SA

Hum

anM

ouse

brai

nSo

uth

Afr

ica

1971

EU

2931

19



.org/D

ownloaded from

http://jvi.asm.org/

Gen

otyp

e5

Eur

opea

nba

tly

ssav

irus

1su

btyp

ea

(EB

LV

-1a)

0724

0FR

AC

at(c

onta

min

ated

byba

t)C

atbr

ain

Fra

nce

2007

EU

6265

52

Eur

opea

nba

tly

ssav

irus

1su

btyp

ea

(EB

LV

-1b)

0834

1FR

AB

at,E

ptes

icus

sero

tinus

Bat

brai

nF

ranc

e20

08G

U81

6009

Eur

opea

nba

tly

ssav

irus

1su

btyp

eb

(EB

LV

-1b)

8918

FR

AB

at,E

ptes

icus

sero

tinus

Mou

sebr

ain

Fra

nce

1989

EU

2931

12

Gen

otyp

e6,

Eur

opea

nba

tly

ssav

irus

2(E

BL

V-2

)90

18H

OL

Bat

,Myo

tisda

sycn

eme

Mou

sebr

ain

Hol

land

1986

EU

2931

14G

enot

ype

7,A

ustr

alia

nba

tly

ssav

irus

(AB

LV

)98

10A

US

Bat

Mou

sebr

ain

Aus

tral

ia?

GU

8160

08G

enot

ype

8(t

enta

tive

spec

ies)

,Dak

arba

tly

ssav

irus

(DB

LV

)U

C04

06SE

N(A

nD42

443)

Bat

,Eid

olon

helv

umM

ouse

brai

nSe

nega

l19

85E

U29

3108

Not

assi

gned

,Wes

tC

auca

sian

bat

viru

s(W

CB

V)

UC

Bat

,Myo

tissc

hrei

bers

iiPl

asm

ide

Rus

sia

2002

EF

6142

58

Ves

icul

oviru

sV

esic

ular

stom

atiti

sIn

dian

avi

rus

(VSI

V)

Ors

ay(0

503F

RA

)?

BSR

cells

f?

?G

U81

6006

Bot

eke

viru

s(B

TK

V)

TS

Dak

ArB

1077

(041

7RC

A)

Coq

uille

ttidi

am

acul

ipen

nis

Mou

sebr

ain

Cen

tral

Afr

ican

Rep

ublic

1968

GU

8160

14

Juro

navi

rus

(JU

RV

)T

SB

eAr

4057

8(0

414B

RE

)H

emag

ogus

speg

azzi

nii

Mou

sebr

ain

Bra

zil

1962

GU

8160

24Po

rton

’svi

rus

(PO

RV

)T

S16

43(0

416M

AL

)M

anso

nia

unifo

rmis

Mou

sebr

ain

Mal

aysi

a(S

araw

ak)

?G

U81

6013

Eph

emer

oviru

sK

oton

kan

viru

sd(K

OT

V)

UA

IbA

r233

80(9

145N

IG)

Cul

icoi

des

spec

ies

Mou

sebr

ain

Nig

eria

1967

AY

8546

38K

imbe

rley

viru

s(K

IMV

)T

SC

S36

8B

osta

urus

Mou

sebr

ain

Aus

tral

ia19

80A

Y85

4637

Oth

eran

imal

rhab

odvi

ruse

sH

art

Park

grou

pK

ames

evi

rus

(KA

MV

)U

AM

P61

86(0

8343

OU

G)

Cul

exan

nulio

risM

ouse

brai

nU

gand

a19

67G

U81

6011

Mos

suri

lvir

us(M

OSV

)U

ASA

Ar

1995

(041

8MO

Z)

Cul

exsi

tiens

Mou

sebr

ain

Moz

ambi

que

1959

GU

8160

12

Kol

ongo

and

Sand

jimba

grou

p,Sa

ndjim

bavi

rus

(SJA

V)

UA

Dak

AnB

373d

(072

44R

CA

)A

croc

epha

lus

scho

enob

aenu

sM

ouse

brai

nC

entr

alA

fric

anR

epub

lic19

70G

U81

6019

Le

Dan

tec

and

Ker

nC

anyo

ngr

oup

Keu

ralib

avi

rus

(KE

UV

)U

AD

akA

nD53

14(9

715S

EN

,042

0SE

N)

Tat

era

kem

piM

ouse

brai

nSe

nega

l19

68G

U81

6021

Nko

lbis

son

viru

s(N

KO

V)

UA

Ar

Y31

/65

(042

5CA

M)

Ere

tmap

odite

sle

ucop

ous

Mou

sebr

ain

Ivor

yC

oast

,C

amer

oon

1965

GU

8160

22

Ung

roup

edG

arba

viru

sg(G

AR

V)

UA

Dak

AnB

439a

(042

2RC

A)

Cor

ytho

rnis

cris

tata

Mou

sebr

ain

Cen

tral

Afr

ican

Rep

ublic

1970

GU

8160

18

Nas

oule

viru

sg(N

ASV

)U

AD

akA

nB42

89a

(041

0RC

A)

And

ropa

dus

vire

nsM

ouse

brai

nC

entr

alA

fric

anR

epub

lic19

73G

U81

6017

Oua

ngo

viru

sg(O

UA

V)

UA

Dak

AnB

1582

a(9

718R

CA

)P

loce

usm

elan

ocep

halu

sM

ouse

brai

nC

entr

alA

fric

anR

epub

lic19

70G

U81

6015

Bim

bovi

rusg

(BB

OV

)U

AD

akA

nB10

54d

(971

6RC

A)

Eup

lect

esaf

erM

ouse

brai

nC

entr

alA

fric

anR

epub

lic19

70G

U81

6016

Ban

gora

nvi

rus

(BG

NV

)U

AD

akA

rB20

53(0

424R

CA

)C

ulex

perf

uscu

sM

ouse

brai

nC

entr

alA

fric

anR

epub

lic19

69G

U81

6010

Gos

sas

viru

sh(G

OSV

)U

AD

akA

nD40

1(0

8344

SEN

)T

adar

ida

spec

ies

Mou

sebr

ain

Sene

gal

1964

NA

i

aU

nles

sst

ated

othe

rwis

e,th

ecl

assi

ficat

ions

and

nam

esof

viru

ses

corr

espo

ndto

appr

oved

viru

sta

xono

my

acco

rdin

gto

ICT

Vdb

.Nam

esin

italic

are

thos

eof

valid

ated

viru

ssp

ecie

s.b

UA

,una

ssig

ned;

TS,

tent

ativ

esp

ecie

s;U

C,u

ncla

ssifi

ed(n

otfo

und

inIC

TV

db).

cPo

sitio

nac

cord

ing

toth

ere

fere

nce

Past

eur

viru

sge

nom

e(N

C_0

0154

2)af

ter

alig

nmen

tofa

llof

the

tiled

sequ

ence

sw

ithth

ere

fere

nce

sequ

ence

(exc

eptf

ortil

edse

quen

ces

from

ungr

oupe

drh

abdo

viru

ses

TU

PVan

dSI

GM

AV

,whi

chw

ere

alig

ned

inde

pend

ently

with

the

refe

renc

ese

quen

ce).

dT

axon

omic

alcl

assi

ficat

ion

acco

rdin

gto

refe

renc

e6.

eA

977-

ntfr

agm

ent

ofth

epo

lym

eras

ege

ne(f

rom

nt70

20to

nt79

97,a

ccor

ding

toth

ere

fere

nce

Past

eur

viru

sge

nom

e�N

C_0

0154

2�)

was

synt

hesi

zed

invi

tro

and

then

clon

edin

topl

asm

idpC

R2.

1(O

pero

n).

fC

lone

ofth

eba

byha

mst

erki

dney

cell

line

BH

K-2

1.g

Not

dete

cted

usin

gPa

thog

enID

v2.0

mic

roar

ray

but

ampl

ified

byPC

Ror

nest

edPC

Rus

ing

cons

ensu

sor

spec

ific

prim

ers.

hN

otde

tect

edus

ing

Path

ogen

IDv2

.0m

icro

arra

yor

ampl

ified

byPC

Ror

nest

edPC

Rus

ing

cons

ensu

sor

spec

ific

prim

ers.

iN

A,n

otap

plic

able

.



.org/D

ownloaded from

http://jvi.asm.org/

tation. If multiple base calls tie for the majority, an undetermined call appears atthis position in the consensus sequence. This procedure generally increases thelength and accuracy of the query sequence for subsequent analysis. Homologysearching of the consensus sequences is performed with BLAST using the pa-rameters previously described, and the taxonomy of the best hit is retrieved as forthe systematic homology searching approach. We tested if the resulting consen-sus sequences had higher identification accuracy than any individual sequence orcould be used to design PCR primers for a characterization of a potential novelisolate.

Sequencing confirmation. Conventional sequencing was undertaken after thePCR amplification of viral targets directly from biological samples (after RNAextraction and RT) or from 10- to 100-fold water-diluted WTA products. Primerdesign was first based on consensus sequences obtained using the consensussequence determination strategy previously described and/or on rhabdovirusnucleotide sequences available in GenBank. Depending on the results obtainedand the virus strain tested, the primer design, the set of primers used, and thePCR conditions used for partial polymerase gene amplification were then ad-justed (list of primers and the PCR conditions are available on request from thecorresponding author). All PCR products were obtained using the proofreadingDNA polymerase ExtTaq (Takara). Sequence assembly and consensus sequenceswere obtained using Sequencher 4.7 (Gene Codes).

Phylogenetic analysis. The data set of 15 newly sequenced rhabdoviruses fromthis study (including the Sandjimba and Kolongo viruses previously only identi-fied on the basis of partial nucleoprotein gene sequences, as well as Piry virus, forwhich the nucleotide sequences of different genes were available) was comparedwith the corresponding block III polymerase amino acid sequences of 91 otherrhabdoviruses collected from GenBank (see Table 6). DNA translation wasperformed with BioEdit software (17), and sequence alignment was performedusing the CLUSTAL W program (39) and then checked for accuracy by eye. Thisresulted in a final alignment of 106 sequences 160 amino acid residues in length.Phylogenetic analysis of these sequences was then undertaken using the Bayesianmethod available in the MRBAYES package (18). This analysis utilized theWAG model of amino acid replacement with a gamma distribution of among-siterate variation. Chains were run for 10 million generations (with a 10% burn in),at which point all of the parameter estimates had converged. The level of supportfor each node is provided by Bayesian posterior probability (BPP) values.

Nucleotide sequence accession numbers. The GenBank accession numbers forthe sequences newly acquired are designated GU815994 to GU816024 and areindicated in Tables 1, 2 and 6.

RESULTS

Identification of lyssaviruses based on two successive Patho-genID microarray generations using a systematic BLASTstrategy. To test whether PathogenID microarrays, and specif-ically the prototype tiled regions, could be used for the iden-

tification of a broad number of viral variants without relying onpredetermined hybridization patterns, representative animalviruses from the family Rhabdoviridae (including unassigned ortentatively classified rhabdoviruses according to ICTVdb) werestudied. The capability of these RMAs to identify and discrim-inate between near phylogenetic neighbors was first testedusing one sequence of the genus Lyssavirus (strain PV, geno-type or species 1) tiled on the first generation of the Patho-genID microarray (Table 1). It was possible to use BLAST to

FIG. 1. Descriptive workflow of the automatic Perl bioinformatics-based analysis of the PathogenID v2.0 data. (A) Systematic BLAST strategy.This strategy consists of filtering of the sequences obtained from the output data of the RMA with filter parameters defined by the user (seeMaterials and Methods for further details), systematic researching of homologues using a local BLAST viral and bacterial database, and finallyretrieval of the taxonomy of the best BLAST hits. (B) Consensus sequence determination strategy. A consensus sequence is generated using amultiple alignment with CLUSTAL W based on the sequences obtained from prototype rhabdovirus sequences tiled on the microarray. With thisprocess, the length and accuracy of the query sequence can be increased. Homology searching of the consensus sequences is performed withBLAST using the previously described parameters and database. The taxonomy of the best BLAST hit is retrieved as for the systematic homologysearching approach.

FIG. 2. Spectrum of detection members of the genus Lyssavirus bythe PathogenID v1.0 microarray according to the natural nucleotidevariation of the virus strains tested. For each lyssavirus strain tested(n � 15, indicated by blue diamonds), results are indicated by thepercentage of nucleotide divergence (compared to the single lyssavirusprototype sequence tiled on the microarray, x axis) according to thepercentage of nucleotide bases determined (call rate, y axis). Thelinear correlation curve between these two values is presented, dem-onstrating a high correlation between these two parameters (correla-tion coefficient of 0.89). All of these 15 virus strains belonged to thesame species as the tiled prototype sequence (species 1) and wereaccurately identified after BLAST analysis (at the species level). Otherspecies (or genotypes) of lyssaviruses were not successfully detected bythe PathogenID v1.0 microarray (nucleotide divergence over 20%;data not shown). For further details concerning the lyssavirus strainsused, see Table 1.



.org/D

ownloaded from

http://jvi.asm.org/

TABLE 3. Level of taxonomic identification of species of the genus Lyssavirus based on sequences tiled on the PathogenID_v2.0 microarray

Species(abbreviation) and

isolate oflyssaviruses and

parameter tested

Result from tiled sequence of Lyssavirus genotype:

1 (RABV),PV

2 (LBV),8619NGA

3 (MOKV),MOKV

4 (DUVV),94286SA

5 (EBLV-1),8918FRA

6 (EBLV-2),9018HOL

7 (ABLV),ABLV

1 (RABV)93127FRA

Base call ratea 95.0 3.8 4.6 6.2 8.0 9.0 6.7Identificationb A C B A A A BDivergencec 0.2 25.6 24.8 22.8 23.2 21.0 22.0

8764THABase call rate 32.6 5.4 5.7 7.0 6.0 3.3 9.0Identification A A B C A A BDivergence 13.7 24.3 24.9 22.7 22.9 21.2 20.7

2 (LBV)8619NIG

Base call rate 2.7 96.6 11.1 6.9 7.1 5.3 6.7Identification Neg A A A Neg Neg BDivergence 25.8 0.0 22.3 22.8 25.2 23.8 23.3

3 (MOKV)86100CAM

Base call rate 2.7 7.2 56.3 8.1 7.0 7.7 3.6Identification A A A A A A BDivergence 24.9 22.5 10.2 22.0 23.6 22.2 24.5

4 (DUVV)86132SA

Base call rate 3.9 1.1 1.4 97.3 0.6 2.5 5.6Identification A Neg Neg A A A NegDivergence 23.2 22.8 22.2 6.0 20.6 21.6 21.9

5 (EBLV-1)8918FRA

Base call rate 8.1 6.9 15.2 13.3 93.8 7.8 4.8Identification B A A A A A NegDivergence 23.8 25.5 23.8 20.7 0.6d 23.4 22.1

6 (EBLV-2)9018HOL

Base call rate 5.7 2.1 3.5 6.5 4.1 98.4 8.7Identification Neg Neg B A B A ADivergence 21.2 23.8 23.5 21.7 23.3 0.0 22.3

7 (ABLV)9810AUS

Base call rate 8.4 8.3 1.4 12.9 3.8 11.3 94.9Identification B A Neg A B B ADivergence 22.5 23.7 24.4 21.6 22.1 22.4 1.6

8e (DBLV)0406SEN

Base call rate 19.3 63.5 29.4 16.3 22.8 18.3 19.5Identification A A A A A A ADivergence 25.0 20.1 21.5 23.4 23.5 22.8 23.8

UnclassifiedWCBV

Base call rate 25.3 28.3 32.7 26.9 26.5 23.8 24.5Identification C A A A A A ADivergence 25.7 23.8 24.2 24.9 24.6 24.8 25.9

a Percentage of base calls generated from full-length tiled sequences.b Taxonomic identification according to the following: A, identification at the species or isolate level when a unique best hit corresponds to the expected species or

isolate; B, identification at the genus level when multiple best viral hits exist and correspond to the genus Lyssavirus; C, identification at the family level when multiplebest viral hits exist and correspond to genera of the family Rhabdoviridae; Neg, negative or inaccurate identification when a BLAST query is not possible or when thereare multiple best hits and some or all of them correspond to other viral families, respectively. Underlined are results obtained using the sequence belonging to the samespecies tiled on the array (homonymous sequence).

c Percentage of nucleotide divergence (based on a 937-nt region of the polymerase gene, positions 7040 to 7977, according to the reference Pasteur virus genome(NC_001542).

d The tiled sequence of 8918FRA corresponds to a preliminary sequencing result, and the complete genome of this virus strain was obtained later (EU293112), whichmay explain the 7-nt difference between those two sequences.

e Tentative species.



.org/D

ownloaded from

http://jvi.asm.org/

successfully identify virus strains with approximately 18% nu-cleotide divergence compared to the prototype (Fig. 2). Thehybridization of 15 virus strains representative of the geneticdiversity found in this species indicated that a single tiledsequence was able to detect all of the variant strains belongingto the same species.

In addition, we evaluated the spectrum of detection of thesecond generation of the PathogenID microarray, which in-cluded one prototype sequence representative of each of theseven described species in the genus Lyssavirus (Table 2). Allof the isolates tested led to the correct species identificationusing a systematic BLAST strategy when hybridizing a targetbelonging to the same species that is tiled on the array (Table3). Moreover, all of the tested isolates of a known genotypewere also recognized by heterospecific tiled sequences (Table3). We also investigated the capacity of this RMA to detectmore distantly related viruses not yet classified into a species.Isolates 0406SEN and WCBV, which have been proposed torepresent new species of the genus Lyssavirus (5, 15), weresurprisingly recognized by almost all of the seven species se-quences tiled on the PathogenID v2.0 microarray (Table 3).This recognition indicates that each sequence tiled on the arrayhas the ability to identify strains that are more than 18%divergent, and up to 25.9% in some cases (Table 3). Thisanalysis also reveals that information on a strain hybridized onPathogenID v2.0 can be obtained from distinct species or iso-lates tiled on the array. Evaluation of the spectrum of detec-tion of this RMA was further extended to two other genera ofthe family Rhabdoviridae—Ephemerovirus and Vesiculovirus(Table 4). Here again, successful identification was achievedusing homospecific sequences tiled on the array, confirmingthe reliability of the identification.

In both experiments (Tables 3 and 4), low base call rate

values were obtained for several combinations of hybridizedand tiled sequences. These values were sufficient for viral iden-tification by BLAST, despite the presence of sequence reads asshort as 14 nt. This indicates that most of these short sequencescorresponded to highly conserved sequence domains. The ac-curacy of these short sequences was checked by comparisonwith those obtained by classical sequencing (data not shown).

Identification of lyssaviruses based on the consensus se-quence determination strategy. A bioinformatic workflow wasdeveloped to gather stretches of sequence reads obtained withmore or less distantly related sequences tiled on PathogenIDv2.0. The aim of this strategy was to enlarge the length of thesequence determined in order to improve the sensitivity of theBLAST analysis compared to previously described methodol-ogies (29). All of the sequence reads obtained from prototypesequences of the genus Lyssavirus (at least 12 nt long with nomore than one undetermined base, whether or not they ini-tially led to a positive BLAST identification) were used togenerate a contiguous sequence. When overlapping fragmentswere identified, a consensus sequence was generated to re-move ambiguous or undetermined base calls. The methodol-ogy used to obtain consensus sequences confirmed the speciesidentification after BLAST analysis in the case of the sevenlyssavirus nucleotide sequences used for hybridization (Table5). Moreover, these consensus sequences were found to bemore powerful in identifying unclassified or new species oflyssaviruses not tiled on the RMA than resequencing datacollected individually from each tiled sequences, as shown forstrains 0406SEN and WCBV. In both cases, an increase in thebase call rate was observed using this consensus sequencestrategy, from 63.5% (best base call rate obtained from indi-vidual prototype sequences) to 75.9% for strain 0406SEN andfrom 32.7% to 60.9% for WCBV (Tables 3 and 5). Once again,

TABLE 4. Levels of taxonomic identification of virus species among the genera Vesiculovirus and Ephemerovirus based on Vesiculovirus andEphemerovirus sequences tiled on the PathogenID_v2.0 microarray

Rhabdovirus genus and species(isolate) and parameter tested

Result from specific rhabdovirus sequence tiled

Vesiculovirus Ephemerovirus LyssavirusRABV(PV)CHPV ISFV PERV SVCV VSIV VSNJV ARV BEFV KIMV KOTV

Vesiculovirus VSIV (0503FRA)Base call ratea 1.2 4.1 1.0 1.0 98.6 2.9 0 0 0 0 0Scoreb Neg Neg Neg Neg A A Neg Neg Neg Neg Neg

EphemerovirusKIMVc (CS 368)

Base call rate 1.9 1.1 0 0 0.3 0.3 9.4 7.3 70.6 9.1 1.4Score Neg Neg Neg Neg Neg Neg Neg Neg A Neg Neg

KOTVd (Ib Ar23380, 9145NIG)Base call rate 6.6 3.8 5.7 3.2 3.7 7.2 8.8 5.2 3.4 100 2.1Score Neg Neg Neg Neg Neg C Neg Neg Neg A Neg

Lyssavirus RABV (93127FRA)Base call rate 0.3 1.2 2.6 1.5 0 0 0.1 0 0.1 2.3 95.0Score Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg A

a Percentage of base calls generated from full-length tiled sequences.b Taxonomic identification according to the following: A, identification at the species or isolate level when a unique best hit corresponds to the expected species or

isolate; C, identification at the family level when multiple best viral hits exist and correspond to genera of the family Rhabdoviridae; Neg, negative or inaccurateidentification when a BLAST query is not possible or when multiple best hits exist and some or all of them correspond to other viral families. Underlined are resultsobtained using the sequence belonging to the same species or isolate tiled on the array (homonymous sequence).

c TS, tentative species according to ICTVdb.d Taxonomic classification according to reference 6.



.org/D

ownloaded from

http://jvi.asm.org/

this increase in nucleotide base determination was associatedwith a relatively high accuracy (91.8% and 97.3% concordancebetween the consensus sequences and the reference sequencesof isolates 0406SEN and WCBV, respectively (Table 5). Tofurther demonstrate the ability of this strategy to detect andidentify novel virus species, consensus sequences were gener-ated based only on six of the seven prototype tiled sequences(excluding the homospecific sequence of the same species tiledon the array). All of the strains of the seven species tested wereaccurately and specifically identified using this restricted ap-proach (Table 5). These results indicate that the consensussequences obtained could improve the detection of a noveldomain(s) not identified using only the closest prototype se-quence tiled on the RMA.

Assessment of clinical specimens. A total of 17 brain biopsysamples originating from experimentally infected mice and var-ious clinical samples (n � 8) obtained from the National Ref-erence Centre for Rabies at the Institut Pasteur were tested forlyssavirus detection and identification using the two versions ofthe PathogenID microarray (Tables 1 and 2). These specimenswere previously collected from humans and animals with clin-ically documented encephalitis and suspected of having rabies.They were used to compare RMA results with conventionalmethods of diagnosis, including the RT-heminested PCR (RT-hnPCR) technique for the intra vitam diagnosis of rabies inhumans (13), the fluorescent-antibody test, the rabies tissueculture inoculation test, and the enzyme-linked immunosor-bent assay for the postmortem diagnosis of humans and ani-mals (8, 47). Among the eight clinical samples, most were brainbiopsy specimens collected from different rabid mammals, in-cluding a bat, a cat, a dog, and two foxes, and from a human.The two other samples comprised a saliva specimen and a skinbiopsy sample collected from two different rabid human pa-tients (Tables 1 and 2). Except for the skin biopsy case, whichwas not recognized, this comparison demonstrated a completeconcordance between our method and conventional methodsfor all of the samples tested. Hence, the accuracy of the se-quences provided with PathogenID microarray was close tothat obtained using classical sequencing (data not shown). Thefailure to detect lyssaviruses in the skin biopsy samples wasprobably due to insufficient sensitivity of the current RMAmethod, as viral RNA was only weakly detected after RT-hnPCR.

In sum, these results demonstrated that the newly developedamplification process by WTA coupled to hybridization to thePathogenID microarray allowed the detection of a large rangeof viral variants from various complex biological samples, in-cluding clinical samples (Tables 1 and 2).

Application of the RMA strategy to characterize new rhab-doviruses. Broad-spectrum detection was demonstrated usingthe consensus sequences-based analysis strategy among virusesof the family Rhabdoviridae, and the more distantly relatedviruses examined included many viruses that are not yet clas-sified as species. Accordingly, 17 different rhabdoviruses weretested by using brain samples from experimentally infectedmice (n � 16) or infected cell suspension. These viruses in-cluded four strains belonging to the genus Vesiculovirus,with Vesicular stomatitis Indiana virus (VSIV) and Boteke(BOTK), Jurona (JURV), and Porton’s (PORV) viruses, thelatter three of which are currently classified as tentative

TABLE 5. Identification of species of the genus Lyssavirus based onLyssavirus sequences tiled on the PathogenID_v2.0 microarray and

using the consensus sequence determination strategy

Lyssavirus species(abbreviation), isolate, and

parameter tested

Result obtained with analysisstrategy of use of:

Prototypesequence

only

Consensus sequence

Including alltiled

sequences

Excludingprototypesequence

1 (RABV)93127FRA

Base call ratea 95.0 96.3 32.7BLAST scoreb 791 801 38Accuracyc 100 99.9 95.9

8764THABase call rate 32.6 47.4 26.7BLAST score 46 64 31Accuracy 94.8 99.1 98.4

2 (LBV), 8619NIGBase call rate 96.6 96.4 28.1BLAST score 816 814 39Accuracy 99.9 99.9 97.7

3 (MOKV), 86100CAMBase call rate 56.3 67.4 28.4BLAST score 66 112 64Accuracy 98.2 99.8 98.5

4 (DUVV), 86132SABase call rate 97.3 97.3 18.1BLAST score 843 833 20Accuracy 99.9 99.8 96.4

5 (EBLV-1), 8918FRABase call rate 93.8 96.0 41.1BLAST score 757 807 83Accuracy 100 100 97.9

6 (EBLV-2), 9018HOLBase call rate 98.4 98.8 26.8BLAST score 871 879 44Accuracy 100 99.9 99.6

7 (ABLV), ABLVBase call rate 94.9 95.6 29.7BLAST score 749 741 40Accuracy 100 99.9 94.5

8e (DBLV), 0406SENBase call rate NAd 75.9 NABLAST score NA 82 NAAccuracy NA 91.8 NA

?, WCBVBase call rate NA 60.9 NABLAST score NA 56 NAAccuracy NA 97.3 NA

a Percentage of base calls generated from full-length tiled sequences.b BLAST score (bit score) obtained after BLAST query on a local viral and

bacterial database using the consensus sequence determination strategy with m(minimum nucleotide length) � 12 and N (maximum undetermined nucleotidecontent) � 10. Default BLAST parameters, except for the minimum word length(7 nt), the expect threshold (increased from the default of 10 to 100,000), and thelow complexity level filter (�F) turned off. All of the BLAST scores indicatecorrect identification at the species or isolate level (i.e., unique best hit corre-sponds to the expected species or isolate).

c Percentage of nucleotides correctly identified, compared to the sequence ob-tained after classical sequencing of the corresponding Lyssavirus species tested.

d NA, not applicable.e Tentative species.



.org/D

ownloaded from

http://jvi.asm.org/

TA

BL

E6.

Des

crip

tions

and

final

clas

sific

atio

nsof

rhab

dovi

ruse

sus

edfo

rph

ylog

enet

ican

alys

is

Gen

usan

dna

mea

(spe

cies

)U

A/T

S/U

Cb

Abb

revi

atio

nSt

rain

Prin

cipa

lhos

tsp

ecie

s/ve

ctor

cSa

mpl

eor

igin

Yr

offir

stis

olat

ion

Gen

Ban

kac

cess

ion

no.

Lys

savi

rus

Rab

ies

viru

s(1

)R

AB

V90

01F

RA

Dog

bitt

enby

bat

Fre

nch

Gui

ana

1990

EU

2931

13R

abie

svi

rus

(1)

RA

BV

9147

FR

AF

oxF

ranc

e19

91E

U29

3115

Rab

ies

viru

s(1

)R

AB

V87

43T

HA

Hum

anT

haila

nd19

83E

U29

3121

Rab

ies

viru

s(1

)R

AB

V97

04A

RG

Bat

,Tad

arid

abr

asili

ensi

sA

rgen

tina

1997

EU

2931

16R

abie

svi

rus

(1)

RA

BV

9706

CH

IV

acci

neA

GC

hina

AY

8546

63R

abie

svi

rus

(1)

RA

BV

9702

IND

Hum

anIn

dia

1997

AY

8546

65L

agos

bat

viru

s(2

)L

BV

8619

NG

AB

at,E

idol

onhe

lvum

Nig

eria

1956

EU

2931

10M

okol

avi

rus

(3)

MO

KV

MO

KV

Cat

Zim

babw

e19

81N

C_0

0642

9M

okol

avi

rus

(3)

MO

KV

8610

0CA

MSh

rew

Cam

eroo

n19

74E

U29

3117

Mok

ola

viru

s(3

)M

OK

V86

101R

CA

Rod

ent

Rep

ublic

ofC

entr

alA

fric

a19

81E

U29

3118

Duv

enha

gevi

rus

(4)

DU

VV

9428

6SA

Bat

,Min

iopt

erus

spec

ies

Sout

hA

fric

a19

81E

U29

3120

Duv

enha

gevi

rus

(4)

DU

VV

8613

2SA

Hum

anSo

uth

Afr

ica

1971

EU

2931

19E

urop

ean

bat

lyss

aviru

s1

(5)

EB

LV

-189

18F

RA

Bat

,Ept

esic

usse

rotin

usF

ranc

e19

89E

U29

3112

Eur

opea

nba

tly

ssav

irus

1(5

)E

BL

V-1

0812

0FR

AB

at,E

ptes

icus

sero

tinus

Fra

nce

2008

EU

6265

51E

urop

ean

bat

lyss

aviru

s2

(6)

EB

LV

-290

18H

OL

Bat

,Myo

tisda

sycn

eme

Net

herl

ands

1986

EU

2931

14E

urop

ean

bat

lyss

aviru

s2

(6)

EB

LV

-293

37SW

IB

at,M

yotis

daub

ento

nii

Switz

erla

nd19

93A

Y85

4657

Aus

tral

ian

bat

lyss

aviru

s(7

)A

BL

VA

BL

hH

uman

Aus

tral

ia19

86A

F41

8014

Aus

tral

ian

bat

lyss

aviru

s(7

)A

BL

VA

BL

b(S

6-12

56)

Bat

,Sac

cola

imus

spec

ies

Aus

tral

ia19

96N

C_0

0324

3D

akar

bat

lyss

avir

us(8

�pro

pose

d�)

UC

DB

LV

0406

SEN

(AnD

4244

3)B

at,E

idol

onhe

lvum

Sene

gal

1985

EU

2931

08

Dak

arba

tly

ssav

irus

(8�p

ropo

sed�

)U

CD

BL

VK

E13

1B

at,E

idol

onhe

lvum

Ken

ya20

07E

U25

9198

Irku

tvi

rus

UC

IRK

VB

at,M

urin

ale

ucog

aste

rR

ussi

a20

02E

F61

4260

Oze

rnoe

viru

sU

CH

uman

Rus

sia

2007

FJ9

0510

5A

rava

nvi

rus

UC

AR

AV

Bat

,Myo

tisbl

ythi

Kyr

gyzs

tan

1991

EF

6142

59K

huja

ndvi

rus

UC

KH

UV

Bat

,Myo

tism

ysta

cinu

sT

ajik

ista

n20

01E

F61

4261

Wes

tC

auca

sian

bat

viru

sU

CW

CB

VB

at,M

inio

pter

ussc

hrei

bers

iiR

ussi

a20

02E

F61

4258

Ves

icul

oviru

sC

hand

ipur

avi

rus

CH

PVI

6535

14H

uman

;do

mes

tican

imal

sd;h

edge

hog,

Ate

lerix

spec

ies;

dipt

eran

,Phl

ebot

omus

spec

ies

Indi

a19

65A

J810

083

Coc

alvi

rus

CO

CV

TR

VL

4023

3L

ives

tock

,equ

ine,

bovi

ne;m

ites,

Gig

anto

lael

aps

spec

ies

Tri

nida

dan

dT

obag

o,T

rini

dad

1961

EU

3736

57

Isfa

han

viru

sIS

FV

9102

6-16

7D

ipte

ran,

Phl

ebot

omus

papa

tasi

Iran

1975

AJ8

1008

4P

iryvi

rus

PIR

YV

BeA

n24

232

(041

3BR

E)

Hum

an;o

poss

um,P

hila

nder

opos

sum

Bra

zil

1960

GU

8160

23V

esic

ular

stom

atiti

sN

ewJe

rsey

viru

sV

SNJV

VSV

NJ-

OSe

vera

lliv

esto

cksp

ecie

s,in

clud

ing

Bos

taur

usan

deq

uine

s;se

vera

ldip

tera

nsp

ecie

s,in

clud

ing

Cul

exni

grip

alpu

s,C

ulic

oide

ssp

ecie

s,an

dM

anso

nia

indu

bita

ns

Uta

h19

49A

Y07

4804

Ves

icul

arst

omat

itis

New

Jers

eyvi

rus

VSN

JVV

SVN

J-H

Seve

rall

ives

tock

spec

ies,

incl

udin

gSu

ssc

rofa

;se

vera

ldip

tera

nsp

ecie

s,in

clud

ing

Cul

exni

grip

alpu

s,C

ulic

oide

ssp

ecie

s,an

dM

anso

nia

indu

bita

ns

Geo

rgia

1952

AY

0748

03

Ves

icul

arst

omat

itis

Indi

ana

viru

sV

SIV

Mud

d-Su

mm

ers

(MS)

Bov

ine,

Bos

taur

usIn

dian

a19

25E

U84

9003

Ves

icul

arst

omat

itis

Indi

ana

viru

sV

SIV

85C

LB

Bov

ine

Col

ombi

a19

85A

F47

3865

Ves

icul

arst

omat

itis

Indi

ana

viru

sV

SIV

98C

OE

Equ

ine

Col

orad

o19

98A

F47

3864

Ves

icul

arst

omat

itis

Ala

goas

viru

sV

SAV

Indi

ana

3E

quin

eliv

esto

ck(m

ule)

,Bos

taur

us;d

ipte

rans

,P

hleb

otom

ussp

ecie

sB

razi

l19

64E

U37

3658

Juro

navi

ruse

TS

JUR

VB

eAr

4057

8(0

414B

RE

)D

ipte

ran,

Hem

agog

ussp

egaz

zini

iB

razi

l19

62G

U81

6024

Peri

net

viru

sT

SPE

RV

Ar

Mg

802

Dip

tera

ns,A

noph

eles

cous

tani

,Cul

exan

tenn

atus

,Cul

exgr

.pip

iens

,Man

soni

aun

iform

is,P

hleb

otom

usbe

rent

ensi

s

Mad

agas

car

1978

AY

8546

52

Pike

fry

rhab

dovi

rus

TS

PFR

VF

4F

ish,

Eso

xlu

cius

Net

herl

ands

1972

FJ8

7282

7



.org/D

ownloaded from

http://jvi.asm.org/

Scop

htha

lmus

max

imus

rhab

dovi

rus

UC

SMR

VQ

Z-2

005

Fis

h,Sc

opht

halm

usm

axim

usC

hina

?A

Y89

5167

Spri

ngvi

rem

iaof

carp

viru

sT

SSV

CV

Fija

n_ce

ll(V

R-1

390,

isol

ated

from

fat

head

min

now

cells

)

Fis

h,C

ypri

nus

carp

ioY

ugos

lavi

a19

71A

J318

079

Spri

ngvi

rem

iaof

carp

viru

sT

SSV

CV

Fija

n_tis

sue

(VR

-139

0,is

olat

edfr

omtis

sues

ofdi

seas

edco

mm

onca

rp)

Fis

h,C

ypri

nus

carp

ioY

ugos

lavi

a19

71U

1810

1

Spri

ngvi

rem

iaof

carp

viru

sT

SSV

CV

BJ0

505-

2F

ish,

Cyp

rinu

sca

rpio

Chi

na20

05E

U17

7782

Eph

emer

oviru

sA

dela

ide

Riv

ervi

rus

AR

VD

PP61

Bov

ine,

Bos

taur

usA

ustr

alia

1981

AY

8546

35B

errim

ahvi

rus

BR

MV

DPP

63B

ovin

e,B

osta

urus

Aus

tral

ia19

81A

Y85

4636

Bov

ine

ephe

mer

alfe

ver

viru

sB

EF

VC

s19

33B

ovin

e,B

osta

urus

Aus

tral

ia19

73A

Y85

4641

Bov

ine

ephe

mer

alfe

ver

viru

sB

EF

VC

s42

Dip

tera

n,A

noph

eles

banc

rofti

Aus

tral

ia19

75A

Y85

4639

Bov

ine

ephe

mer

alfe

ver

viru

sB

EF

VB

B77

21B

ovin

e,B

osta

urus

Aus

tral

ia19

68N

C_0

0252

6K

imbe

rley

viru

sT

SK

IMV

CS

368

Bov

ine,

Bos

taur

usA

ustr

alia

1980

AY

8546

37K

oton

kan

viru

sU

AK

OT

VIb

Ar

2338

0D

ipte

ran,

Cul

icoi

des

spec

ies

Nig

eria

1967

AY

8546

38A

lmpi

war

grou

pA

lmpi

war

viru

sU

AA

LM

VM

RM

4059

Mam

mal

s,d

bovi

ne,e

quin

e,ov

ine,

kang

aroo

,ba

ndic

oot,

hum

an;b

irds

;dliz

ard,

Abl

epha

rus

bout

onii

virg

atus

and

othe

rsk

inks

d

Aus

tral

ia19

66A

Y85

4645

Cha

rlev

ille

viru

sU

AC

HV

VC

h98

24H

uman

;ddi

pter

an,P

hleb

otom

usan

dL

asio

hele

asp

ecie

sA

ustr

alia

1969

AY

8546

44

Cha

rlev

ille

viru

sU

AC

HV

VC

h98

47H

uman

;ddi

pter

an,P

hleb

otom

usan

dL

asio

hele

asp

ecie

sA

ustr

alia

1969

AY

8546

72

Hum

pty

doo

viru

sU

AH

DO

OV

CS

79D

ipte

rans

,Las

iohe

lea

spec

ies,

Cul

icoi

des

mar

ksi

Aus

tral

ia19

75A

Y85

4643

Har

tPa

rkgr

oup

Ban

gora

nvi

ruse

UA

BG

NV

Dak

ArB

2053

(042

4RC

A)

Bir

d,T

urdu

slib

onya

nus;

dipt

eran

,Cul

expe

rfus

cus

Cen

tral

Afr

ican

Rep

ublic

1969

GU

8160

10

Fla

nder

svi

rus

UA

FL

AN

V61

-748

4B

irds

,Sei

urus

auro

capi

llus,

Age

laiu

sph

oeni

ceus

;dip

tera

ns,C

ulis

eta

mel

anur

a,C

ulex

spec

ies

New

Yor

k19

61A

F52

3199

Kam

ese

viru

seU

AK

AM

VM

P61

86(0

8343

OU

G)

Dip

tera

ns,A

edes

afric

anus

,Cul

exsp

ecie

s,in

clud

ing

Cul

exan

nulio

ris

Uga

nda

1967

GU

8160

11

Mos

suri

lvir

use

UA

MO

SVSA

Ar

1995

(041

8MO

Z)

Bir

ds,A

ndro

padu

svi

rens

,Col

iusp

asse

rm

acro

urus

;dip

tera

ns,A

edes

abno

rmal

is,

Cul

exsp

ecie

s,in

clud

ing

Cul

exsi

tiens

Moz

ambi

que

1959

GU

8160

12

Nga

inga

nvi

rus

UA

NG

AV

MR

M14

556

Mam

mal

s,d

wal

labi

es,k

anga

roos

,bov

ines

;di

pter

an,C

ulic

oide

sbr

evita

rsis

Aus

tral

ia19

70A

Y85

4649

Parr

yC

reek

viru

sU

APC

RV

OR

189

Dip

tera

n,C

ulex

annu

liros

tris

Aus

tral

ia19

72A

Y85

4647

Port

on’s

viru

seT

S(V

SV)

POR

V16

43(0

416M

AL

)D

ipte

ran,

Man

soni

aun

iform

isM

alay

sia

(Sar

awak

)?

GU

8160

13W

onga

belv

irus

UA

WO

NV

CS

264

Sea

bird

s;d

dipt

eran

,Cul

icoi

des

aust

ropa

lpal

isA

ustr

alia

1979

AY

8546

48

Le

Dan

tec

grou

pF

ukuo

kavi

rus

UA

(Ker

nC

anyo

nG

roup

)F

UK

VF

UK

-11

Bov

ine;

dipt

eran

,Cul

icoi

des

punc

tatu

s,C

ulex

trita

enio

rhyn

chus

Japa

n19

82A

Y85

4651

Keu

ralib

avi

ruse

UA

KE

UV

Dak

AnD

5314

(971

5SE

N,0

420S

EN

)R

oden

ts,T

ater

asp

ecie

s,in

clud

ing

Tat

era

kem

pi,T

ater

illus

spec

ies

Sene

gal

1968

GU

8160

21

Le

Dan

tec

viru

sU

AL

DV

Dak

HD

763

Hum

anSe

nega

l19

65A

Y85

4650

Nko

lbis

son

viru

seU

A(K

ern

Can

yon

Gro

up)

NK

OV

Ar

YM

31/6

5(0

425C

AM

)D

ipte

ran,

Aed

essp

ecie

s,E

retm

apod

ites

spec

ies,

incl

udin

gE

retm

apod

ites

leuc

opou

s,C

ulex

tele

silla

Cam

eroo

n19

65G

U81

6022

Con

tinue

don

follo

win

gpa

ge



.org/D

ownloaded from

http://jvi.asm.org/

TA

BL

E6—

Con

tinue

d

Gen

usan

dna

mea

(spe

cies

)U

A/T

S/U

Cb

Abb

revi

atio

nSt

rain

Prin

cipa

lhos

tsp

ecie

s/ve

ctor

cSa

mpl

eor

igin

Yr

offir

stis

olat

ion

Gen

Ban

kac

cess

ion

no.

Mou

ssa

grou

pM

ouss

avi

rus

UC

MO

USV

C23

Dip

tera

n,C

ulex

dece

nsIv

ory

Coa

st20

04F

J985

748

Mou

ssa

viru

sU

CM

OU

SVD

24D

ipte

ran,

Cul

exsp

ecie

sIv

ory

Coa

st20

04F

J985

749

Sand

jimba

grou

pB

imbo

viru

seU

AB

BO

VD

akA

nB10

54d

(971

6RC

A)

Bir

d,E

uple

ctes

afer

Cen

tral

Afr

ican

Rep

ublic

1970

GU

8160

16

Bot

eke

viru

seT

S(V

SV)

BT

KV

Dak

ArB

1077

(041

7RC

A)

Dip

tera

n,C

oqui

lletti

dia

mac

ulip

enni

sC

entr

alA

fric

anR

epub

lic19

68G

U81

6014

Gar

bavi

ruse

UA

GA

RV

Dak

AnB

439a

(042

2RC

A)

Bir

ds,C

oryt

horn

iscr

ista

ta,N

ecta

rina

pulc

hella

Cen

tral

Afr

ican

Rep

ublic

1970

GU

8160

18

Kol

ongo

viru

sU

AK

OL

VD

akA

nB10

94d

(971

7RC

A)

Bir

ds,E

uple

ctes

afer

,Plo

ceus

cucu

llatu

sC

entr

alA

fric

anR

epub

lic19

70G

U81

6020

Nas

oule

viru

seU

AN

ASV

Dak

AnB

4289

a(0

410R

CA

)B

ird,

And

ropa

dus

vire

nsC

entr

alA

fric

anR

epub

lic19

73G

U81

6017

Oak

-Val

evi

rus

UA

OV

RV

CS

1342

Fer

ralp

igs;

ddi

pter

an,A

edes

vigi

lax,

Cul

exsp

ecie

s,in

clud

ing

(Cul

exed

war

dsi)

Aus

tral

ia19

81A

Y85

4670

Oua

ngo

viru

seU

AO

UA

VD

akA

nB15

82a

(971

8RC

A)

Bir

d,P

loce

usm

elan

ocep

halu

sC

entr

alA

fric

anR

epub

lic19

70G

U81

6015

Sand

jimba

viru

sU

ASJ

AV

Dak

AnB

373d

(072

44R

CA

)B

ird,

Acr

ocep

halu

ssc

hoen

obae

nus

Cen

tral

Afr

ican

Rep

ublic

1970

GU

8160

19

Sigm

agr

oup

Dro

soph

ilaaf

finis

sigm

avi

rus

UC

DA

ffSV

10D

ipte

rian

,Dro

soph

ilaaf

finis

New

Con

nect

icut

2007

GQ

4109

80D

roso

phila

mel

anog

aste

rsi

gma

viru

sU

ASI

GM

AV

(DM

elSV

)A

P30

Dip

teri

an,D

roso

phila

mel

anog

aste

rF

lori

da20

05N

C_0

1313

5

Dro

soph

ilam

elan

ogas

ter

sigm

avi

rus

UA

SIG

MA

V(D

Mel

SV)

HA

P23

Dip

teri

an,D

roso

phila

mel

anog

aste

rF

ranc

e?

GQ

3752

58

Dro

soph

ilaob

scur

asi

gma

viru

sU

CD

Obs

SV10

AD

ipte

rian

,Dro

soph

ilaob

scur

aU

nite

dK

ingd

om20

07G

Q41

0979

Sini

star

grou

pSi

nipe

rca

chua

tsir

habd

ovir

usU

CSC

RV

Fis

h,Si

nipe

rca

chua

tsi

Chi

na?

NC

_008

514

Star

ryflo

unde

rrh

abdo

viru

sU

CSF

RV

Fis

h,P

latic

hthy

sst

ella

tus

Was

hing

ton

2000

AY

4506

44

Tib

roga

rgan

grou

pT

ibro

garg

anvi

rus

UA

TIB

VC

S13

2B

ovin

es,d

wat

erbu

ffalo

es,c

attle

;dip

tera

n,C

ulic

oide

sbr

evita

rsis

Aus

tral

ia19

76A

Y85

4646

Tup

aia

viru

sT

S(V

SV)

TU

PVT

RV

1591

Tre

esh

rew

,Tup

aia

bela

nger

iT

haila

nd?

NC

_007

020

Nov

irhab

dovi

rus

Hira

me

rhab

dovi

rus

HIR

RV

CA

9703

Fis

h,in

clud

ing

cult

ured

Kor

ean

flou

nder

s,P

aral

icht

hys

oliv

aceu

s,P

leco

glos

sus

altiv

elis

,M

ilio

mac

roce

phal

us,a

ndSe

bast

esin

erm

is

Kor

ea19

97N

C_0

0509

3

Infe

ctio

ushe

mat

opoi

etic

necr

osis

viru

sIH

NV

HV

7601

AB

2316

60

Infe

ctio

ushe

mat

opoi

etic

necr

osis

viru

sIH

NV

WR

AC

Fis

h,in

clud

ing

salm

onid

Onc

orhy

nchu

sts

chaw

ytsc

haId

aho

NC

_001

652

Snak

ehea

drh

abdo

viru

sSH

RV

Fis

h,in

clud

ing

Oph

icep

halu

sst

riat

us,C

laria

sbr

atac

hus,

and

Oxy

eleo

tism

arm

orat

usT

haila

ndN

C_0

0090

3

Vira

lhem

orrh

agic

sept

icem

iavi

rus

VH

SVK

RR

V98

22F

ish,

Japa

nese

flou

nder

Japa

nA

B17

9621

Vira

lhem

orrh

agic

sept

icem

iavi

rus

VH

SV07

-71

Fis

h,O

ncor

hync

hus

myk

iss

Fra

nce

AJ2

3339

6V

iralh

emor

rhag

icse

ptic

emia

viru

sV

HSV

JF00

Ehi

1F

ish,

Par

alic

hthy

sol

ivac

eus

Japa

n20

00A

B49

0792

Vira

lhem

orrh

agic

sept

icem

iavi

rus

VH

SV14

-58

Fis

h,O

ncor

hync

hus

myk

iss

Fra

nce

AF

1438

63

Nuc

leor

habd

oviru

sM

aize

mos

aic

viru

sM

MV

Plan

ts(h

ost)

,Gra

min

ae,i

nclu

ding

Zea

may

s;he

mip

tera

ns(v

ecto

r),D

elph

acid

aeU

nite

dSt

ates

NC

_005

975



.org/D

ownloaded from

http://jvi.asm.org/

Ric

eye

llow

stun

tvi

rus

RY

SVPl

ant

(hos

t),O

ryza

sativ

a;ho

mop

tera

ns(v

ecto

r),C

icad

ellid

aeN

C_0

0374

6

Sonc

hus

yello

wne

tvi

rus

SYN

VPl

ants

(hos

t),A

ster

acea

e,in

clud

ing

Sonc

hus

oler

aceu

s;he

mip

tera

ns(v

ecto

r),A

phid

idae

NC

_001

615

Iran

ian

mai

zem

osai

cnu

cleo

rhab

dovi

rus

UC

IMM

NV

Plan

ts(h

ost)

,Gra

min

ae,i

nclu

ding

Zea

may

s;he

mip

tera

ns(v

ecto

r),D

elph

acid

aeIr

anN

C_0

1154

2

Mai

zefin

est

reak

viru

sU

CM

FSV

Plan

ts(h

ost)

,Gra

min

ae,i

nclu

ding

Zea

may

s;ho

mop

tera

ns(v

ecto

r),C

icad

ellid

aeG

eorg

ia19

99N

C_0

0597

4

Orc

hid

fleck

viru

sfU

CO

FV

SoPl

ants

(hos

t),O

rchi

dace

ae,i

nclu

ding

Cym

bidi

umsp

ecie

s;ac

arid

(vec

tor)

,B

revi

palp

usca

lifor

nicu

s

Japa

nN

C_0

0960

9

Tar

ove

inch

loro

sis

viru

sU

CT

aVC

VPl

ant

(hos

t),C

oloc

asia

escu

lent

aF

ijiIs

land

sN

C_0

0694

2

Cyt

orha

bdov

irus

Bar

ley

yello

wst

riate

mos

aic

BY

SMV

Zan

jan-

1Pl

ants

(hos

t),G

ram

inae

,inc

ludi

ngT

ritic

umsp

ecie

s;he

mip

tera

ns(v

ecto

r),D

elph

acid

aeIr

anF

J665

628

Let

tuce

necr

otic

yello

ws

viru

sL

NY

V31

8Se

vera

l(ho

st)

plan

tfa

mili

esan

dsp

ecie

s,in

clud

ing

Alli

umsa

tivum

and

Lac

tuca

sativ

a;he

mip

tera

ns(v

ecto

r),A

phid

idae

Aus

tral

iaN

C_0

0764

2

Nor

ther

nce

real

mos

aic

viru

sN

CM

VPl

ants

(hos

t),G

ram

inae

,inc

ludi

ngH

orde

umvu

lgar

e;he

mip

tera

ns(v

ecto

r),D

elph

acid

aeJa

pan

NC

_002

251

Stra

wbe

rry

crin

kle

viru

sSC

VH

B-A

1Pl

ant

(hos

t),F

raga

ria

spec

ies;

hem

ipte

rans

(vec

tor)

,Aph

idid

aeA

Y33

1389

Stra

wbe

rry

crin

kle

viru

sSC

V37

-2Pl

ant

(hos

t),F

raga

ria

spec

ies;

hem

ipte

rans

(vec

tor)

,Aph

idid

aeA

Y33

1388

Stra

wbe

rry

crin

kle

viru

sSC

V37

-1Pl

ant

(hos

t),F

raga

ria

spec

ies;

hem

ipte

rans

(vec

tor)

,Aph

idid

aeA

Y33

1387

Let

tuce

yello

wm

ottle

viru

sU

CL

YM

oVPl

ant

(hos

t),L

actu

casa

tiva

Fra

nce

1998

NC

_011

532

Taa

stru

pgr

oup,

Taa

stru

pvi

rus

UC

TV

Hem

ipte

ran

(pot

entia

lvec

tor)

,Psa

mm

otet

tixal

ienu

sF

ranc

e19

96A

Y42

3355

aN

ames

ofvi

ruse

sin

italic

sco

rres

pond

toap

prov

edsp

ecie

sac

cord

ing

toth

eIn

tern

atio

nalC

omm

ittee

onT

axon

omy

ofV

irus

esda

taba

se.

bU

A,u

nass

igne

d;T

S,te

ntat

ive;

UC

,unc

lass

ified

(not

foun

din

ICT

Vdb

).c

Inbo

ldis

the

host

spec

ies

from

whi

chth

evi

rus

was

first

isol

ated

,if

that

info

rmat

ion

isav

aila

ble.

dSe

rolo

gica

ldet

ectio

non

ly.

eF

irst

iden

tifica

tion

base

don

nucl

eic

acid

dete

rmin

atio

nan

dcl

assi

ficat

ion

base

don

phyl

ogen

ican

alys

is(t

his

stud

y).

fA

lso

tent

ativ

ely

clas

sifie

din

toth

ene

wge

nus

Dic

horh

abdo

viru

sac

cord

ing

toits

unus

ualb

ipar

tite

geno

me

(20)

.



.org/D

ownloaded from

http://jvi.asm.org/

species; two strains belonging to the genus Ephemerovirus,the Kimberley (KIMV) and kotonkan (KOTV) viruses, cor-responding to a tentative and an unassigned species, respec-tively; and 11 presently unassigned rhabdoviruses, namely,the Kamese (KAMV), Mossuril (MOSV), Sandjimba

(SAJV), Keuraliba (KEUV), Nkolbisson (NKOV), Garba(GARV), Nasoule (NASV), Ouango (OUAV), Bimbo(BBOV), Bangoran (BGNV), and Gossas (GOSV) viruses(virus taxonomy according to ICTVdb) (Table 2).

In the first step, successful detection and identification of

FIG. 3. Phylogenetic relationships of the Rhabdoviridae based on a 160-amino-acid alignment of the polymerase gene. The phylogenetic analysisof 106 amino acid sequences of block III of the polymerase (160 amino acid residues in length) of rhabdoviruses was performed using a Bayesianmethod based on the WAG model of amino acid replacement with a gamma distribution of rate variation among sites. Chains were run for 10million generations (with a 10% burn in), at which point all of the parameter estimates had converged. The level of support for each node isprovided by BPP values. The genera (black font) and groups (red font) of the family Rhabdoviridae are indicated, along with their associated BPPvalues. All of the horizontal branch lengths are drawn to a scale of amino acid replacements per residue. The tree is midpoint rooted for clarityonly. Sequences tiled on the array or closely related sequences (�, 9147FRA instead of PV) are indicated in blue font. Sequences correspondingto lyssavirus species 1 and positively detected by PathogenID v1.0 are indicated by a red line (#). Sequences detected by PathogenID v2.0 areindicated by red squares.



.org/D

ownloaded from

http://jvi.asm.org/

these viruses using the PathogenID v2.0 microarray was ob-tained for 12 (70.5%) out of 17 viruses; accurate taxonomicpositioning—that is, within the family Rhabdoviridae—was alsoachieved, and for some, the corresponding genus (when avail-able) was also matched accurately (data not shown). In thesecond step, specific and consensus primers were designedbased on the stretches of sequences identified by the microar-ray using the consensus sequence determination strategy andthen subsequently used for PCR and classical sequencing ofthe amplified target nucleotide sequences. For four (GARV,NASV, OUAV, and BBOV) of the five rhabdoviruses notdetected by the microarray, a region of 1,000 nt of the poly-merase gene encompassing that tiled on the array was success-fully amplified by PCR and sequenced using the primers de-scribed above. The only exception was the GOSV isolate,which remained undetected by either the microarray or PCR.Further, two other rhabdoviruses not previously tested with thePathogenID v2.0 microarray—Kolongo virus (KOLV, an un-classified species) and Piry virus (PIRYV, a vesiculovirus)—were also amplified and sequenced using these primers.

All of the newly sequenced nucleotide regions of the poly-merase gene were further translated into protein sequencesand aligned with 88 sequences of animal or plant rhabdovi-ruses obtained from GenBank, producing a total data set of106 sequences 160 amino acid residues in length. A Bayesianphylogenetic analysis of these sequences tentatively distin-guished 15 groups of viruses based on their strongly supportedmonophyly (Table 6 and Fig. 3). The members of the sixgenera—Ephemerovirus, Lyssavirus, Vesiculovirus, Cytorhab-dovirus, Nucleorhabdovirus, and Novirhabdovirus—fall intowell-supported monophyletic groups (BPP value, �0.97) (Fig.3). Interestingly, this analysis suggested the existence of at leastnine more groups of currently unclassified rhabdoviruses,which reflect important biological characteristics of the virusesin question. Five of these groups have been proposed previ-ously and were further supported by our analysis (data avail-able at the CRORA database website [http://www.pasteur.fr/recherche/banques/CRORA/]) (6, 27; reviewed in reference7). The first group, tentatively named the Hart Park group,contains the previously described Parry Creek (PCRV),Wongabel (WONV), Flanders (FLANV), and Ngaingan(NGAV) viruses added to the newly identified viruses BGNV,KAMV, MOSV, and PORV. This group has a large distribu-tion that encompasses Africa, Australia, Malaysia, and theUnited States. These viruses have a wide host range, as theyhave been found to infect dipterans, birds, and mammals. Thesecond group is the Almpiwar group, containing four mem-bers—two strains of Charleville (CHVV) virus, i.e., CHVV_Ch9824 and CHVV_Ch9847—and the Almpiwar (ALMV) andHumpty doo (HDOOV) viruses. Viruses of this group wereisolated in Australia and are associated with infections ofdipterans and lizards but also birds and mammals, includinghumans. Another group, herein referred to as the Le Dantecgroup, was also seen to form a distinct cluster with Le Dantecvirus (LDV), Fukuoka virus (FUKV), and the two newly mo-lecularly identified viruses KEUV and NKOV. Members wereisolated in Japan and Africa, where they were shown toinfect dipterans and mammals, including humans. Thefourth group has been tentatively named the Tibrogargangroup and includes the Tupaia (TUPV) and Tibrogargan

(TIBV) viruses. These viruses were isolated in SoutheastAsia, Australia, and New Guinea from dipterans and mam-mals. Finally, we observed the Sigma group as previouslydescribed (27). It includes Drosophila affinis (DAffSV), Dro-sophila obscura (DObsSV), and two strains of Drosophilamelanogaster (SIGMAV_AP30 and SIGMAV_HAP23)sigma viruses, infecting Drosophila flies which were found inthe United States and Europe.

In addition, four other tentative groups of viruses are newlydescribed in this study. The Sandjimba group includes the firstmolecularly classified viruses BBOV, BTKV, NASV, GARV,and OUAV and the previously described Oak-Vale virus(OVRV), SJAV, and KOLV (identification of the latter twobased only on a limited region of the nucleoprotein gene).These viruses were isolated from birds and dipterans from theCentral African Republic and Australia (data available at http://www.pasteur.fr/recherche/banques/CRORA/) (6, 9). Interest-ingly, all of the African members of this group clusteredclosely, whereas the sole Australian virus was more divergent,suggesting a potential geographical segregation. Second, theSinistar group includes the Siniperca chuatsi rhabdovirus(SCRV) isolated from mandarin fish in China (37) and thestarry flounder rhabdovirus (SFRV) from starry flounder inthe United States (30). These two viruses appear to be moreclosely related to the Le Dantec group than to viruses in thegenus Vesiculovirus, in which several other fish rhabdovirusesare classified. The third one is the Moussa group, including twoisolates of Moussa virus (MOUV_D24 and MOUV_C23) col-lected from mosquitoes in Ivory Coast (34). Finally, a phylo-genetic analysis suggests the presence of another group withinthe plant rhabdoviruses: the Taastrup group, which comprisesthe single isolate Taastrup virus (TV) isolated from leafhop-pers (Psammotettix alienus) originally collected in France (28).All of these groups were strongly supported by the Bayesiananalysis (BPP value, �0.98), with the exception of the Sigmagroup, which exhibits a BPP value of 0.88.

In addition, classification of some uncharacterized rhab-doviruses from our phylogenetic analysis diverged from thatpreviously suggested by serology (according to ICTVdb) andwill probably need further investigation to determine theirprecise taxonomic positions within the family Rhabdoviridae(Table 6) (9, 38). In particular, PORV and BTKV, previ-ously identified as vesiculoviruses, were included within theHart Park and Sandjimba groups, respectively, and NKOVwas classified into the Le Dantec group instead of the KernCanyon group. Moreover, in contrast to a previous phylo-genetic study (22), TUPV was found to be more closelyrelated to TIBV than to any other isolates in the Sandjimbagroup. Finally, our study confirmed the previous serology-based classification of JURV and the recently identifiedScophthalmus maximus rhabdovirus (SMRV) within the Ve-siculovirus genus (38, 49).

DISCUSSION

We have analyzed the capacity of viral detection and iden-tification of two versions of a newly described RMA, termedPathogenID, which was designed specifically for multiplepathogen detection using database similarity searching (1). Toevaluate this microarray, we focused on one of the largest and



.org/D

ownloaded from

http://jvi.asm.org/

most diverse viral families described to date, the Rhabdoviridae(ICTVdb, reviewed in reference 7). All of the virus strainstested (except WCBV) were extracted from biological samplesand amplified using a nonspecific and unbiased WTA step aspreviously described (3). Rhabdovirus-targeted sequenceswere selected among blocks of conservation within the poly-merase gene (6). This region was chosen so as to encompass asufficient number of homologous but also polymorphic sites.The key advantage of this RMA strategy is that it does notrequire a specific match between the samples tested and tiledsequences; indeed, mismatches add value as they allow precisetyping of the unknown genetic resequenced element. In ourcase, the conserved nature of the target region of the polymer-ase gene (block III) and the capability of detection of the RMAallows a precise taxonomic identification (i.e., family, genus,species) and also provides key information on phylogeneticrelationships for some unclassified, unassigned, or tentativespecies of rhabdoviruses. For example, results obtained by thePathogenID v1.0 microarray evaluation demonstrated thatmost of the intraspecies nucleotide diversity found in the genusLyssavirus can be covered by a single prototype sequence tiledon the microarray. Using the second version of PathogenIDwhich included one prototype sequence of each of the sevenspecies recognized thus far within the genus Lyssavirus, weextended the spectrum of detection of the RMA to potentiallyall of the known or unknown lyssaviruses (i.e., positive detec-tion of virus isolates presenting up to 25.9% nucleotide diver-gence with the tiled sequence considered), which is greaterthan that previously reported (24–26, 43, 44).

This study also indicates that accurate viral identificationmay still be possible even when only shorter sequences areobtained from individual tiled prototype sequences. Indeed,taken individually, these short stretches of nucleotide sequencecould not give positive results during the initial BLAST query.However, when used in the consensus sequence determinationstrategy employed here, they improved the identification ofvirus strains distantly related with that tiled on the RMA. Forexample, we were able to test and detect rhabdoviruses basedon sequence data obtained with tiled sequences that originatedfrom other viral genera.

The strategy developed here also allowed the potential de-tection of genetically diverse rhabdoviruses previously identi-fied or unknown by using a limited number of sequences tiledon the microarray. Using the PathogenID v2.0 microarray, wewere able to identify 30 rhabdoviruses in total. This included 12viruses currently unclassified, unassigned, or assigned as ten-tative species within the family Rhabdoviridae (according toICTVdb). Moreover, the consensus sequence-based analysis ofRMA results was shown to be accurate compared to sequencesobtained through classical sequencing (Table 5 and data notshown). Sequence data provided by the PathogenID v2.0 mi-croarray were also extremely helpful in the design of specificprimers to further sequence the targeted region of the viralpolymerase gene of some other rhabdoviruses. Finally, thisapproach allowed us to undertake the largest phylogeneticanalysis of the family Rhabdoviridae (Table 6 and Fig. 3), eventhough it is important to note that the list of viruses andpotential taxa described here is still incomplete and more vi-ruses will clearly be characterized in the near future. Despitethese phylogenetic divisions, all of the viruses included in these

proposed groups are closely related to vesiculoviruses andephemeroviruses and were found to infect a large spectrum ofanimals, included dipterans and mammals (and previously re-ferred to as the dimarhabdovirus supergroup (6) but also liz-ards (Almpiwar group), birds (especially the Sandjimba groupbut also with Hart Park group), and fish (Sinistar group) (Ta-ble 6).

Although promising, inadequate sequence selection for thedesign of the RMA, and consequently a lack of coverage of theviral sequence space, represents an important limitation. Aproper selection of blocks of conserved sequence across taxo-nomic subdivisions in the viral world could be similarly definedand targeted by the RMA assay, and in doing so improve thedetection power of this tool and therein greatly aid in theidentification of members of the family Rhabdoviridae or evenother viral families. The results presented here validated theusefulness of the design methodology. It emphasizes the gainin identification using a consensus sequence strategy determi-nation compared to a systematic BLAST strategy (29). Indeed,this strategy allows us to use and accurately analyze the RMAoutput data, even if only short subsequences with a high basecall rate are obtained. It provides an informative alternative tocurrent molecular methods, such as classical or multiplex PCR,for the rapid identification of viral pathogens. It is currentlybeing applied to assist in a new generation of RMA aimed atthe detection and identification of genetically diverse and un-known viral pathogens and more broadly of any virus presentin a clinical specimen. In contrast to conventional microarrays,it is not limited by the requirement of prior knowledge of theidentities of viruses present in biological samples and it is notrestricted to the detection of a limited number of candidateviruses. As such, this strategy has a great potential for beingimplemented as a high-throughput platform to identify moredivergent viral organisms. This technology could be especiallyuseful in clinical diagnosis or in surveillance programs fordetecting uncharacterized viral pathogens or highly variablevirus strains in the same taxonomic genus or family, which isfrequently the case for RNA viruses (2). The potential appli-cations of such a methodology therefore appear to be numer-ous: differential diagnostics for illnesses with multiple potentialcauses (for example, central nervous diseases like encephalitisand meningitis), tracking of emergent pathogens, the distinc-tion of biological threats from harmless phylogenetic neigh-bors, and the broader-scale investigation of biodiversity in theviral world.

ACKNOWLEDGMENTS

This work was supported by grant UC1 AI062613 (G. C. Kennedy)from the U.S. National Institute of Allergy and Infectious Diseases,National Institute of Health; the Programme Transversal de Recher-che (PTR DEVA 246) from the Institut Pasteur, Paris, France; theEuropean Commission, through the VIZIER Integrated Project(LSHG-CT-2004-511966); and the Institut Pasteur International Net-work Actions Concertees InterPasteuriennes (2003/687). We thank thesponsorship of the Total-Institut Pasteur for financial support.

We are grateful to D. Blondel, H. Zeller, and the CRORA databasefor having provided some of the rhabdovirus isolates tested in thisstudy. We are also grateful to the technical staff of the Genotyping ofPathogens and Public Health Technological Platform for their pa-tience and their excellent work in the sequencing of the differentrhabdoviruses.



.org/D

ownloaded from

http://jvi.asm.org/

REFERENCES

1. Berthet, N., P. Dickinson, I. Filliol, A. K. Reinhardt, C. Batejat, T. Vallaeys,K. A. Kong, C. Davies, W. Lee, S. Zhang, Y. Turpaz, B. Heym, G. Coralie, L.Dacheux, A. M. Burguiere, H. Bourhy, I. G. Old, J. M. Manuguerra, S. T.Cole, and G. C. Kennedy. 2007. Massively parallel pathogen identificationusing high-density microarrays. Microb. Biotechnol. 1:79–86.

2. Berthet, N., I. Leclercq, A. Dublineau, S. Shigematsu, A. M. Burguiere, C.Filippone, A. Gessain, and J. C. Manuguerra. 2010. High-density rese-quencing DNA microarrays in public health emergencies. Nat. Biotech-nol. 28:25–27.

3. Berthet, N., A. K. Reinhardt, I. Leclercq, S. van Ooyen, C. Batejat, P.Dickinson, R. Stamboliyska, I. G. Old, K. A. Kong, L. Dacheux, H. Bourhy,G. C. Kennedy, C. Korfhage, S. T. Cole, and J. C. Manuguerra. 2008. Phi29polymerase based random amplification of viral RNA as an alternative torandom RT-PCR. BMC Mol. Biol. 9:77.

4. Bodrossy, L., and A. Sessitsch. 2004. Oligonucleotide microarrays in micro-bial diagnostics. Curr. Opin. Microbiol. 7:245–254.

5. Botvinkin, A. D., E. M. Poleschuk, I. V. Kuzmin, T. I. Borisova, S. V.Gazaryan, P. Yager, and C. E. Rupprecht. 2003. Novel lyssaviruses isolatedfrom bats in Russia. Emerg. Infect. Dis. 9:1623–1625.

6. Bourhy, H., J. A. Cowley, F. Larrous, E. C. Holmes, and P. J. Walker. 2005.Phylogenetic relationships among rhabdoviruses inferred using the L poly-merase gene. J. Gen. Virol. 86:2849–2858.

7. Bourhy, H., A. Gubala, R. P. Weir, and D. Boyle. 2008. Animal rhabdovi-ruses, p. 111–121. In B. W. J. Mahy and M. H. V. Van Regenmortel (ed.),Encyclopedia of virology, vol. 1. Elsevier, Oxford, United Kingdom.

8. Bourhy, H., P. E. Rollin, J. Vincent, and P. Sureau. 1989. Comparative fieldevaluation of the fluorescent-antibody test, virus isolation from tissue cul-ture, and enzyme immunodiagnosis for rapid laboratory diagnosis of rabies.J. Clin. Microbiol. 27:519–523.

9. Calisher, C. H., N. Karabatsos, H. Zeller, J. P. Digoutte, R. B. Tesh, R. E.Shope, A. P. Travassos da Rosa, and T. D. St. George. 1989. Antigenicrelationships among rhabdoviruses from vertebrates and hematophagousarthropods. Intervirology 30:241–257.

10. Chiu, C. Y., A. A. Alizadeh, S. Rouskin, J. D. Merker, E. Yeh, S. Yagi, D.Schnurr, B. K. Patterson, D. Ganem, and J. L. DeRisi. 2007. Diagnosis of acritical respiratory illness caused by human metapneumovirus by use of apan-virus microarray. J. Clin. Microbiol. 45:2340–2343.

11. Chiu, C. Y., A. L. Greninger, K. Kanada, T. Kwok, K. F. Fischer, C. Runckel,J. K. Louie, C. A. Glaser, S. Yagi, D. P. Schnurr, T. D. Haggerty, J. Parson-net, D. Ganem, and J. L. DeRisi. 2008. Identification of cardioviruses relatedto Theiler’s murine encephalomyelitis virus in human infections. Proc. Natl.Acad. Sci. U. S. A. 105:14124–14129.

12. Chiu, C. Y., A. Urisman, T. L. Greenhow, S. Rouskin, S. Yagi, D. Schnurr,C. Wright, W. L. Drew, D. Wang, P. S. Weintrub, J. L. Derisi, and D. Ganem.2008. Utility of DNA microarrays for detection of viruses in acute respiratorytract infections in children. J. Pediatr. 153:76–83.

13. Dacheux, L., J. M. Reynes, P. Buchy, O. Sivuth, B. M. Diop, D. Rousset, C.Rathat, N. Jolly, J. B. Dufourcq, C. Nareth, S. Diop, C. Iehle, R. Rajerison,C. Sadorge, and H. Bourhy. 2008. A reliable diagnosis of human rabies basedon analysis of skin biopsy specimens. Clin. Infect. Dis. 47:1410–1417.

14. Delarue, M., O. Poch, N. Tordo, D. Moras, and P. Argos. 1990. An attemptto unify the structure of polymerases. Protein Eng. 3:461–467.

15. Delmas, O., E. C. Holmes, C. Talbi, F. Larrous, L. Dacheux, C. Bouchier,and H. Bourhy. 2008. Genomic diversity and evolution of the lyssaviruses.PLoS One 3:e2057.

16. Hacia, J. G. 1999. Resequencing and mutational analysis using oligonucle-otide microarrays. Nat. Genet. 21:42–47.

17. Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignmenteditor and analysis program for Windows 95/98/NT. Nucleic Acids Symp.Ser. 41:95–98.

18. Huelsenbeck, J. P., and F. Ronquist. 2001. MRBAYES: Bayesian inferenceof phylogenetic trees. Bioinformatics 17:754–755.

19. Kistler, A., P. C. Avila, S. Rouskin, D. Wang, T. Ward, S. Yagi, D. Schnurr,D. Ganem, J. L. DeRisi, and H. A. Boushey. 2007. Pan-viral screening ofrespiratory tract infections in adults with and without asthma reveals unex-pected human coronavirus and human rhinovirus diversity. J. Infect. Dis.196:817–825.

20. Kondo, H., T. Maeda, Y. Shirako, and T. Tamada. 2006. Orchid fleck virusis a rhabdovirus with an unusual bipartite genome. J. Gen. Virol. 87:2413–2421.

21. Kothapalli, R., S. J. Yoder, S. Mane, and T. P. Loughran, Jr. 2002. Microar-ray results: how accurate are they? BMC Bioinformatics 3:22.

22. Kuzmin, I. V., G. J. Hughes, and C. E. Rupprecht. 2006. Phylogeneticrelationships of seven previously unclassified viruses within the familyRhabdoviridae using partial nucleoprotein gene sequences. J. Gen. Virol.87:2323–2331.

23. Leski, T. A., B. Lin, A. P. Malanoski, Z. Wang, N. C. Long, C. E. Meador, B.Barrows, S. Ibrahim, J. P. Hardick, M. Aitichou, J. M. Schnur, C. Tibbetts,and D. A. Stenger. 2009. Testing and validation of high density resequencingmicroarray for broad range biothreat agents detection. PLoS One 4:e6569.

24. Lin, B., K. M. Blaney, A. P. Malanoski, A. G. Ligler, J. M. Schnur, D.Metzgar, K. L. Russell, and D. A. Stenger. 2007. Using a resequencingmicroarray as a multiple respiratory pathogen detection assay. J. Clin. Mi-crobiol. 45:443–452.

25. Lin, B., A. P. Malanoski, Z. Wang, K. M. Blaney, A. G. Ligler, R. K. Rowley,E. H. Hanson, E. von Rosenvinge, F. S. Ligler, A. W. Kusterbeck, D. Metz-gar, C. P. Barrozo, K. L. Russell, C. Tibbetts, J. M. Schnur, and D. A.Stenger. 2007. Application of broad-spectrum, sequence-based pathogenidentification in an urban population. PLoS One 2:e419.

26. Lin, B., Z. Wang, G. J. Vora, J. A. Thornton, J. M. Schnur, D. C. Thach,K. M. Blaney, A. G. Ligler, A. P. Malanoski, J. Santiago, E. A. Walter, B. K.Agan, D. Metzgar, D. Seto, L. T. Daum, R. Kruzelock, R. K. Rowley, E. H.Hanson, C. Tibbetts, and D. A. Stenger. 2006. Broad-spectrum respiratorytract pathogen identification using resequencing DNA microarrays. GenomeRes. 16:527–535.

27. Longdon, B., D. J. Obbard, and F. M. Jiggins. 2010. Sigma viruses fromthree species of Drosophila form a major new clade in the rhabdovirusphylogeny. Proc. Biol. Sci. 277:35–44.

28. Lundsgaard, T. 1997. Filovirus-like particles detected in the leafhopperPsammotettix alienus. Virus Res. 48:35–40.

29. Malanoski, A. P., B. Lin, Z. Wang, J. M. Schnur, and D. A. Stenger. 2006.Automated identification of multiple micro-organisms from resequencingDNA microarrays. Nucleic Acids Res. 34:5300–5311.

30. Mork, C., P. Hershberger, R. Kocan, W. Batts, and J. Winton. 2004. Isolationand characterization of a rhabdovirus from starry flounder (Platichthys stel-latus) collected from the northern portion of Puget Sound, Washington,USA. J. Gen. Virol. 85:495–505.

31. Paez, J. G., M. Lin, R. Beroukhim, J. C. Lee, X. Zhao, D. J. Richter, S.Gabriel, P. Herman, H. Sasaki, D. Altshuler, C. Li, M. Meyerson, and W. R.Sellers. 2004. Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification. NucleicAcids Res. 32:e71.

32. Palacios, G., P. L. Quan, O. J. Jabado, S. Conlan, D. L. Hirschberg, Y. Liu,J. Zhai, N. Renwick, J. Hui, H. Hegyi, A. Grolla, J. E. Strong, J. S. Towner,T. W. Geisbert, P. B. Jahrling, C. Buchen-Osmond, H. Ellerbrok, M. P.Sanchez-Seco, Y. Lussier, P. Formenty, M. S. Nichol, H. Feldmann, T.Briese, and W. I. Lipkin. 2007. Panmicrobial oligonucleotide array for diag-nosis of infectious diseases. Emerg. Infect. Dis. 13:73–81.

33. Poch, O., I. Sauvaget, M. Delarue, and N. Tordo. 1989. Identification of fourconserved motifs among the RNA-dependent polymerase encoding ele-ments. EMBO J. 8:3867–3874.

34. Quan, P. L., S. Junglen, A. Tashmukhamedova, S. Conlan, S. K. Hutchi-son, A. Kurth, H. Ellerbrok, M. Egholm, T. Briese, F. H. Leendertz, andW. I. Lipkin. 2010. Moussa virus: a new member of the Rhabdoviridaefamily isolated from Culex decens mosquitoes in Cote d’Ivoire. VirusRes. 147:17–24.

35. Quan, P. L., G. Palacios, O. J. Jabado, S. Conlan, D. L. Hirschberg, F. Pozo,P. J. Jack, D. Cisterna, N. Renwick, J. Hui, A. Drysdale, R. Amos-Ritchie, E.Baumeister, V. Savy, K. M. Lager, J. A. Richt, D. B. Boyle, A. Garcia-Sastre,I. Casas, P. Perez-Brena, T. Briese, and W. I. Lipkin. 2007. Detection ofrespiratory viruses and subtype identification of influenza A viruses byGreeneChipResp oligonucleotide microarray. J. Clin. Microbiol. 45:2359–2364.

36. Taitt, C. R., A. P. Malanoski, B. Lin, D. A. Stenger, F. S. Ligler, A. W.Kusterbeck, G. P. Anderson, S. E. Harmon, L. C. Shriver-Lake, S. K. Pol-lack, D. M. Lennon, F. Lobo-Menendez, Z. Wang, and J. M. Schnur. 2008.Discrimination between biothreat agents and ‘near neighbor’ species using aresequencing array. FEMS Immunol. Med. Microbiol. 54:356–364.

37. Tao, J. J., G. Z. Zhou, J. F. Gui, and Q. Y. Zhang. 2008. Genomic sequenceof mandarin fish rhabdovirus with an unusual small non-transcriptionalORF. Virus Res. 132:86–96.

38. Tesh, R. B., A. P. Travassos Da Rosa, and J. S. Travassos Da Rosa. 1983.Antigenic relationship among rhabdoviruses infecting terrestrial vertebrates.J. Gen. Virol. 64(Pt. 1):169–176.

39. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment throughsequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 22:4673–4680.

40. Vora, G. J., C. E. Meador, D. A. Stenger, and J. D. Andreadis. 2004. Nucleicacid amplification strategies for DNA microarray-based pathogen detection.Appl. Environ. Microbiol. 70:3047–3054.

41. Wang, D., L. Coscoy, M. Zylberberg, P. C. Avila, H. A. Boushey, D. Ganem,and J. L. DeRisi. 2002. Microarray-based detection and genotyping of viralpathogens. Proc. Natl. Acad. Sci. U. S. A. 99:15687–15692.

42. Wang, D., A. Urisman, Y. T. Liu, M. Springer, T. G. Ksiazek, D. D. Erdman,E. R. Mardis, M. Hickenbotham, V. Magrini, J. Eldred, J. P. Latreille, R. K.Wilson, D. Ganem, and J. L. DeRisi. 2003. Viral discovery and sequencerecovery using DNA microarrays. PLoS Biol. 1:E2.

43. Wang, Z., L. T. Daum, G. J. Vora, D. Metzgar, E. A. Walter, L. C. Canas,A. P. Malanoski, B. Lin, and D. A. Stenger. 2006. Identifying influenzaviruses with resequencing microarrays. Emerg. Infect. Dis. 12:638–646.

44. Wang, Z., A. P. Malanoski, B. Lin, C. Kidd, N. C. Long, K. M. Blaney, D. C.



.org/D

ownloaded from

http://jvi.asm.org/

Thach, C. Tibbetts, and D. A. Stenger. 2008. Resequencing microarray probedesign for typing genetically diverse viruses: human rhinoviruses and entero-viruses. BMC Genomics 9:577.

45. Wilson, W. J., C. L. Strout, T. Z. DeSantis, J. L. Stilwell, A. V. Carrano, andG. L. Andersen. 2002. Sequence-specific identification of 18 pathogenic mi-croorganisms using microarray technology. Mol. Cell. Probes 16:119–127.

46. Xiong, Y., and T. H. Eickbush. 1990. Origin and evolution of retroelementsbased upon their reverse transcriptase sequences. EMBO J. 9:3353–3362.

47. Xu, G., P. Weber, Q. Hu, H. Xue, L. Audry, C. Li, J. Wu, and H. Bourhy.

2007. A simple sandwich ELISA (WELYSSA) for the detection of lyssavirusnucleocapsid in rabies suspected specimens using mouse monoclonal anti-bodies. Biologicals 35:297–302.

48. Yoo, S. M., J. Y. Choi, J. K. Yun, J. K. Choi, S. Y. Shin, K. Lee, J. M. Kim,and S. Y. Lee. 2010. DNA microarray-based identification of bacterial andfungal pathogens in bloodstream infections. Mol. Cell. Probes 24:44–52.

49. Zhang, Q. Y., J. J. Tao, L. Gui, G. Z. Zhou, H. M. Ruan, Z. Q. Li, and J. F.Gui. 2007. Isolation and characterization of Scophthalmus maximus rhab-dovirus. Dis. Aquat. Organ. 74:95–105.



.org/D

ownloaded from

http://jvi.asm.org/

Application of Broad-Spectrum Resequencing …jvi.asm.org/content/84/18/9557.full.pdf... Lyssavirus...

Documents

Transcript of Application of Broad-Spectrum Resequencing …jvi.asm.org/content/84/18/9557.full.pdf... Lyssavirus...