research article real-time rt-pcr for the detection of...

Research ArticleReal-Time RT-PCR for the Detection of Lyssavirus Species

A Deubelbeiss M-L Zahno M Zanoni D Bruegger and R Zanoni

Institute of Virology and Immunology 3012 Berne Switzerland

Correspondence should be addressed to R Zanoni zanonivetsuisseunibech

Received 21 July 2014 Revised 19 September 2014 Accepted 24 September 2014 Published 16 October 2014

Academic Editor Masanori Tohno

Copyright copy 2014 A Deubelbeiss et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The causative agents of rabies are single-stranded negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae consistingof twelve classified and three as yet unclassified species including classical rabies virus (RABV) Highly neurotropic RABV causesrapidly progressive encephalomyelitis with nearly invariable fatal outcome Rapid and reliable diagnosis of rabies is highly relevantfor public and veterinary health Due to growing variety of the genus Lyssavirus observed the development of suitable molecularassays for diagnosis and differentiation is challenging This work focused on the establishment of a suitable real-time RT-PCRtechnique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standardfor diagnosis and confirmationThe real-time RT-PCRwas adapted with the goal to detect the whole spectrum of lyssavirus speciesfor nine of which synthesized DNA fragments were used For the detection of species seven probes were developed Serial dilutionsof the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR Using apanel of thirty-one lyssaviruses representing four species the suitability of the protocol could be shown Phylogenetic analysis ofthe sequences obtained by heminested PCR allowed correct classification of all viruses used

1 Introduction

Rabies diagnosis is based on fluorescent antibody testing(FAT) in brain smears and inoculation of brain suspensioneither inmouse neuroblastoma cell cultures or intracerebrallyin mice as confirmatory assays with high sensitivity andspecificity for postmortem diagnosis [1ndash3] These techniquesarewell suited for rapid and reliable routine diagnosis if brainmaterial is available For other diagnostic specimens sampledintra vitam in case of clinical suspicion for example salivacerebrospinal fluid or skin biopsies more suited moleculartechniqueswith excellent sensitivity have been developed andvalidated mostly targeting the conserved nucleoprotein geneof the lyssavirus genome for example [4ndash10] Among thesethere are protocols both for classical RT-PCR and for real-time RT-PCR which adds speed efficiency contaminationsafety and reliability to the technique combined with thepotential to quantify the viral load [11 12] Amajor advantageoffered by these molecular techniques is the characterisationof viral isolates by sequencing the given amplification prod-ucts followed by phylogenetic or phylogeographic analysis[13ndash15] The greatest challenge for these powerful novel

techniques is the ever growing spectrum of known lyssavirusspeciesgenotypes (GT) probably all of which having thepotential to cause animal and human rabies fatalities [16 17]So far 12 lyssavirus species and three as yet unclassifiedspecies have been identified [17]The development of classicalsimple or (hemi)nested RT-PCR methods with particularemphasis on the diagnosis of a broad spectrumof lyssavirusesspecies has been published in a number of studies [18ndash23] Also several real-time RT-PCR protocols suited for thebroad detection or differentiation of several genotypes havebeen developed [9 24ndash28] The work of Wakeley et al[25] is of particular interest for the rabies epidemiology inEurope concerning the European bat lyssaviruses types 1 and2 [29ndash32] apart from classical rabies [33 34] This assay usesgenotype (GT) specific probes and fluorophore signals fordirect differentiation of GT1 GT5 and GT6 in a single-tubereaction The protocol proposed by Nadin-Davis et al [8] ishighly suited for the detection of a broad range of classicalrabies viruses

The goal of this work was based on a comprehensivereview of the rich literature on the theme and adaptingthereof to develop establish and validate classical and

Hindawi Publishing CorporationJournal of Veterinary MedicineVolume 2014 Article ID 476091 12 pageshttpdxdoiorg1011552014476091

2 Journal of Veterinary Medicine

real-time RT-PCR protocols for (intravitam) diagnosis ofrabies and molecular-epidemiological characterisation ofviral strains with the main emphasis on the growing numberof known speciesgenotypes

2 Materials and Methods

21 Samples

211 Cell Culture Supernatant of Viral Strains All operationswith potentially infectiousmaterial apart from centrifugationin closed tubes were performed in a class II biologicalsafety cabinet Frozen cell culture supernatants of rabiesvirus strains propagated previously on BHK-21 cells (babyhamster kidney cells ATCC Manassas USA) were thawedcentrifuged at 2000 g for 10min at 4∘C and filtrated usingthe Millex-HA 450 nm Filter (Millipore Cork Ireland)

212 Brain Suspension Approximately 1 g of cerebellummedulla oblongata and hippocampus from unfixed freshlyobtained brain material was homogenized to a 20 brainsuspension using a mortar and pestle after addition ofapproximately 1 g of quartz sand (Merck KGaA DarmstadtGermany) and 5mL of Modified Eagle Medium with Earlersquossalts with 22 gL NaHCO

3(Bioswisstec AG Schaffhausen

Switzerland) supplemented with 5 penicillin 10000 IUmL(Bioswisstec AG Schaffhausen Switzerland) and 20 foetalcalf serum (FCS PAA Laboratories GmbH Pasching Aus-tria) The suspension was then decanted into a 5mL tube(5mL Polystyrene Round-Bottom Tube BD BiosciencesErembodegem Belgium) to let it sediment for 1 hour at4∘C and subsequently centrifuged at 1400 g for 10min andfiltrated as above Frozen brain suspensions from formermouse inoculation tests [39] were handled like frozen cellculture supernatant

213 Saliva and Oral Swabs 500120583L of RNA Storage Solution(Ambion Foster City USA)was added to 100ndash200120583L of freshor previously frozen saliva samples or swabs which weresubsequently vortexed (Vortex Genie 2 TEWIS LaborbedarfAG Berne Switzerland) for 1min and centrifuged in aBiofuge Pico (Heraeus Holding GmbH Hanau Germany) at5000 rpm for 10min

214 Cerebrospinal Fluid Cerebrospinal fluid (CSF) sampleswere diluted 1 2 to 1 4 with RNA Storage Solution (AmbionFoster City USA)

215 Skin Biopsies Skin biopsies were shaved cut into smallpieces with a sterile pair of scissors and further processed asdescribed for brain suspension

22 Viral Strains and Propagation in Cell Culture A panelof 31 lyssaviruses (including 4 species) was used to testthe diagnostic performance of our PCR (Table 1) Stocks ofviral strains were produced in neuroblastoma cells (MNA4213) as described for the rabies tissue culture infectiontest (RTCIT described below) The maintenance medium

used for the first passage was Dulbeccorsquos MEM supple-mented with 05 neomycin 50 IUmL (Bioswisstec AGSchaffhausen Switzerland) 5 tryptose phosphate (BioCon-cept Ltd Amimed Allschwil Switzerland) 1 nonessen-tial amino acids (Bioswisstec AG Schaffhausen Switzer-land) 3 foetal calf serum and 1 L-Glutamine 200mM(Bioswisstec AG Schaffhausen Switzerland) to which 1Diethylaminoethyl-Dextran (DEAE-Dextran Sigma-AldrichCorporation St Louis USA) was added Dulbeccorsquos MEMwith 10 foetal calf serum was used for the 3 consecutivepassages Staining of the cells was performed after eachpassage using Rabies DFA Reagent (Millipore LivingstonUK) as a conjugate

23 FAT and RTCIT The standard fluorescent antibody test(FAT) was performed with brain tissue samples as previouslydescribed [40] Rabies tissue culture infection test (RTCIT)using four consecutive passages on murine neuroblastomacells [41ndash43] was applied to clinical specimens like brainspecimens liquor saliva or skin biopsies

24 Primers Probes and Synthetic DNA Primers probesand synthetic DNA were obtained from Microsynth (Micro-synth GmbH Balgach Switzerland) The location of suit-able N-directed primers and probes for heminested RT-PCR and real-time RT-PCR respectively was chosen andevaluated based on published work [8 18 22 25] Usingmultiple sequence alignment (ClustalX 203 program [44ndash46]) of the N gene region of the available lyssavirusspecies RABV (33 sequences) LBV (4 sequences) MOKV(5 sequences) DUVV (4 sequences) EBLV-1 (17 sequences)EBLV-2 (14 sequences) ABLV Aravan Khujand Irkut andWest Caucasian bat (each one) from GenBank (NationalCenter for Biotechnology Information and National Libraryof Medicine Rockville Pike USA) variable positions ofprimers and probes were adjusted with wobble positionsfor a potentially broader match Alternatively primers withsinglewobbles or substitutionsweremixed Several probes forreal-time RT-PCR for broadening the spectrum of detectablespecies were designed in this work (Table 2) As internalcontrol for conventional RT-PCR amplification of GAPDH(glyceraldehyde 3-phosphate dehydrogenase) was used [37]As internal inhibition control for real-time RT-PCR primersand probes for the amplification of Sendai virus (ATCCManassas USA [38]) which was added to the samples beforeRNA isolation were used (Table 2)

Synthetic DNA with a length of 125 bases encompassingpositions 48ndash172 according to the Pasteur virus genome(X03673) of the following lyssavirus species were obtainedfrom Microsynth Aravan virus (ARAV) EF614259 Khujandvirus (KHUV) EF614261 Bokeloh bat lyssavirus (BBLV)JF311903 Australian bat lyssavirus (ABLV) AF418014 Irkutvirus (IRKV) EF614260 Lagos bat virus (LBV) EU293110Mokola virus (MOKV) 293117 Shimoni bat virus (SHIV)GU170201 andWest Caucasian bat virus (WCBV) EF614258

25 RNA Extraction RNA was isolated from 140 120583L ofsample supernatant using the QIAamp Viral RNA Mini Kit

Journal of Veterinary Medicine 3

Table1Ra

bies

virusesu

sed

Species

Designatio

nHostspecies

Orig

inyearo

fisolatio

nreceipta

Material

Accessionnu

mberb

EBLV

-1Ba

tStade

Bat(E

serotin

us)

Germany(Stade)1970

Mou

sebrainsuspensio

nKF

831524K

F831550

EBLV

-1Ba

tHam

burg

RV9

Bat(E

serotin

us)

Germany(H

ambu

rg)1968

Mou

sebrainsuspensio

nKF

831526K

F831552

EBLV

-2Ba

tFinland

RV8

Hum

anbatexpo

sure

Finland1986

Mou

sebrainsuspensio

nKF

831528K

F831553

EBLV

-1Ba

tHolland

RV31

Bat

Netherla

nds1988

aMou

sebrainsuspensio

nKF

831525K

F831551

RABV

Dog

Tunisia

Dog

Tunisia

1986

BHK-

21cellcultu

resupernatant

KF831519K

F831545

EBLV

-1Ba

tSpain

RV119

Bat

Spain

1989

aBH

K-21

cellcultu

resupernatant

KF831527

RABV

BatF

lorid

aYello

wbat

USA

(Florid

a)1986a

BHK-

21cellcultu

resupernatant

KF831522K

F831548

EBLV

-1Ba

tDenmark

Bat(E

serotin

us)

Denmark1986

aBH

K-21

cellcultu

resupernatant

KF831523K

F831549

RABV

Raccoo

ndo

gPo

land

Raccoo

ndo

gPo

land

1985

Mou

sebrainsuspensio

nKF

831518K

F831544

RABV

Raccoo

nFlorida

Raccoo

nUSA

(Florid

a)1986

BHK-

21cellcultu

resupernatant

KF831521K

F831547

RABV

BatC

hileRV

108

Bat

Chile1988a

Mou

sebrainsuspensio

nKF

831520K

F831546

RABV

JackalZimbabw

eJackal

Zimbabw

e1990

aMou

sebrainsuspensio

nKF

831529K

F831554

RABV

Cattle

Zimbabw

eCattle

Zimbabw

e1990

aMou

sebrainsuspensio

nKF

831555

RABV

Canada

RedFo

xRe

dFo

xCa

nada

(Ontario)1986

aMou

sebrainsuspensio

nKF

831530K

F831556

RABV

LEP

Hum

anUSA

(Georgia)1939

BHK-

21cellcultu

resupernatant

KF831531K

F831557

RABV

HEP

Hum

anUSA

1939

BHK-

21cellcultu

resupernatant

KF831532K

F831568

RABV

Dog

NepalNr96

Dog

Nepal1989a

BHK-

21cellcultu

resupernatant

KF831534K

F831559

RABV

SriL

anka

dog121

Dog

SriL

anka1986

BHK-

21cellcultu

resupernatant

KF831535K

F831572

RABV

Eurofox91287

Fox

Switzerland

1987

BHK-

21cellcultu

resupernatant

KF831538K

F831562

DUVV

Duvenhage

RV6

Hum

anSouthAfrica1988

aBH

K-21

cellcultu

resupernatant

KF831533K

F831558

RABV

NYC

58Labo

ratory

strain

USA

1987a

Mou

sebrainsuspensio

nKF

831539K

F831563

RABV

Dog

Lima

Dog

Peru

(Lim

a)1985a

Mou

sebrainsuspensio

nKF

831540

KF831564

EBLV

-1Ba

tBremerhavenRV

11Ba

tGermany(Bremerhaven)1989a

BHK-

21cellcultu

resupernatant

KF831541K

F831565

EBLV

-1Ba

tB24

Bat

Europe1986a

Mou

sebrainsuspensio

nKF

831536K

F831560

RABV

Skun

kCa

nada

Strip

edskun

kCa

nada

(Ontario)1986

BHK-

21cellcultu

resupernatant

KF831537K

F831561

RABV

SADBe

rnER

Alowast

USA

1935(received1976)

BHK-

21cellcultu

resupernatant

KF831542K

F831566

RABV

CSSR

A-viru

sRo

dentlaboratorystr

ain

CSSR

(Prague)1987a

BHK-

21cellcultu

resupernatant

KF831543K

F831567

RABV

Challengev

irusstand

ard(C

VS-11)AT

CCVR959

Labo

ratory

strain

France

(CNEV

A)1995

aBH

K-21

cellcultu

resupernatant

GU992321

EBLV

-2TW

181492

Bat(Mdaubentonii)

Switzerland

(Plaffeien)1992

Na4

213

cellcultu

resupernatant

KF831569

EBLV

-2TW

139293

Bat(Mdaubentonii)

Switzerland

(Versoix)1993

Na4

213

cellcultu

resupernatant

KF831570

EBLV

-2TW

11802

Bat(Mdaubentonii)

Switzerland

(Geneva)2002

Na4

213

cellcultu

resupernatant

KF831571

Lyssaviru

seso

f4different

speciesw

eretested

a Yearo

freceipt

attheS

wiss

rabies

center

b Accessio

nnu

mbersof

220b

pand543b

pNfragments

respectiv

elysequenced

inthiswork

lowast

Reference[36]


Table2Prim

ersa

ndprob

es

Metho

dNam

eSequ

ence

Leng

thPo

sitiona

Prod

uct

Reference

hnRT

-PCR

JW12-F

ATGTA

ACAC

CYCT

ACAAT

G19

55ndash73

Pann

ingetal2010

[22]

JW6A

S1-R1

CAAT

TGGCA

CACA

TTTT

GTG

2066

0ndash64

160

6bp

JW6A

S2-R1

CAGTT

AGCG

CACA

TCTT

ATG

2066

0ndash64

160

6bp

JW10AS1-R2

GTC

ATCA

ATGTG

TGAT

GTT

C20

636ndash

617

582b

pJW

10AS2-R2

GTC

ATTA

GAG

TATG

GTG

CTC

20636ndash

617

582b

p

Clon

ingRT

-PCR

TWclo

n-F

ACGCT

TAAC

RACM

AAAC

CAG

201ndash20

Thiswork

TWclo

n-R

TGKA

TGAART

AAG

AGTG

WGGRA

C23

933ndash911

933b

p

Con

trol1

GAPD

H-F

GGCA

AGTT

CCAT

GGCA

CAGT

2058ndash72b

Ravazzoloetal2006

[37]

GAPD

H-R

ACGTA

CTCA

GCA

CCAG

CATC

AC22

161ndash182b

125b

p

Real-timeR

T-PC

R

RABV

D1-F

ATGTA

ACAC

CYCT

ACAAT

G19

55ndash73

Nadin-D

avisetal2009

[8]

RABV

D1-R

GCM

GGRT

AYTT

RTAY

TCAT

A20

165ndash146

111b

pNadin-D

avisetal2009

[8]

RABV

D1-P

51015840-FAM-C

CGAY

AAG

ATTG

TATT

YAARG

TCAAKA

ATCA

GGT-BH

Q1-31015840

3478ndash111

Nadin-D

avisetal2009

[8]

LysG

T5-P

51015840-YY-AAC

ARG

GTT

GTT

TTYA

AGGTC

CATA

A-BH

Q1-31015840

2680ndash105

Wakele

yetal2005

[25]

LysG

T6-P

51015840-C

y5-ACA

RAAT

TGTC

TTCA

ARG

TCCA

TAAT

CAG-BHQ2-31015840

2981ndash109

Wakele

yetal2005

[25]

ivvW

CBV-P

51015840-FAM-TCG

GAT

ATCA

CTTC

GGGTT

TGAG

AGTC

A-BH

Q1-31015840

28141ndash114

Thiswork

ivvM

ok-P

51015840-YY-TT

GTG

TTCA

AGGTG

AAY

AAY

CAAG

T-BH

Q1-31015840

2587ndash111

Thiswork

ivvA

BLV2n

-P51015840Cy

5-AT

TGTC

TTTA

AGGTC

AAC

AAT

CAGTT

-BHQ2-31015840

2686ndash111

Thiswork

ivvL

BV-P

51015840-FAM-ATT

GTT

TTCA

AAG

TYCA

YAAT

CAGGTM

GTG

TC-BHQ1-31015840

3286ndash117

Thiswork

ivvK

hujand

-P51015840-YY-AC

AGAAT

TGTC

TTYA

AAG

TYMAKA

ATCA

-BHQ1-31015840

2881ndash108

Thiswork

ivvSBL

V2-P

51015840-C

y5-TCW

GAG

ATTA

TRTC

TGGCT

TCAAAG

ACAC

-BHQ2-31015840

29141ndash113

Thiswork

Con

trol1

Send

ai-F

GTC

ATGGAT

GGGCA

GGAG

TC20

8553ndash8572b

Kaise

r2001

[38]

Send

ai-R

CGTT

GAAG

AGCC

TTAC

CCAG

A21

8788ndash8768b

236b

pSend

ai-P

51015840-FAM-C

AAAAT

TAGGAAC

GGAG

GAT

TGTC

CCCT

C-Tamra-31015840

298720ndash8748b

Changedbasesreferrin

gto

ther

eference

areu

nderlin

edW

obbled

positions

area

sfollowsY=CTXN=GA

TCW

=ATS

=CGR

=AG

M=ACK

=GTH

=ACTand

B=CGT

a Rabiesp

rimer

andprob

eposition

sare

givenaccordingto

theP

asteur

virusg

enom

e(accessionnu

mberX

03673)

b Position

softhe

GAPD

H-prim

ersrefer

totheG

enBa

nksequ

ence

AJ431207position

softhe

Send

ai-prim

ersrefer

totheG

enBa

nksequ

ence

M30202

1 Con

trolsforc

lassicalandreal-timeR

T-PC

RFA

M6-carbo

xyflu

orescein

repo

rter

dyeBH

Q-1B

lack

HoleQ

uencher-1YY

Yakim

aYellow

Cy5Cy

anine5

BHQ-2B

lack

HoleQ

uencher-2andTamracarbo

xy-te

tram

ethyl-rho

damine


(QIAGEN Germantown USA) according to the manufac-turerrsquos instructions except the usage of RNA Storage Solution(Ambion Foster City USA) for elution Extracted RNA wasstored at minus20∘C until use

26 Heminested RT-PCR Heminested RT-PCR was per-formed as previously described [22] using the OneStep RT-PCR Kit Briefly for the first round 3 120583L of extracted RNAwas amplified in the RT-PCR mix prepared according tomanufacturerrsquos instructions supplemented with 06 120583M ofeach primer (JW12-F JW6AS1-R1 and JW6AS2-R1 Table 2)and 5U RNase inhibitor (RNasin Plus 40U120583L PromegaMadison USA) with the following cycling conditions 50∘Cfor 30min and 95∘ for 15min for reverse transcription andsubsequent activation of the polymerase followed by 10 cyclesof 95∘C for 20 s 60∘C for 30 s (1∘C decreasecycle) followedby 72∘C for 30 s and 35 cycles of 95∘C for 20 s 52∘C for 30 sfollowed by 72∘C for 30 s For the second round the TaqDNAPolymerase Kit (QIAGEN Germantown USA) was usedwith 2 120583L of the first round product 25 120583L 10x PCR buffer12mM final dNTP concentration (Invitrogen Foster CityUSA) 04 120583M of heminested primers (JW12-F JW10AS1-R2and JW10AS2-R2 Table 2) 05U of Taq DNA polymeraseand 1935 120583L Nuclease Free Water (Ambion Foster CityUSA) Cycling conditions were as follows 95∘C for 5minfollowed by 35 cycles of 94∘C for 20 s 52∘C for 20 s followedby 72∘C for 30 s Amplifications were performed in a 2720Thermal Cycler Internal controls using GAPDH primers asdescribed in the one-round PCR were run in parallel in thefirst round

27 Gel Electrophoresis for Sequencing Gel electrophoresiswas performedusing a 15TAE agarose gel (agarose LE ana-lytical grade PromegaMadisonUSA) stainedwith ethidiumbromide (Eurobio Courtaboeuf France) For sequencing thedesired band was excised under UV light [47]

28 Real-Time RT-PCR (NDWD) Real-time RT-PCR(NDWD) adapted fromNadin-Davis et al andWakeley et al[8 25] was performed using the QuantiTect Probe RT-PCRKit (QIAGEN Germantown USA) The 25 120583L reactionvolume consisted of 95 120583L RNase-free water or 9 120583L in themultiplex mix 125 120583L 2x QuantiTect RT-PCR Master Mix025 120583L of the RABVD1 forward and reverse primer (04 120583Mfinal concentration each) 025120583L of the probes RABVD1LysGT5 and LysGT6 together in the multiplex mix or025 120583L of the probes ivvWCBV-P ivvMok-P ivvABLV2n-PivvLBV-P ivvKhujand-P and ivvSBLV2-P (Table 2) alone ina mix (all 02 120583M final concentration) 025120583L of QuantiTectRT Mix and 2 120583L of sample RNA Additionally for theinternal Sendai virus control 5 120583L of sample RNA wasadded to a mix containing 6625 120583L of RNase-free water125 120583L 2x QuantiTect RT-PCR Master Mix 0625 120583L ofSendai forward primer reverse primer and probe mix(Sendai-F Sendai-R and Sendai-P 8 120583M Table 2) and025 120583L of QuantiTect RT Mix The reactions were carriedout in MicroAmp Fast Optical 96-Well Reaction Plates withBarcode 01mL (Applied Biosystems Foster City USA) in a

7500 Fast Real-Time PCR System v131 (Applied BiosystemsFoster City USA) using the following cycling conditions 1cycle of 50∘C for 30min and 95∘C for 15min followed by 45cycles of 95∘C for 15 sec and 50∘C for 1min Amplificationcurve analysis was performed using the 7500 Software v206(Applied Biosystems Foster City USA)

29 Cloning for Analytical Sensitivity Analysis For cloninga segment of viral genome covering a part of the N gene(positions 1ndash933 according to the Pasteur virus genomeaccession number X03673) of CVS-11 was generated usingprimers designed for this purpose (TWclon-F and TWclon-R Table 2) Amplification was performed using the OneStepRT-PCR Kit with the master mix consisting of 2925120583L ofRNase-free water 10 120583L 5xQIAGENOneStep RT-PCR buffer04mM of each dNTP 06 120583M of the cloning primers 10URNasin (40U120583L) and 2 120583L of QIAGEN OneStep RT-PCREnzyme mixed with 5 120583L of extracted RNA to a total volumeof 50120583L Amplifications were performed in a Veriti 96-WellThermal Cycler (Applied Biosystems Foster City USA) usingthe following cycling conditions 30min at 50∘C followed by15min at 95∘C and 40 repetitive cycles of 1min at 94∘C 1minwith a temperature gradient from 56∘C to 46∘C in steps of2∘C and 1min at 72∘C Elongation at 72∘Cwas extended for 10additional minutes in the last cycle After gel electrophoresisexcised PCR fragments with a length of 933 bp were elutedwith the QIAquick Gel Extraction Kit (QIAGEN German-town USA) and cloned into the pCR-II-TOPO vector usingthe TOPO TA Cloning Kit (Invitrogen Foster City USA)according to the manufacturerrsquos instructions

210 Sequencing Sequencing reactions were performed byMicrosynthAG Balgach Switzerland For this purpose reac-tion mixtures containing 225 ng DNA per 100 bp 30 pmol ofeach primer used for the amplification (Table 2) and DEPCtreated water (Ambion Foster City USA) to a final volumeof 15 120583L were prepared Sequences were edited using theSeqMan II v501 Software (DNASTAR Madison USA) andsubsequently evaluated using the Clone Manager 9 software(Scientific amp Educational Software Cary USA)

211 Phylogenetic Reconstruction The 543 bp nucleotidesequences of the nucleoprotein gene obtained from the hem-inested PCR reaction together with additional sequencesretrieved from GenBank were saved in Fasta file formatThe sequences were then aligned using the ClustalX 203Software and theGeneDoc Software v250 [48] Phylogenetictrees were constructed with the MEGA5 software usingKimura 2-parameter distances [35]

3 Results

31 Cell Culture Thirty-one lyssaviruses (19x RABV 7xEBLV-1 4x EBLV-2 and 1x DUVV) were analysed in RTCITAll isolates were viable in murine neuroblastoma cells andcould be visualized using Rabies DFA Reagent as a conjugate


1000

500

100

Ladder A B C D E F G H

(bp)

Figure 1 Amplification of a CVS-11 serial dilution using hnRT-PCR Ethidium bromide stained gel after amplification of the CVS-11 strain using dilutions from 10minus1 to 10minus8 (AndashH) The amplificationproduct of 582 bp is clearly visible up to a dilution of 10minus5 (lane E)Ladder = standard for determination of amplification product size

32 Sensitivity of PCR Methods For the comparison withinfectious titres RNA of the classical rabies virus strain CVS-11 was extracted from cell culture supernatants (BHK-21 cellsor neuroblastoma cells MNA 4213) and serially diluted from10minus1 to 10minus8 The infectious titre was determined according tothe Spearman-Karber formula [49 50] in fluorescent focusforming doses 50 (FFD

50) In the heminested RT-PCR

(hnRT-PCR) the amplification product of 582 bp was visibleup to a dilution of 10minus5 corresponding to a detection limit of04 FFD

50(10minus04 FFD

50 Figure 1) In the real-time RT-PCR

(NDWD) the serial dilution was performed in triplicate Thelimit of detection was defined as the last dilution at which atleast 2 out of 3 replicates were positive Using this definitionCVS-11 was detectable up to a dilution of 10minus7 (once ata dilution of 10minus8) The amplification plot showed regularintervals of the curves with CT-values from 181 (dilution10minus1) to 400 (dilution 10minus7) Relating to infectious titre real-timeRT-PCR reached a detection limit of 0003 FFD

50(10minus26)

for CVS-11The efficiency of the real-time RT-PCR (NDWD) was

determined using linear regression of the CT-values and thetitres of the samples (Figure 2) On the basis of the slopecoefficient the efficiency can be inversely derived as 119864 =10(minus1slope coefficient)

minus 1 The efficiency for CVS-11 was 945with a slope coefficient of minus346

33 Analytical Sensitivity The limit of detection in terms ofDNA copy numbers was determined using 10-fold serial dilu-tions of a cloned 933 bp segment of N of CVS-11 containing106 to 100 DNA copiesPCR reaction In the hnPCR the limitof detection was 100 DNA copies3120583L of starting material ofthe CVS-11 plasmid In the real-time PCR (NDWD) the 10-fold serial dilution of theCVS-plasmidwas tested in triplicateThe limit of detection was 10 DNA copies2 120583L of the CVS-11plasmid with CT-values between 386 and 475The efficiencywas at 828 with a slope coefficient of minus382 (not shown)The intra-assay repeatability of the real-time PCR (NDWD)

15

18

21

24

27

30

33

36

39

42

minus40 minus30 minus20 minus10 00 10 20 30 40

Mea

n CT

-val

ue

Log titre FFD50

Figure 2 Efficiency of the real-time RT-PCR (NDWD) The linearregression of the CT-values (ordinate) and the titres of seriallydiluted CVS-11 (abscissa) exhibited a slope coefficient of minus346corresponding to an efficiency of 945 (119864 = 10(minus1slope coefficient)

minus 1)

proved to be excellent with very low coefficients of variationup to 102 DNA copies (018ndash137) with and outlier at 101copies (1205 Table 3)

34 Diagnostic Broadness of PCR Methods A total of 31lyssaviruses (19 identified as classical RABV 7 as EBLV-1 4 as EBLV-2 and 1 as DUVV) were used to test thediagnostic performance of the PCR (Table 1) All viruses usedwere detected with the expected band size in the hnRT-PCR technique (Table 4) The specificity of the amplifiedproduct was confirmed as RABV EBLV-1 EBLV-2 or DUVVby sequencing in each case Host GAPDH (glyceraldehyde3-phosphate dehydrogenase) was detected in all of the cellculture supernatants and mouse brain suspensions as inter-nal control indicating that sample material was presentand RNA isolation reverse transcription and amplificationwere not inhibited All samples were also tested positiveby the real-time RT-PCR assay (Table 4) Sendai virus asinternal control for inhibition was run in parallel using thesame cycling conditions In the real-time RT-PCR (NDWD)with an annealing temperature of 50∘C some samples weredetected with more than one probe With the exceptionof the sample derived from a raccoon dog from Polandthe probe for the homologous genotype always yielded thelowest CT-value In this exceptional case the probe for thedetection of genotype 6 was slightly lower than the onefor genotype 1 (150 versus 161 Table 4) Comparison ofthese two probes with the sequence of the rabies variantin question showed 1 and 2 mismatches with the RABVD1-P and LysGT6-P probe respectively (Figure 3) Duvenhagevirus also designated as genotype 4 was detectable withthe probe adapted for genotype 6 in spite of 3 mismatches(LysGT6-P)Theprobe LysGT6-Pwas able to detect syntheticDNA fragments of BBLV ARAV and IRKV although con-taining one or two mismatches respectively The syntheticDNA fragments of KHUV ABLV LBV MOKV SHIBV andWCBV which exhibited from four (ABLVKHUV) up toten (WCBV) mismatches to the best fitting probe for GT1


Table 3 Serial dilutions of the CVS-11 plasmid in real-time PCR (NDWD)

Dilution Copy numbers2 120583L CT-value 1 CT-value 2 CT-value 3 Mean plusmn SD CV ()10minus1 106 226 220 224 223 plusmn 031 13710minus2 105 251 252 253 252 plusmn 010 04010minus3 104 291 288 287 289 plusmn 021 07210minus4 103 329 329 328 329 plusmn 006 01810minus5 102 364 362 359 362 plusmn 025 07010minus6 101 475 386 390 417 plusmn 503 120510minus7 100 mdash mdash mdash mdash mdashSD standard deviation CV coefficient of variation

Table 4 PCR-results of rabies viruses tested

Real-time RT-PCR (NDWD)Sample Species hnRT-PCR CT-valuea

FAM (GT1) YY (GT5) Cy5 (GT6)1 Bat Stade (1970) EBLV-1 + 147 1732 Bat Hamburg RV 9 (1968) EBLV-1 + 202 148 1643 Bat Finland RV 8 (1986) EBLV-2 + 1514 Bat Holland RV 31 EBLV-1 + 202 147 1685 Dog Tunisia (1986) RABV + 246 272 3326 Bat Spain RV 119 EBLV-1 + 334 276 3167 Bat Florida RABV + 242 4168 Bat Denmark EBLV-1 + 265 211 2249 Raccoon dog Poland (1985) RABV + 161 15010 Raccoon Florida (1986) RABV + 25811 Bat Chile RV 108 RABV + 14412 Jackal Zimbabwe RABV + 14613 Cattle Zimbabwe RABV + 18014 Canada Red Fox RABV + 17915 LEP (Flury 1939) RABV + 17716 HEP (Flury) RABV + 28517 Dog Nepal Nr 96 RABV + 21718 Sri Lanka dog 121 (1986) RABV + 23219 Eurofox 91287 RABV + 28320 Duvenhage RV6 DUVV + 27021 NYC 58 RABV + 16522 Dog Lima RABV + 20023 Bat Bremerhaven RV11 EBLV-1 + 27024 Bat B24 EBLV-1 + 169 20625 Skunk Canada RABV + 23226 SAD Bern 1935 RABV + 18827 CSSRA-virus RABV + 18528 Challenge virus standard (CVS-11) ATCC VR 959 RABV + 19829 TW 181492 EBLV-2 +30 TW 139293 EBLV-2 +31 TW 11802 EBLV-2 +aOnly for samples with positive reactionFAM 6-carboxyfluorescein reporter dye to detect RABV (GT1) YY Yakima Yellow to detect EBLV-1 (GT5) Cy5 Cyanine 5 to detect EBLV-2 (GT6) NDnot done


ccgayaagattgtattyaargtcaakaatcaggt

ccact

RABVD1-P 5998400-78

RABV raccoon dog111-3998400

(a)

acaraattgtcttcaargtccataatcag

agaa

5998400-81 109-3998400LysGT6-P

RABV raccoon dog

(b)

Figure 3 Alignment of probes RABVD1-P (a) and LysGT6-P (b) with a RABV strain isolated from a raccoon dog from Poland Matchesin nondegenerated positions are displayed as dots Matches in wobbled positions are highlighted in yellow Mismatches are highlighted inturquoise

GT6 or GT5 respectively were all detected with the corre-sponding species-specific new probe (Table 2) 1052 copiesof the synthetic DNA were detected at CT-values of 279ndash335 for ABLV MOKV SHIBV and WCBV (not shown) Theefficiency of detection of KHUV which showed an atypicalamplification plot at the highest number of 10102 copies ofthe synthetic DNA and that of LBV was lower with CT-values of 400 and 405 at a copy number of 1052 respectively(not shown) Multiplexing of combinations of these probes(ivvLBV-P (FAM 6-carboxyfluorescein) ivvKhujand-P (YYYakima Yellow) and ivvSBLV2-P (Cy5 Cyanine 5) as wellas ivvWCBV-P (FAM) ivvMok-P (YY) and ivvABLV2n-P(Cy5)) as for the species 1 5 and 6 was not successful

35 Sequencing of Amplification Products and PhylogeneticAnalysis The 543 bp amplification products of the nucle-oprotein gene obtained in the heminested PCR reactionswere sequenced (Table 1) and analysed phylogenetically withadditional sequences retrieved from GenBank represent-ing all known lyssaviruses The similarity of the 543 bpnucleotide sequences of the nucleoprotein gene (positions74ndash616 according to the Pasteur virus genome) among thelyssaviruses included ranged from646 to 901 (Hammingdistance) Similarity at the amino acid level was 680ndash95The resulting phylogenetic tree obtained by the neighbor-joining method implemented in the MEGA5 software ispresented in Figure 4 All known genotypes were resolvedwith high bootstrap confidence with our samples groupingexpectedly

36 Clinical Specimens Clinical specimens like brain sus-pensions (1x human 12x mouse) skin biopsies from thenape of the neck (3x human 1x mouse) saliva (4x human)and cerebrospinal fluids (6x human) were used for theestablishment of the assays Both hnRT-PCR and real-timeRT-PCR (NDWD) were shown to work properly on allthese samples without significant inhibition usingGAPDHasinternal control for conventional RT-PCR and Sendai virusfor real-time RT-PCR (NDWD) which was mixed to thesamples before RNA isolation Skin biopsies taken from theneck and lip of a mouse (white Swiss mouse at an age of 3weeks) euthanized 2 weeks after intracerebral infection withSAD Bern virus 20 years ago were positive with the hnRT-PCR and real-time PCR (NDWD) Skin biopsies taken fromother locations on the head were positive with the real-timePCR (NDWD) and weakly positive in hnRT-PCR whereassamples taken around the vibrissae were weakly positive inreal-time PCR (NDWD) only Sequencing of amplification

products excluded a contamination with the positive CVS-11control (not shown)

Brain suspension from an imported human rabies casein 2012 was already strongly positive after one round of thehnRT-PCR Phylogenetic analysis of the 543 bp nucleotidefragment revealed its close relationship to the classical rabiesvirus strains circulating in the insectivorous Mexican free-tailed bat Tadarida brasiliensis a species common in thesouthern United States and Mexico This allowed identifica-tion of the origin of the patientrsquos infection who had travelledextensively and did not report any previous biting incident[51 52]

4 Discussion

Based on a large amount of published work we were ableto establish and quantitatively characterise RT-PCR proto-cols for the detection of lyssaviruses in clinical samplesWell suited real-time protocols [8 25] for the detection ofclassical rabies virus and the genotypes 5 and 6 (EBLV-1and EBLV-2) were adapted and extended for the detectionof at least 13 lyssavirus species To this goal 6 species-specific probes (KHUV ABLV LBV MOKV SHIBV andWCBV) were designed and verified on synthetic DNA frag-ments encompassing the targeted sequence of the lyssaviralnucleoprotein gene Multiplexing the probes in a single-tubereaction as described for the genotypes 1 5 and 6 was notpossible with the newly designed probes probably due tofalse priming andor interference within the mix of reagentsand synthetic target sequence [53] As far as evaluated withthe multiplexed probes for the genotypes 1 5 and 6 using 31viral isolates which were all detectable with high sensitivitydirect differentiation of targeted genotypes on the base ofthe quantitative reaction (CT-values) was mostly possibleFurthermore a single probe for genotype 6 (species EBLV-2)was able to detect up to 7 genotypesspeciesWe consider thistype of cross hybridization as an advantage of the techniqueusing degenerate primers and probes in terms of a broaddetectability of lyssavirus rather than a lack of accuracy ofdiscrimination In an approach using SYBRGreen qPCRwithsimilarly degenerate primers spanning the same part of Nas in this work [28] the detection of all lyssavirus speciesknown at that time was achieved Using several genotype-specific probes in a TaqMan real-time protocol we wereable to add more intrinsic confidence to the specificity ofthe analysis [54] Quantitative characterisation of the assayusing the probe for genotypes 1 5 and 6 on CVS-11 showedexcellent sensitivity and repeatability with a detection limitof as low as an infectious dose of 0003 FFD

50combined


00U22852maHu00U22643dzDg

TunesPantnDgZimbaPanzwJcZimbaPanzwCt

CSSRaPanczRoGU992321frCV

NYC58usRoHEPBHPanusHuLEPBHPanusHu

DQ837480trDgEU086163saFx

EU086199omFxDQ837430ilDg

AF045664EUFxPolanPanplRd

EU293115frFxCHVulPanchFxDQ837462egDg

LimadPanpeDg00X03673frPa

EF206710cSADSADbePanusDg

NepalPannpDgCanadPancaFxCanadPancaSkSriLaPanlkDgEU086204phDg

EU086182cnDgEU086173cnDgEU086165mmDg

EU086193laDgAB116579vnDgAB299033vnDgEU086172khDg

USAFlPanusRaFloriPanusBtAF351834caBtTW045PanusHuEU293116arBtChilePanclBt00U27221usRa

AF418014auHuAF006497auBtT1392PanchBtAY863407chBtT1814PanchBtTW118PanchBtAY863408chBt

EF157977ukHuEU293114nlBtGU002399fiBt00U22846fiHuFinHuPanfiHu

EF614261tjBtJF311903deBt

EF614259kgBtEF614260ruBt

DuvenPanzaHu00U22848zaHuEU293112frBtAY863402frBt00U22844plBtEU293109frBtHambuPandeBtEF157976deBtHollaPannlBtDenmaPandkBtStadePandeBtBremePandeBtB24EvPandeBt

S59448Moko00U22843zwCaY09762Moko

EU293118cfRoEU293117cmSh

GU170201KeBtGU170202keBt

EU293110ngBt00U22842ngBt

EU259198keBtEU293108snBt

EF614258ruBtJX193798tzCi

JX402204esBt

100

100

100

100

71

91

74

99

100

100

71

100

100

100

100

100

100

92

100

100

91

80

73

100

8

9479

100

73

100

98

92100

100

100

8479

100

100

100

92

9298

97

99

98

82

7386

86

7588

92

005

RABV

ABLV

EBLV-2

KHUVBBLVARAVIRKVDUVV

EBLV-1

MOKV

SHIBV

LBV

WCBVIKOVLLEBV

Figure 4 Phylogenetic tree with 543 bp fragments of N Phylogenetic tree obtained with MEGA5 software [35] The length of branches(horizontal lines) corresponds to phylogenetic distance between different sequences (scale bar in substitutions per site) Numbers proximalto nodes indicate bootstrap confidence of subjacent groups Lyssavirus strains used in this study are depicted in red Sequences are given withGenBank or own designation followed by the two-letter country code and a two-letter code for the source species as follows Bt bat Ca catCi African civet Dg dog Fx fox Hu human Jc jackal Ra raccoon Rd raccoon dog Ro rodent Sh shrew Sk skunk except for Pa cSADand CV which are standing for Pasteur strain Street Alabama Dufferin strain and challenge virus standard respectively The currently usedabbreviations for species are shown next to the tree RABV rabies virus ABLV Australian bat lyssavirus WCBV West Caucasian bat virusIKOV Ikoma virus LLEBV Lleida bat lyssavirus MOKV Mokola virus SHIBV Shimoni bat virus LBV Lagos bat virus KHUV Khujandvirus BBLV Bokeloh bat lyssavirus ARAV Aravan virus DUVV Duvenhage virus EBLV European bat lyssavirus IRKV Irkut virus


with an analytical sensitivity of 10 DNA copies at efficienciesof 945 and 828 respectively Considering the amountof degeneration in both primers and probes the efficienciesdetermined must be considered satisfactory As far as theabsolute detection limit in terms of target copies is concernedthe usage of plasmid DNA rather than RNA must be kept inmind Since reverse transcription cannot be assumed as 100efficient and reproducible [55] the limit for RNA might besomewhat lower

For further characterisation of samples with a posi-tive reaction in real-time RT-PCR excellent simple and(hemi)nested RT-PCR protocols are available With theprotocol used for this work [22] all 31 viral strains usedcould be amplified and sequenced confirming the diagnosticbroadness of the assay Phylogenetic analysis of these partialnucleotide sequences belonging to genotypes 1 4 5 and6 along with known sequences confirmed the suitabilityof this relatively conserved genomic region for molecular-epidemiological characterisation of lyssaviruses circulatingworldwide [56ndash58]This was also confirmed in the context ofa human rabies case imported to Switzerland in 2012 whichcould be attributed unequivocally to an exposure to a bat ofthe species Tadarida brasiliensis in California USA

5 Conclusion

In this work we could show and validate the suitability of anadapted and further developed real-time RT-PCR protocolfor the rapid efficient andhighly sensitive intravitamdiagno-sis of a wide spectrum of lyssavirus species followed by rapidmolecular-epidemiological characterisation of viral isolatesrespective to origin of the virus and source of exposureusing heminested RT-PCR This new tool is also particularlypromising for active surveillance of European bat lyssavirusesusing oral swabs in live-captured bats

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to acknowledge the critical review ofthe paper of PD Dr Philippe Plattet The help of AudreyMegali for her advice in the molecular techniques is greatlyacknowledged

References

[1] Laboratory Techniques in Rabies World Health OrganizationGeneva Switzerland 4th edition 1996

[2] WHO ldquoWHO expert consultation on rabiesrdquo Second ReportWHO Geneva Switzerland 2013

[3] O I E Rabies Manual of Standards for Diagnostic Tests andVaccines for Terrestrial Animals World Health OrganizationParis France 2013

[4] D Sacramento H Bourhy and N Tordo ldquoPCR technique as analternative method for diagnosis and molecular epidemiology

of rabies virusrdquoMolecular and Cellular Probes vol 5 no 3 pp229ndash240 1991

[5] H Bourhy P Sureau and J A Montano Hirose Methodes deLaboratoire pour le Diagnostic de la Rage Institut Pasteur ParisFrance 1990

[6] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004

[7] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006

[8] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009

[9] S Wacharapluesadee and T Hemachudha ldquoAnte- and post-mortem diagnosis of rabies using nucleic acid-amplificationtestsrdquo Expert Review of Molecular Diagnostics vol 10 no 2 pp207ndash218 2010

[10] M Fischer K Wernike C M Freuling et al ldquoA step forwardin molecular diagnostics of lyssavirusesmdashresults of a ring trialamong European laboratoriesrdquo PLoS ONE vol 8 no 3 ArticleID e58372 2013

[11] C FreulingAVosN Johnson et al ldquoExperimental infection ofserotine bats (Eptesicus serotinus) with European bat lyssavirustype 1ardquo Journal of General Virology vol 90 no 10 pp 2493ndash2502 2009

[12] K Schutsky D Curtis E K Bongiorno et al ldquoIntramuscularinoculation ofmice with the live-attenuated recombinant rabiesvirus TriGAS results in a transient infection of the draininglymph nodes and a robust long-lasting protective immuneresponse against rabiesrdquo Journal of Virology vol 87 no 3 pp1834ndash1841 2013

[13] H Bourhy J-M Reynes E J Dunham et al ldquoThe originand phylogeography of dog rabies virusrdquo Journal of GeneralVirology vol 89 no 11 pp 2673ndash2681 2008

[14] D T S Hayman N Johnson D L Horton et al ldquoEvolutionaryhistory of rabies in Ghanardquo PLoS Neglected Tropical Diseasesvol 5 no 4 Article ID e1001 2011

[15] S A Nadin-Davis and L A Real ldquoMolecular phylogenetics ofthe lyssaviruses-insights from a coalescent approachrdquo Advancesin Virus Research vol 79 pp 203ndash238 2011

[16] C A Hanlon and J E Childs ldquoEpidemiologyrdquo in RabiesScientific Basis of Disease and its Management A C JacksonEd pp 61ndash121 Academic Press New York NY USA 2013

[17] S A Nadin-Davis ldquoMolecular epidemiologyrdquo in Rabies Scien-tific Basis of Disease and Its Management A C Jackson Ed pp123ndash177 Academic press Amsterdam The Netherlands 2013

[18] P R Heaton P Johnstone L M McElhinney R CowleyE OrsquoSullivan and J E Whitby ldquoHeminested PCR assay fordetection of six genotypes of rabies and rabies-related virusesrdquoJournal of Clinical Microbiology vol 35 no 11 pp 2762ndash27661997

[19] H Bourhy ldquoLyssavirusesmdashspecial emphasis on rabies virusrdquo inDiagnostic Virology Protocols J R Stephenson and A WarnesEds vol 12 of Methods in Molecular Medicine pp 129ndash142Humana Press Totowa NJ USA 1998


[20] F Cliquet and E Picard-Meyer ldquoRabies and rabies-relatedviruses a modern perspective on an ancient diseaserdquo RevueScientifique et Technique de LlsquoOffice International des Epizootiesvol 23 no 2 pp 625ndash642 2004

[21] S Vazquez-Moron A Avellon and J E Echevarrıa ldquoRT-PCRfor detection of all seven genotypes of Lyssavirus genusrdquo Journalof Virological Methods vol 135 no 2 pp 281ndash287 2006

[22] M Panning S Baumgarte S Pfefferle T Maier A Martensand C Drosten ldquoComparative analysis of rabies virus reversetranscription-PCR and virus isolation using samples from apatient infected with rabies virusrdquo Journal of Clinical Microbi-ology vol 48 no 8 pp 2960ndash2962 2010

[23] P de Benedictis C de Battisti L Dacheux et al ldquoLyssavirusdetection and typing using pyrosequencingrdquo Journal of ClinicalMicrobiology vol 49 no 5 pp 1932ndash1938 2011

[24] E M Black J P Lowings J Smith P R Heaton and LM McElhinney ldquoA rapid RT-PCR method to differentiate sixestablished genotypes of rabies and rabies-related viruses usingTaqMan (TM) technologyrdquo Journal of Virological Methods vol105 pp 25ndash35 2002

[25] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005

[26] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010

[27] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety formolecular diagnosis of classical rabies viruses by use ofa TaqMan real-time reverse transcription-PCR ldquodouble checkrdquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010

[28] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011

[29] T Muller N Johnson C M Freuling A R Fooks T Selhorstand A Vos ldquoEpidemiology of bat rabies in Germanyrdquo Archivesof Virology vol 152 no 2 pp 273ndash288 2007

[30] A C Banyard N Johnson K Voller et al ldquoRepeated detectionof European bat lyssavirus type 2 in dead bats found at a singleroost site in the UKrdquo Archives of Virology vol 154 no 11 pp1847ndash1850 2009

[31] A Megali G Yannic M-L Zahno et al ldquoSurveillance forEuropean bat lyssavirus in Swiss batsrdquo Archives of Virology vol155 no 10 pp 1655ndash1662 2010

[32] WWMuller ldquoReview of reported rabies case data in Europe tothe WHO Collaborating Centre Tubingen from 1977 to 1988rdquoRabies Bulletin Europe vol 4 pp 16ndash19 1977

[33] C Freuling T Selhorst A Kliemt and T Muller ldquoRabiessurveillance in Europe 2004ndash2007rdquo Rabies Bulletin Europe vol31 no 4 pp 7ndash8 2004

[34] P Demetriou and J Moynagh ldquoThe European Union strategyfor external cooperation with neighbouring countries on rabiescontrolrdquo Rabies Bulletin Europe vol 35 no 1 pp 5ndash7 2011

[35] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[36] M K Abelseth ldquoAn attenuated rabies vaccine for domestic ani-mals produced in tissue culturerdquo Canadian Veterinary Journalvol 5 no 11 pp 279ndash286 1964

[37] A P Ravazzolo C Nenci H-R Vogt et al ldquoViral load organdistribution histopathological lesions and cytokine mRNAexpression in goats infected with a molecular clone of thecaprine arthritis encephalitis virusrdquo Virology vol 350 no 1 pp116ndash127 2006

[38] D Kaiser Entwicklung und Evaluation eines RT-TaqMan-PCR-Testverfahrens zum Nachweis des BVD-Virus [PhD disserta-tion] Universitat Bern 2001

[39] H Koprowski ldquoThe mouse inoculation testrdquo in LaboratoryTechniques in Rabies MM Kaplan andH Koprowski Eds pp85ndash93 World Health Organization Geneva Switzerland 1973

[40] D J Dean and M K Abelseth ldquoThe fluorescent antibodytestrdquo in Laboratory Techniques in Rabies M M Kaplan andH Koprowski Eds pp 73ndash83 World Health OrganizationGeneva Switzerland 1973

[41] A Gerhardt Teilvalidierung einer Zellkulturmethode als Alter-native zum Tierversuch fur den Nachweis von Tollwutvirusaus Hirnmaterial [dissertation thesis] VeterinarmedizinischeUniversitat Wien Universitat Bern 1995

[42] R J Rudd and C V Trimarchi ldquoDevelopment and evaluationof an in vitro virus isolation procedure as a replacement for themouse inoculation test in rabies diagnosisrdquo Journal of ClinicalMicrobiology vol 27 no 11 pp 2522ndash2528 1989

[43] P Stohr K Stohr H Kiupel and E Karge ldquoImmunofluo-rescence investigations of rabies field viruses in comparisonof several fluorescein-labelled antiserardquo Tierarztliche Umschauvol 47 pp 813ndash818 1992

[44] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[45] D G Higgins and P M Sharp ldquoCLUSTAL a package forperforming multiple sequence alignment on a microcomputerrdquoGene vol 73 no 1 pp 237ndash244 1988

[46] M A Larkin G Blackshields N P Brown et al ldquoClustalW andclustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[47] T Maniatis E F Fritsch and J Sambrook Molecular CloningA Laboratory Manual Cold Spring Harbor Laboratory ColdSpring Harbor NY USA 1982

[48] K B Nicholas H B J Nicholas and D W Deerfield IIldquoGeneDoc analysis and visualization of genetic variationrdquoEmbnew News vol 4 article 14 1997

[49] G Kaerber ldquoBeitrag zur kollektiven Behandlung pharmakol-ogischer Reihenversucherdquo Archiv for Experimentelle Pathologieund Pharmakologie vol 162 pp 480ndash487 1931

[50] C Spearman ldquoThe method of ldquoright or wrong casesrdquo (constantstimuli) withoutGaussrsquos formulaerdquoBritish Journal of Psychologyvol 2 pp 227ndash242 1908

[51] A N Deubelbeiss C Trachsel E B Bachli et al ldquoImportedhuman rabies in Switzerland 2012 a diagnostic conundrumrdquoJournal of Clinical Virology vol 57 no 2 pp 178ndash181 2013

[52] S Farley S Zarate E Jenssen et al ldquoUS-acquired human rabieswith symptomonset and diagnosis abroad 2012rdquoMorbidity andMortality Weekly Report vol 61 no 39 pp 777ndash781 2012

[53] A Apte and S Daniel PCR Primer Design Cold Spring HarborProtocols 2009


[54] C J Smith and A M Osborn ldquoAdvantages and limitationsof quantitative PCR (Q-PCR)-based approaches in microbialecologyrdquo FEMS Microbiology Ecology vol 67 no 1 pp 6ndash202009

[55] R Sanders D J Mason C A Foy and J F Huggett ldquoEvaluationof digital PCR for absolute RNA quantificationrdquo PLoS ONE vol8 no 9 Article ID e75296 2013

[56] E C Holmes C H Woelk R Kassis and H Bourhy ldquoGeneticconstraints and the adaptive evolution of rabies virus in naturerdquoVirology vol 292 no 2 pp 247ndash257 2002

[57] A J Foord H G Heine L I Pritchard et al ldquoMolecular diag-nosis of lyssaviruses and sequence comparison of Australian batlyssavirus samplesrdquo Australian Veterinary Journal vol 84 no 7pp 225ndash230 2006

[58] T Scott R Hassel and L Nel ldquoRabies in kudu (Tragelaphusstrepsiceros)rdquo Berliner und Munchener Tierarztliche Wochen-schrift vol 125 no 5-6 pp 236ndash241 2012

Submit your manuscripts athttpwwwhindawicom

Veterinary MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International



International Journal of

Microbiology


AnimalsJournal of

EcologyInternational Journal of


PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of


Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

Biotechnology Research International


Agronomy




Journal of Parasitology Research

Hindawi Publishing Corporation httpwwwhindawicom


Volume 2014

Zoology

GenomicsInternational Journal of


InsectsJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


VirusesJournal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of



Case Reports in Veterinary Medicine


real-time RT-PCR protocols for (intravitam) diagnosis ofrabies and molecular-epidemiological characterisation ofviral strains with the main emphasis on the growing numberof known speciesgenotypes

2 Materials and Methods

21 Samples

211 Cell Culture Supernatant of Viral Strains All operationswith potentially infectiousmaterial apart from centrifugationin closed tubes were performed in a class II biologicalsafety cabinet Frozen cell culture supernatants of rabiesvirus strains propagated previously on BHK-21 cells (babyhamster kidney cells ATCC Manassas USA) were thawedcentrifuged at 2000 g for 10min at 4∘C and filtrated usingthe Millex-HA 450 nm Filter (Millipore Cork Ireland)

212 Brain Suspension Approximately 1 g of cerebellummedulla oblongata and hippocampus from unfixed freshlyobtained brain material was homogenized to a 20 brainsuspension using a mortar and pestle after addition ofapproximately 1 g of quartz sand (Merck KGaA DarmstadtGermany) and 5mL of Modified Eagle Medium with Earlersquossalts with 22 gL NaHCO

3(Bioswisstec AG Schaffhausen

Switzerland) supplemented with 5 penicillin 10000 IUmL(Bioswisstec AG Schaffhausen Switzerland) and 20 foetalcalf serum (FCS PAA Laboratories GmbH Pasching Aus-tria) The suspension was then decanted into a 5mL tube(5mL Polystyrene Round-Bottom Tube BD BiosciencesErembodegem Belgium) to let it sediment for 1 hour at4∘C and subsequently centrifuged at 1400 g for 10min andfiltrated as above Frozen brain suspensions from formermouse inoculation tests [39] were handled like frozen cellculture supernatant

213 Saliva and Oral Swabs 500120583L of RNA Storage Solution(Ambion Foster City USA)was added to 100ndash200120583L of freshor previously frozen saliva samples or swabs which weresubsequently vortexed (Vortex Genie 2 TEWIS LaborbedarfAG Berne Switzerland) for 1min and centrifuged in aBiofuge Pico (Heraeus Holding GmbH Hanau Germany) at5000 rpm for 10min

214 Cerebrospinal Fluid Cerebrospinal fluid (CSF) sampleswere diluted 1 2 to 1 4 with RNA Storage Solution (AmbionFoster City USA)

215 Skin Biopsies Skin biopsies were shaved cut into smallpieces with a sterile pair of scissors and further processed asdescribed for brain suspension

22 Viral Strains and Propagation in Cell Culture A panelof 31 lyssaviruses (including 4 species) was used to testthe diagnostic performance of our PCR (Table 1) Stocks ofviral strains were produced in neuroblastoma cells (MNA4213) as described for the rabies tissue culture infectiontest (RTCIT described below) The maintenance medium

used for the first passage was Dulbeccorsquos MEM supple-mented with 05 neomycin 50 IUmL (Bioswisstec AGSchaffhausen Switzerland) 5 tryptose phosphate (BioCon-cept Ltd Amimed Allschwil Switzerland) 1 nonessen-tial amino acids (Bioswisstec AG Schaffhausen Switzer-land) 3 foetal calf serum and 1 L-Glutamine 200mM(Bioswisstec AG Schaffhausen Switzerland) to which 1Diethylaminoethyl-Dextran (DEAE-Dextran Sigma-AldrichCorporation St Louis USA) was added Dulbeccorsquos MEMwith 10 foetal calf serum was used for the 3 consecutivepassages Staining of the cells was performed after eachpassage using Rabies DFA Reagent (Millipore LivingstonUK) as a conjugate

23 FAT and RTCIT The standard fluorescent antibody test(FAT) was performed with brain tissue samples as previouslydescribed [40] Rabies tissue culture infection test (RTCIT)using four consecutive passages on murine neuroblastomacells [41ndash43] was applied to clinical specimens like brainspecimens liquor saliva or skin biopsies

24 Primers Probes and Synthetic DNA Primers probesand synthetic DNA were obtained from Microsynth (Micro-synth GmbH Balgach Switzerland) The location of suit-able N-directed primers and probes for heminested RT-PCR and real-time RT-PCR respectively was chosen andevaluated based on published work [8 18 22 25] Usingmultiple sequence alignment (ClustalX 203 program [44ndash46]) of the N gene region of the available lyssavirusspecies RABV (33 sequences) LBV (4 sequences) MOKV(5 sequences) DUVV (4 sequences) EBLV-1 (17 sequences)EBLV-2 (14 sequences) ABLV Aravan Khujand Irkut andWest Caucasian bat (each one) from GenBank (NationalCenter for Biotechnology Information and National Libraryof Medicine Rockville Pike USA) variable positions ofprimers and probes were adjusted with wobble positionsfor a potentially broader match Alternatively primers withsinglewobbles or substitutionsweremixed Several probes forreal-time RT-PCR for broadening the spectrum of detectablespecies were designed in this work (Table 2) As internalcontrol for conventional RT-PCR amplification of GAPDH(glyceraldehyde 3-phosphate dehydrogenase) was used [37]As internal inhibition control for real-time RT-PCR primersand probes for the amplification of Sendai virus (ATCCManassas USA [38]) which was added to the samples beforeRNA isolation were used (Table 2)

Synthetic DNA with a length of 125 bases encompassingpositions 48ndash172 according to the Pasteur virus genome(X03673) of the following lyssavirus species were obtainedfrom Microsynth Aravan virus (ARAV) EF614259 Khujandvirus (KHUV) EF614261 Bokeloh bat lyssavirus (BBLV)JF311903 Australian bat lyssavirus (ABLV) AF418014 Irkutvirus (IRKV) EF614260 Lagos bat virus (LBV) EU293110Mokola virus (MOKV) 293117 Shimoni bat virus (SHIV)GU170201 andWest Caucasian bat virus (WCBV) EF614258

25 RNA Extraction RNA was isolated from 140 120583L ofsample supernatant using the QIAamp Viral RNA Mini Kit


Table1Ra

bies

virusesu

sed

Species

Designatio

nHostspecies

Orig

inyearo

fisolatio

nreceipta

Material

Accessionnu

mberb

EBLV

-1Ba

tStade

Bat(E

serotin

us)

Germany(Stade)1970

Mou

sebrainsuspensio

nKF

831524K

F831550

EBLV

-1Ba

tHam

burg

RV9

Bat(E

serotin

us)

Germany(H

ambu

rg)1968

Mou

sebrainsuspensio

nKF

831526K

F831552

EBLV

-2Ba

tFinland

RV8

Hum

anbatexpo

sure

Finland1986

Mou

sebrainsuspensio

nKF

831528K

F831553

EBLV

-1Ba

tHolland

RV31

Bat

Netherla

nds1988

aMou

sebrainsuspensio

nKF

831525K

F831551

RABV

Dog

Tunisia

Dog

Tunisia

1986

BHK-

21cellcultu

resupernatant

KF831519K

F831545

EBLV

-1Ba

tSpain

RV119

Bat

Spain

1989

aBH

K-21

cellcultu

resupernatant

KF831527

RABV

BatF

lorid

aYello

wbat

USA

(Florid

a)1986a

BHK-

21cellcultu

resupernatant

KF831522K

F831548

EBLV

-1Ba

tDenmark

Bat(E

serotin

us)

Denmark1986

aBH

K-21

cellcultu

resupernatant

KF831523K

F831549

RABV

Raccoo

ndo

gPo

land

Raccoo

ndo

gPo

land

1985

Mou

sebrainsuspensio

nKF

831518K

F831544

RABV

Raccoo

nFlorida

Raccoo

nUSA

(Florid

a)1986

BHK-

21cellcultu

resupernatant

KF831521K

F831547

RABV

BatC

hileRV

108

Bat

Chile1988a

Mou

sebrainsuspensio

nKF

831520K

F831546

RABV

JackalZimbabw

eJackal

Zimbabw

e1990

aMou

sebrainsuspensio

nKF

831529K

F831554

RABV

Cattle

Zimbabw

eCattle

Zimbabw

e1990

aMou

sebrainsuspensio

nKF

831555

RABV

Canada

RedFo

xRe

dFo

xCa

nada

(Ontario)1986

aMou

sebrainsuspensio

nKF

831530K

F831556

RABV

LEP

Hum

anUSA

(Georgia)1939

BHK-

21cellcultu

resupernatant

KF831531K

F831557

RABV

HEP

Hum

anUSA

1939

BHK-

21cellcultu

resupernatant

KF831532K

F831568

RABV

Dog

NepalNr96

Dog

Nepal1989a

BHK-

21cellcultu

resupernatant

KF831534K

F831559

RABV

SriL

anka

dog121

Dog

SriL

anka1986

BHK-

21cellcultu

resupernatant

KF831535K

F831572

RABV

Eurofox91287

Fox

Switzerland

1987

BHK-

21cellcultu

resupernatant

KF831538K

F831562

DUVV

Duvenhage

RV6

Hum

anSouthAfrica1988

aBH

K-21

cellcultu

resupernatant

KF831533K

F831558

RABV

NYC

58Labo

ratory

strain

USA

1987a

Mou

sebrainsuspensio

nKF

831539K

F831563

RABV

Dog

Lima

Dog

Peru

(Lim

a)1985a

Mou

sebrainsuspensio

nKF

831540

KF831564

EBLV

-1Ba

tBremerhavenRV

11Ba


BHK-

21cellcultu

resupernatant

KF831541K

F831565

EBLV

-1Ba

tB24

Bat

Europe1986a

Mou

sebrainsuspensio

nKF

831536K

F831560

RABV

Skun

kCa

nada

Strip

edskun

kCa

nada

(Ontario)1986

BHK-

21cellcultu

resupernatant

KF831537K

F831561

RABV

SADBe

rnER

Alowast

USA

1935(received1976)

BHK-

21cellcultu

resupernatant

KF831542K

F831566

RABV

CSSR

A-viru

sRo

dentlaboratorystr

ain

CSSR

(Prague)1987a

BHK-

21cellcultu

resupernatant

KF831543K

F831567

RABV

Challengev

irusstand

ard(C

VS-11)AT

CCVR959

Labo

ratory

strain

France

(CNEV

A)1995

aBH

K-21

cellcultu

resupernatant

GU992321

EBLV

-2TW

181492

Bat(Mdaubentonii)

Switzerland

(Plaffeien)1992

Na4

213

cellcultu

resupernatant

KF831569

EBLV

-2TW

139293

Bat(Mdaubentonii)

Switzerland

(Versoix)1993

Na4

213

cellcultu

resupernatant

KF831570

EBLV

-2TW

11802

Bat(Mdaubentonii)

Switzerland

(Geneva)2002

Na4

213

cellcultu

resupernatant

KF831571

Lyssaviru

seso

f4different

speciesw

eretested

a Yearo

freceipt

attheS

wiss

rabies

center

b Accessio

nnu

mbersof

220b

pand543b

pNfragments

respectiv

elysequenced

inthiswork

lowast

Reference[36]


Table2Prim

ersa

ndprob

es

Metho

dNam

eSequ

ence

Leng

thPo

sitiona

Prod

uct

Reference

hnRT

-PCR

JW12-F

ATGTA

ACAC

CYCT

ACAAT

G19

55ndash73

Pann

ingetal2010

[22]

JW6A

S1-R1

CAAT

TGGCA

CACA

TTTT

GTG

2066

0ndash64

160

6bp

JW6A

S2-R1

CAGTT

AGCG

CACA

TCTT

ATG

2066

0ndash64

160

6bp

JW10AS1-R2

GTC

ATCA

ATGTG

TGAT

GTT

C20

636ndash

617

582b

pJW

10AS2-R2

GTC

ATTA

GAG

TATG

GTG

CTC

20636ndash

617

582b

p

Clon

ingRT

-PCR

TWclo

n-F

ACGCT

TAAC

RACM

AAAC

CAG

201ndash20

Thiswork

TWclo

n-R

TGKA

TGAART

AAG

AGTG

WGGRA

C23

933ndash911

933b

p

Con

trol1

GAPD

H-F

GGCA

AGTT

CCAT

GGCA

CAGT

2058ndash72b

Ravazzoloetal2006

[37]

GAPD

H-R

ACGTA

CTCA

GCA

CCAG

CATC

AC22

161ndash182b

125b

p

Real-timeR

T-PC

R

RABV

D1-F

ATGTA

ACAC

CYCT

ACAAT

G19

55ndash73

Nadin-D

avisetal2009

[8]

RABV

D1-R

GCM

GGRT

AYTT

RTAY

TCAT

A20

165ndash146

111b

pNadin-D

avisetal2009

[8]

RABV

D1-P

51015840-FAM-C

CGAY

AAG

ATTG

TATT

YAARG

TCAAKA

ATCA

GGT-BH

Q1-31015840

3478ndash111

Nadin-D

avisetal2009

[8]

LysG

T5-P

51015840-YY-AAC

ARG

GTT

GTT

TTYA

AGGTC

CATA

A-BH

Q1-31015840

2680ndash105

Wakele

yetal2005

[25]

LysG

T6-P

51015840-C

y5-ACA

RAAT

TGTC

TTCA

ARG

TCCA

TAAT

CAG-BHQ2-31015840

2981ndash109

Wakele

yetal2005

[25]

ivvW

CBV-P

51015840-FAM-TCG

GAT

ATCA

CTTC

GGGTT

TGAG

AGTC

A-BH

Q1-31015840

28141ndash114

Thiswork

ivvM

ok-P

51015840-YY-TT

GTG

TTCA

AGGTG

AAY

AAY

CAAG

T-BH

Q1-31015840

2587ndash111

Thiswork

ivvA

BLV2n

-P51015840Cy

5-AT

TGTC

TTTA

AGGTC

AAC

AAT

CAGTT

-BHQ2-31015840

2686ndash111

Thiswork

ivvL

BV-P

51015840-FAM-ATT

GTT

TTCA

AAG

TYCA

YAAT

CAGGTM

GTG

TC-BHQ1-31015840

3286ndash117

Thiswork

ivvK

hujand

-P51015840-YY-AC

AGAAT

TGTC

TTYA

AAG

TYMAKA

ATCA

-BHQ1-31015840

2881ndash108

Thiswork

ivvSBL

V2-P

51015840-C

y5-TCW

GAG

ATTA

TRTC

TGGCT

TCAAAG

ACAC

-BHQ2-31015840

29141ndash113

Thiswork

Con

trol1

Send

ai-F

GTC

ATGGAT

GGGCA

GGAG

TC20

8553ndash8572b

Kaise

r2001

[38]

Send

ai-R

CGTT

GAAG

AGCC

TTAC

CCAG

A21

8788ndash8768b

236b

pSend

ai-P

51015840-FAM-C

AAAAT

TAGGAAC

GGAG

GAT

TGTC

CCCT

C-Tamra-31015840

298720ndash8748b


gto

ther

eference

areu

nderlin

edW

obbled

positions

area

sfollowsY=CTXN=GA

TCW

=ATS

=CGR

=AG

M=ACK

=GTH

=ACTand

B=CGT

a Rabiesp

rimer

andprob

eposition

sare

givenaccordingto

theP

asteur

virusg

enom

e(accessionnu

mberX

03673)

b Position

softhe

GAPD

H-prim

ersrefer

totheG

enBa

nksequ

ence

AJ431207position

softhe

Send

ai-prim

ersrefer

totheG

enBa

nksequ

ence

M30202

1 Con

trolsforc


T-PC

RFA

M6-carbo

xyflu

orescein

repo

rter

dyeBH

Q-1B

lack

HoleQ

uencher-1YY

Yakim

aYellow

Cy5Cy

anine5

BHQ-2B

lack

HoleQ


xy-te

tram

ethyl-rho

damine










3 Results



1000

500

100


(bp)





50(10minus04 FFD



50(10minus26)





15

18

21

24

27

30

33

36

39

42


Mea

n CT

-val

ue

Log titre FFD50


minus 1)











ccact

RABVD1-P 5998400-78


(a)


agaa

5998400-81 109-3998400LysGT6-P

RABV raccoon dog

(b)







4 Discussion


50combined



























JX402204esBt

100

100

100

100

71

91

74

99

100

100

71

100

100

100

100

100

100

92

100

100

91

80

73

100

8

9479

100

73

100

98

92100

100

100

8479

100

100

100

92

9298

97

99

98

82

7386

86

7588

92

005

RABV

ABLV

EBLV-2


EBLV-1

MOKV

SHIBV

LBV

WCBVIKOVLLEBV





5 Conclusion




Acknowledgments


References





































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







Table1Ra

bies

virusesu

sed

Species

Designatio

nHostspecies

Orig

inyearo

fisolatio

nreceipta

Material

Accessionnu

mberb

EBLV

-1Ba

tStade

Bat(E

serotin

us)

Germany(Stade)1970

Mou

sebrainsuspensio

nKF

831524K

F831550

EBLV

-1Ba

tHam

burg

RV9

Bat(E

serotin

us)

Germany(H

ambu

rg)1968

Mou

sebrainsuspensio

nKF

831526K

F831552

EBLV

-2Ba

tFinland

RV8

Hum

anbatexpo

sure

Finland1986

Mou

sebrainsuspensio

nKF

831528K

F831553

EBLV

-1Ba

tHolland

RV31

Bat

Netherla

nds1988

aMou

sebrainsuspensio

nKF

831525K

F831551

RABV

Dog

Tunisia

Dog

Tunisia

1986

BHK-

21cellcultu

resupernatant

KF831519K

F831545

EBLV

-1Ba

tSpain

RV119

Bat

Spain

1989

aBH

K-21

cellcultu

resupernatant

KF831527

RABV

BatF

lorid

aYello

wbat

USA

(Florid

a)1986a

BHK-

21cellcultu

resupernatant

KF831522K

F831548

EBLV

-1Ba

tDenmark

Bat(E

serotin

us)

Denmark1986

aBH

K-21

cellcultu

resupernatant

KF831523K

F831549

RABV

Raccoo

ndo

gPo

land

Raccoo

ndo

gPo

land

1985

Mou

sebrainsuspensio

nKF

831518K

F831544

RABV

Raccoo

nFlorida

Raccoo

nUSA

(Florid

a)1986

BHK-

21cellcultu

resupernatant

KF831521K

F831547

RABV

BatC

hileRV

108

Bat

Chile1988a

Mou

sebrainsuspensio

nKF

831520K

F831546

RABV

JackalZimbabw

eJackal

Zimbabw

e1990

aMou

sebrainsuspensio

nKF

831529K

F831554

RABV

Cattle

Zimbabw

eCattle

Zimbabw

e1990

aMou

sebrainsuspensio

nKF

831555

RABV

Canada

RedFo

xRe

dFo

xCa

nada

(Ontario)1986

aMou

sebrainsuspensio

nKF

831530K

F831556

RABV

LEP

Hum

anUSA

(Georgia)1939

BHK-

21cellcultu

resupernatant

KF831531K

F831557

RABV

HEP

Hum

anUSA

1939

BHK-

21cellcultu

resupernatant

KF831532K

F831568

RABV

Dog

NepalNr96

Dog

Nepal1989a

BHK-

21cellcultu

resupernatant

KF831534K

F831559

RABV

SriL

anka

dog121

Dog

SriL

anka1986

BHK-

21cellcultu

resupernatant

KF831535K

F831572

RABV

Eurofox91287

Fox

Switzerland

1987

BHK-

21cellcultu

resupernatant

KF831538K

F831562

DUVV

Duvenhage

RV6

Hum

anSouthAfrica1988

aBH

K-21

cellcultu

resupernatant

KF831533K

F831558

RABV

NYC

58Labo

ratory

strain

USA

1987a

Mou

sebrainsuspensio

nKF

831539K

F831563

RABV

Dog

Lima

Dog

Peru

(Lim

a)1985a

Mou

sebrainsuspensio

nKF

831540

KF831564

EBLV

-1Ba

tBremerhavenRV

11Ba


BHK-

21cellcultu

resupernatant

KF831541K

F831565

EBLV

-1Ba

tB24

Bat

Europe1986a

Mou

sebrainsuspensio

nKF

831536K

F831560

RABV

Skun

kCa

nada

Strip

edskun

kCa

nada

(Ontario)1986

BHK-

21cellcultu

resupernatant

KF831537K

F831561

RABV

SADBe

rnER

Alowast

USA

1935(received1976)

BHK-

21cellcultu

resupernatant

KF831542K

F831566

RABV

CSSR

A-viru

sRo

dentlaboratorystr

ain

CSSR

(Prague)1987a

BHK-

21cellcultu

resupernatant

KF831543K

F831567

RABV

Challengev

irusstand

ard(C

VS-11)AT

CCVR959

Labo

ratory

strain

France

(CNEV

A)1995

aBH

K-21

cellcultu

resupernatant

GU992321

EBLV

-2TW

181492

Bat(Mdaubentonii)

Switzerland

(Plaffeien)1992

Na4

213

cellcultu

resupernatant

KF831569

EBLV

-2TW

139293

Bat(Mdaubentonii)

Switzerland

(Versoix)1993

Na4

213

cellcultu

resupernatant

KF831570

EBLV

-2TW

11802

Bat(Mdaubentonii)

Switzerland

(Geneva)2002

Na4

213

cellcultu

resupernatant

KF831571

Lyssaviru

seso

f4different

speciesw

eretested

a Yearo

freceipt

attheS

wiss

rabies

center

b Accessio

nnu

mbersof

220b

pand543b

pNfragments

respectiv

elysequenced

inthiswork

lowast

Reference[36]


Table2Prim

ersa

ndprob

es

Metho

dNam

eSequ

ence

Leng

thPo

sitiona

Prod

uct

Reference

hnRT

-PCR

JW12-F

ATGTA

ACAC

CYCT

ACAAT

G19

55ndash73

Pann

ingetal2010

[22]

JW6A

S1-R1

CAAT

TGGCA

CACA

TTTT

GTG

2066

0ndash64

160

6bp

JW6A

S2-R1

CAGTT

AGCG

CACA

TCTT

ATG

2066

0ndash64

160

6bp

JW10AS1-R2

GTC

ATCA

ATGTG

TGAT

GTT

C20

636ndash

617

582b

pJW

10AS2-R2

GTC

ATTA

GAG

TATG

GTG

CTC

20636ndash

617

582b

p

Clon

ingRT

-PCR

TWclo

n-F

ACGCT

TAAC

RACM

AAAC

CAG

201ndash20

Thiswork

TWclo

n-R

TGKA

TGAART

AAG

AGTG

WGGRA

C23

933ndash911

933b

p

Con

trol1

GAPD

H-F

GGCA

AGTT

CCAT

GGCA

CAGT

2058ndash72b

Ravazzoloetal2006

[37]

GAPD

H-R

ACGTA

CTCA

GCA

CCAG

CATC

AC22

161ndash182b

125b

p

Real-timeR

T-PC

R

RABV

D1-F

ATGTA

ACAC

CYCT

ACAAT

G19

55ndash73

Nadin-D

avisetal2009

[8]

RABV

D1-R

GCM

GGRT

AYTT

RTAY

TCAT

A20

165ndash146

111b

pNadin-D

avisetal2009

[8]

RABV

D1-P

51015840-FAM-C

CGAY

AAG

ATTG

TATT

YAARG

TCAAKA

ATCA

GGT-BH

Q1-31015840

3478ndash111

Nadin-D

avisetal2009

[8]

LysG

T5-P

51015840-YY-AAC

ARG

GTT

GTT

TTYA

AGGTC

CATA

A-BH

Q1-31015840

2680ndash105

Wakele

yetal2005

[25]

LysG

T6-P

51015840-C

y5-ACA

RAAT

TGTC

TTCA

ARG

TCCA

TAAT

CAG-BHQ2-31015840

2981ndash109

Wakele

yetal2005

[25]

ivvW

CBV-P

51015840-FAM-TCG

GAT

ATCA

CTTC

GGGTT

TGAG

AGTC

A-BH

Q1-31015840

28141ndash114

Thiswork

ivvM

ok-P

51015840-YY-TT

GTG

TTCA

AGGTG

AAY

AAY

CAAG

T-BH

Q1-31015840

2587ndash111

Thiswork

ivvA

BLV2n

-P51015840Cy

5-AT

TGTC

TTTA

AGGTC

AAC

AAT

CAGTT

-BHQ2-31015840

2686ndash111

Thiswork

ivvL

BV-P

51015840-FAM-ATT

GTT

TTCA

AAG

TYCA

YAAT

CAGGTM

GTG

TC-BHQ1-31015840

3286ndash117

Thiswork

ivvK

hujand

-P51015840-YY-AC

AGAAT

TGTC

TTYA

AAG

TYMAKA

ATCA

-BHQ1-31015840

2881ndash108

Thiswork

ivvSBL

V2-P

51015840-C

y5-TCW

GAG

ATTA

TRTC

TGGCT

TCAAAG

ACAC

-BHQ2-31015840

29141ndash113

Thiswork

Con

trol1

Send

ai-F

GTC

ATGGAT

GGGCA

GGAG

TC20

8553ndash8572b

Kaise

r2001

[38]

Send

ai-R

CGTT

GAAG

AGCC

TTAC

CCAG

A21

8788ndash8768b

236b

pSend

ai-P

51015840-FAM-C

AAAAT

TAGGAAC

GGAG

GAT

TGTC

CCCT

C-Tamra-31015840

298720ndash8748b


gto

ther

eference

areu

nderlin

edW

obbled

positions

area

sfollowsY=CTXN=GA

TCW

=ATS

=CGR

=AG

M=ACK

=GTH

=ACTand

B=CGT

a Rabiesp

rimer

andprob

eposition

sare

givenaccordingto

theP

asteur

virusg

enom

e(accessionnu

mberX

03673)

b Position

softhe

GAPD

H-prim

ersrefer

totheG

enBa

nksequ

ence

AJ431207position

softhe

Send

ai-prim

ersrefer

totheG

enBa

nksequ

ence

M30202

1 Con

trolsforc


T-PC

RFA

M6-carbo

xyflu

orescein

repo

rter

dyeBH

Q-1B

lack

HoleQ

uencher-1YY

Yakim

aYellow

Cy5Cy

anine5

BHQ-2B

lack

HoleQ


xy-te

tram

ethyl-rho

damine










3 Results



1000

500

100


(bp)





50(10minus04 FFD



50(10minus26)





15

18

21

24

27

30

33

36

39

42


Mea

n CT

-val

ue

Log titre FFD50


minus 1)











ccact

RABVD1-P 5998400-78


(a)


agaa

5998400-81 109-3998400LysGT6-P

RABV raccoon dog

(b)







4 Discussion


50combined



























JX402204esBt

100

100

100

100

71

91

74

99

100

100

71

100

100

100

100

100

100

92

100

100

91

80

73

100

8

9479

100

73

100

98

92100

100

100

8479

100

100

100

92

9298

97

99

98

82

7386

86

7588

92

005

RABV

ABLV

EBLV-2


EBLV-1

MOKV

SHIBV

LBV

WCBVIKOVLLEBV





5 Conclusion




Acknowledgments


References





































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







Table2Prim

ersa

ndprob

es

Metho

dNam

eSequ

ence

Leng

thPo

sitiona

Prod

uct

Reference

hnRT

-PCR

JW12-F

ATGTA

ACAC

CYCT

ACAAT

G19

55ndash73

Pann

ingetal2010

[22]

JW6A

S1-R1

CAAT

TGGCA

CACA

TTTT

GTG

2066

0ndash64

160

6bp

JW6A

S2-R1

CAGTT

AGCG

CACA

TCTT

ATG

2066

0ndash64

160

6bp

JW10AS1-R2

GTC

ATCA

ATGTG

TGAT

GTT

C20

636ndash

617

582b

pJW

10AS2-R2

GTC

ATTA

GAG

TATG

GTG

CTC

20636ndash

617

582b

p

Clon

ingRT

-PCR

TWclo

n-F

ACGCT

TAAC

RACM

AAAC

CAG

201ndash20

Thiswork

TWclo

n-R

TGKA

TGAART

AAG

AGTG

WGGRA

C23

933ndash911

933b

p

Con

trol1

GAPD

H-F

GGCA

AGTT

CCAT

GGCA

CAGT

2058ndash72b

Ravazzoloetal2006

[37]

GAPD

H-R

ACGTA

CTCA

GCA

CCAG

CATC

AC22

161ndash182b

125b

p

Real-timeR

T-PC

R

RABV

D1-F

ATGTA

ACAC

CYCT

ACAAT

G19

55ndash73

Nadin-D

avisetal2009

[8]

RABV

D1-R

GCM

GGRT

AYTT

RTAY

TCAT

A20

165ndash146

111b

pNadin-D

avisetal2009

[8]

RABV

D1-P

51015840-FAM-C

CGAY

AAG

ATTG

TATT

YAARG

TCAAKA

ATCA

GGT-BH

Q1-31015840

3478ndash111

Nadin-D

avisetal2009

[8]

LysG

T5-P

51015840-YY-AAC

ARG

GTT

GTT

TTYA

AGGTC

CATA

A-BH

Q1-31015840

2680ndash105

Wakele

yetal2005

[25]

LysG

T6-P

51015840-C

y5-ACA

RAAT

TGTC

TTCA

ARG

TCCA

TAAT

CAG-BHQ2-31015840

2981ndash109

Wakele

yetal2005

[25]

ivvW

CBV-P

51015840-FAM-TCG

GAT

ATCA

CTTC

GGGTT

TGAG

AGTC

A-BH

Q1-31015840

28141ndash114

Thiswork

ivvM

ok-P

51015840-YY-TT

GTG

TTCA

AGGTG

AAY

AAY

CAAG

T-BH

Q1-31015840

2587ndash111

Thiswork

ivvA

BLV2n

-P51015840Cy

5-AT

TGTC

TTTA

AGGTC

AAC

AAT

CAGTT

-BHQ2-31015840

2686ndash111

Thiswork

ivvL

BV-P

51015840-FAM-ATT

GTT

TTCA

AAG

TYCA

YAAT

CAGGTM

GTG

TC-BHQ1-31015840

3286ndash117

Thiswork

ivvK

hujand

-P51015840-YY-AC

AGAAT

TGTC

TTYA

AAG

TYMAKA

ATCA

-BHQ1-31015840

2881ndash108

Thiswork

ivvSBL

V2-P

51015840-C

y5-TCW

GAG

ATTA

TRTC

TGGCT

TCAAAG

ACAC

-BHQ2-31015840

29141ndash113

Thiswork

Con

trol1

Send

ai-F

GTC

ATGGAT

GGGCA

GGAG

TC20

8553ndash8572b

Kaise

r2001

[38]

Send

ai-R

CGTT

GAAG

AGCC

TTAC

CCAG

A21

8788ndash8768b

236b

pSend

ai-P

51015840-FAM-C

AAAAT

TAGGAAC

GGAG

GAT

TGTC

CCCT

C-Tamra-31015840

298720ndash8748b


gto

ther

eference

areu

nderlin

edW

obbled

positions

area

sfollowsY=CTXN=GA

TCW

=ATS

=CGR

=AG

M=ACK

=GTH

=ACTand

B=CGT

a Rabiesp

rimer

andprob

eposition

sare

givenaccordingto

theP

asteur

virusg

enom

e(accessionnu

mberX

03673)

b Position

softhe

GAPD

H-prim

ersrefer

totheG

enBa

nksequ

ence

AJ431207position

softhe

Send

ai-prim

ersrefer

totheG

enBa

nksequ

ence

M30202

1 Con

trolsforc


T-PC

RFA

M6-carbo

xyflu

orescein

repo

rter

dyeBH

Q-1B

lack

HoleQ

uencher-1YY

Yakim

aYellow

Cy5Cy

anine5

BHQ-2B

lack

HoleQ


xy-te

tram

ethyl-rho

damine










3 Results



1000

500

100


(bp)





50(10minus04 FFD



50(10minus26)





15

18

21

24

27

30

33

36

39

42


Mea

n CT

-val

ue

Log titre FFD50


minus 1)











ccact

RABVD1-P 5998400-78


(a)


agaa

5998400-81 109-3998400LysGT6-P

RABV raccoon dog

(b)







4 Discussion


50combined



























JX402204esBt

100

100

100

100

71

91

74

99

100

100

71

100

100

100

100

100

100

92

100

100

91

80

73

100

8

9479

100

73

100

98

92100

100

100

8479

100

100

100

92

9298

97

99

98

82

7386

86

7588

92

005

RABV

ABLV

EBLV-2


EBLV-1

MOKV

SHIBV

LBV

WCBVIKOVLLEBV





5 Conclusion




Acknowledgments


References





































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of















3 Results



1000

500

100


(bp)





50(10minus04 FFD



50(10minus26)





15

18

21

24

27

30

33

36

39

42


Mea

n CT

-val

ue

Log titre FFD50


minus 1)











ccact

RABVD1-P 5998400-78


(a)


agaa

5998400-81 109-3998400LysGT6-P

RABV raccoon dog

(b)







4 Discussion


50combined



























JX402204esBt

100

100

100

100

71

91

74

99

100

100

71

100

100

100

100

100

100

92

100

100

91

80

73

100

8

9479

100

73

100

98

92100

100

100

8479

100

100

100

92

9298

97

99

98

82

7386

86

7588

92

005

RABV

ABLV

EBLV-2


EBLV-1

MOKV

SHIBV

LBV

WCBVIKOVLLEBV





5 Conclusion




Acknowledgments


References





































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







1000

500

100


(bp)





50(10minus04 FFD



50(10minus26)





15

18

21

24

27

30

33

36

39

42


Mea

n CT

-val

ue

Log titre FFD50


minus 1)











ccact

RABVD1-P 5998400-78


(a)


agaa

5998400-81 109-3998400LysGT6-P

RABV raccoon dog

(b)







4 Discussion


50combined



























JX402204esBt

100

100

100

100

71

91

74

99

100

100

71

100

100

100

100

100

100

92

100

100

91

80

73

100

8

9479

100

73

100

98

92100

100

100

8479

100

100

100

92

9298

97

99

98

82

7386

86

7588

92

005

RABV

ABLV

EBLV-2


EBLV-1

MOKV

SHIBV

LBV

WCBVIKOVLLEBV





5 Conclusion




Acknowledgments


References





































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of














ccact

RABVD1-P 5998400-78


(a)


agaa

5998400-81 109-3998400LysGT6-P

RABV raccoon dog

(b)







4 Discussion


50combined



























JX402204esBt

100

100

100

100

71

91

74

99

100

100

71

100

100

100

100

100

100

92

100

100

91

80

73

100

8

9479

100

73

100

98

92100

100

100

8479

100

100

100

92

9298

97

99

98

82

7386

86

7588

92

005

RABV

ABLV

EBLV-2


EBLV-1

MOKV

SHIBV

LBV

WCBVIKOVLLEBV





5 Conclusion




Acknowledgments


References





































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of
































JX402204esBt

100

100

100

100

71

91

74

99

100

100

71

100

100

100

100

100

100

92

100

100

91

80

73

100

8

9479

100

73

100

98

92100

100

100

8479

100

100

100

92

9298

97

99

98

82

7386

86

7588

92

005

RABV

ABLV

EBLV-2


EBLV-1

MOKV

SHIBV

LBV

WCBVIKOVLLEBV





5 Conclusion




Acknowledgments


References





































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of









5 Conclusion




Acknowledgments


References





































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






















































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






research article real-time rt-pcr for the detection of...

Documents