rabies diagnosis is based on fluorescent antibody testing ( fat ) in brain smears and inoculation of brain suspension either in mouse neuroblastoma cell cultures or intracerebrally in mice as confirmatory assays with high sensitivity and specificity for postmortem diagnosis [ 13 ] . these techniques are well suited for rapid and reliable routine diagnosis , if brain material is available . for other diagnostic specimens sampled intra vitam in case of clinical suspicion , for example , saliva , cerebrospinal fluid , or skin biopsies , more suited molecular techniques with excellent sensitivity have been developed and validated , mostly targeting the conserved nucleoprotein gene of the lyssavirus genome , for example , [ 410 ] . among these , there are protocols both for classical rt - pcr and for real - time rt - pcr , which adds speed , efficiency , contamination safety , and reliability to the technique combined with the potential to quantify the viral load [ 11 , 12 ] . a major advantage offered by these molecular techniques is the characterisation of viral isolates by sequencing the given amplification products followed by phylogenetic or phylogeographic analysis [ 1315 ] . the greatest challenge for these powerful novel techniques is the ever growing spectrum of known lyssavirus species / genotypes ( gt ) , probably all of which having the potential to cause animal and human rabies fatalities [ 16 , 17 ] . so far , 12 lyssavirus species and three as yet unclassified species have been identified . the development of classical simple or ( hemi)nested rt - pcr methods with particular emphasis on the diagnosis of a broad spectrum of lyssaviruses species has been published in a number of studies [ 1823 ] . also several real - time rt - pcr protocols suited for the broad detection or differentiation of several genotypes have been developed [ 9 , 2428 ] . is of particular interest for the rabies epidemiology in europe concerning the european bat lyssaviruses types 1 and 2 [ 2932 ] apart from classical rabies [ 33 , 34 ] . this assay uses genotype ( gt ) specific probes and fluorophore signals for direct differentiation of gt1 , gt5 , and gt6 in a single - tube reaction . the goal of this work was , based on a comprehensive review of the rich literature on the theme and adapting thereof , to develop , establish , and validate classical and real - time rt - pcr protocols for ( intravitam ) diagnosis of rabies and molecular - epidemiological characterisation of viral strains with the main emphasis on the growing number of known species / genotypes . all operations with potentially infectious material apart from centrifugation in closed tubes were performed in a class ii biological safety cabinet . frozen cell culture supernatants of rabies virus strains propagated previously on bhk-21 cells ( baby hamster kidney cells , atcc , manassas , usa ) were thawed , centrifuged at 2,000  g for 10  min at 4c , and filtrated using the millex - ha 450  nm filter ( millipore , cork , ireland ) . approximately 1  g of cerebellum , medulla oblongata , and hippocampus from unfixed , freshly obtained brain material was homogenized to a 20% brain suspension using a mortar and pestle , after addition of approximately 1  g of quartz sand ( merck kgaa , darmstadt , germany ) and 5  ml of modified eagle medium with earle 's salts with 2.2  g / l nahco3 ( bioswisstec ag , schaffhausen , switzerland ) supplemented with 5% penicillin 10,000  iu / ml ( bioswisstec ag , schaffhausen , switzerland ) and 20% foetal calf serum ( fcs ; paa laboratories gmbh , pasching , austria ) . the suspension was then decanted into a 5  ml tube ( 5  ml polystyrene round - bottom tube ; bd biosciences , erembodegem , belgium ) to let it sediment for 1 hour at 4c and subsequently centrifuged at 1,400  g for 10  min and filtrated as above . frozen brain suspensions from former mouse inoculation tests   were handled like frozen cell culture supernatant . 500  l of rna storage solution ( ambion , foster city , usa ) was added to 100200  l of fresh or previously frozen saliva samples or swabs , which were subsequently vortexed ( vortex genie 2 , tewis laborbedarf ag , berne , switzerland ) for 1  min and centrifuged in a biofuge pico ( heraeus holding gmbh , hanau , germany ) at 5,000  rpm for 10  min . cerebrospinal fluid ( csf ) samples were diluted 1  :  2 to 1  :  4 with rna storage solution ( ambion , foster city , usa ) . skin biopsies were shaved , cut into small pieces with a sterile pair of scissors , and further processed as described for brain suspension . a panel of 31 lyssaviruses ( including 4 species ) was used to test the diagnostic performance of our pcr ( table 1 ) . stocks of viral strains were produced in neuroblastoma cells ( mna 42/13 ) as described for the rabies tissue culture infection test ( rtcit , described below ) . the maintenance medium used for the first passage was dulbecco 's mem supplemented with 0.5% neomycin 50  iu / ml ( bioswisstec ag , schaffhausen , switzerland ) , 5% tryptose phosphate ( bioconcept ltd . amimed , allschwil , switzerland ) , 1% nonessential amino acids ( bioswisstec ag , schaffhausen , switzerland ) , 3% foetal calf serum , and 1% l - glutamine 200  mm ( bioswisstec ag , schaffhausen , switzerland ) to which 1% diethylaminoethyl - dextran ( deae - dextran ; sigma - aldrich corporation , st . dulbecco 's mem with 10% foetal calf serum was used for the 3 consecutive passages . staining of the cells was performed after each passage using rabies dfa reagent ( millipore , livingston , uk ) as a conjugate . the standard fluorescent antibody test ( fat ) was performed with brain tissue samples as previously described . rabies tissue culture infection test ( rtcit ) using four consecutive passages on murine neuroblastoma cells [ 4143 ] was applied to clinical specimens like brain specimens , liquor , saliva , or skin biopsies . primers , probes , and synthetic dna were obtained from microsynth ( microsynth gmbh , balgach , switzerland ) . the location of suitable n - directed primers and probes for heminested rt - pcr and real - time rt - pcr , respectively , was chosen and evaluated based on published work [ 8 , 18 , 22 , 25 ] . using multiple sequence alignment ( clustalx 2.0.3 program [ 4446 ] ) of the n gene region of the available lyssavirus species rabv ( 33 sequences ) , lbv ( 4 sequences ) , mokv ( 5 sequences ) , duvv ( 4 sequences ) , eblv-1 ( 17 sequences ) , eblv-2 ( 14 sequences ) , ablv , aravan , khujand , irkut , and west caucasian bat ( each one ) from genbank ( national center for biotechnology information and national library of medicine , rockville pike , usa ) , variable positions of primers and probes were adjusted with wobble positions for a potentially broader match . several probes for real - time rt - pcr for broadening the spectrum of detectable species were designed in this work ( table 2 ) . as internal control for conventional rt - pcr , amplification of gapdh ( glyceraldehyde 3-phosphate dehydrogenase ) was used . as internal inhibition control for real - time rt - pcr , primers and probes for the amplification of sendai virus ( atcc , manassas , usa ) , which was added to the samples before rna isolation , were used ( table 2 ) . synthetic dna with a length of 125 bases encompassing positions 48172 according to the pasteur virus genome ( x03673 ) of the following lyssavirus species were obtained from microsynth : aravan virus ( arav ) ef614259 , khujand virus ( khuv ) ef614261 , bokeloh bat lyssavirus ( bblv ) jf311903 , australian bat lyssavirus ( ablv ) af418014 , irkut virus ( irkv ) ef614260 , lagos bat virus ( lbv ) eu293110 , mokola virus ( mokv ) 293117 , shimoni bat virus ( shiv ) gu170201 , and west caucasian bat virus ( wcbv ) ef614258 . rna was isolated from 140  l of sample supernatant using the qiaamp viral rna mini kit ( qiagen , germantown , usa ) according to the manufacturer 's instructions , except the usage of rna storage solution ( ambion , foster city , usa ) for elution . heminested rt - pcr was performed as previously described   using the onestep rt - pcr kit . briefly , for the first round 3  l of extracted rna was amplified in the rt - pcr mix prepared according to manufacturer 's instructions , supplemented with 0.6  m of each primer ( jw12-f , jw6as1-r1 , and jw6as2-r1 ; table 2 ) and 5  u rnase inhibitor ( rnasin plus , 40  u/l ; promega , madison , usa ) , with the following cycling conditions : 50c for 30  min and 95 for 15  min for reverse transcription and subsequent activation of the polymerase , followed by 10 cycles of 95c for 20  s , 60c for 30  s ( 1c decrease / cycle ) , followed by 72c for 30  s and 35 cycles of 95c for 20  s , 52c for 30  s , followed by 72c for 30  s. for the second round , the taq dna polymerase kit ( qiagen , germantown , usa ) was used with 2  l of the first round product , 2.5  l 10x pcr buffer , 1.2  mm final dntp concentration ( invitrogen , foster city , usa ) , 0.4  m of heminested primers ( jw12-f , jw10as1-r2 , and jw10as2-r2 ; table 2 ) , 0.5  u of taq dna polymerase , and 19.35  l nuclease free water ( ambion , foster city , usa ) . cycling conditions were as follows : 95c for 5  min followed by 35 cycles of 94c for 20  s , 52c for 20  s , followed by 72c for 30  s. amplifications were performed in a 2720 thermal cycler . internal controls using gapdh primers , as described in the one - round pcr , were run in parallel in the first round . gel electrophoresis was performed using a 1.5% tae agarose gel ( agarose le , analytical grade ; promega , madison , usa ) stained with ethidium bromide ( eurobio , courtaboeuf , france ) . for sequencing , the desired band was excised under uv light . real - time rt - pcr ( ndwd ) adapted from nadin - davis et al . and wakeley et al . [ 8 , 25 ] was performed using the quantitect probe rt - pcr kit ( qiagen , germantown , usa ) . the 25  l reaction volume consisted of 9.5  l rnase - free water , or 9  l in the multiplex mix , 12.5  l 2x quantitect rt - pcr master mix , 0.25  l of the rabvd1 forward and reverse primer ( 0.4  m final concentration each ) , 0.25  l of the probes rabvd1 , lysgt5 , and lysgt6 together in the multiplex mix or 0.25  l of the probes ivvwcbv - p , ivvmok - p , ivvablv2n - p , ivvlbv - p , ivvkhujand - p , and ivvsblv2-p ( table 2 ) alone in a mix ( all 0.2  m final concentration ) , 0.25  l of quantitect rt mix , and 2  l of sample rna . additionally , for the internal sendai virus control , 5  l of sample rna was added to a mix containing 6.625  l of rnase - free water , 12.5  l 2x quantitect rt - pcr master mix , 0.625  l of sendai forward primer , reverse primer , and probe mix ( sendai - f , sendai - r , and sendai - p , 8  m ; table 2 ) , and 0.25  l of quantitect rt mix . the reactions were carried out in microamp fast optical 96-well reaction plates with barcode 0.1  ml ( applied biosystems , foster city , usa ) in a 7500 fast real - time pcr system v1.3.1 ( applied biosystems , foster city , usa ) using the following cycling conditions : 1 cycle of 50c for 30  min and 95c for 15  min followed by 45 cycles of 95c for 15  sec and 50c for 1  min . amplification curve analysis was performed using the 7500 software v2.0.6 ( applied biosystems , foster city , usa ) . for cloning , a segment of viral genome covering a part of the n gene ( positions 1933 according to the pasteur virus genome accession number x03673 ) of cvs-11 was generated using primers designed for this purpose ( twclon - f and twclon - r , table 2 ) . amplification was performed using the onestep rt - pcr kit with the master mix consisting of 29.25  l of rnase - free water , 10  l 5x qiagen onestep rt - pcr buffer , 0.4  mm of each dntp , 0.6  m of the cloning primers , 10  u rnasin ( 40  u/l ) , and 2  l of qiagen onestep rt - pcr enzyme mixed with 5  l of extracted rna to a total volume of 50  l . amplifications were performed in a veriti 96-well thermal cycler ( applied biosystems , foster city , usa ) using the following cycling conditions : 30  min at 50c , followed by 15  min at 95c and 40 repetitive cycles of 1  min at 94c , 1  min with a temperature gradient from 56c to 46c in steps of 2c , and 1  min at 72c . after gel electrophoresis excised pcr fragments with a length of 933  bp were eluted with the qiaquick gel extraction kit ( qiagen , germantown , usa ) and cloned into the pcr - ii - topo vector using the topo ta cloning kit ( invitrogen , foster city , usa ) according to the manufacturer 's instructions . sequencing reactions were performed by microsynth ag , balgach , switzerland . for this purpose , reaction mixtures containing 22.5  ng dna per 100  bp , 30  pmol of each primer used for the amplification ( table 2 ) , and depc treated water ( ambion , foster city , usa ) to a final volume of 15  l were prepared . sequences were edited using the seqman ii v5.01 software ( dnastar , madison , usa ) and subsequently evaluated using the clone manager 9 software ( scientific & educational software , cary , usa ) . the 543  bp nucleotide sequences of the nucleoprotein gene obtained from the heminested pcr reaction , together with additional sequences retrieved from genbank , were saved in fasta file format . the sequences were then aligned using the clustalx 2.0.3 software and the genedoc software v2.5.0 . thirty - one lyssaviruses ( 19x rabv , 7x eblv-1 , 4x eblv-2 , and 1x duvv ) were analysed in rtcit . all isolates were viable in murine neuroblastoma cells and could be visualized using rabies dfa reagent as a conjugate . for the comparison with infectious titres , rna of the classical rabies virus strain cvs-11 was extracted from cell culture supernatants ( bhk-21 cells or neuroblastoma cells mna 42/13 ) and serially diluted from 10 to 10 . the infectious titre was determined according to the spearman - karber formula [ 49 , 50 ] in fluorescent focus forming doses 50% ( ffd50 ) . in the heminested rt - pcr ( hnrt - pcr ) , the amplification product of 582  bp was visible up to a dilution of 10 corresponding to a detection limit of 0.4 ffd50 ( 10 ffd50 ; figure 1 ) . in the real - time rt - pcr ( ndwd ) the limit of detection was defined as the last dilution at which at least 2 out of 3 replicates were positive . using this definition , cvs-11 was detectable up to a dilution of 10 ( once at a dilution of 10 ) . the amplification plot showed regular intervals of the curves with ct - values from 18.1 ( dilution 10 ) to 40.0 ( dilution 10 ) . relating to infectious titre real - time rt - pcr reached a detection limit of 0.003 ffd50 ( 10 ) for cvs-11 . the efficiency of the real - time rt - pcr ( ndwd ) was determined using linear regression of the ct - values and the titres of the samples ( figure 2 ) . on the basis of the slope coefficient the limit of detection in terms of dna copy numbers was determined using 10-fold serial dilutions of a cloned 933  bp segment of n of cvs-11 containing 10 to 10 dna copies / pcr reaction . in the hnpcr , the limit of detection was 100 dna copies/3  l of starting material of the cvs-11 plasmid . in the real - time pcr ( ndwd ) , the 10-fold serial dilution of the cvs - plasmid was tested in triplicate . the limit of detection was 10 dna copies/2  l of the cvs-11 plasmid with ct - values between 38.6 and 47.5 . the efficiency was at 82.8% with a slope coefficient of 3.82 ( not shown ) . the intra - assay repeatability of the real - time pcr ( ndwd ) proved to be excellent with very low coefficients of variation up to 10 dna copies ( 0.181.37% ) with and outlier at 10 copies ( 12.05% ; table 3 ) . a total of 31 lyssaviruses ( 19 identified as classical rabv , 7 as eblv-1 , 4 as eblv-2 , and 1 as duvv ) were used to test the diagnostic performance of the pcr ( table 1 ) . all viruses used were detected with the expected band size in the hnrt - pcr technique ( table 4 ) . the specificity of the amplified product was confirmed as rabv , eblv-1 , eblv-2 , or duvv by sequencing in each case . host gapdh ( glyceraldehyde 3-phosphate dehydrogenase ) was detected in all of the cell culture supernatants and mouse brain suspensions as internal control , indicating that sample material was present and rna isolation , reverse transcription , and amplification were not inhibited . all samples were also tested positive by the real - time rt - pcr assay ( table 4 ) . sendai virus as internal control for inhibition was run in parallel using the same cycling conditions . in the real - time rt - pcr ( ndwd ) with an annealing temperature of 50c , some samples were detected with more than one probe . with the exception of the sample derived from a raccoon dog from poland , the probe for the homologous genotype always yielded the lowest ct - value . in this exceptional case , the probe for the detection of genotype 6 was slightly lower than the one for genotype 1 ( 15.0 versus 16.1 ; table 4 ) . comparison of these two probes with the sequence of the rabies variant in question showed 1 and 2 mismatches with the rabvd1-p and lysgt6-p probe , respectively ( figure 3 ) . duvenhage virus , also designated as genotype 4 , was detectable with the probe adapted for genotype 6 in spite of 3 mismatches ( lysgt6-p ) . the probe lysgt6-p was able to detect synthetic dna fragments of bblv , arav , and irkv , although containing one or two mismatches , respectively . the synthetic dna fragments of khuv , ablv , lbv , mokv , shibv , and wcbv , which exhibited from four ( ablv / khuv ) up to ten ( wcbv ) mismatches to the best fitting probe for gt1 , gt6 , or gt5 , respectively , were all detected with the corresponding species - specific new probe ( table 2 ) . 10 copies of the synthetic dna were detected at ct - values of 27.933.5 for ablv , mokv , shibv , and wcbv ( not shown ) . the efficiency of detection of khuv , which showed an atypical amplification plot at the highest number of 10 copies of the synthetic dna , and that of lbv was lower with ct - values of 40.0 and 40.5 at a copy number of 10 , respectively ( not shown ) . multiplexing of combinations of these probes ( ivvlbv - p ( fam : 6-carboxyfluorescein ) , ivvkhujand - p ( yy : yakima yellow ) , and ivvsblv2-p ( cy5 : cyanine 5 ) , as well as ivvwcbv - p ( fam ) , ivvmok - p ( yy ) , and ivvablv2n - p ( cy5 ) ) as for the species 1 , 5 , and 6 was not successful . the 543  bp amplification products of the nucleoprotein gene obtained in the heminested pcr reactions were sequenced ( table 1 ) and analysed phylogenetically with additional sequences retrieved from genbank representing all known lyssaviruses . the similarity of the 543  bp nucleotide sequences of the nucleoprotein gene ( positions 74616 according to the pasteur virus genome ) among the lyssaviruses included ranged from 64.6% to 90.1% ( hamming distance ) . the resulting phylogenetic tree obtained by the neighbor - joining method implemented in the mega5 software is presented in figure 4 . clinical specimens like brain suspensions ( 1x human , 12x mouse ) , skin biopsies from the nape of the neck ( 3x human , 1x mouse ) , saliva ( 4x human ) , and cerebrospinal fluids ( 6x human ) were used for the establishment of the assays . both hnrt - pcr and real - time rt - pcr ( ndwd ) were shown to work properly on all these samples without significant inhibition using gapdh as internal control for conventional rt - pcr and sendai virus for real - time rt - pcr ( ndwd ) , which was mixed to the samples before rna isolation . skin biopsies taken from the neck and lip of a mouse ( white swiss mouse at an age of 3 weeks ) euthanized 2 weeks after intracerebral infection with sad bern virus 20 years ago were positive with the hnrt - pcr and real - time pcr ( ndwd ) . skin biopsies taken from other locations on the head were positive with the real - time pcr ( ndwd ) and weakly positive in hnrt - pcr whereas samples taken around the vibrissae were weakly positive in real - time pcr ( ndwd ) only . sequencing of amplification products excluded a contamination with the positive cvs-11 control ( not shown ) . brain suspension from an imported human rabies case in 2012 was already strongly positive after one round of the hnrt - pcr . phylogenetic analysis of the 543  bp nucleotide fragment revealed its close relationship to the classical rabies virus strains circulating in the insectivorous mexican free - tailed bat , tadarida brasiliensis , a species common in the southern united states and mexico . this allowed identification of the origin of the patient 's infection , who had travelled extensively and did not report any previous biting incident [ 51 , 52 ] . based on a large amount of published work , we were able to establish and quantitatively characterise rt - pcr protocols for the detection of lyssaviruses in clinical samples . well suited real - time protocols [ 8 , 25 ] for the detection of classical rabies virus and the genotypes 5 and 6 ( eblv-1 and eblv-2 ) were adapted and extended for the detection of at least 13 lyssavirus species . to this goal , 6 species - specific probes ( khuv , ablv , lbv , mokv , shibv , and wcbv ) were designed and verified on synthetic dna fragments encompassing the targeted sequence of the lyssaviral nucleoprotein gene . multiplexing the probes in a single - tube reaction as described for the genotypes 1 , 5 , and 6 was not possible with the newly designed probes , probably due to false priming and/or interference within the mix of reagents and synthetic target sequence . as far as evaluated with the multiplexed probes for the genotypes 1 , 5 , and 6 using 31 viral isolates , which were all detectable with high sensitivity , direct differentiation of targeted genotypes on the base of the quantitative reaction ( ct - values ) was mostly possible . furthermore , a single probe for genotype 6 ( species eblv-2 ) was able to detect up to 7 genotypes / species . we consider this type of cross hybridization as an advantage of the technique using degenerate primers and probes in terms of a broad detectability of lyssavirus rather than a lack of accuracy of discrimination . in an approach using sybr green qpcr with similarly degenerate primers spanning the same part of n as in this work , the detection of all lyssavirus species known at that time was achieved . using several genotype - specific probes in a taqman real - time protocol , we were able to add more intrinsic confidence to the specificity of the analysis . quantitative characterisation of the assay using the probe for genotypes 1 , 5 , and 6 on cvs-11 showed excellent sensitivity and repeatability with a detection limit of as low as an infectious dose of 0.003 ffd50 combined with an analytical sensitivity of 10 dna copies at efficiencies of 94.5% and 82.8% , respectively . considering the amount of degeneration in both primers and probes , the efficiencies determined must be considered satisfactory . as far as the absolute detection limit in terms of target copies is concerned , the usage of plasmid dna rather than rna must be kept in mind . since reverse transcription can not be assumed as 100% efficient and reproducible , the limit for rna might be somewhat lower . for further characterisation of samples with a positive reaction in real - time rt - pcr , excellent simple and ( hemi)nested rt - pcr protocols are available . with the protocol used for this work , all 31 viral strains used could be amplified and sequenced , confirming the diagnostic broadness of the assay . phylogenetic analysis of these partial nucleotide sequences belonging to genotypes 1 , 4 , 5 , and 6 along with known sequences confirmed the suitability of this relatively conserved genomic region for molecular - epidemiological characterisation of lyssaviruses circulating worldwide [ 5658 ] . this was also confirmed in the context of a human rabies case imported to switzerland in 2012 , which could be attributed unequivocally to an exposure to a bat of the species tadarida brasiliensis in california , usa . in this work we could show and validate the suitability of an adapted and further developed real - time rt - pcr protocol for the rapid , efficient , and highly sensitive intravitam diagnosis of a wide spectrum of lyssavirus species followed by rapid molecular - epidemiological characterisation of viral isolates respective to origin of the virus and source of exposure , using heminested rt - pcr . this new tool is also particularly promising for active surveillance of european bat lyssaviruses using oral swabs in live - captured bats .
the causative agents of rabies are single - stranded , negative - sense rna viruses in the genus lyssavirus of rhabdoviridae , consisting of twelve classified and three as yet unclassified species including classical rabies virus ( rabv ) . 
 highly neurotropic rabv causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome . 
 rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health . due to growing variety of 
 the genus lyssavirus observed , the development of suitable molecular assays for diagnosis and differentiation is challenging . 
 this work focused on the establishment of a suitable real - time rt - pcr technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation . 
 the real - time rt - pcr was adapted with the goal to detect the whole spectrum of lyssavirus species , for nine of which synthesized dna fragments were used . for the detection of species , seven probes were developed . 
 serial dilutions of the rabies virus strain cvs-11 showed a 100-fold higher sensitivity of real - time pcr compared to heminested rt - pcr . using a panel of thirty - one lyssaviruses representing four species , 
 the suitability of the protocol could be shown . 
 phylogenetic analysis of the sequences obtained by heminested pcr allowed correct classification of all viruses used .