Download Detection of yellow fever virus: a comparison of

Journal of Virological Methods 110 (2003) 185 /191
www.elsevier.com/locate/jviromet
Detection of yellow fever virus: a comparison of quantitative
real-time PCR and plaque assay
Hi-Gung Bae a,1, Andreas Nitsche a,b,*,1, Anette Teichmann a, Stefan S. Biel a,2,
Matthias Niedrig a
b
a
Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany
Medizinische Klinik II m.S. Onkologie und Hämatologie, Charité, Humboldt Universität, Berlin, Germany
Received 25 February 2003; received in revised form 31 March 2003; accepted 31 March 2003
Abstract
Yellow fever virus quantitation is performed routinely by cultivation of virus containing samples using susceptible cells. Counting
of the resulting plaques provides a marker for the number of infectious particles present in the sample. This assay usually takes up to
5 days before results are obtained and must be carried out under L2 or L3 laboratory conditions, depending on the yellow fever virus
strain used. For clinical diagnosis of yellow fever virus infections the cell culture-based approach takes too long and is of limited
practical relevance. Recently, due to its considerable sensitivity, PCR has become a promising method for virus detection. However,
whilst PCR can detect virus-specific nucleic acids, it does not allow conclusions to be drawn regarding the infectious potential of the
virus detected. Nonetheless, for diagnostic purposes, a rapid, specific and sensitive virus PCR is preferable. Therefore, two
independent yellow fever virus-specific real-time PCR assays were established and compared the viral RNA loads to the results of a
traditional plaque assay. The estimated ratio of yellow fever virus genomes to infectious particles was between 1000:1 and 5000:1;
both approaches displayed a comparable precision of B/45%. A significant correlation between genome number as determined by
real-time PCR and the corresponding number of plaques in paired samples was found with a Pearson coefficient of correlation of
r /0.88 (P B/0.0001).
# 2003 Elsevier Science B.V. All rights reserved.
Keywords: Yellow fever virus; 17D; Plaque assay; Real-time PCR; TaqMan
1. Introduction
Yellow fever virus is the prototype of the family
Flaviviridae (Westaway et al., 1985) which comprises
approximately 70 strains (Calisher et al., 1989). The
viruses of this family contain non-segmented, positivestranded RNA genome of 10 862 nucleotides with a
5?cap structure and a non-polyadenylated 3?end. The
yellow fever virus genome encodes a polyprotein of 3411
amino acids which is cleaved by proteolytic processing
into 11 viral polypeptides (Chambers et al., 1990).
* Corresponding author. Tel.: /49-30-4547-2313; fax: /49-304547-2605.
E-mail address: [email protected] (A. Nitsche).
1
These authors contributed equally to this work.
2
Present address: Beiersdorf AG, Hamburg, Germany.
Yellow fever infection in man can result in inapparent
infection through to fulminant disease which is invariably fatal, provoking great public health concern for
centuries (Fields et al., 2001). However, yellow fever
epidemics are still a major problem in endemic areas
where suitable vaccination regimes are lacking.
Sporadically, cases of yellow fever virus infection are
imported to countries where yellow fever virus is not
endemic, and therefore, rapid diagnosis of these infections is an important task, especially to rule out other
agents of hemorraghic fever requiring patient management. The last two yellow fever virus cases imported to
Europe clearly proved that a rapid diagnosis is a
prerequisite for efficient medical treatment (Teichmann
et al., 1999; Colebunders et al., 2002). In these two cases,
yellow fever virus detection by conventional RT-PCR
and real time RT-PCR was the method of choice for
rapid diagnosis, permitting rapid decision regarding the
0166-0934/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0166-0934(03)00129-0
186
H.-G. Bae et al. / Journal of Virological Methods 110 (2003) 185 /191
patient’s condition as well as the management and
control of viral hemorraghic fever (Drosten et al., 2002).
Usually, the diagnosis of yellow fever virus is carried
out by virus cultivation on susceptible cells followed by
immune-fluorescence detection, ELISA, immunohistochemical examination or PCR (Deubel et al., 1997; De
Brito et al., 1992; Monath et al., 1989; Monath and
Nystrom, 1984). For quantifying the load of infectious
particles either in cell culture or serum samples, the
plaque assay is still a method used commonly. However,
it is a very time-consuming technique that takes at least
5 days (Porterfield, 1959), and moreover, it has to be
undertaken under L2 conditions for yellow fever virus
strain 17D or L3 conditions for yellow fever virus wild
type isolates (WHO, 1971).
With the recent development of quantitative real-time
PCR, a more feasible approach to quantifying viral load
is available (Mackay et al., 2002). However, from the
biological point of view, the exclusive detection of viral
nucleic acids provides no information regarding the
infectious capability of the virion. A correlation between
plaque assay results and quantitation of yellow fever
virus genomic RNA could simplify and accelerate the
diagnosis of yellow fever virus. Therefore, two novel
quantitative real-time PCR assays for the quantitation
of yellow fever virus RNA based on the 5?-nuclease
chemistry (TaqMan; Holland et al., 1991; Livak et al.,
1995) were developed and evaluated. The benefits and
pitfalls of a real-time time PCR approach to viral
diagnostics have been discussed previously (Mackay et
al., 2002). The overall precision of the real-time PCR
and plaque assay were determined. Finally, the results
obtained by the real-time PCR were compared with the
results obtained by the plaque assay carried out
routinely, with the aim of assessing a correlation
between viral RNA load and plaque assay.
2. Material and methods
2.1. Virus cultivation
Various cell lines from vertebrates (Vero B4, STC-1,
U-937) and insects (C6/36) were infected with yellow
fever virus 17D using a multiplicity of infection (MOI)
of 0.5 and cultivated in RPMI 1640 medium (Gibco,
BRL) for a period of 8 days. Lyophilised yellow fever
virus 17D was obtained from yellow fever virus-vaccine
Batch No. 189/00/1, 190/00/1 and 191/00/1 (Robert
Koch Institute Berlin, Germany). Assays were carried
out either with freshly reconstituted yellow fever virus
YFV vaccine ampoules, with frozen yellow fever virusladen cell culture supernatants or with human sera.
Cell culture supernatant samples were taken daily
from infected cell cultures, and the concentration of
yellow fever virus was determined using both the plaque
assay and the quantitative real-time PCR assay.
Serum from yellow fever virus infected patients was
obtained by gravity sedimentation of peripheral blood
as described previously (Nitsche et al., 2000). Viral RNA
was prepared from serum as described below for cell
culture supernatants.
2.2. Plaque assay
The plaque assay was carried out as a modified
version of the assay described by De Madrid and
Porterfield (De Madrid and Porterfield, 1969). Briefly,
6 /105 porcine kidney cells in 200 ml RPMI 1640 were
seeded in each well of a 24-well plate. Serial dilutions
(1:20 000, 1:40 000 and 1:80 000) of the different viral
suspensions were added to the wells (200 ml each). After
an incubation period of 4 h, overlay medium (1.6%
carboxymethyl-cellulose, 3% fetal calf serum in RPMI)
was added, and the plates were incubated for 5 days at
37 8C.
After a 15 min fixation step with 4% formalin in PBS,
the cells were stained with Naphtalin Black for 20 min.
The plaques caused by lysis of infected cells were
counted and the calculation of plaque forming units
(pfu) was carried out according to Reed and Münch
(1938).
2.3. Viral RNA isolation and reverse transcription of
yellow fever virus genome
Yellow fever virus 17D RNA was isolated from 140 ml
cell culture supernatants and serum using the QIAamp
Viral RNA Mini Kit (Qiagen, Hilden). RNA was eluted
with 60 ml of sterile water. Total cDNA was produced by
reverse transcription (RT) of 2 ml of purified RNA, 2 ml
of 10 /buffer, 2 ml of 10 mM DTT, 5 ml of 2.5 mM
dNTP (each), 0.5 ml of random hexamers and 6 ml of
sterile water. Samples were heated to 72 8C for 3 min
and subsequently chilled on ice for 3 min to allow primer
hybridisation. After the addition of 1 ml of 20 U RNAse
inhibitor and 0.5 ml 200 U/ml reverse transcriptase,
samples were incubated for 40 min at 42 8C for RT.
Subsequently, the RT reaction was terminated by
enzyme inactivation at 83 8C for 5 min.
2.4. Primers and TaqMan probes
The oligonucleotide primers and probes were designed according to the general guidelines for real-time
PCR primer and probe design and synthesised by TIB
MOLBIOL, Berlin, Germany. To increase the reliability
of the real-time PCR diagnosis, two independent PCR
assays were established, targeting highly conserved
regions within the NS3 region and the 3?UTR region
of the yellow fever virus genome, respectively. Both
H.-G. Bae et al. / Journal of Virological Methods 110 (2003) 185 /191
187
Table 1
Sequence, location and annealing temperature of oligonucleotides used for real-time PCR
NS3 sense
NS3 antisense
NS3 TaqMan
3?UTR sense
3?UTR antisense
3?UTR TaqMan
Sequence of primer/TaqMan probe
K02749
Tm (8C)
AggTCCAgTTgATCgCggC
gAgCgACAgCCCCgATTTCT
F-TggTCAACgTCCAgACAAAACCgAgC T Tg p
¯
AACCCACACATgCAggACAA
GTTgCAggTCAgCATCCACA
F-CCATTTAgTCATCCATCgTATCCgAACgC T p
¯
4857-1875
4961-4942
4893-4921
10 109-128
10 337-318
10 237-266
62.1
62.2
71.7
58.8
59.1
68.2
Abbreviations: F, 6-carboxyfluorescein attached to 5?-terminus (FAM), T, 5-carboxytetramethylrhodamine (5-TAMRA) attached to 5¯ method as described elsewhere (Breslauer et al., 1986).
ethylamino-dThymidin. Melting temperature was calculated by nearest neighbour
assays were optimised to be carried out simultaneously
under identical conditions. Primer sequences, location
and annealing temperatures are shown in Table 1.
tion and plaque number was determined by Pearson
analysis.
2.5. Real-time PCR
3. Results
Quantitative real-time PCR was performed in a
Perkin/Elmer 7700 Sequence Detection System (Applied Biosystems, Foster City). Amplifications were
carried out in 50 ml reaction mixtures containing 2 ml
of template cDNA, 0.5 mM of each primer, 0.1 mM of
TaqMan probe, 0.2 mM of each dNTP and 4 mM
MgCl2. As a passive fluorescent reference dye 1 mM
ROX (6-carboxy-X-rhodamine) was added to the mixture. Cycling conditions were as follows: initial denaturation at 94 8C for 3 min, 45 cycles with 94 8C for 30 s
and 60 8C for 1 min.
For calibration of the real-time PCR, two independent plasmids pYFV-NS3amp and pYFV-3?UTRamp
were constructed by cloning the respective amplicons of
104 and 228 bp into a TOPO TA Cloning vector,
according to the manufacturer’s instructions (Invitrogen, Leek, The Netherlands). Serial dilutions of 108 /101
plasmids/ml were used to generate calibration curves as
described previously (Nitsche et al., 1999).
3.1. Reproducibility of the real-time PCR assay
Two quantitative real-time PCR assays for the detection of yellow fever virus were designed and evaluated.
First, to allow quantitation of yellow fever virus
genomic RNA, calibration curves were generated by
amplification of serial dilutions of the plasmids pYFVNS3amp and pYFV-3?UTRamp. CT values were determined in quadruplicate and plotted against the plasmid
copy number. Fig. 1 presents the resulting calibration
curves from both assays, indicating a PCR efficiency of
98.7%, a linear detection range between 108 and 101
plasmids and a high correlation of R2 /0.999 between
cycle number and plasmid number for both independent
PCR assays.
Real-time PCR reproducibility was determined by
repeated amplification of two 10-fold dilutions of the
2.6. Test evaluation
Several independent steps can affect the reproducibility and sensitivity of an RT-PCR reaction. Therefore,
the variability of the complete RT-PCR procedure was
determined by undertaking 10 RT reactions in parallel
on aliquots of one identical RNA sample. This was
repeated four times. Subsequently, the cDNA was
quantified in duplicate by real-time PCR. To compare
real-time PCR and plaque assay results, three yellow
fever virus samples were examined in parallel with
plaque assay and the real-time PCR using three different
sample dilutions (non-diluted, 1:100, 1:1000). The three
dilutions were split up in six aliquots to determine the
intra-assay variability and experiments were repeated on
4 consecutive days to determine the inter-assay variability. The correlation between viral genome concentra-
Fig. 1. Calibration curves of the two independent real-time PCR
assays NS3 (black circles) and 3?UTR (open triangles). Plasmid copy
numbers are plotted against the corresponding CT value. The slope
indicates a PCR efficiency of /98.7% for both assays.
188
H.-G. Bae et al. / Journal of Virological Methods 110 (2003) 185 /191
plasmids pYFV-NS3amp and pYFV-3?UTRamp and by
amplification of two 10-fold dilutions of three different
yellow fever virus 17D vaccine batches. The intra-assay
precision could be determined as B/20% for the vaccine
samples and B/13% for the plasmid dilutions for both
assays. Only ten plasmid copies, the lowest number
detectable, showed a reduced reproducibility of B/34%
for both assays. The inter-assay precision could be
determined as B/30%; for both real-time PCR assays.
Only low copy numbers showed a lower reproducibility
of B/45%. However, although a reduced reproducibility
in the template range close to the detection limit was
observed, 100% of the samples containing ten plasmid
copies could be detected, indicating a high reliability
even in the low range of low template concentrations
(Peccoud and Jacob, 1996).
Representative amplification plots of the NS3 assay
carried out on yellow fever virus 17D in two serial 10fold dilutions on the first day tested are shown in Fig. 2.
All samples were measured in quadruplicate. Based on
the calibration curves shown in Fig. 1 results obtained
from both real-time PCR assays were averaged. yellow
fever viral could be quantified in the three vaccine
batches as follows: batch 189/1: 2.2 /109, batch 190/1:
3.0 /109 and batch 191/1: 3.9 /109 genome equivalents
(GE)/ml. Serial serum samples of a patient suffering
from fulminant yellow fever virus infection showed a
decrease in yellow fever virus genome load from 1.6 /
108 to 9.5 /106 GE/ml serum on the last 3 days before
his death (data not shown).
All yellow fever virus control samples were detected as
positive by both real-time PCR assays from cell culture
as well as from diagnostic human serum samples.
Yellow fever virus free cell culture supernatants as well
as plasma samples of healthy blood donors remained
negative in all PCR reactions carried out. In addition,
there were no cross reactions with other flaviviruses
including Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus and the dengue viruses 1/
4.
3.2. Reproducibility of the RT reaction
In order to measure the extent of variation in the RT
reaction, the cDNA concentration of ten independent
RT reactions were determined in duplicate by employing
real-time PCR. Applying the calibration curves as
described above, quantitation by real-time PCR resulted
in genome loads of 1.4 /106 GE/assay (S.D./16%) for
the NS3 assay and 1.8 /106 GE/assay (S.D./12%) for
the 3?UTR assay, respectively. When reducing RNA
concentration 20-fold prior to cDNA synthesis, variations of 9.1 /104 GE/assay (S.D. /16%) for the NS3
assay and 1.0 /105 GE/assay (S.D. /10%) for the
3?UTR assay, respectively, were obtained. Variation
fell within the range of the overall assay variation
observed with both real-time PCR assays.
3.3. Reproducibility of the plaque assay
The plaque assay results of the three yellow fever virus
17D vaccine batches (189/1, 190/1, 191/1) were 8.2 /105,
7.2 /105 and 9.2 /105 pfu/ml. Determining the intraassay variability, B/33% variation for the three independent yellow fever virus 17D vaccine batches was
observed. Maximum inter-assay variation was B/44%.
Fig. 2. Representative amplification plots of the NS3 real-time PCR assay. Two serial 10-fold dilutions of yellow fever virus 17D vaccine batch 189
were measured in quadruplicate. The high reproducibility can be seen by the small variation in CT values.
H.-G. Bae et al. / Journal of Virological Methods 110 (2003) 185 /191
3.4. Correlation between plaque assay and real-time PCR
assay
To elucidate if there was a correlation between the
quantitative results of the real-time PCR assays and the
plaque assay, an NS3-specific real-time PCR assay and a
plaque assay were carried out on the same samples of
different yellow fever virus concentrations. In general,
viral genome numbers determined by real-time PCR
were 1000/5000-fold higher than the infectious particles
that were determined by the plaque assay. A significant
correlation of r/0.88 with P B/0.0001 was found
plotting the viral genome concentration as determined
by real-time PCR versus plaque number (Fig. 3).
4. Discussion
The detection of viruses by rapid and reliable
techniques is still one of the most demanding tasks in
virus diagnosis. Cell culture-based methods which
determine the number of infectious virus particles, are
time consuming and complex. The need for alternative
fast and sensitive diagnostic tools has provided PCR
(Niesters, 2001). Beside many qualitative approaches,
quantitative PCR assays have evolved rapidly during
recent years. In addition to the option of reliable,
accurate quantification, attributes including speed, sensitivity and a reduced risk of amplicon carry-over
contamination are restricted to real-time PCR-based
methods. Therefore, two independent real-time PCRbased assays have been developed to be carried out
under identical conditions. These assays demonstrated
all benefits of real-time PCR: speed (B/3 h including
sample preparation), a very low detection limit of ten
Fig. 3. Correlation between concentration of yellow fever virus nucleic
acids and plaque number of yellow fever virus containing cell culture
supernatants (n/36). Results obtained with the real-time PCR assay
are plotted against the pfu values, as determined by the plaque assay.
Pearson analysis revealed a significant of P B/0.001 and R/0.88.
189
GE per reaction and a linear quantification range of
seven orders of magnitude which should encompass the
general clinical requirements. Despite the different
amplicon lengths produced by the two assays (104 vs.
228 bp), detection limits and precision were nearly
identical under optimised conditions. Both assays produced a high overall precision of B/30% and still B/45%
in the lowest detection range of ten GE per assay. This
precision is comparably high, considering that the RT
reaction can vary from 5 to 95% efficiency (Ferré et al.,
1994). An increased variation in low template concentration ranges close to the cetection limit can be caused
solely by statistical distribution (De Vries et al., 1999).
However, it has been suggested, that the problem of
inter-assay variation is mainly attributed to RNA
preparation and sample processing, not to the PCR
reaction itself (Keilholz et al., 1998). Considering all the
different steps included in the real-time PCR detection,
an overall precision of B/45% is high.
Although each assay was constructed to amplify
several different yellow fever virus strains, the use of
two independent PCR assays, spanning approximately
5000 nt, reduced the risk of false positive results due to
sequence heterogeneity in the primer or probe binding
regions. However, results obtained from both assays
were comparable consistently and no assay failures with
the different templates tested were found. Considering
that absolute quantification of RT-PCR reactions is still
demanding, the use of an external calibration curve in
separate reactions was preferred. Although external
calibration curves are not perfectly quantitative because
of uncontrolled and unmonitored inter-tube variations
(Mackay et al., 2002), the use of an internal standard
within the PCR reactions can influence the detection
limit, and therefore, reduce the diagnostic potential of
the real-time PCR assays. The use of the internal
reference dye ROX controls at least for tube-to-tube
variations.
However, one of the main drawbacks of (real-time)
PCR in viral diagnosis is the lack of a croealtion
between viral nucleic acid detection and the presence
of infectious particles. Unfortunately, infectious particles can only be demonstrated by virus amplification
using laborious cell culture systems. To determine
whether there is a correlation between the concentrations of genomes and the number of infectious particles,
results from quantitative real-time PCR were compared
with the results obtained from the plaque assay carried
out routinely on identical samples. As expected, values
for the genome detecting real-time PCR were approximately 1000/5000-fold higher compared with the infectious particle detecting plaque assay. This difference
may result from the production of defective noninfectious particles, as previously shown for HTLV-1
(Morozov and Weiss, 1999). The extent of this production varies from virus to virus and even within virions of
190
H.-G. Bae et al. / Journal of Virological Methods 110 (2003) 185 /191
the same stock the ratio between infectious particles and
viral genome copies can vary significantly. The overall
precision of the plaque assay was comparable to the
precision of the real-time PCR assays. Despite a distinct
correlation between genomic load and plaque number
being unlikely, the amount of yellow fever virus genome
on the one hand was compared with the number of
infectious virus particles on the other. Pearson analysis
revealed a significant correlation between genomic load
and plaque number.
Because real-time PCR can only detect a biological
molecule, i.e. the genome, but not a biological feature,
i.e. the infectious potential of a cell, PCR can never
replace the plaque assay. However, due to comparable
precision, quantitative PCR has the additional benefits
of flexibility, feasibility and speed. The last yellow fever
infection cases have shown, that a fast and sensitive
yellow fever virus detection assay that includes quantification can be an important factor for adequate
treatment of the patient. Hence, real-time PCR has
proven to be the method of choice for monitoring of
antiviral treatment (Held et al., 2000). Whenever the
identification of infectious virus particles is required, for
example during vaccine production and control, the
plaque assay, although very time consuming and
laborious, is appropriate. In this regard, the real-time
PCR assay described now is a very useful addition to
diagnostic procedures but cannot replace the plaque
assay.
Acknowledgements
The authors thank Ian M. Mackay for critical reading
of the manuscript and H. Emmel for excellent technical
assistance.
References
Breslauer, K.J., Frank, R., Blocker, H., Marky, L.A., 1986. Predicting
DNA duplex stability from the base sequence. Proceedings of the
National Academy Sciences of the United States of America 83
(11), 3746 /3750.
Calisher, C.H., Karabatsos, N., Dalrymple, J.M., Shope, R.E.,
Porterfield, J.S., Westaway, E.G., Brandt, W.E., 1989. Antigenic
relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. Journal of General Virology
70, 37 /43.
Chambers, T.J., Hahn, C.S., Galler, R., Rice, C.M., 1990. Flavivirus
genome organisation, expression and replication. Annual Review
of Microbiology 44, 649 /688.
Colebunders, R., Mariage, J.L., Coche, J.C., Pirenne, B., Kempinaire,
S., Hantson, P., Van Gompel, A., Niedrig, M., Van Esbroeck, M.,
Bailey, R., Drosten, C., Schmitz, H., 2002. A Belgian traveler who
acquired yellow fever in the Gambia. Clinical Infectious Diseases
35 (10), 113 /116.
De Madrid, A.T., Porterfield, J.S., 1969. A simple micro-culture
method for the study of group B arboviruses. Bulletin of World
Health Organisation 40, 113 /121.
De Brito, T., Siqueira, S.A., Santos, R.T., Nassar, E.S., Coimbra,
T.L., Alves, V.A., 1992. Human fatal yellow fever. Immunohistochemical detection of viral antigens in the liver, kidney and heart.
Pathology Research and Practice 188, 177 /181.
De Vries, T.J., Fourkour, A., Punt, C.J., van de Locht, L.T., Wobbes,
T., van den Bosch, S., De Rooij, M.J., Mensink, E.J., Ruiter, D.J.,
van Muijen, G.N., 1999. Reproducibility of detection of tyrosinase
and MART-1 transcripts in the peripheral blood of melanoma
patients: a quality control study using real-time quantitative RTPCR. British Journal of Cancer 80 (5-6), 883 /891.
Deubel, V., Huerre, M., Cathomas, G., Drouet, M.T., Wuscher, N., Le
Guenno, B., Widmer, A.F., 1997. Molecular detection and
characterization of yellow fever virus in blood and liver specimens
of a non-vaccinated fatal human case. Journal of Medical Virology
53, 212 /217.
Drosten, C., Gottig, S., Schilling, S., Asper, M., Panning, M., Schmitz,
H., Gunther, S., 2002. Rapid detection and quantification of rna of
ebola and marburg viruses, lassa virus, crimean-congo hemorrhagic
fever virus, rift valley fever virus, dengue virus and yellow fever
virus by real-time reverse transcription-pcr. Journal of Clinical
Microbiology 40, 2323 /2330.
Ferré, F., Marchese, A., Pezzoli, P., Griffin, S., Buxton, E., Boyer, V.,
1994. Quantitative PCR: an overview. In: Mullis, K.B., Ferré, F.,
Gibbs, R.A. (Eds.), The Polymerase Chain Reaction. Birkhäuser,
Boston, pp. 67 /88.
Fields, B.N., Howley, P.M., Griffin, D.E., 2001. Fields-Virology,
fourth ed.
Held, T.K., Biel, S.S., Nitsche, A., Kurth, A., Chen, S., Gelderblom,
H.R., Siegert, W., 2000. Treatment of BK virus-associated hemorrhagic cystitis and simultaneous CMV reactivation with cidofovir.
Bone Marrow Transplantation 26 (3), 347 /350.
Holland, P.M., Abraham, R.D., Watson, R., Gelfand, D.H., 1991.
Detection of specific polymerase chain reaction product by utilizing
the 5?† ?3? exonuclease activity of thermus aquaticus DNA polymerase. Proceedings of the National Academy Sciences of the
United States of America 88, 7276 /7280.
Keilholz, U., Willhauck, M., Rimoldi, D., Brasseur, F., Dummer, W.,
Rass, K., de Vries, T., Blaheta, J., Voit, C., Lethe, B., Burchill, S.,
1998. Reliability of reverse transcription-polymerase chain reaction
(RT-PCR) based assays for the detection of circulating tumour
cells: a quality-assurance initiative of the EORTC Melanoma
Cooperative Group. European Journal of Cancer 34, 750 /753.
Livak, K.J., Flood, J.A., Marmato, J., Giusti, W., Deetz, K., 1995.
Oligonucleotides with fluorescent dyes at opposite ends provide a
quenched probe system useful for detecting PCR product and
nucleic acid hybridization. PCR Methods and Applications 4, 357 /
362.
Mackay, I.M., Arden, K.E., Nitsche, A., 2002. Real-time PCR in
virology. Nucleic Acids Research 30 (6), 1292 /1305.
Monath, T.P., Nystrom, R.R., 1984. Detection of yellow fever virus in
serum by enzyme immunoassay. American Journal of Tropical
Medicine and Hygiene 33, 151 /157.
Monath, T.P., Ballinger, M.E., Miller, B.R., Salaun, J.J., 1989.
Detection of yellow fever viral rna by nucleic acid hybridization
and viral antigen by immunocytochemistry in fixed human liver.
American Journal of Tropical Medicine and Hygiene 40,
663 /668.
Morozov, V.A., Weiss, R.A., 1999. Two types of HTLV-1 particles are
released from MT-2 cells. Virology 255 (2), 279 /284 (March 15).
Niesters, H.G.M., 2001. Quantitation of viral load using real-time
amplification techniques. Methods 25 (4), 419 /429.
Nitsche, A., Steuer, N., Schmidt, C.A., Landt, O., Siegert, W., 1999.
Different real-time PCR formats compared for the quantitative
H.-G. Bae et al. / Journal of Virological Methods 110 (2003) 185 /191
detection of human cytomegalovirus DNA. Clinical Chemistry 45
(11), 1932 /1937.
Nitsche, A., Steuer, N., Schmidt, C.A., Landt, O., Ellerbrok, H., Pauli,
G., Siegert, W., 2000. Detection of human cytomegalovirus DNA
by real-time quantitative PCR. Journal of Clinical Microbiology 38
(7), 2734 /2737.
Peccoud, J., Jacob, C., 1996. Theoretical uncertainty of measurements
using quantitative polymerase chain reaction. Biophysics Journal
71, 101 /108.
Porterfield, J.S., 1959. A plaque technique for the titration of yellow
fever virus and antisera. Transactions of the Royal Society of
Tropical Medicine and Hygiene 53, 458 /465.
191
Reed, C.J., Münch, H., 1938. A simple method of estimating fifty
percent endpoints. American Journal of Hygiene 27, 493 /497.
Teichmann, D., Grobusch, M.P., Wesselmann, H., TemmesfeldWollbrück, B., Breuer, T., Dietel, M., Emmerich, P., Schmitz,
H., Suttorp, N., 1999. A haemorrhagic fever from the Côte
d’Ivoire. Lancet 354, 1608.
Westaway, E.G., Brinton, M.A., Gaidamovich, S.Y., Horzinek, M.C.,
Igarishi, A., Kääriäinen, L., Lvov, D.K., Porterfield, J.S., Russel,
P.K., Trent, D.W., 1985. Flaviviridae Intervirology 24, 183 /192.
WHO Expert Committee on Yellow Fever. Third Report of the WHO
Expert Committee, World Health Organization, 1971. Technical
Report Series No 479, ISBN: 92 4 120479 6.