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Journal of Clinical Microbiology, May 2001, p. 1922-1927, Vol. 39, No. 5
Unité de Virologie, Centre de
Recherches du Service de Santé des Armées (CRSSA) Emile
Pardé, Grenoble,1
Hôpital Pasteur, Colmar,2 and
Université Louis Pasteur,
Strasbourg,3 France
Received 14 August 2000/Returned for modification 29 December
2000/Accepted 1 March 2001
Arthropod-transmitted flaviviruses are responsible for considerable
morbidity and mortality, causing severe encephalitic, hemorrhagic, and
febrile illnesses in humans. Because there are no specific clinical
symptoms for infection by a determined virus and because different
arboviruses could be present in the same area, a genus diagnosis by PCR
would be a useful first-line diagnostic method. The six published
Flavivirus genus primer pairs localized in the NS1, NS3,
NS5, and 3' NC regions were evaluated in terms of specificity and
sensitivity with flaviviruses (including the main viruses pathogenic
for humans) at a titer of 105 50% tissue culture
infectious doses (TCID50s) ml Flaviviruses are
arthropod-transmitted viruses that belong to the
Flaviviridae family. The genus Flavivirus
includes more than 70 single-stranded RNA viruses sharing common
antigenic determinants, and the group is divided into eight
serosubgroups and nine individual serotypes. Flaviviruses are
responsible for considerable morbidity and mortality and may cause
severe encephalitic, hemorrhagic, hepatic, and febrile illness in
vertebrates, including humans. The pathogenic viruses in this genus
include the Yellow fever virus (YF), Dengue
viruses (DEN-1, -2, -3, and -4), Tick-borne encephalitis
virus (TBE), Japanese encephalitis virus (JE),
St. Louis encephalitis virus (SLE), and West Nile
encephalitis virus (WN) (25).
Conventional flavivirus diagnosis is based on serology tests screening
for the presence of virus-specific antibodies in the patient serum,
which often require documentation of a rise in antibody concentration
from an acute-phase blood sample to a convalescent-phase sample.
In August 1999, an outbreak of arboviral encephalitis was first
recognized in New York City (2) and has since been
identified in a neighboring state. This outbreak resulted in 61 human infections and seven deaths (5). Although initially
attributed to SLE virus based on positive serologic findings in
cerebrospinal fluid and serum samples using a virus-specific
immunoglobulin M-capture enzyme-linked immunosorbent assay (ELISA), the
cause of the outbreak has been confirmed as a WN virus (JE virus
complex) based on the identification of the virus in human, avian, and
mosquito samples by molecular tools (1, 18, 22).
The exceptional sensitivity of the PCR method allowed rapid detection
and identification of flaviviruses (9) in mosquitoes and
clinical samples (11, 21), in which virus culture is
difficult or time-consuming and when early diagnosis is necessary for
clinical treatment (28) and has implications for
vaccination and mosquito control. The aim of this study was to develop,
after comparing the specificity and sensitivity of the published
primers for genus diagnosis of the main human pathogenic flaviviruses,
a more sensitive technique with new primers. After having selected the
best primers, we used the widely accepted heminested method to increase
sensitivity, designing a new degenerate primer likely to be used
in patient samples.
Viruses and isolation of viral RNA.
All viruses were
manipulated in a level 3 facility. With the exception of hepatitis C
virus (HCV), all viral strains (Table 1)
were obtained from mouse brain tissues and propagated in Vero cells as
previously described (10). HCV was obtained from an HCV-positive patient's plasma, GR416. Its genotype, determined with
the Inno Lipa HCV II kit (Innogenetics, Zwijndrecht, Belgium), was 1b,
and its viral load, quantified with the Monitor HCV RNA assay (Roche
Diagnostics System, Meylan, France), was 0.5 × 106 copies/ml. RNA was extracted with silica gel
membrane spin columns (QIAmp Viral RNA 250; Qiagen SA, Courtaboeuf,
France) from 280-µl samples obtained either from cell culture
supernatant for arboviruses or from human serum for HCV. The extracted
nucleic acid was stored at
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.1922-1927.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Comparison of Flavivirus Universal Primer Pairs
and Development of a Rapid, Highly Sensitive Heminested Reverse
Transcription-PCR Assay for Detection of Flaviviruses Targeted to a
Conserved Region of the NS5 Gene Sequences
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1 with a common
identification step by agarose gel electrophoresis. Only one NS5
primer pair allowed the detection of all tested flaviviruses with the
sensitivity limit of 105 TCID50s
ml1. Using a heminested PCR with new primers designed in
the same region after an alignment of 30 different flaviviruses, the
sensitivity of reverse transcription-PCR was improved and allowed the
detection of about 200 infectious doses ml1 with all of
the tick- and mosquito-borne flaviviruses tested. It was confirmed that
the sequenced amplified products in the NS5 region allowed
predictability of flavivirus species by dendrogram, including the New
York 99 West Nile strain. This technique was successfully performed
with a cerebrospinal fluid sample from a patient hospitalized with West
Nile virus encephalitis.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
80°C and eventually diluted with
ultrapure water (pretreated with diethyl procarbonate [DEPC] in a
1:1,000 dilution; Sigma, St. Quentin Fallavier, France).
TABLE 1.
Arboviruses used, virus titer before extraction, and
equivalent after dilution
Primers.
The primers used in this study (Table
2) were either published previously or
were designed with Primer Premier software (Primer Premier v4.1;
Premier Biosoft International, Palo Alto, Calif.) after alignment of
the flavivirus sequences with DNASIS software (DNASIS v2.6 for
networks; Hitachi Software Engineering Europe S.A., Ardon, France). The
locations of heminested primers are given according to the 17D YF virus
sequence (GenBank accession no. X03700). They were synthesized by
GIBCO-BRL (Life Technologies SARL, Cergy Pontoise, France).
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RT-PCR. Reverse transcription (RT) reactions were performed in 30 µl containing 6 µl of RT buffer, 2 µl of 0.1 M dithiothreitol, 2.5 µl of 2.5 mM deoxynucleoside triphosphate (dNTP; final concentration, 200 µM), 1 µl of RNase (RNase out; GIBCO BRL), 10 µl of RNA template, 2.5 µl of reverse primer (10 µM; final concentration, 0.75 µM) and 5 µl of ultrapure DEPC-pretreated water.
Each sample was boiled for 5 min and cooled on wet ice. One microliter of Moloney murine leukemia virus (MMLV) reverse transcriptase (GIBCO BRL) was added. The reaction mixture was incubated at 37°C for 1 h and at 95°C for 10 min. The PCRs were performed in 100 µl containing 10 µl of 10× Taq buffer, 3 µl of 50 mM MgCl2 (final concentration, 1.5 mM), 2 µl of 10 mM dNTP (final concentration, 200 µM), 2.5 µl of 10 µM reverse primer, and 2.5 µl of 10 µM sense primer (final concentration, 500 nM), 3 U of Taq DNA polymerase (GIBCO BRL), and 70 µl of ultrapure DEPC-pretreated H2O. Then, 10 µl of cDNA was added. The heminested PCRs were performed with 100 µl of mixture containing 10 µl of 10× Taq buffer, 3 µl of 50 mM MgCl2 (final concentration, 1.5 mM), 2 µl of 10 mM dNTP (final concentration, 200 µM), 2.5 µl of 10 µM reverse primer, 2.5 µl of 10 µM sense primer (final concentration, 500 nM), 3 U of Taq DNA polymerase (GIBCO BRL), and 75 µl of pure H2O. Then, 5 µl of PCR products was added. The PCR thermal cycling incubations used for cFD2 and MAMD amplimers were performed as follows: initial amplification of 25 cycles of incubation at 94, 53, and 72°C for 1 min each; and amplification with nested primers cFD2 and FS 778 with 35 cycles of incubation at 94, 54, and 72°C for 1 min each. All thermal cycling was performed with PE Applied Biosystems 2400 machines. The amplification products were identified by their molecular weights analyzed by electrophoresis in a 2% agarose gel, and the separated fragments were stained with ethidium bromide and visualized under UV light transillumination (28).Light cycler amplification. A PCR quantitative instrument (LightCycler instrument, Roche Molecular Biochemicals, Meylan, France) PCR amplification was performed by using the same primers, but with a rapid cycle (denaturation, 1 s; annealing, 5 s; extension, 10 s), with results given in real time and with a master mix optimized for this machine containing the following in a final 20-µl volume: a 0.2 mM concentration of each of the dNTPs, 4 mM MgCl2, 0.6 µM concentrations of the cFD2 and FS 778 primers, and 0.16 µl of Taq DNA polymerase (Taq Start antibody; Ozyme, St. Quentin/Yvelines, France), stained with 2 µl of Sybr Green (LightCycler-DNA Master Sybr Green 1; Roche Molecular Biochemicals, Meylan, France). Analysis of the melting curve of specific PCR products was performed by slowly raising the temperature of the thermal chamber from 54°C to 95°C by means of regular fluorescence measurements.
Sequencing. Products of PCR amplification in the laboratory were sequenced. The sequencing reaction was performed by PCR amplification in a final volume of 20 µl with 100 ng of PCR products, 5 pmol of primer, and 8 µl of BigDye Terminators premix according to the Applied Biosystems protocol. After being heated to 94°C for 2 min, the reaction mixture underwent 25 cycles of 30 s at 94°C, 30 s at 55°C, and 4 min at 60°C (Perkin-Elmer 9600 thermal cycler). Excess of BigDye Terminators was removed with exclusion columns. The samples were dried in a vacuum centrifuge and dissolved with 2 µl of deionized formamide-EDTA (5/1 ratio) (pH 8.0). The samples were loaded onto an Applied Biosystems 373XL sequencer and run for 12 h on a 4.5% denaturing acrylamide gel. Other sequences were obtained from GenBank. All sequences were compared by using DNASIS software that contains the Higgins and Sharp algorithm CLUSTAL 4 (16). This program takes a dendrogram as input, produced by applying the unweighted pair group method with arithmetic mean (UPGMA) to a matrix of similarity scores for all of the aligned sequences. The similarity scores are calculated as the number of exactly matched residues (top diagonals = 5) in a Wilbur and Lipman alignment between two sequences, minus a fixed penalty of 10 for every gap (23). The floating gap penalty was 10, and the K-tuple was 2.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Comparison between different published Flavivirus
genus RT-PCR assays.
The flaviviruses were tested with six
different primer pairs, and each result was confirmed three times. One
HCV strain, three viruses of the Bunyaviridae family, and
three viruses of the Togaviridae family were used as
negative controls, and nonspecific amplifications were not found. DEN
viruses, one WN strain, and Zika virus were amplified (Table
3) by amplimers of Meiyu (DJA and DJS,
Table 2) and Chow (DV1 and DV3, Table 2). Using the primer pair of Chang (CFDJ 9977 and FUDJ 9166, Table 2), we detected DEN-1, DEN-2, and
WN viruses; the Fulop (FG1 and FG2, Table 2) primer pair allowed us to
amplify DNA fragments for many viruses, except JE, WN, Wesselsbron, and
YF FNV. The best result was obtained with the primer pair
proposed by Kuno (cFD2 and MA, Table 2), since we amplified every
flavivirus tested (both mosquito- and tick-transmitted flaviviruses).
However, the sensitivity limit is about 105 50%
tissue culture infectious doses (TCID50s)
ml1: there is no detection of a virus titer
lower than 104 TCID50s
ml1.
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New sensitive heminested technique for Flavivirus
genus detection.
NS5 gene sequences of 30 different flaviviruses
were aligned in order to design new primers allowing a heminested PCR
(Table 4). cFD2 (location,
NS5, 9232 to 9258) was not changed because it allowed the detection of every flavivirus in vitro. MA was modified
in MAMD (location, NS5, 9006 to 9029): the primer
was shifted toward the 5'-end region and was extended to 23 bases to
increase the melting temperature. The inner sense primer (location, NS5, 9044 to 9066) allowing the heminested PCR
was designed in an internal consensus region. After a cFD2 RT step, the
first PCR was performed with the cFD2 reverse primer (6)
and MAMD sense primer. The fragments of the expected 250-bp target size were successfully amplified from all mosquito- and tick-transmitted flaviviruses tested. The heminested PCR, performed with cFD2 reverse primer and a new FS 778 sense primer, allowed the detection of 200 infectious doses ml1 (Fig.
1). The PCR in real time with a PCR
quantitative instrument confirmed the DNA amplification of every
flavivirus, and in a shorter time, the amplified products with the same
titer of 105 TCID50
ml1 were detected between 20 and 40 min after
30 and 55 cycles. The melting curve confirms that the heterogeneity of
the amplified product depends on the degenerate primers on the
different sequences targeted: peaks were not observed at the same
temperature.
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Alignment of 12 sequences amplified by the cFD2-FS 778 primer pair
and construction of the dendrogram
The alignment
of the different sequences of PCR products did not show any complete
homology between the different species, confirming the absence of
cross-contamination. It also permitted us to verify that amplimers had
amplified the expected products in NS5 location. In drawing
the phylogenetic tree, it was possible to predict all tested
Flavivirus serocomplexes and species (Fig. 2), including the most pathogenic
viruses, such as the DEN-1, DEN-2, DEN-4, JE, YF, WN, and TBE viruses
(20, 25).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Several RT-PCRs have been developed for detection of flavivirus
RNA by using different pairs of primers for differentiating between
species of viruses (12), including flavi-universal primers for mosquito-borne flaviviruses (29) and six published
primer pairs permitting complete detection of the Flavivirus
genus (7, 8, 15, 19, 24, 26). Our purpose was first to
compare these flavivirus genus diagnosis references. To clarify their ability to detect the genus, we used both mosquito- and tick-borne viruses, including some strains of all of the virus groups of major
public health importance, at a virus titer of 105
TCID50s ml1. We tested
specificity by using other viruses, either because of their potential
epidemiological or clinical homology (other arboviruses) or because of
a close relationship in the Flaviviridae family (HCV). In
order to compare the actual levels of performance of the primers, we
chose to use agarose gel electrophoresis and simple ethidium bromide
staining to eliminate any possible identification variation factor.
Sequence similarity calculations revealed that the NS5 proteins are the most highly conserved of the flavivirus nonstructural proteins (17). The best result was obtained with the cFD2-MA pair (designed on the basis of the NS5 gene), which was the only pair able to detect all of the targeted viruses, and there was no problem of specificity towards other tested viruses. Tests carried out with cFD2 and FG1 primers gave positive results, except for the Wesselsbron and WN viruses.
These results do not mean that the other primers will completely fail when their own detection methods are used: only three groups (15, 19, 24) proposed an analysis of amplification products by electrophoresis on an agarose gel, while some others used a more sensitive technique, e.g., ELISA revelation of amplification products stained by digoxigenin (7, 26).
However, even with the cFD2-MA amplimers described by Kuno
(19), the detection limit was only
105 TCID50s
ml1. This is sufficient to ensure virus
identification by cell culture, because this titer is usually obtained
after a few passages on Vero or C6/36 cells. However, this sensitivity
is often insufficient to detect the virus in a serum (or cerebrospinal
fluid) sample from symptomatic patients, whose samples are often
obtained after the viremia peak. Consequently, we tried to increase
this sensitivity by using a heminested approach.
MAMD and FS 778 were designed after determining the consensus base
region on the basis of an alignment of 30 NS5 flavivirus sequences
extracted from GenBank. The Langat virus was used to complete the
specificity analysis with a detection of a tick-borne virus. The
percentage of similarities of Langat virus NS5 amino acid sequences and
those of other flaviviruses exceed 70% (17). These
amplimers allowed the detection of the 12 viruses at a minimum of 200 infectious doses ml1. This sensitivity could be
improved by using a better detection method.
The detection of the positive specimens (Table 1) was accomplished by determining the size of amplified DNA by agarose gel electrophoresis. Each product amplified by the cFD2 and MAMD primers was sequenced to eliminate any false-positive result by cross-contamination (data not shown). Phylogenetic trees of flaviviruses derived from NS5 gene sequences have been described previously (20, 22). Furthermore, the phylogenetic tree designed by comparison of the amplification of the heminested products in the NS5 location (220 bp) allowed us to find all of the complexes of medical interest: the DEN, JE, YF, and TBE groups. Interestingly, by using this method, we were able to identify flaviviruses in two patient samples.
In the first case, a virus was isolated on Vero cells from serum from a febrile patient in Senegal. The virus obtained from the cell culture was difficult to identify because the polyclonal hyperimmune antisera showed anti-DEN virus cross-reaction, but the virus was able to kill 3-week-old Swiss albino mice after intraperitoneal infection, which contradicted the DEN virus characteristics. The specific PCR product was visible at approximately 220 bp with the heminested cFD2-MAMD and cFD2-FS778 pairs (Fig. 1). In the second case, the amplified product was directly obtained from a cerebrospinal fluid sample from a patient suffering from severe encephalitis. In these two cases, sequencing and comparison of the NS5 amplified products classified the viruses among the WN species on the phylogenetic tree (WNc; Fig. 2). The identification was then confirmed by a species-specific PCR targeting the envelope gene of WN viruses (3).
An outbreak of arboviral encephalitis associated with mosquitoes was recognized in New York City in late August 1999. SLE virus was identified initially as the causative agent because of compatible clinical symptoms (neurological disease including fatal encephalitis) and positive serological laboratory tests (13, 14). However, RT-PCR and sequencing first permitted the identification of Kunjin-WN-like flavivirus in brains of deceased patients (4), before identifying the WN lineage (18, 22). We confirmed that the NS5 amplification, sequencing, and phylogenetic analysis of the heminested amplified product would be able to provide a first key to identify the New York 99 WN strain (Fig. 2), even though these products are too short to ensure a definitive phylogenetic analysis. This genus PCR procedure could be used as a first-line diagnostic PCR screening test for an unknown virus, indicating the relatedness of the poorly characterized viruses to the pathogenic members of genus Flavivirus after cell culture. A definitive identification obviously requires both complete sequencing and the appropriate expertise in flavivirus identification. Because it would take only a few hours, PCR detection of a flavivirus directly from patient samples could help the physician choose the appropriate first-line treatment. The few successes obtained with amplification directly from patient samples without a cell culture step must be confirmed on a larger scale. The use of a single-step RT-PCR can shorten the reaction time. The PCR amplification in real time allowed a quicker diagnosis and could allow quantitative detection with fluorogenic hybridization probes. A quick and ultrasensitive technique is key to allowing diagnosis directly from patient samples.
Diagnosis of flavivirus infections by this method requires a subsequent stage to allow rapid identification of the virus species. This could be achieved by species-specific PCR or by hybridization (27) of specific probes of each serogroup (30) with fragments produced by these RT-PCR procedures.
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ACKNOWLEDGMENTS |
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This work was supported by research grants from the Service de Santé des Armées (SSA).
We thank Corinne Rothlisberger, Danielle Gratier, Josette Guimet, and Henri Blancquaert for technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: CRSSA, 24 Avenue des Maquis du Grésivaudan, BP87, 38702 La Tronche Cedex, France. Phone: 33-476-63-68-44. Fax: 33-476-63-69-17. E-mail: Daniel.Garin{at}wanadoo.fr.
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Discussion
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Journal of Clinical Microbiology, May 2001, p. 1922-1927, Vol. 39, No. 5
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.1922-1927.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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