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Journal of Veterinary Diagnostic Investigation Vol. 18 Issue 5, 459-462
Copyright © 2006 by the American Association of Veterinary Laboratory Diagnosticians
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Brief Communication

A New Fluorogenic Real-time Rt-pcr Assay for Detection of Lineage 1 and Lineage 2 West Nile Viruses

Miguel Angel Jiménez-Clavero1, Montserrat Agüero, Gema Rojo and Concepción Gómez-Tejedor

Correspondence: 1 Corresponding Author: Miguel Angel Jiménez-Clavero, Departamento de Enfermedades Emergentes, Laboratorio Central de Veterinaria, Ctra. Algete, km 8, 28110, Algete (Madrid), Spain, e-mail: majimenez{at}mapya.es


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West Nile virus represents an emerging threat for animal and human health worldwide. This virus exhibits a marked genetic variation, with at least 2 distinct evolutionary lineages. Lineage 1 has been recognized in Africa, Asia, Europe, Oceania, and more recently in the Americas, whereas lineage 2 is restricted to Africa. Perhaps for this reason, the available real-time RT-PCR methods for detecting West Nile virus genome have mainly focused on lineage 1. However, both viruses may potentially be spread beyond their endemic areas by migratory birds. This report describes a new real-time reverse transcription-PCR (RT-PCR) method based on a 5'-Taq nuclease-3' minor groove binder DNA probe (TaqMan MGB©) that allows the detection of a wide range of West Nile virus isolates, including both lineages 1 and 2. This method was able to detect West Nile viruses from different origins (North and Central Africa, Middle East, Europe, and North America), whereas other flaviviruses (Usutu, Dengue, Yellow fever) analyzed in parallel remained negative. The sensitivity achieved by this assay was 10–2-10–3 pfu/tube. This method, which can be performed in 96-well format, could be suitable for the large-scale surveillance of West Nile virus in areas where both lineages can potentially spread.

Key Words: Fluorogenic • polymerase chain reaction • probes • real-time • West Nile virus

West Nile virus (WNV) is a member of the Flavivirus genus, within the family Flaviviridae, belonging to the Japanese encephalitis group, such as Japanese encephalitis virus (JEV), Usutu virus (USUV), Saint Louis encephalitis virus (SLEV), and Murray Valley Fever virus (MVFV). Transmitted by mosquito bites, WNV affects a wide range of vertebrates, including mammals, birds, reptilians, and amphibians. Birds are considered the main reservoir host of WNV, and migratory birds play an important role in its spread.9 WNV can be pathogenic in both domestic and wild birds. Horses are also highly susceptible, and mortality in these animals can be observed in WNV outbreaks. Humans infected by WNV may develop a variety of symptoms, from a mild fever to more severe illness, including acute encephalitis, meningitis, and hepatitis, which can be fatal in a small percentage (<1%) of cases.

West Nile virus exhibits 2 genetically distinct evolutionary lineages. Whereas strains from lineage 2 have been exclusively found in Africa,2 strains belonging to lineage 1 are widely distributed, with 2 different sublineages, or clades: clade 1a, present in Africa, Europe, Asia, and America, where it was recently introduced, and clade 1b, also known as Kunjin variants, found in Oceania. The emergence and re-emergence of WNV in different parts of the world make it necessary to implement surveillance programs aimed at detecting circulating virus in a variety of samples, including mosquitoes, human and animal tissues, cerebrospinal fluid, serum, blood, and blood derivatives. The most widely used methodology for WNV detection is based on RT-PCR, for which a variety of methods, including nested4 and real-time RT-PCR6,8,12 have been described. Although highly sensitive, nested RT-PCR is slow, cumbersome, and likely to result in cross-contamination, which restrains this technique from application in surveillance programs, where the analysis of a high number of samples in a short period of time is required. Real-time RT-PCR (RRT-PCR) methodology overcomes these difficulties by achieving a high sensitivity with a faster turnaround time. This methodology relies upon fluorogenic 5'-nuclease (TaqMan) probes, molecular beacons, FRET probes, or SYBR green fluorescent dyes.1 Of these, TaqMan-based assays have become the most widely used real-time PCR methods for virus detection, because they are more specific than SYBR green-based assays, and, in addition, they use hybridization probes of shorter length than molecular beacons and FRET probes, increasing the likelihood of locating a suitable target sequence within a highly variable viral RNA genome. An improvement in TaqMan assays is the introduction of a minor groove binder (MGB), a 3'-labelling group that, in addition to acting as a quencher, increases the binding affinity between the probe and its target sequence,5 allowing the selection of shorter probe sequence targets. The current OIE (World Organization for Animal Health) manual references highly specific and very sensitive nested and real-time RT-PCR methods for the detection of WNV, although none of these methods has been designed to detect lineage 2 WNV.7 This paper addresses the issue of whether the use of TaqMan-MGB probes can improve the performance of the real-time fluorogenic RT-PCR assays currently available for WNV diagnosis by broadening the spectrum of detection of WNV variants, with no significant loss in sensitivity.

The primers and probe set were designed using the Primer Express (version 2.0.0) software,a directed to a highly conserved sequence within the 3'NC region of the WNV genome, which was chosen using multiple alignments of previous and recent WNV sequences (Table 1). A second TaqMan-MGB probe was included to detect lineage 2 isolates of WNV whose genomes differ in 2 nucleotide positions from that of lineage 1 (Table 1). Virus isolates analyzed in this study included 6 WNV isolates: WNV Eg101, (Egypt,1951); WNV DAK (strain Ar B 310, Central African Republic, 1967), WNV NY99 (strain 99-34940-31A, New York State, 1999), WNV ISR (Israel, 1950)b; and WNV MOR'03 (Morocco, 2003)c and 1 isolate of Usutu virus (USUV, strain SAAR 1776).d All viruses were grown in Vero cellse except WNV MOR'03, which was grown in the BSR clone of BHK-21 (baby hamster kidney) cells.f Viruses were titrated by a standard limiting dilution assay.10 The remaining viruses analyzed in this study, comprising WNV representatives of lineages 1a (isolate NY'99), 1b (Kunjin), 2 (B956 prototype strain), and other flaviviruses (Dengue virus and yellow fever virus), were supplied as plasma-diluted, lyophilized material, quantified by quantitative real-time RT-PCR, as part of an External Quality Assessment for West Nile Virus diagnosis performed in 2005 by the European Network of Emerging Viral Diseases (ENIVD).g Tenfold dilutions of the clarified infection supernatants of each virus, or 0.1-ml suspensions of the lyophilized infected plasma, were subjected to nucleic acid extraction using High Pure Viral (HPV) Nucleic Acid extraction kith following manufacturer's instructions. The TaqMan MGB-RRT-PCR was carried out using a commercial kit (QuantiTect Probe RT-PCRi). Briefly, 2 µl of isolated RNA was mixed with 12.5 µl of 2x QuantiTect Probe RT-PCR Master Mix, 0.625 µl of QuantiTect RT-mix, WNV-specific primers (WN-LCV-F1 and WN-LCV-R1, Table 1) at 0.4 µM final concentration, each of the fluorogenic TaqMan probes (WN-LCV-S1 and WN-LCV-S2, Table 1) at 0.25 µM final concentration, and RNase-free water up to 25 µl. Amplification conditions consisted of a first reverse-transcription step at 50°C for 30 minutes, followed by 15 minutes at 95°C ("hot start") and 45 cycles of 15 seconds at 95°C and 1 minute at 60°C. The reaction was carried out in Smart Cycler II equipment and software.j The same samples were analyzed in parallel with the TaqMan-RRT-PCR method described by Lanciotti et al.6 (3'NC primers and probe set), which uses TAMRA (tetramethylrhodamine) as a quencher, with minor modifications, using the Smart Cycler II equipment and software.


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  		Table 1 Oligonucleotide primers and probes used in the Taqman-MGB real-time RT-PCR described in this work.

 
We tested the ability of both RRT-PCR methods, the previously described Taqman-TAMRA6 and the Taqman-MGB method described in this work, to detect a range of WNV isolates, comprising representatives of the main lineages of this virus. The sensitivity of both methods with WNV isolate NY'99 is shown in Fig. 1. By analyzing known quantities of the viral RNA, it can be observed that both methods performed comparably in terms of sensitivity (100 RNA copies/ml), linearity, and efficiency of reaction. Next we assessed the range of detection and the specificity of both methods by analyzing 10-fold dilutions of 7 different WNV isolates, as well as 3 non-WNV flaviviruses. The results are summarized in Table 2. As a result, both methods were successful at detecting all 4 WNV isolates belonging to clade 1a with comparable sensitivities; however, Kunjin (clade 1b within lineage 1) and B956 (lineage 2) WNV isolates were only detected by the new TaqMan MGB-RRT-PCR method described here, with a sensitivity similar to that obtained for lineage 1, which is within the range previously described.6


Figure 18050501
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  		Figure 1 Comparative sensitivity analysis of WNV (isolate NY'99) RNA, using 2 protocols: 1) TaqMan-MGB real-time RT-PCR (TaqMan-MGB method), closed circles) and 2) TaqMan-TAMRA RRT-PCR (TaqMan-TAMRA method, open circles). Tenfold virus dilutions were subjected to analysis by both methods in parallel. Threshold cycle values (cycles at which the fluorescence signal begins to be detectable) are plotted against viral RNA copies/ml on a log scale. The regression function and correlation coefficient (R2) corresponding to the TaqMan-MGB method data is inserted in the plot.

 

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  		Table 2 Analytical sensitivity and specificity of the TaqMan-MGB and TaqMan-TAMRA RRT-PCR methods for WNV detection compared in this study.

 
Highly variable virus genomes, such as those found in RNA viruses, represent a difficulty when a real-time RT-PCR protocol is developed, since a single mismatch in the hybridization region can cause significant loss of sensitivity.3 Hence, TaqMan-based assays often detect some virus variants but not others, as was the case with one of the methods compared in this study.6 This latter assay was the first described real-time TaqMan-based assay for WNV detection, targeting a sequence within the 3' noncoding region (3'NC) of the virus genome. Although highly sensitive for lineage 1 WNV (0.1 pfu/tube for NY'99 isolate of WNV), it is totally insensitive for both Kunjin variants and lineage 2 West Nile viruses. Recently, a new TaqMan-based assay has been described12 that can detect a wide range of WNV strains, including representatives of both lineages 1 and 2, using degenerate primers targeting the cap region, although the sensitivity for WNV NY'99 isolate decreases 2–3 orders of magnitude with respect to the previous method.6 The new method described here, which employs 2 TaqMan-MGB probes, can detect all the known range of West Nile viruses without loss of sensitivity. The method demonstrates that TaqMan-MGB fluorogenic probes may be more useful for detection of highly variable RNA genomes than other TaqMan-based assays.

Surveillance programs must target all WNV variants likely to occur in a given area. Bearing in mind the potential of the RRT-PCR technology for high-throughput screening,11 the method described here constitutes a valuable tool for surveillance of West Nile virus circulation in areas where both WNV lineages can be expected. However, the method still needs further validation with field samples in order to assess its suitability for diagnosis.


   Acknowledgments
 
We thank A. Tenorio for helpful discussions, A. Buckley for providing WNV isolates NY'99 and DAK as well as Usutu SAAR 1776 virus, M. Halevy for providing WNV isolate Isr, H. Zeller for providing WNV Eg101 isolate, M. El-Harrak for providing WNV isolate Mor-2003 and BSR cells, and M. Niedrig (ENIVD) for providing WNV NY'99, Kunjin, and B956 as well as yellow fever and dengue viruses. M.A. Jiménez-Clavero is a member of the EVITAR network, a group funded by FIS (G03/059).


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From the Laboratorio Central de Veterinaria, Ctra. Algete, km 8, 28110, Algete (Madrid), Spain. Back

a. Primer Express (version 2.0.0) software, Applied Biosystems, Branchburg, NJ. Back

b. WNV DAK (strain Ar B 310, Central African Republic, 1967), WNV NY'99 (strain 99-34940-31A), Usutu virus (Strain SAAR 1776): Center for Ecology and Hydrology (CEH), Oxford, UK. Back

c. WNV-ISR: Israel Institute for Biological Research, Ness-Ziona, Israel. Back

d. WNV MOR'03: Biopharma, Rabat, Morocco. Back

e. Vero cells (ATCC-CCL 81): American Type Culture collection, Manassas, VA. Back

f. BSR clone of BHK-21 cells: Biopharma, Rabat, Morocco. Back

g. WNV NY'99, WNV 1b (Kunjin), WNV B956 prototype strain, Dengue virus and yellow fever virus RNAs: European Network of Emerging Viral Diseases (ENIVD). Back

h. High Pure Viral (HPV) Nucleic Acid extraction kit, Roche Diagnostics, Indianapolis, IN. Back

i. QuantiTect Probe RT-PCR, QIAGEN, Valencia, CA. Back

j. Smartcycler II, Cepheid, Sunnyvale, CA. Back


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  1. Arya M., Shergill I.S., Williamson M., et al.: 2005, Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn 5:209–219.[Medline]
  2. Burt F.J., Grobbelaar A.A., Leman P.A., et al.: 2002, Phylogenetic relationships of southern African West Nile virus isolates. Emerg Infect Dis 8:820–826.[Medline]
  3. Jimenez-Clavero M.A., Agüero M., San Miguel E., et al.: 2006, High throughput detection of bluetongue virus by a new real time fluorogenic reverse transcription-polymerase chain reaction: application on clinical samples from current Mediterranean outbreaks. J Vet Diagn Invest 18:6–16.
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  5. Kutyavin I.V., Afonina I.A., Mills A., et al.: 2000, 3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucl Acids Res 28:655–661.[Abstract/Free Full Text]
  6. Lanciotti R.S., Kerst A.J., Nasci R.S., et al.: 2000, Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay. J Clin Microbiol 38:4066–4071.[Abstract/Free Full Text]
  7. Office International des Epizooties, World Organisation for Animal Health: 2004, West Nile encephalitis. In: Manual of diagnostic tests and vaccines for terrestrial animals, ed. OIE Standards Commission, 5th ed. ed, Chapter 2.10.7, pp. 1064–1071. Office International des Epizooties, Paris, France.
  8. Papin J.F., Vahrson W., Dittmer D.P.: 2004, SYBR green-based real-time quantitative PCR assay for detection of West Nile Virus circumvents false-negative results due to strain variability. J Clin Microbiol 42:1511–1518.[Abstract/Free Full Text]
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  11. Shi P.Y., Kauffman E.B., Ren P., et al.: 2001, High-throughput detection of West Nile virus RNA. J Clin Microbiol 39:1264–1271.[Abstract/Free Full Text]
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