Abstract
Background: Variola virus (family Poxviridae, genus Orthopoxvirus) and the closely related cowpox, vaccinia, and monkeypox viruses can infect humans. Efforts are mounting to replenish the smallpox vaccine stocks, optimize diagnostic methods for poxviruses, and develop new antivirals against smallpox, because it is feared that variola virus might be used as a weapon of bioterrorism.
Methods: We developed an assay for the detection of variola virus DNA. The assay is based on TaqMan chemistry targeting the 14-kD protein gene. For the 1st stage of the assay we used genus consensus primers and a mixture of 2 probes (14-kD POX and 14-kD VAR) spanning the 14-kD protein-encoding gene for detection of all human pathogenic orthopoxviruses. We then tested positive samples with the specific orthopoxvirus-specific probe 14-kD POX to identify monkeypox, cowpox, and vaccinia viruses and with the 14-kD VAR probe to identify variola viruses. The assay was established on 4 different PCR cycler platforms. It was assessed in a study with 85 different orthopoxvirus species and strains that included variola, camelpox, cowpox, monkeypox, and vaccinia viruses at concentrations ranging from 100 ng/L to 1 μg/L.
Results: The assay detected as little as 0.05 fg of DNA, corresponding to 25 copies of DNA, and enabled differentiation of variola virus from the other orthopoxviruses.
Conclusions: This real-time PCR assay provides a rapid method for the early detection and differentiation of smallpox and other human pathogenic orthopoxvirus infections.
The WHO officially declared smallpox eradicated from the world in 1980 (1). Efforts are underway, however, to replenish the vaccine stocks and develop new drugs and diagnostic methods for this virus because of the possibility that the variola virus will be used as a weapon of mass destruction. The family Poxviridae and the subfamily Chordopoxvirinae (poxviruses of vertebrates) contain 8 genera. Variola virus and the closely related cowpox virus (CPV)1 , vaccinia virus (VV), and monkeypox virus (MKP) can all infect humans and are classified in the genus Orthopoxvirus. These DNA viruses are large brick-shaped particles that vary in size (length, 220–450 nm; width, 140–260 nm); the genome of variola virus consists of 186 kb of linear double-stranded DNA (2). DNA-based techniques have been developed for the early detection and identification of several orthopoxviruses. These methods include PCR, nucleic acid sequencing, and restriction fragment length polymorphism analysis (2)(3)(4)(5)(6)(7)(8)(9). Sequencing and restriction fragment length polymorphism analysis provide high specificity, but only PCR amplification offers high sensitivity and the needed lower limit of detection. Indeed, PCR methods allow rapid detection and identification of many viruses in clinical and environmental samples (10)(11)(12). Several PCR-based methods with gel-based analyses of PCR products have been described for the identification of orthopoxviruses (4)(6)(7)(8)(9)(13)(14) but they require laborious and time-consuming postPCR processing. One of the most promising approaches for rapid and sensitive diagnosis is the real-time 5′ nuclease PCR assay that includes the TaqMan assay.
We report the development of a real-time TaqMan assay based on the gene encoding the 14-kD protein for detection of orthopoxviruses in clinical material.
Materials and Methods
viruses, clinical samples, and dna preparation
To calibrate the method, we first used DNA isolated from CPV (Brighton strain), VV (Copenhagen, Lister and MVA strains), varicella-zoster virus (VZV, LES 2770 and PER 2762 strains), and monkeypox (Zaïre-96), and a synthesized variola plasmid. CPV and VV were produced on Vero cells (ATCC CCL-81), which were propagated in medium 199 glutamax (M199) with 5% heat inactivated fetal calf serum (FCS), penicillin, and streptomycin at 37 °C in a 5% CO2 atmosphere. VZV was grown on MRC-5 cells (ATCC CCL-171) in RPMI 1640 glutamax with 5% FCS, penicillin, and streptomycin at 37 °C in a 5% CO2 atmosphere. Virus titers were determined by the 50% tissue culture infective dose (TCID50) method (16)(17). Viral DNA was extracted from 100 μL of tissue culture suspensions by use of the automated MagNAPure LC® device with the DNA Isolation Kit I (both from Roche Diagnostics) according to the manufacturer’s instructions.
To obtain clinical samples, we infected mice via the intranasal route with CPV (Brighton strain). Spleen and lungs were homogenized with 1 mL RPMI 1640 with 4 mL FCS/L, freeze-thawed 3 times, and centrifuged for 10 min at 750g. Viral DNA was extracted from supernatants as described above. The viral titer of the supernatants was determined in plaque-forming units and genomic quantification in DNA copies per milliliter.
We then evaluated the TaqMan assay using 85 different orthopoxvirus strains from both our in-house laboratory strain collection and from the WHO collaborating center for variola and other poxviruses at the CDC (Atlanta, GA). A smallpox diagnostic panel compiled by Inger Damon’s laboratory at the CDC included 12 different variola virus isolates (VAR), 13 different strains and isolates of camelpox, cowpox (CPV), ectromelia, herpes, monkeypox, Staphylococcus aureus, vaccinia (VV), and varicella-zoster viruses (VZV). The concentrations of DNA used in the test panel ranged from 1 ng/L to 1 μg/L and included total viral and cellular DNA from cell lysates and crude material as well as purified viral DNA.
pcr primers, target sequences, and fluorogenic probes
The sequences of orthopox consensus genus primers GF (5′-GCC AGA GAT ATC ATA GCC GCT C-3′), and GR (5′-CAA CGA CTA ACT AAT TTG GAA AAA AAG AT-3′) and orthopoxvirus consensus genus probe 14-kD POX (5′-TTT TCC AAC CTA AAT AGA ACT TCA TCG TTG CGT T-3′), and specific variola virus 14-kD VAR probe (5′-TTT TCC AAC CTA AAT AGA ACG TCA TCA TTG CGT T-3′) were designed after alignment of all known gene sequences of the 14-kD protein from different orthopoxvirus species (open reading frame A30L with the variola virus India strain as reference according to the nomenclature). The gene sequence alignments of selected representative human pathogenic orthopoxviruses and camelpox virus, the most closely related to variola virus (18) are shown in Fig. 1⇓ . Probes were designed in an area that shows several bases specific for the virus group vaccinia–cowpox–monkeypox viruses, differentiating them from variola virus. Primers and probes were synthesized by Genset Oligos. Probes contained 6-carboxy-fluorescein at the 5′-end and 6-carboxytetramethylrhodamine linked to a phosphate residue at the 3′-end.
Sequence alignment of the first 150 bases of the 14-kD protein gene of Variola virus (VAR, India strain) and of other orthopoxvirus locating primers (GF and GR) and probes (14-kD POX and 14-kD VAR) used in the assay.
14-kD VAR is the variola-specific probe and 14-kD POX is the orthopoxvirus consensus genus probe. The sequences were obtained from EMBL/GenBank, accession numbers: X69198 (VAR India, A30L), NC000900 (VAR Garcia, A31L), M35027 (vaccinia virus, Copenhagen strain, A27L), X75158 (cowpox virus, Brighton strain, V162), Z99061 (monkeypox virus, Zaire A29L), and X75156 (camelpox virus, cp-1, 144L).
plasmid preparation
Positive DNA controls from members of the genus Orthopoxvirus were cloned with the pGEM®-T Easy Vector Cloning Kit (Promega Corp.) from the 14-kD protein gene of vaccinia (GenBank accession no. M35027), cowpox (X75158), and monkeypox (Z99061) viruses. The respective PCR amplicons were then inserted into separate plasmid vectors. Identity of the inserted sequence was verified by sequencing. The vaccinia virus (Lister strain) plasmid was named pVACV-List143, the cowpox virus (Brighton strain) plasmid named pCPXV-V162, and the monkeypox virus Zaire strain plasmid named pMPXV-A29L.
A positive control for variola virus (X69198) was generated by multisite-directed mutagenesis from the gene encoding the 14-kD protein of the cowpox virus (permission from WHO was granted by Dr Guénaël Rodier, n°S2-180-4, July 1, 2003) using the QuikChange Multisite-Directed Mutagenesis Kit (Stratagene). The cowpox gene was partially mutated so that this recombinant positive control plasmid (pVARV-A30L) could be distinguished from variola DNA by sequencing in the case of false-positive results due to plasmid contamination of the sample. The sequences of the primers for the multisite-directed mutagenesis were 1 (5′-GCT CTT AGA GTT TCA GCG TGA TTT TCC AAC-3′), 2 (5′-CTA AAT AGA ACG TCA TCA TTG CGT TTA CAA CAC TTT TC-3′), 3 (5′-GTT GTT ACA TTA GTA ATT TTT TTT TCC AAA TTA GTT AGT CGT TG-3′), 4 (5′-GAG AGT TTC TTC ATT ATT GTC TCC ATC GGC TTT AAC AAT TGC TTC G-3′), and 5 (5′-GAA TTG CAA GAT CAT CAT CTC CAG GGA AAA GAG TTC-3′).
5′ nuclease pcr assay
The 5′ nuclease PCR assay and amplification conditions were optimized according to the standard protocols used in our laboratory by adjusting primers, probes, and MgCl2 concentrations as well as the thermal cycling temperatures and duration. The 14-kD POX and 14-kD VAR probes were tested either in the same PCR tube or separately in different PCR tubes in the same PCR run. The assay was assessed on 4 different PCR cycler platforms as follows: the reactions were carried out in 30-μL total volume with the SmartCycler instrument (Cepheid) and in 20 μL with the LightCycler (Roche Diagnostics), the MX 4000 (Stratagene), and the 7000 SDS (ABI Prism) instruments. PCR reactions were performed with LightCycler-FastStart DNA Master Hybridization Probes for both the LightCycler and the SmartCycler instruments and TaqMan Universal PCR Master Mix for the 7000 SDS and the MX 4000 instruments. LightCycler concentrations were 0.5 μmol/L for each primer, 0.5 μmol/L for each TaqMan probe, and 4 mmol/L for MgCl2. TaqMan concentrations were 0.3 μmol/L for each primer and 0.2 μmol/L for each TaqMan probe, and the MgCl2 concentration was already optimized by the manufacturer. For all reactions, 5 μL of template DNA was added. Thermal cycling for the LightCycler and the SmartCycler instruments was performed as follows: 1 cycle at 95 °C for 10 min, followed by 45 cycles each of 95 °C for 15 s, followed by 62 °C for 60 s. For the 7000 SDS and the MX 4000 instruments, thermal cycling was performed as follows: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles each of 95 °C for 15 s, followed by 60 °C for 60 s. Data acquisition and analysis were carried out with the respective platform company data analysis software: LightCycler (version 3.5.3), Cepheid SmartCycler (version 1.2d), ABI Prism 7000 SDS (version 1.0.1), and MX 4000 (version 3.01).
All PCR reactions were performed on coded samples with at least 1 well that contained 0.05 fg (25 DNA copies equivalent) of purified pVARV-A30L plasmid as a positive control sample, as well as 1 no-template control, and uninfected cellular extracts as negative controls. Baseline fluorescence was given by the respective program of the different PCR cyclers. The threshold cycle (Ct) was defined for each sample as the number of cycles necessary for the fluorescence emitted by the sample to cross above baseline fluorescence. A sample was considered negative, below the limit of detection (LOD) of the assay, if the emitted fluorescence was found to be below baseline on 2 separate occasions (i.e., the sample contained <25 gene copies). For comparison, the curves were classified as high or low according to the Fmax value, defined as the maximum fluorescence emitted by the positive control after a specific PCR run. High curves were between Fmax and Fmax/3 and low curves were below Fmax/3 but above baseline fluorescence. The LOD of the assay was determined on the LightCycler instrument. For the 14-kD POX probe (orthopoxvirus consensus genus probe) the LOD was determined from serial dilutions of both pCPXV-V162 and pVACV-List143. For the 14-kD VAR probe (variola specific) the LOD was determined from serial dilutions of pVARV-A30L.
Results
limit of detection and dynamic range of the assay
The LOD was 0.05 fg DNA for both pVACV-List143 and pCPXV-V162 plasmid, which represents ∼25 copies of the 14-kD protein-encoding gene (Fig. 2A⇓ ). The LOD was also 0.05 fg of pVARV-A30L plasmid DNA (Fig. 2B⇓ ). The Figs. show the expected linearity. The dynamic range (Fig. 2C⇓ and 2D⇓ ) was 8 orders of magnitude and represented ∼25 to 2.5 × 108 copies for pVACV-List143 and 14-kD POX (Fig. 2C⇓ ) and pVARV-A30L with 14-kD VAR (Fig. 2D⇓ ). Similar results were obtained on the 7000 SDS instrument (data not shown) and with other orthopoxviruses plasmids (e.g., MKP, CPV).
LOD of the assay expressed in copy numbers (Fig. 2A⇓ and 2B⇓ ).
Eight-fold serial dilutions of purified plasmid DNA (100 pg/μL to 0.01 fg/μL) were tested on the LightCycler instrument. (A), pVACV-List143 plasmid with the TaqMan probe 14-kD POX; (B), pVARV-A30L plasmid with the TaqMan probe 14-kD VAR. Each curve represents the mean fluorescence value of 5 different PCR runs. Since 5 μL of DNA was used for each concentration in each PCR run, the corresponding number of copies were 2.5 × 101, 2.5 × 102, 2.5 × 103, 2.5 × 104, 2.5 × 105, 2.5 × 106, 2.5 × 107, and 2.5 × 108.
Dynamic range of the assay (Fig. 2C⇓ and 2D⇓ ).
Regression analysis was performed on the Ct against log copy numbers produced from a total of 24 samples of purified DNA plasmids with concentrations ranging from 0.01 fg/μL (25 copies) to 100 pg/μL (2.5 × 108 copies). (C), pVACV-List143 plasmid with the TaqMan probe, 14-kD POX. The actual and predicted values are shown with the linearity coefficient (r = −1.00; Error = 0.0719). (D), pVARV-A30L plasmid with the TaqMan probe, 14-kD VAR. The actual and predicted values are shown with the linearity coefficient (r = −1.00; Error = 0.0632).
With purified DNA, the LOD with the LightCycler instrument was 5 fg per run and 200 fg for the MX4000 instrument. With crude cell extracts, the LOD with the LightCycler instrument was 50 fg per run and 1 pg for the MX4000 instrument, corresponding to ∼1 TCID50 dose.
selectivity (specificity) of the assay
A representative curve obtained with variola, monkeypox, cowpox and vaccinia virus with the 2 probes mixed in the same PCR mixture is shown in Fig. 3⇓ . Each virus produced a single curve with a Ct different from the others. Nonpoxvirus samples were always negative. Analysis of the curves generated by the virus samples with the 2 probes used separately was necessary to differentiate variola from other orthopoxviruses (Fig. 4⇓ and Table 1⇓ ). A high curve with the 14-kD VAR probe compared to 14-kD POX was considered to indicate the presence of the variola virus, and a high curve obtained with the 14-kD POX probe compared to 14-kD VAR the presence of other human pathogenic orthopoxviruses, such as monkeypox, cowpox or vaccinia viruses, in human clinical samples.
Detection of variola virus DNA (d) and other orthopoxviruses [monkeypox (a), variola (b) and cowpox (c) viruses] with the mixed probes 14-kD POX and 14-kD VAR.
The different observed Cts depend on the different amount of viruses present in the sample. Baseline fluorescence and the respective Cts for each virus are shown on the graph.
Detection of variola, monkeypox, cowpox and vaccinia viruses with the probes, 14-kD VAR (variola specific) and 14-kD POX, used separately.
The variola virus is easily differentiated from the other orthopoxviruses. The different observed Cts depends on the different amount of viruses present in each tube. (A), with 14-kD VAR (variola-specific), the variola virus produces a high curve (fluorescence superior to Fmax /3), from which the Fmax is calculated, whereas the other orthopoxviruses produce either a low curve (monkeypox virus; fluorescence inferior to Fmax /3) or a negative result (cowpox and vaccinia viruses; fluorescence inferior to baseline). (B), with 14-kD POX, the variola virus produces a low curve (fluorescence inferior to Fmax/3) whereas the other orthopoxviruses produce high curves (fluorescence superior to Fmax/3).
Keys to analysis of results and conclusion.
The assay was evaluated on 85 samples of orthopoxvirus genomic DNA. DNA genomes of herpes and varicella-zoster viruses, BSC40 cells, staphylococcus aureus, and human genomic DNA were not detected (Table 2⇓ ⇓ ). With the 14-kD VAR probe, variola virus DNA was detected only with the 12 variola strains tested. The same assay specificity was obtained with the 4 tested PCR instruments. Using the LightCycler instrument the method also successfully detected orthopoxvirus species in lungs, spleen, and blood of mice infected with CPV: 21 days after intranasal infection with CPV, there were ∼12 × 106 DNA copies in the lungs, 9.5 × 102 in the spleen, and no copies were detected in blood.
Identification of orthopoxviruses with the 14-kD POX and 14-kD VAR TaqMan probes used separately.
Continued
Discussion
Smallpox eradication renders the variola virus one of the most feared weapons in biological warfare or bioterrorism scenarios (19)(20)(21)(22). It is crucial that reliable diagnostic tests be developed for the early detection and confirmation of suspected variola contamination as a first line of defense. Apart from the variola virus, a number of other orthopoxviruses and viruses belonging to other families cause diseases with similar symptoms (fever and papulovesicular rash). Differentiation between these viruses is an important part of any early smallpox detection system, so that specific measures to counteract the spread of the disease can be put in place. Timely action depends on early diagnosis and rapid characterization of the virus responsible for the contamination (23). Several PCR-based assays have been designed for the identification of orthopoxviruses, but they require analysis to determine the sizes of the PCR products from gel electrophoresis (4)(6)(7)(8)(9)(13)(14) after restriction endonuclease digestion. Although these methods provide high specificity and allow species and sometimes strain differentiation, they are very labor-intensive and time-consuming and thus may delay diagnosis. Newer approaches that use species-specific oligonucleotide hybridization on a microchip (24) and fluorescence resonance energy transfer probes on the LightCycler instrument (25)(26) have also been described. Another approach, the 5′ nuclease PCR or the TaqMan assay, first developed by Holland et al. (27) and improved by Lee et al. (28), allows for the simultaneous amplification and detection of nucleic acids in real time. Some assays targeting the 14-kD protein gene (29) or the rpo18 gene (30) and relying on the concept of melting temperature analyses correctly discriminate nonvariola orthopoxviruses from variola virus, but they can not be used on all real-time PCR platforms.
We describe a real-time PCR assay that targets the 14-kD protein gene and allows easy and rapid diagnosis of all human pathogenic orthopoxviruses by use of a mixture of 2 probes in a first-screening test without modifying the performance of the test. If the sample is positive, variola virus can then be differentiated from monkeypox, cowpox, and vaccinia viruses by using the same probes in separate runs with LightCycler, 7000 SDS, MX 4000, and SmartCycler instruments. Although TaqMan technology cannot generate melting curves to enable comparison of the 2 amplification product reactions, similar relevant information can be easily obtained by the simple comparison of the amplification curves obtained with each of the 2 probes. In the gene area targeted by both probes, we observed 3 mismatches (Fig. 1⇑ ). Guanine at position 62 is specific for variola virus and adenine at position 68 is observed in the majority of variola strains. When these 2 bases are present at their respective positions, the variola-specific 14-kD VAR probe is able to hybridize with a high efficiency: the fluorescence intensity is higher than that observed with the 14-kD POX probe. However, if only 1 specific base is present (as in the Alastrim variola strain, Garcia), then both probes are able to hybridize the DNA template. On the other hand, when thymidine at position 62 and guanine at position 68 are targeted specifically by the orthopoxvirus consensus probe 14-kD POX, it is possible to conclude that the sample is from another orthopoxvirus. Guanine at position 59 is specific for camelpox virus, and this mismatch could explain the lack of sensitivity obtained with the 14-kD POX probe with this virus. This was not considered to be a problem because the objective of the test was the detection of orthopoxviruses in human clinical samples, thus making the camelpox virus, which is not pathogenic in humans, less relevant for the purpose of the assay. However, the method perfectly detected and differentiated variola from monkeypox virus (endemic in Africa), from cowpox virus (endemic in Europe but responsible for only limited cutaneous lesions in immunocompetent people), and also from vaccinia virus, thus helping to ascertain the diagnosis of complications of a smallpox vaccination in clinical practice (e.g., eczema vaccinatum). The probes were also able to detect ectromelia virus but with a lower sensitivity (data not shown). The detection limit assessed with DNA plasmids was 0.05 fg, corresponding to ∼25 gene copies for each probe. The 2 probes perfectly detected the orthopoxviruses in all 99 samples of the WHO repository panel that included variola virus-infected cells and tissues, purified variola virus DNA, and the DNA of 13 different strains and isolates of camelpox, cowpox, ectromelia, monkeypox, vaccinia viruses, and none of the nonorthopoxviruses, including varicella-zoster and herpes viruses as well as Staphylococcus aureus.
The assays carried out on the SmartCycler instrument and on the 7000SDS instrument showed comparable sensitivity and specificity. These assays showed lower sensitivity with the MX 4000 instrument, which could be explained by the fact that the initial development of the assay was standardized on the LightCycler and the 7000 SDS instruments without specific adaptation for the MX4000 instrument. It is possible that the sensitivity of the assay on this PCR platform could be improved with time, as could that of the SmartCycler, for which the assay has not been fully evaluated for sensitivity.
In conclusion, we describe a consensus orthopoxvirus PCR assay with 2 built-in steps that allow the 2nd stage of the assay to differentiate variola virus from other human pathogenic orthopoxviruses with only 2 probes. Positive control plasmids have a different gene sequence from the variola virus gene sequence and can be provided to any hospital or research institution. In contrast to fluorescence resonance energy transfer technology, the TaqMan technology can be used on all real-time PCR platforms now available. As part of a prompt defense against bioterrorism, the assay we describe must be followed by a more detailed analysis with other specific PCR smallpox reagent sets (26)(31) to customize vaccination and treatment to the contaminant virus, because it is possible that a bioattack could be carried out not only with wild-type virus but also with genetically modified pathogenic orthopoxviruses (32).
Acknowledgments
Grant/funding support: This work was supported by grants from the Service de Santé des Armées (SSA), the Délégation Générale pour l’Armement (DGA) and association ARAMI.
Financial disclosures: none declared.
Acknowledgements: We thank the CDC Poxvirus Program, and specifically acknowledge Inger Damon, Miriam Laker, and Joanne Patton for design of the experiment, preparation of the reagents, and facilitation of the variola nucleic acid testing. This work was done in conjunction with the Global Health Security Action Group’s laboratory exercise. We also thank Danielle Gratier, Josette Guimet, Henri Blancquaert, and Mary O’Brien for their technical assistance and Bernard Souberbielle for critically reviewing the manuscript.
Footnotes
Negative curve: curve fluorescence below baseline fluorescence. Positive curve: curve fluorescence above baseline fluorescence. Positive–low: fluorescence below Fmax/3. Positive–high: fluorescence above Fmax/3 (see Fig. 4⇑ ).
Results were reported after comparing the respective amplification curves of the samples listed in this table with each probe (see Table 1⇑ for keys to results analysis and Fig. 4⇑ for representative curves).
1 NA, not applicable; –, negative sample; +, H: Positive sample, high curve; +, L: Positive sample, low curve; VAR: variola virus
↵1 Nonstandard abbreviations: CPV, cowpox virus; VV, vaccinia virus; MKP, monkeypox virus; FCS, fetal calf serum; TCID 50, 50% tissue culture inefective dose; Ct, threshold cycle; LOD, limit of detection.
- © 2007 The American Association for Clinical Chemistry
References
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