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Journal of Clinical Microbiology, May 2004, p. 1940-1946, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.1940-1946.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Real-Time PCR System for Detection of Orthopoxviruses and Simultaneous Identification of Smallpox Virus

Victoria A. Olson,1 Thomas Laue,2 Miriam T. Laker,1 Igor V. Babkin,3 Christian Drosten,4 Sergei N. Shchelkunov,3 Matthias Niedrig,5 Inger K. Damon,1 and Hermann Meyer6*

Poxvirus Section, Centers for Disease Control and Prevention, Atlanta, Georgia,1 Artus Biotech,2 Bernhard Nocht Institute for Tropical Medicine, Hamburg,4 Robert Koch Institut, Berlin,5 Institute of Microbiology of the German Armed Forces, Munich, Germany,6 State Research Center of Virology and Biotechnology ("Vector"), Koltsovo, Russia3

Received 15 September 2003/ Returned for modification 31 October 2003/ Accepted 21 January 2004


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ABSTRACT
 
A screening assay for real-time LightCycler (Roche Applied Science, Mannheim, Germany) PCR identification of smallpox virus DNA was developed and compiled in a kit system under good manufacturing practice conditions with standardized reagents. In search of a sequence region unique to smallpox virus, the nucleotide sequence of the 14-kDa fusion protein gene of each of 14 variola virus isolates of the Russian World Health Organization smallpox virus repository was determined and compared to published sequences. PCR primers were designed to detect all Eurasian-African species of the genus Orthopoxvirus. A single nucleotide mismatch resulting in a unique amino acid substitution in smallpox virus was used to design a hybridization probe pair with a specific sensor probe that allows reliable differentiation of smallpox virus from other orthopoxviruses by melting-curve analysis. The applicability was demonstrated by successful amplification of 120 strains belonging to the orthopoxvirus species variola, vaccinia, camelpox, mousepox, cowpox, and monkeypox virus. The melting temperatures (Tms) determined for 46 strains of variola virus (Tms, 55.9 to 57.8°C) differed significantly (P = 0.005) from those obtained for 11 strains of vaccinia virus (Tms, 61.7 to 62.7°C), 15 strains of monkeypox virus (Tms, 61.9 to 62.2°C), 40 strains of cowpox virus (Tms, 61.3 to 63.7°C), 8 strains of mousepox virus (Tm, 61.9°C), and 8 strains of camelpox virus (Tms, 64.0 to 65.0°C). As most of the smallpox virus samples were derived from infected cell cultures and tissues, smallpox virus DNA could be detected in a background of human DNA. By applying probit regression analysis, the analytical sensitivity was determined to be 4 copies of smallpox virus target DNA per sample. The DNAs of several human herpesviruses as well as poxviruses other than orthopoxviruses were not detected by this method. The assay proved to be a reliable technique for the detection of orthopoxviruses, with the advantage that it can simultaneously identify variola virus.


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INTRODUCTION
 
The genus Orthopoxvirus, family Poxviridae, comprises morphologically and antigenically closely related viruses, including variola virus (VAR; smallpox virus) and several pathogens of veterinary and zoonotic importance (21). Orthopoxviruses (OPVs) are allocated into 11 species, 8 Eurasian-African species (VAR, monkeypox virus [MPX], vaccinia virus [VAC], cowpox virus [CPX], camelpox virus [CML], ectromelia, taterapox, and Uasin Gishu disease viruses) and 3 North American species (raccoon poxvirus [RCN], volepox virus [VPX], and skunkpox virus [SKN]). The most notorious member is VAR, the causative agent of smallpox (14, 22). As a result of international collaboration under the World Health Organization (WHO) eradication program, smallpox was declared eradicated in 1980. Nevertheless, VAR is considered a potential threat agent or bioterrorist weapon: it is naturally transmissible by large respiratory droplets and experimentally by the aerosol route, it causes high rates of high morbidity and mortality, and much of the human population is now susceptible because of the cessation of routine smallpox vaccination (7). Therefore, the identification of a single suspected case of smallpox must be treated as an international health emergency. A potentially confusing diagnosis is monkeypox, the agent of which is zoonotic and endemic to the African rain forest and which can cause a disease indistinguishable from smallpox (16). Recently, several mild cases of human monkeypox occurred in the United States due to transmission from infected prairie dogs which had become infected at pet stores handling imported exotic African rodent species (1). Two other orthopoxvirus species, namely, CPX and VAC, can cause localized skin lesions in humans; and generalized infections are possible in immunocompromised hosts. Historically, differentiation of OPVs into single species has been achieved by biological means, such as pock morphology on the chorioallantoic membrane or ceiling growth temperature, because serological tests proved to be of limited value (3). DNA maps and several other molecular biologic features, including full genome sequencing, have been applied and have provided definitive information for virus classification (4, 24, 25, 26). Various PCR methods have been used to identify and subtype the available OPVs by using consensus primers combined with restriction cleavage and/or sequencing of amplicons (6, 10, 13, 18, 23) or oligonucleotide-based microchip technology (11, 12, 20). Today, real-time PCR is even more efficient because it combines amplification and detection of target DNA in one vessel, thereby eliminating any time-consuming post-PCR procedures and potentially limiting possible contamination events (5, 8, 9). Because of the serious consequences of a diagnosis of smallpox or even the consequences of a misdiagnosis, there is a need to be able to unambiguously and reliably identify smallpox and to differentiate it from other similar clinical entities. In the first international quality assessment study on the rapid detection of viral agents of bioterrorism, only 6 of 22 laboratories provided good results for OPV diagnostic PCR regarding sensitivity and specificity (21a). One option for the design of a PCR assay is to identify VAR-specific insertions or deletions. However, it has been shown that sequences previously considered VAR specific (10) are conserved in some cowpox virus strains (15). In addition, a VAR-specific assay would require VAR DNA as a positive control, which might lead to serious consequences if cross-contamination were to occur. Studies of OPV species to date have shown that unique sequences are very rare, but species-specific single nucleotide polymorphisms are observed in many genes. Therefore, the rationale of the assay design described here is based on a VAR-specific single nucleotide polymorphism that results in an amino acid substitution. This real-time PCR assay was validated by using a large number of different specimens (n = 123) containing OPV DNA, including VAR DNA. On the basis of melting-curve analysis, lesions or specimen containing VAR could be clearly differentiated from those containing other OPVs.


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MATERIALS AND METHODS
 
Viruses and DNA extraction. Table 1 summarizes the designations, origins, and years of isolation of the OPV strains used. Seventy-four strains belonging to the OPV species MPX, VAC, CPX, CML, or mousepox virus (ECT) were grown on an African Green Monkey kidney cell line, MA 104. In addition, DNA from 46 VAR strains or scabs and from one strain each of RCN, SKN, and VPX had been prepared at the WHO smallpox repository at the Centers for Disease Control and Prevention, Atlanta, Ga. (8) with an Aquapure Genomic DNA Isolation kit (Bio-Rad). The concentrations of the VAR DNAs used in the test panel ranged from 100 fg per µl to 1 ng per µl and included total viral and cellular DNAs from cell lysates and crust material as well as purified viral DNAs. Nucleic acids of species other than VAR were extracted either from infected tissue culture cells or from purified preparations by using conventional chemistry (QiaAmp DNA Mini kit; Qiagen, Hilden Germany) or as described earlier (12, 18). Parapoxvirus strain D 1701, avipoxvirus strain HP-1, and tanapoxvirus TP-1, as well as one strain each of herpes simplex virus types 1 and 2, Epstein-Barr virus, varicella-zoster virus, and cytomegalovirus, were also included.


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  		TABLE 1. Designations and origins of OPV strains and isolates investigated

Sequencing and sequence alignment. The 14-kDa fusion protein gene sequences of 14 VAR strains which are part of the collection held at the WHO smallpox repository at the State Research Center of Virology and Biotechnology ("Vector"), Koltsovo, Russia, were determined following amplification of a 602-bp fragment containing the entire gene. The sequences of the following strains are deposited in GenBank under accession nos. AY223482 to AY223495, respectively (origins or years of isolation, or both, are given in parentheses): TajBarin (Pakistan, 1970), India 378 (India), India 164 (India), M-Sur-60 (Russia, 1960), M-Sok-60 (Russia, 1960), M-N-60 (Russia, 1960), M-A-60 (Russia, 1960), M-Bl-60 (Russia, 1960), Aslam (Pakistan, 1970), Khateen (Pakistan, 1970), 6/58 (Pakistan, 1958), Butler (United Kingdom, 1952), Brazil 128 (Brazil), and Brazil 131 (Brazil). The sequences of Aslam, India 164, India 378, and Khateen was determined from DNA extracted from scabs, whereas the others were derived from infected cell culture material. Sequences of the 14-kDa fusion protein genes of OPVs were downloaded from GenBank (www.ncbi.nlm.nih.gov) and aligned by use of the Mac Vector software package (Accelrys Inc., Cambridge, United Kingdom).

Primer and probe design. Oligonucleotides were designed with Primer Express software supplied by Applied Biosystems (Darmstadt, Germany). Primer and probe sequences were checked against those in the GenBank and EMBL databases by use of the BLAST (www.ncbi.nlm.nih.gov/BLAST) and FASTA (www.ebi.ac.uk/fasta33/) algorithms.

LightCycler PCR. The LightCycler instrument (Roche Diagnostics, Mannheim, Germany) was used to amplify a 110-bp region of the 14-kDa fusion protein gene. During amplification the fluorescence was continuously monitored at the annealing step of PCR. Fluorescence was generated by specific hybridization of two oligonucleotides inside the developing PCR fragment. Different from conventional hybridization probe assays, the anchor probe contained LC 640 fluorophore (TIB Molbiol, Berlin, Germany) at the 5' end and a fluorescein sensor probe at the 3' end. The sensor probe was additionally covalently linked to a minor groove binder (MGB; Applied Biosystems) and a dark quencher at the 5' end. This design allows a much higher discrimination power in melting-curve analysis compared to that obtained with conventional hybridization pairs. PCR was performed in a total volume of 20 µl of a mixture containing 2 µl of MgSO4 solution (50 mM) and 13 µl of a PCR master mixture containing Taq polymerase reaction buffer, a deoxynucleoside triphosphate mixture, bovine serum albumin (Artus, Hamburg, Germany), and 5 pmol each of two forward primers (Table 2), two reverse primers, the anchor probe, and the sensor probe (Aplera, Darmstadt, Germany). After distribution of 15 µl of the master mixture, 5 µl of template DNA solution was added to each glass capillary (Roche Diagnostics), centrifuged, and placed in the LightCycler sample carousel. LightCycler amplification involved a first denaturation at 95°C for 10 s, followed by amplification of the target DNA for 45 cycles (95°C for 5 s, 55°C for 15 s, and 72°C for 15 s) with a temperature transition rate of 20°C/s. All PCR runs included appropriate negative controls to exclude the possibility of DNA contamination and positive controls to ensure high PCR performance. In some experiments, 10 ng of human genomic DNA was added to the master mixture.


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  		TABLE 2. Designations and sequences of primers and probes

Melting-curve analysis. Differentiation of VAR from all other members of the genus Orthopoxvirus was achieved by melting-curve analysis. This feature makes use of the different melting behaviors of oligonucleotides, which results in specific shifts of the melting temperature (Tm) due to a single nucleotide mismatch. The melting-curve analysis was performed by denaturation at 95°C for 10 s, followed by a cooling step at 40°C for 20 s and heating to 80°C at a linear temperature transition rate of 0.1°C/s. For improved determination of Tm, the first derivative of the initial melting curve was plotted against the temperature.

Positive control. A positive control (TL-1), consisting of linearized plasmid DNA (pcr Skript; Stratagene, La Jolla, Calif.) with the 14-kDa fusion protein gene of VAR Bangladesh synthesized in vitro, was generated by overlap extension amplification with 80-bp oligonucleotides (Metabion, Munich, Germany). The DNA concentration was quantified by photometric and fluorescence photometric methods. TL-1 was used as a high- and a low-copy-number control (10,000 and 100 copies, respectively) to facilitate initial evaluation and optimization of the assay.

Statistical analysis. To determine the analytical sensitivity of the assay, we used purified and photometrically quantified VAC (strain Copenhagen) and VAR (strain Variolator 4) DNA preparations. The DNA was diluted in Tris-EDTA (or water for VAR) and was tested in 24 replicates with DNA concentrations of 100, 20, 4, 0.8, and 0.16 fg as inputs. A probit analysis as a model of nonlinear regression was done with a commercial software suite (SPSS, version 11.0, for Mac OSX; SPSS, Inc., Chicago, Ill.). The software determines a continuous 95% confidence interval of the probability of achieving a positive result at any given input DNA concentration within the concentration range of the experiment.


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RESULTS AND DISCUSSION
 
The nucleotide sequences of the 14-kDa fusion protein genes of 14 VAR strains from the Russian repository were determined and aligned to sequences of various OPVs deposited in GenBank. All VAR sequences showed a stable mismatch at nucleotide position 231 of the 14-kDa fusion protein gene (A27L) compared to the sequences of all other OPV strains. This results in a change from amino acid Glu-77 to Asp. This unique change was selected for by designing a hybridization probe pair that would discriminate a single nucleotide mismatch by Tm. Conventional hybridization probe pairs showed a maximum discrimination power of 4°C; thus, we decided to use a new approach and selected a relatively short sensor probe (the probe which hybridizes to the mutated site) with a covalently linked MGB molecule (Table 2). MGB-linked oligonucleotides are more sensitive to nucleotide mismatches, and thus, the power of discrimination of a site with a single nucleotide polymorphism is increased. Since the probe was designed to be specific for Eurasian-African OPV species other than VAR, such as VAC, MPX, and CPX, primer-probe hybrids specific for these species should melt at temperatures higher than those for hybrids specific for VAR. In order to prove this assumption, a total of 123 OPVs (listed in Table 1) were investigated. Amplification was seen for all 46 VAR strains (Table 3) and 82 other isolates belonging to the Eurasian-African OPV species, whereas the DNAs of three strains representing the North American OPV species (RCN, SKN, and VPX) were not amplified. The results of melting-curve analyses of one LightCycler run for VAR and VAC are shown in Fig. 1. The results of all analyses are summarized in Table 4. A clear discrimination of smallpox virus is possible: all 46 VAR strains displayed lower Tms (Tms, 55.9 to 57.8°C) than the 11 VAC strains (Tms, 61.7 to 62.7°C), the 15 MPX strains (Tms, 61.9 to 62.2°C), the 40 CPX strains (Tms, 61.3 to 63.7°C), the 8 ECT strains (Tm, 61.9°C), and the 8 CML strains (Tms, 64.0 to 65.0°C). Melting point differences between OPV species were statistically analyzed by one-way analysis of variance (with five different post hoc tests). The differences in the Tms between VAR and the other OPV species were significant (P = 0.005). Although no amplification of VPX was observed, 1 ng of genomic VPX DNA was sufficient to cross-react with the probe and produce a melting curve of a much lower temperature (Tm, 46.09°C) than those for all other OPVs tested (Table 4). Furthermore, the primers and probes did not cross-react with the DNA of parapoxvirus, avipoxvirus, or tanapoxvirus strains or with the DNA of human herpesviruses (cytomegalovirus, Epstein-Barr virus, herpes simplex virus types 1 and 2, and varicella-zoster virus).


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  		TABLE 3. Investigation of different amounts of total DNA prepared from VAR-infected cell lysate material or scabs by LightCycler PCR combined with melting-curve analysis



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  		FIG. 1. Melting curves generated after LightCycler amplification of 100, 10, and 1 fg of DNA prepared from VAR (strain Kali Mathu)-infected cell culture material. Each DNA concentration was run in triplicate. The melting curve after amplification of 100 fg of VAC (strain MVA) is also given. Fl, fluorescence.


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  		TABLE 4. Comparison of Tms of different OPV species

The analytical sensitivity of the assay was determined by probit regression analysis with purified VAR DNA and 24 replicate amplification reactions. Amplification was positive in all 24 replicate reactions containing 100, 20, and 4 fg of VAR input DNA. When 0.8 and 0.16 fg of VAR DNA was input, amplification resulted in 23 and 16 positive reactions, respectively. Figure 2 shows the results of the probit analysis for VAR. The concentration at which 95% of results are expected to be positive was calculated to be 0.7 fg of DNA. On the basis of the number of VAR base pairs (ca. 186 kbp), this corresponds to about 4 genome copies per assay. By using the same statistical approach with purified VAC DNA, the sensitivity was calculated to be about 6 genome copies (data not shown). However, these results were achieved with purified DNA from only two OPV species. Crude viral-cellular lysates derived from infected cell cultures may more faithfully correspond to the nucleic acid material present in a clinical specimen, as both viral and host cell DNA are present in samples of DNA extracted directly from infected tissue cultures. In these samples (a total of 40 samples were examined; Table 3) the assay was able to detect VAR DNA in samples in which as little as 10 fg of total input nucleic acid was present. Investigation of four archival human smallpox specimens (Table 3) yielded positive amplification with an input nucleic acid concentration of 100 fg per reaction mixture. Given that 106 pock-forming units can be isolated from skin lesions by day 2 of the smallpox rash (3) and that a greater number of viral particles are present than can be quantified as pock-forming units, at a minimum, 200 pg of VAR DNA can be extracted per skin lesion by day 2 of the rash. This amount of viral DNA exceeds the detection limit of the assay by several orders of magnitude and is certainly sufficient to diagnose smallpox at this stage.



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  		FIG. 2. Predicted proportion of positive amplification results versus the input concentration of purified VAR (strain Variolator 4) DNA, as determined by probit regression analysis. The DNA concentration at which 95% of results are expected to be positive was calculated to be 0.7 fg, which corresponds to ca. 4 copies/assay.

The linearity of the assay was established by using a dilution series of purified VAR DNA from both the Variolator 4 and the Bangladesh strains. The cycle at which the fluorescence crossed the threshold increased in a linear manner from 1 ng to 1 fg of VAR DNA (Table 5). The Tms varied slightly, depending on the concentration of viral DNA, and increased up to 1.3°C over a 106-fold dilution series. Limiting amounts (1 ng to 1 fg) of purified VAR DNA (strain Bangladesh) were amplified in the presence of 10 ng of purified human genomic DNA/reaction mixture. This did not inhibit the detection of VAR DNA, nor did it affect the VAR Tm (Table 5).


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  		TABLE 5. Investigation of different amounts of purified VAR DNA by LightCycler PCR combined with melting-curve analysis

All reagents used in this assay were produced under good manufacturing practice conditions and were assembled into a kit in order to standardize OPV diagnostics. The kit contains an internal control, which consists of 100 copies of a 400-bp artificial DNA fragment and which is detected in the F3 channel of the LightCycler instrument. The internal control is designed in a way that the amplification efficiency of the internal control reaction is generally weaker than that of the specific amplification assay. Thus, problems with DNA extraction or the presence of inhibitors would affect the internal control earlier than they would affect the specific amplification signal. Spiking experiments with known PCR inhibitors (e.g., heparin) demonstrated that this type of internal control inhibits the reaction at 1/10 of the concentration actually required to inhibit the amplification of the test target gene (data not shown). To monitor inhibition of the polymerase, the internal control was included in the master mixture used in each amplification assay. The internal control was amplified in all experiments except when a great excess of target viral DNA was present. In such instances, the internal control was not amplified due to the sequestering of resources for amplification of the viral DNA.

The assay was developed on the basis of sequences of the 14-kDa fusion protein gene. This gene, named open reading frame A27L, plays an important role in virulence and immunogenicity (2, 19). We confirmed by successful amplification of 120 Eurasian-African OPVs that A27L sequences are highly conserved and thereby useful for establishment of an OPV consensus PCR. Furthermore, we demonstrated by sequencing that the unique amino acid change (Glu-77 to Asp) of VAR is conserved in 14 different VAR strains of the Russian WHO collection. To our knowledge, this is the highest number of different VAR strains (n = 60) and OPVs other than VAR (n = 74) used to evaluate a PCR assay for the genus Orthopoxvirus. We stress that the screening of large OPV strain collections is essential to demonstrate the usefulness and to establish the performance characteristics of assays being developed. In a recent paper (5), the investigators stated that mismatches in the fluorescence resonance energy transfer probes used in their assay enabled discrimination of VAR from other OPVs by DNA melting-curve analysis. Due to the new OPV sequences in GenBank, the fluorescence resonance energy transfer probes also display identity to CML and some CPX strains. Analysis of such strains must be conducted to prove whether the reliable identification of VAR is still possible. In the assay described here, we included a rather large number (n = 40) of CPX strains, since viruses of this species displayed considerable genetic heterogeneity (17) and have a large genome (ca. 220 kbp) containing sequences previously considered VAR specific (15).

Nevertheless, we want to stress that a positive PCR result for VAR must be confirmed by amplifying other parts of the OPV genome. In this respect, the hemagglutinin gene has been the most comprehensively characterized, and a VAR-specific real-time PCR assay was recently described (8). In addition, the use of classical techniques, such as viral culture and electron microscopy, will enhance the confidence in the final diagnostic results.


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ACKNOWLEDGMENTS
 
We thank Gudrun Zoeller, Sabine Raith, and Matthias Wagner for valuable technical help.

A part of this work was supported by ISTC (project 1987p).


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Mikrobiologie der Bundeswehr, Neuherbergstr. 11, 80937 Munich, Germany. Phone: 49-89-3168-3910. Fax: 49-89-3168-3292. E-mail: hermann1meyer{at}bundeswehr.org. Back


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Journal of Clinical Microbiology, May 2004, p. 1940-1946, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.1940-1946.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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