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Technical Briefs |
1 Robert Koch-Institut, Zentrum für Biologische Sicherheit 1, Berlin, Germany;2 Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Oberschleissheim, Germany;3 Institut für Tierhygiene und Öffentliches Veterinärwesen, Leipzig, Germany;4 Bundeswehr Institute of Microbiology, München, Germany;
aaddress correspondence to this author at: Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany; fax 49-30-4547-2605, e-mail nitschea{at}rki.de
Abstract
Background: Detection of parapoxviruses is important in various animals as well as in humans as zoonotic infections. Reliable detection of parapoxviruses is fundamental for the exclusion of other rash-causing illnesses, for both veterinarians and medical practitioners. To date, however, no real-time PCR assay for the detection of parapoxviruses has been reported.
Methods: A minor groove binder–based quantitative real-time PCR assay targeting the B2L gene of parapoxviruses was developed on the ABI Prism and the LightCycler platforms.
Results: The real-time PCR assay successfully amplified DNA fragments from a total of 41 parapoxvirus strains and isolates representing the species orf virus, bovine papular stomatitis virus, pseudocowpoxvirus, and sealpoxvirus. Probit analysis gave a limit of detection of 4.7 copies per assay (95% confidence interval, 3.7–6.8 copies per reaction). Scabs contain a sufficient amount of parapoxvirus DNA and can therefore be used for PCR without any DNA preparation step. No cross-reactivity to human, bovine, or sheep genomic DNA or other DNA viruses, including orthopoxviruses, molluscum contagiosum viruses, and yaba-like disease viruses, was observed.
Conclusion: The presented assay is suitable for the detection of parapoxvirus infections in clinical material of human and animal origin.
Parapoxviruses (PPVs) form a unique genus within the subfamily Chordopoxvirinae and are differentiated into the following species: orf virus (ORFV), bovine papular stomatitis virus (BPSV), pseudocowpoxvirus (PCPV), and PPV of the red deer in New Zealand. Enveloped virions present with an ovoid shape and can be clearly distinguished by electron microscopy (EM) from orthopoxviruses (OPV) because of their regular surface structure (1). The viral genome consists of a linear double-stranded DNA 130 000–150 000 nucleotides in length covalently cross-linked at the ends (1).
Ruminants are the main hosts that become infected with PPV worldwide. Affected animals such as sheep, goats, and cattle develop proliferative dermatitis in the region of the mouth, teats, and skin. In young, stressed, or immunosuppressed sheep, formation of severe bloody lesions can be fatal. A recently published report (2) described an infection of harbor seals with PPV that was significantly different from known PPV species. In addition, infections of several animal species, including squirrels (3), gazelles(4), and nonhuman primates such as pygmy chimpanzees, with PPV/PPV-like viruses have been described, indicating an even broader host range for PPVs. Occasionally, humans are affected by PPV after direct contact with lesions of infected animals, with infection rates of up to 34% in individuals at risk (5). The well-known milkers’ node is a typical human clinical picture observed after infection with PCPV. Human PPV infection occurs most commonly on the index finger. The characteristic lesion resembles a tumor and resolves spontaneously, usually without complications, but rare cases with complications have been reported. Interestingly, the infection of a marine mammal research technician with sealpoxvirus (SPV) was reported recently (6), confirming that all currently known PPV species can be transmitted to humans.
Because human PPV infection can resemble a localized infection with orthopoxviruses, such as cowpoxvirus or vaccinia virus infection, a fast and reliable diagnosis is required to assess the risk rapidly and to prevent a potentially fatal infection with OPV, especially in immunosuppressed individuals (7).
All published cases of PPV infection were diagnosed by EM (2)(8)(9), histopathology (6)(10), or in some cases by conventional PCR (8)(9)(11). Diagnostic EM is rapid and clearly distinguishes PPV from OPV particles; however, EM facilities with experienced personnel are not universally available at present. Even if an EM diagnostic laboratory is available, EM diagnoses require high viral loads of 
106 particles/mL with an intact morphology (12). Such a load usually occurs in fresh lesions and crusts of infected individuals, but in cases of lower viral loads or material not preserved according to EM requirements, EM may produce false-negative results with a certain probability (13).
PCR is well recognized to be a stable, fast, and sensitive diagnostic method for the detection of nucleic acids, although it cannot confirm the presence of complete or infectious particles (14). However, in combination with a typical clinical picture, detection of the respective viral nucleic acid must be considered as evidence.
To date, a modern PPV-specific diagnostic technique such as real-time PCR has not been published; we therefore developed a real-time PCR-based assay with all of the advantages that real-time PCR offers: It is fast, specific, and reliable, with a decreased risk of carryover contamination, and it makes large-scale diagnoses possible.
The PPV real-time PCR assay was designed as a consensus PCR to specifically amplify DNA from all PPV species: ORFV, PCPV, BPSV, and SPV. A gene region highly conserved among 59 available sequences of the major envelope protein gene (B2L) was chosen for the location of two primers and one 5'-nuclease minor groove-binder (MGB) probe (see Table 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol52/issue2) that amplify a 95-bp product.
Reaction conditions for the PPV assay were established for the Applied Biosystems 7700/7900/7500 Sequence Detection System series in a total reaction volume of 25 µL containing 1x PCR buffer, 5 mM MgCl2, 1 mM deoxynucleotide triphosphate mixture with dUTP, 1 µM ROX, 1 U of Platinum Taq polymerase (Invitrogen), 7.5 pmol each of the primers, 2.5 pmol of the MGB probe, and 5 µL of template DNA or (5 µL of) crust material without the preceding preparation step. Cycling conditions were set to 5 min at 95 °C and 40 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 30 s.
On the LightCycler (Roche), PCR was performed in glass capillaries in a total reaction volume of 20 µL containing 7.5 pmol of each primer, 2.5 pmol of MGB probe, 2 µL of LightCycler-Fast Start DNA Master Hybridization Probes Mix (Roche), 5 mM Mg2+, and 5 µL of template DNA. Cycling conditions were 10 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 10 s at 55 °C, and 10 s at 72 °C.
We assessed the performance of the PPV real-time PCR assay with, as calibrators, 4 plasmids that were cloned according to routine procedures and contained 487 bp of the B2L gene of ORFV (strain B006, pORFV), PCPV (strain B021, pPCPV), BPSV (strain B177, pBPSV), and SPV (pSPV), including the original sequence of these isolates in the primer-binding (PPV up, PPV do) and probe-binding (PPV TMGB) region (cloning primers are shown in supplemental material Table 1 in the online Data Supplement). Amplification of 10-fold serial dilutions of each plasmid in a constant background of 1 ng/µL 
DNA revealed a linear detection range from 106 to 101 copies per reaction, with a correlation (R2) of 0.99 and a PCR efficiency >97%, indicating an efficiency close to the ideal amplification factor of 2 per PCR cycle. There were no significant differences among the 4 plasmids tested, indicating adequate binding of primers and probe to ORFV, BPSV, PCPV, and SPV DNA (Fig. 1
). We performed a probit analysis, which indicated that the limit of detection (LOD) was 4.7 copies per assay (95% confidence interval, 3.7–6.8 copies/reaction). We determined the overall variability of the assay by repeat amplifications of 3 different plasmid concentrations (106, 104, and 102) and 3 genomic PPV DNA dilutions with DNA concentrations similar to those of the plasmids. Variability was <30% for concentrations close to the detection limit (102 copies) and <15% for higher DNA concentrations (104 and 106 copies).
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We evaluated the specificity of the PPV real-time PCR assay by use of DNA from several viruses, including orthopoxviruses (vaccinia, cowpox, monkeypox, ectromelia, and camelpox virus), molluscum contagiosum virus, yaba-like disease virus, human herpesviruses types 1 to 8, and adenoviruses. Neither amplification products, as shown by gel analysis, nor fluorescence signals were observed, indicating the required specificity for PPV (data not shown).
Finally, to confirm applicability of the PPV real-time PCR in clinical diagnostics, we prepared DNA from 41 clinical specimens, crusts, or lesions from infected humans or animals (Table 1
) with the Qiagen DNA Tissue Kit (Qiagen) according to the manufacturer’s instructions, and subjected the DNA to the PPV real-time PCR and to the 18S real-time PCR (see material Table 1 in the online Data Supplement). Both 18S PCR and PPV PCR showed positive reactions for all 41 (100%) specimens, with viral loads ranging from 5.7 x 107 to 2.9 x 104 per 106 copies of the DNA for reference gene 18S. Although 18S has frequently been reported to be a poor reference gene for quantitative expression analysis (15), its DNA is highly conserved among various animal species, and the applied PCR assay detects DNA from sheep, goats, cattle, and camels and can easily serve as a control of DNA quality. Subsequent amplification and calibrated sequence analysis of all specimens with the PPV primers PPP-1 and PPP-4 (see material Table 1 in the online Data Supplement) allowed us to clearly identify the respective PPV species.
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To evaluate whether more rapid diagnosis was possible, we used small pieces of dry crust material, usually containing high viral loads, directly as templates in real-time PCR without the DNA preparation step. The expected amplification curves were obtained for 3 individual crust specimens, allowing clear detection of PPV but with lower product yields, as indicated by lower fluorescence yields in the PCR. However, accurate quantification in this crude material seems difficult, and we recommend the use of crust material for qualitative analysis only.
Several PCR assays for the detection of PPV DNA have been published to date (11)(16), but these assays were based on only the few sequences available at the time of publication or were used for typing of PPV or amplifying long DNA fragments; the sensitivities of these PCR assays were therefore reduced.
To close this gap, we developed an assay with a short amplicon of 95 bp to enable sensitive and reliable detection of PPV DNA, even in samples of moderate DNA quality, such as DNA extracted from paraffin- or formalin-fixed tissue. Although there is a divergence of up to 15.6% in the B2L gene sequences of different PPV species, careful selection of primers and probe gave satisfactory and comparable amplification efficiency as well as a detection limit for the PPV species ORFV, BPSV, PCPV, and SPV, which was confirmed by analysis of 41 clinical samples from humans or animals infected with these PPV species.
In summary, the PPV real-time PCR assay is a rapid and useful diagnostic tool to identify PPV in clinical specimens. This may be important in both veterinary and human diagnostics in cases when differential diagnosis from other skin tropic infections, especially with OPV, is mandatory for risk assessment.
Acknowledgments
DNA from sealpoxvirus was kindly provided by Paul Becher (Institut für Virologie, Justus-Liebig Universitat, Giessen, Germany).
References
The following articles in journals at HighWire Press have cited this article:
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  V. Balamurugan, K. D. Jayappa, M. Hosamani, V. Bhanuprakash, G. Venkatesan, and R. K. Singh Comparative efficacy of conventional and taqman polymerase chain reaction assays in the detection of capripoxviruses from clinical samples J Vet Diagn Invest, March 1, 2009; 21(2): 225 - 231. [Abstract] [Full Text] [PDF]
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 B. J. Hindson, S. M. Reid, B. R. Baker, K. Ebert, N. P. Ferris, L. F. B. Tammero, R. J. Lenhoff, P. Naraghi-Arani, E. A. Vitalis, T. R. Slezak, et al. Diagnostic Evaluation of Multiplexed Reverse Transcription-PCR Microsphere Array Assay for Detection of Foot-and-Mouth and Look-Alike Disease Viruses J. Clin. Microbiol., March 1, 2008; 46(3): 1081 - 1089. [Abstract] [Full Text] [PDF]
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