Detection of monkeypox virus with real-time PCR assays

Background Human monkeypox, a zoonotic disease, was first reported outside of Africa during the 2003 US outbreak. Objectives We present two real-time PCR assays critical for laboratory diagnosis of monkeypox during the 2003 US outbreak. Study design A TaqMan-based assay (E9L-NVAR) targets the orthopoxvirus DNA polymerase gene and detects Eurasian orthopoxviruses other than Variola. A hybridization assay, utilizing a MGB Eclipse™ (Epoch Biosciences) probe, targets an envelope protein gene (B6R) and specifically detects monkeypox virus (MPXV). Assays were validated using coded orthopoxvirus DNA samples and used to evaluate lesion samples from five confirmed US monkeypox cases. Results E9L-NVAR did not detect variola (48 strains), North American orthopoxviruses (2), or DNA derived from non-poxviral rash illnesses. The assay reproducibly identified various concentrations of 13 Eurasian orthopoxvirus strains and was sensitive to 12.5 vaccinia genomes. The B6R assay recognized 15 different MPXV strains, while other orthopoxvirus (9) and bacteria (15) strains did not cross-react. Of the 13 human samples tested from confirmed cases, both assays identified 100% as containing MPXV DNA. Conclusions E9L-NVAR and B6R assays demonstrate 100% specificity for non-variola Eurasian orthopoxvirus and MPXV, respectively. Using two discrete viral gene targets, these assays together provide a reliable and sensitive method for quickly confirming monkeypox infections.


Introduction
Orthopoxvirus monkeypox, first isolated in 1958 from captive primate rash specimens (Magnus et al., 1959), was recognized to cause human illness in 1970 during smallpox eradication campaign intensification (Jezek and Fenner, 1988;Ladnyi et al., 1972). Between 1970 and1986, >400 monkeypox cases were reported in Africa, 95% within Zaire (now the Democratic Republic of Congo, DRC) (Jezek and Fenner, 1988). Until the 2003 US outbreak (Reed et al., 2004), no human cases had been reported outside of Africa. Human monkeypox, as described in the Congo Basin, typically presents with symptoms similar to discrete, ordinary smallpox. After about a 2-week, asymptomatic incubation period, infected individuals develop fever followed by disseminated rash. Both illnesses are transmissible between humans and can result in death. The case fatality rate for monkeypox (∼0-10%) (Jezek and Fenner, 1988) is much lower than for variola major (10-30%) (Fenner et al., 1998), as is the rate of human-to-human transmissibility. Control measures for these two diseases would differ, and recent events emphasize the need for monkeypox diagnostics. After discontinuation of smallpox vaccination, susceptibility to zoonotic monkeypox increased, likely contributing to increased disease reports in DRC (Hutin et al., 2001;Meyer et al., 2002;Mwanbal et al., 1997). The US monkeypox outbreak demonstrated the ability of the virus to exploit new hosts and move globally (Reed et al., 2004).
Several nucleic acid test methods have been developed for monkeypox virus (MPXV) detection and characterization (Kulesh et al., 2004;Meyer et al., 2004). Compared with other diagnostic methods, real-time PCR has the advantages of fast, high-quantity throughput and increased sensitivity. This is the first report of real-time PCR assays used to diagnose human monkeypox from clinical rash samples. The assays target different orthopoxvirus genes: DNA polymerase (E9L) and envelope protein (B6R). We report the analytic sensitivity and specificity of both assays and demonstrate their utility for US monkeypox outbreak human rash specimens. These assays represent two sensitive, rapid diagnostic tools for identification of orthopoxviral infection within clinical samples.
Clinical samples were obtained from vesicular lesions as skin biopsies (scab or vesicle roof), vesicular fluid slide ("touch prep"), or vesicular fluid swab. Recommendations for lesion sampling can be found at http://www.bt.cdc.gov/agent/ smallpox/response-plan/files/guide-d.pdf. In brief, samples were processed under biosafety containment conditions to form homogenates suitable for DNA extraction: 1. Skin biopsies were homogenized in sterile water or PBS (500 L) by freezing, disruption with a disposable pestle, and vortexing. If physical disruption was insufficient, the sample underwent further freeze-thaw/grinding cycles. Finally, samples, in closed tubes, were sonicated (cup-horn sonicator, 40% maximum output). 2. Nuclease-free water (100 L) was added to each spot on the slide, scraped, and recovered into a sterile tube. Water addition/scraping was repeated twice and pooled into the same tube. 3. Shafts of vesicular fluid swabs were broken near the top of the swab material. The swab was hydrated (300 L PBS) for 5-10 min in a sterile tube and then transferred to a Swab Extraction Tube System tube (Roche Applied Science, Indianapolis, IN). The swab/tube was spun for 1 min to rinse the swab and collect the eluent.

E9L non-variola (NVAR) assay
The primer/probe sequences were selected from the DNA polymerase gene (E9L; GenBank L22579) with Primer Express (version 1.5; Applied Biosystems). These included E9L forward primer (5 -TCA ACT GAA AAG GCC ATC TAT GA-3 ), E9L reverse primer (5 -GAG TAT AGA GCA CTA TTT CTA AAT CCC A-3 ), and E9L-NVAR probe (5 TET-CCA TGC AAT ATA CGT ACA AGA TAG TAG CCA AC-3 ). Primers and probe were synthesized in the Biotechnology Core Facility (CDC, Atlanta GA), utilizing standard phosphoramidite chemistry. The detection probe contained 5 reporter molecule (TET) and 3 aminomodifier (Glen Research, Sterling, VA). A 3 quencher molecule, QSY7 (Molecular Probes, Eugene, OR), was conjugated to the 3 amino group after synthesis. PCR assay conditions were optimized according to standard protocols (protocol 04304449, Applied Biosystems, Foster City, CA) by adjusting primer and probe concentrations, and thermal cycling temperatures/duration. Each reaction (50 L) contained 1× TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), 0.4 mol/L each primer, 200 nmol/L TaqMan probe, and 2 L of template DNA. Thermal cycling conditions for the ABI7700 (Applied Biosystems, Foster City, CA): one cycle of 95 • C for 10 min; followed by 40 cycles of 95 • C for 10 s, and 60 • C for 40 s. PCR amplification is based on fluorescent emission after annealing/elongation (60 • C).

B6R MPXV-specific assay
The primer sequences were selected from the extracellular enveloped virus protein gene (B6R; GenBank L22579) using Primer Express (version 1.5; Applied Biosystems, Foster City, CA). These included B6R forward primer (5 -ATT GGT CAT TAT TTT TGT CAC AGG AAC A-3 ), and B6R reverse primer (5 -AAT GGC GTT GAC AAT TAT GGG TG-3 ). The MPXV-specific probe (5 -MGB/ DarkQuencher-AGA GAT TAG AAA TA-FAM3 ) was selected from the B6R sequence with the aid of MGB Eclipse TM By Design software (Epoch Biosciences, Bothell, WA) and has a conjugated minor groove binding (MGB) ligand and a dark quencher at the 5 -end, with the fluorophore at the 3 -end. Fluorescence of the single-stranded probe is efficiently quenched by the interaction of the terminal dye and quencher groups when not hybridized (Afonina et al., 2002a,b).

Statistical probit analysis
Analytical sensitivity was determined using purified, photometrically quantified vaccinia DNA diluted in water (24 replicates of 5 concentrations). Probit analysis as a model of non-linear regression was accomplished with commercial software (SPSS 11.0, for Mac ® OS X; SPSS, Inc., Chicago, IL). 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.

E9L-NVAR orthopoxvirus assay
Several TaqMan-based real-time PCR assays have been developed as rapid orthopoxvirus diagnostics. One diagnostic assay for orthopoxviral infections other than variola targets the viral DNA polymerase gene (E9L), amplifying a conserved gene segment within all Eurasian orthopoxviruses. The probe, however, targets 32 bases within the E9L gene containing a three nucleotide difference between variola and other orthopoxviruses (Fig. 1), and thus efficiently anneals to Eurasian orthopoxviruses other than variola. This orthopoxviral diagnostic can be used without raising concern that variola has been detected.
The specificity and sensitivity of the assay, designated E9L-NVAR, was determined utilizing a coded panel of multiple orthopoxviruses. Each sample was tested singly, and positive samples are denoted by the cycle where fluorescence crossed the threshold (Ct) ( Table 2). E9L-NVAR identified all non-variola Eurasian orthopoxviruses (13 species) at concentrations between 2 pg and 20 fg of viral DNA, depending upon DNA quality. The assay detected 20 fg of partially purified MPXV DNA (∼100 genomes). Partially purified cowpox, ectromelia, and vaccinia were identified at similar efficiencies (Table 2). Partially purified camelpox and taterapox demonstrated a diminished interaction with the E9L-NVAR probe (Table 2) due to a single base difference between these viruses and variola in the probe target region (Fig. 1).
Vaccinia and MPXV sequences are identical in this region (Fig. 1), and serially diluted purified vaccinia DNA established linearity of the E9L-NVAR assay from 2 ng to 20 fg (83% reaction efficiency) ( Table 2). Probit regression analysis determined assay sensitivity using the same preparation of purified DNA in 24 replicate amplification reactions. Amplification was positive in all 24 replicate reactions containing 20, 10, and 5 fg of vaccinia input DNA. Only 22 of 24 replicates containing 2.5 fg were detected, while no reactions containing 1.25 fg vaccinia DNA were positive (data not shown). Therefore, 2.54 fg viral DNA (∼12.5 genomes) is the calculated detection limit for 95% confidence.
The E9L-NVAR assay is specific for six non-variola Eurasian orthopoxviruses, not cross-reacting with variola (48 strains) or North American orthopoxviruses (2 strains). Furthermore, E9L-NVAR assay did not cross-react with any DNA derived from rash illnesses potentially confused with orthopoxviral infection, such as herpesvirus and rickettsial infections, or with human cellular DNA (Table 2), even at high concentrations (2 ng). Overall, the E9L-NVAR assay can reproducibly detect as few as 12.5 genomes of purified vaccinia or MPXV DNA without giving false positive results.

Monkeypox-specific B6R assay
Although the E9L-NVAR assay reliably detects Eurasian orthopoxviruses, other than variola, it is unable to make a species-specific identification. Due to the low G + C content (∼30%) and 90% sequence similarity to other Eurasian orthopoxviruses, it is difficult to design a monkeypox-specific TaqMan assay. To improve the reliability of a MPXV-specific assay, we utilized the MGB-based real-time PCR technology. Linking a DNA double helix MGB protein to the probe permits use of shorter probe sequences, which can detect single nucleotide polymorphisms (SNPs) (Afonina et al., 2002a,b;Belousov et al., 2004) such as within the MPXV envelope protein gene (B6R) (Fig. 1). The probe 5 -MGB molecule stabilizes probe-template interaction (Afonina et al., 2002a,b) and enhances assay specificity and sensitivity.
A coded test panel containing orthopoxviral and bacterial DNAs was assayed in triplicate using the iCycler platform (Bio-Rad, Hercules, CA) to monitor reproducibility and specificity of the B6R assay (Table 3). MPXV DNA was reproducibly detected in a linear fashion to ∼10 viral copies (2 fg). The B6R assay did not cross-react with any other orthopoxviral DNA (variola, cowpox, camelpox, and vaccinia) or with 15 bacterial species. Although certain Grampositive bacterial DNAs were less efficiently extracted, all a Ability of the assay to detect multiple strains of monkeypox virus. Each sample was tested in triplicate; all three assays were positive and the average Ct value is shown. b Assay limit of detection for various quantities of purified monkeypox DNA. Each sample was tested in triplicate using either freshly diluted DNA (fresh) or diluted DNA that had undergone multiple freeze-thaw cycles (old). Where all three assays were positive, the average Ct value is shown. ND, not detected. bacterial samples mimicked conditions expected within clinical samples. Furthermore, the presence of bacterial 16S DNA was confirmed using real-time PCR (data not shown). The B6R assay should not cross-react with these potential contaminating bacteria within clinical samples. The B6R assay detects multiple monkeypox strains; all 15 isolates of MPXV DNA were detected at 10 ng (Table 4), an amount often found within rash samples. Assay linearity and sensitivity were determined using purified MPXV DNA; all quantities from 2 ng to 2 fg cross-reacted with the B6R probe in a linear fashion (Table 4). The reaction efficiency was affected by the freshness of diluted DNA and B6R assay probe and primers: freshly diluted DNA produced higher reaction efficiency (97%) than DNA that had undergone multiple freeze-thaw cycles (67%). Overall, the B6R assay demonstrates a sensitive, specific, and rapid method for identifying MPXV.
The B6R probe did not detect any of our stocks of cowpox DNA (strain BRT (Table 3) and German isolates [GER2 and GER3] (data not shown)). However, the published cowpox GRI90 sequence is unusual in that it contains more monkeypox SNPs than cowpox signatures, including within the B6R probe region. Based on available sequence information, the B6R assay would likely cross-react with cowpox GRI90, but not other diverse cowpox isolates. At this time, we are unable to obtain cowpox GRI90 DNA to experimentally assess B6R probe cross-reactivity.

Diagnostic use of real-time PCR assays during the 2003 US monkeypox outbreak
The first US human monkeypox cases were recognized in Wisconsin (Reed et al., 2004). From five of these cases, we received a variety of rash-lesion samples at a vesicular stage of development. Samples (skin, slides, and swabs) were inoculated into tissue culture to evaluate for viral cytopathic effect and DNA extracted for various PCR-based assays. Previously established standard PCR techniques amplified the HA and ATI genes of all orthopoxviruses with subsequent Restriction Fragment Length Polymorphism RFLP analysis providing species-specific identification (Meyer et al., 1997;Ropp et al., 1995), or amplified only MPXV using monkeypox-specific primers of the E9L gene Multiplex assay (Dhar et al., 2004). Samples were assayed with both well-established assays and new real-time PCR assays (E9L-NVAR and B6R) and their performance compared.
For real-time PCR assays, a positive control (20 fg MPXV DNA) within each run established the Ct cut-off value for positivity. All monkeypox-positive samples by standard PCR or tissue culture were also detected by real-time PCR (Table 5). In fact, real-time PCR assays identified low levels of MPXV DNA, undetectable by standard PCR (Table 5, cases 2003-038 and 2003-040). Both real-time PCR assays detected viral DNA from various sample types: vesicle fluid (slides or swabs), and vesicle skin. As evidenced by different Ct values and days to evident cytopathic effect, viral DNA load varied between samples, possibly due to differences in sampling technique, disease stage, and/or sample type; vesicle skin having the highest viral amounts. MPXV-positive rash samples were collected anywhere from day 1 to day 22 after rash onset, indicating assay usefulness throughout multiple disease stages. Clinical assay specificity was demonstrated by samples (case 2003-072) which were orthopoxvirus negative by our PCR assays (Table 5), despite sample adequacy indicated by detection of human DNA (␤-actin) (data not shown), but were confirmed to contain varicella zoster virus by PCR (National Varicella Reference Laboratory, CDC, personal communication). These real-time PCR diagnostic assays proved to be highly sensitive and specific during clinical application.

Summary
Two rapid real-time PCR assays for the detection of orthopoxvirus and MPXV DNA have been developed. The E9L-NVAR and B6R assays target orthopoxvirus DNA poly-merase and extracellular enveloped protein genes, respectively. These assays are highly sensitive (2 fg or ∼10 viral genomes) and specific. The E9L-NVAR assay detects 13 Eurasian orthopoxviruses but not variola or North American orthopoxviruses, and the B6R assay detects MPXV isolates but no other orthopoxviruses. Neither assay gave false positives with other rash illness-causing viruses or bacteria. The E9L-NVAR assay, initially developed upon the ABI7700, has provided similar results with other real-time PCR platforms such as the Lightcycler (Roche) and iCycler (data not shown). Similarly, the B6R assay, validated on the iCycler, is compatible with the ABI7700 real-time PCR technology (data not shown). Although validation was executed upon specific real-time PCR platforms, both assays are flexible and capable of using most available real-time PCR technologies and platforms, thereby allowing compatibility with laboratories regardless of which real-time PCR platform they possess.
During the 2003 US monkeypox outbreak, the E9L-NVAR and B6R assays provided reliable and sensitive identification of human monkeypox infections within a clinical context. Assay compatibility with multiple real-time PCR platforms allowed simultaneous testing of suspect samples for orthopoxvirus (E9L-NVAR) and MPXV (B6R) DNA. Interestingly, US MPXV isolates were distinct from previously characterized African Congo Basin isolates in both clinical manifestations and genomic sequences, more closely matching monkeypox isolates from West Africa (Likos et al., 2005;Reed et al., 2004). These relationships correlated well with the Ghanaian origin of the US monkeypox outbreak (Reed et al., 2004). The B6R assay was designed to detect Congo Basin MPXV; West African/US MPXV has one SNP within the B6R probe (Fig. 1). The lack of complete homology to the US monkeypox isolates did not adversely affect the detection of MPXV DNA within human samples (Table 5), confirming the B6R assay diagnostic utility for both known MPXV clades (Likos et al., 2005). Furthermore, monkeypox spread outside of Africa suggests these diagnostic assays may be relevant worldwide for identification of smallpox-like orthopox diseases.