Use of Internally Controlled Real-Time Genome Amplification for Detection of Variola Virus and Other Orthopoxviruses Infecting Humans

Abstract

Smallpox, once a devastating disease caused by Variola virus, a member of the Orthopoxvirus genus, was eradicated in 1980. However, the importance of variola virus infections has been stressed widely in the last few years, particularly following recent social events in the world. Today, variola virus is considered to be one of the most significant agents with potential use as a biological weapon. In this study we developed an internally controlled real-time PCR assay for rapid detection and simultaneous differentiation of variola virus from other orthopoxviruses. The assay is based on TaqMan 3-minor groove binder (MGB) chemistry and uses generic primers, designed in highly conserved genomic regions of the crmB gene, and three TaqMan MGB probes designed to identify orthopoxviruses, variola virus, and an internal control. The results obtained suggest that the assay is rapid, sensitive, specific, and suitable for the generic detection of orthopoxviruses and the identification of variola virus and avoids false-negative results in a single reaction tube.Variola virus (VARV) is the etiological agent of smallpox, a severe and deadly disease that is considered one of the most serious human infections in the history of mankind. Humans are the only known reservoir of VARV, and no animal or insect reservoirs have been identified. VARV is a member of the family Poxviridae, subfamily Chordopoxvirinae, and genus Orthopoxvirus. Its genome consists of 186 kb of linear doublestranded DNA (25). The Orthopoxvirus genus includes other human pathogen viruses: Vaccinia virus (VACV) (used for immunization against smallpox disease), Monkeypox virus (MPXV), Cowpox virus (CPXV), and Buffalopox virus. Following the eradication of smallpox through a global immunization effort, there are still stocks of VARV held in the United States and the former Soviet Union (7). The importance of smallpox has been stressed widely in past years; moreover, VARV is currently considered to be a major biological weapon (2,14,26).Several molecularly based diagnostic methods to detect the DNA of VARV and differentiate it from other orthopoxviruses were published before September 2001. These methods were based on the analysis of the pattern lengths of the restriction endonuclease digestion products of previously amplified genomes (13,22).Soon after this date, as an alternative to these time-consuming methods, several real-time PCR protocols for the detection and identification of orthopoxvirus DNA were described (5,8,17,18,19,24).In this study we describe a real-time PCR based on TaqMan 3Ј-minor groove binder (MGB) chemistry (Applied Biosystems) to simultaneously detect VARV and differentiate it from other orthopoxviruses in a single reaction tube. Moreover the method uses an internal control to detect false negatives.A careful review of previously published methods for the real-time amplification of variola virus is also described. MATERIALS AND METHODSCell culture and viral strains. VACV, Western Reserve strain, was grown for 2 days on CV1 cell monolayers and...

Characterization of Two Historic Smallpox Specimens from a Czech Museum

“…Two real-time PCR assays amplifying sequences of the crmB and the fusion protein gene yielded VARV-positive signals. Specificity was achieved by use of a VARV-specific probe [ 18 ] and by using hybridization probes followed by melting curve analysis [ 19 ]. Based on the C t -values, the number of VARV copies was determined to be about 25,000/assay.…”

“…DNA was analyzed using Qubit 2.0 fluorometer (Thermo Fisher Scientific, Darmstadt, Germany) and a 2% agarose gel using standard protocols. One µL was used as template in two real-time PCR assays targeting sequences of the 14 kDa fusion protein gene and the cytokine response modifier B gene, respectively [ 18 , 19 ] and in a standard PCR assay targeting sequences of the hemagglutinin gene [ 20 ].…”

Synergistic Efficacy of the Combination of ST-246 with CMX001 against Orthopoxviruses

“…Delayed treatment may be the most important determining factor for the selection of antiviral compounds to pursue in light of the anticipated response time following bioterror events. If confirmed release of smallpox were to occur, detection may be accomplished in a matter of hours, but analysis of the susceptibilities of the isolates to antiviral drugs may take days to determine (6,25). Therefore, combination therapy would be highly useful to improve the likelihood of providing an effective therapeutic approach.…”

Variola: Smallpox

“…Fedele and colleagues [16] proposed an assay that targets the cytokine response modifier B (crmB) gene with OPV generic primers and two 5 nuclease probes modified by MGB. One MGB probe is designed to recognize VARV only, whereas the other detects OPVs generically.…”