key: cord-261134-zarq507s
authors: Pulford, David; Meyer, Hermann; Brightwell, Gale; Damon, Inger; Kline, Richard; Ulaeto, David
title: Amplification refractory mutation system PCR assays for the detection of variola and Orthopoxvirus
date: 2004-02-13
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2004.01.001
sha: 
doc_id: 261134
cord_uid: zarq507s

PCR assays that can identify the presence of variola virus (VARV) sequences in an unknown DNA sample were developed using principles established for the amplification refractory mutation system (ARMS). The assay’s specificity utilised unique single nucleotide polymorphisms (SNP) identified among Orthopoxvirus (OPV) orthologs of the vaccinia virus Copenhagen strain A13L and A36R genes. When a variola virus specific primer was used with a consensus primer in an ARMS assay with different Orthopoxvirus genomes, a PCR product was only amplified from variola virus DNA. Incorporating a second consensus primer into the assay produced a multiplex PCR that provided Orthopoxvirus generic and variola-specific products with variola virus DNA. We tested two single nucleotide polymorphisms with a panel of 43 variola virus strains, collected over 40 years from countries across the world, and have shown that they provide reliable markers for variola virus identification. The variola virus specific primers did not produce amplicons with either assay format when tested with 50 other Orthopoxvirus DNA samples. Our analysis shows that these two polymorphisms were conserved in variola virus genomes and provide a reliable signature of Orthopoxvirus species identification.

The World Health Organisation (WHO) announced the eradication of variola virus (VARV), the causative agent of smallpox in 1980 and subsequently recommended that global vaccination should cease (Fenner et al., 1988) . Reference stocks of variola virus are currently maintained in WHO licensed repositories at the Centers for Disease Control & Prevention (CDC), Atlanta and Vector Laboratories, Novosibirsk. There is no information on possible unlicensed stocks that may be held elsewhere in the world, however the deliberate releases of a virulent anthrax strain in the USA in 2001 has highlighted the hazards that unlicensed stocks may represent. Today the majority of children and adults are not vaccinated against smallpox, and the consequences of a re-emergence of variola virus, by whatever means, would be far reaching without effective public health interventions (Gani and Leach, 2001; Meltzer et al., 2001) .

The clinical presentation of ordinary, haemorrhagic and flat forms of smallpox have historically been confused with monkeypox, chickenpox, meningococcal and other diseases that produce generalized skin lesions (Henderson et al., 1999; Breman and Henderson, 2002) . Laboratory diagnosis of smallpox can take several days if virus culturing is performed requiring biosafety level IV laboratories. A simple, reliable and sensitive diagnostic assay would offer improvement for effective medical and public health countermeasures in the event of a release. The first genetic techniques employed for specific identification of variola virus utilised restriction fragment length polymorphisms (RFLP) of viral genomic DNA (Mackett and Archard, 1979; Esposito and Knight, 1985; Dumbell et al., 1999) . This approach has been used extensively to differentiate between other Orthopoxvirus (OPV) species including vaccinia, cowpox, camelpox, monkeypox, mousepox or ectromelia viruses and for other members of the genus. RFLP of genomic DNA requires lengthy virus culture to generate suitable quantities of high quality DNA. Today, PCR based methods offer considerable improvement in sensitivity and specificity for diagnosis and the subsequent sequencing of a large number of Orthopoxvirus genomes (Goebel et al., 1990; Smith et al., 1991; Massung et al., 1994; Shchelkunov, 1995; Antoine et al., 1998; Shchelkunov et al., 1998 Shchelkunov et al., , 2000 Shchelkunov et al., , 2002 Gubser and Smith, 2002; Afonso et al., 2002) , has significantly increased the potential for developing new genetic based assays.

RFLP analysis of PCR products has been widely adopted for Orthopoxvirus species identification. RFLP of amplified A-type inclusion body protein gene sequences have been employed for differentiation of Orthopoxvirus species (Meyer et al., 1994 Neubauer et al., 1997 Neubauer et al., , 1998 . A collection of assays have been developed around genetic studies of the haemagglutinin (HA) protein, another Orthopoxvirus non-essential gene (Ropp et al., 1995) . RFLP examination of the cytokine response modifier B gene has also demonstrated that polymorphisms can be exploited by restriction endonuclease digestion (Loparev et al., 2001 ). An extensive RFLP survey of 45 strains has also been performed on a set of 10 kb pair amplicons that represent the entire virus genome (LeDuc et al., 2002) .

Recently, Lightcycler PCR of the HA gene was employed to amplify products from members of the genus, and could differentiate from other Orthopoxvirus species on the basis of melt curve analysis (Espy et al., 2002) . However, only a very limited number of strains were investigated in this study. Rapid assays based on fluorogenic Taqman PCR of the HA gene have demonstrated the utility of single nucleotide polymorphisms (SNP) for Orthopoxvirus identification (Ibrahim et al., 1997 (Ibrahim et al., , 1998 Sofi Ibrahim et al., 2003) . Rapid fluorogenic assays offer benefits for rapid diagnosis but require expensive equipment and reagents.

A variola-specific PCR assay was described that could differentiate between variola major and alastrim minor isolates based on the size of the amplified product . However, a recent evaluation of this PCR revealed that a subset of cowpox viruses also produced amplicons of the same size described for variola minor strains . Consequently, more sequence information on variola, vaccinia, cowpox, camelpox and monkeypox virus isolates is required to demonstrate the existence of species-specific sequences. Studies of Orthopoxvirus species to date have shown that unique genes are very rare, (Shchelkunov et al., 1998 (Shchelkunov et al., , 2000 (Shchelkunov et al., , 2002 Gubser and Smith, 2002) but species-specific SNPs are frequently observed in most genes. We have sequenced several genes from a large collection of Orthopoxviruses to identify virus specific polymorphisms and have observed that SNPs are usually the only reliable genetic elements for virus identification (Pulford et al., 2002) . Frequently no restricticion endonucleases exist that can exploit these unique markers by RFLP. To test this hypothesis, we constructed PCR assays based upon the amplification refractory mutation system (ARMS) (Newton et al., 1989) which is a sensitive technique for interrogating single base pair differences between DNA templates. ARMS has been used to detect a number of different variants of the hepatitis B virus (Gramegna et al., 1993; Liang et al., 1994) as well as identify genetic profiles of virulent, attenuated or vaccine strains of transmissible gastroenteritis virus and porcine respiratory coronavirus (Lai et al., 1995) . Using a modification of ARMS we have developed novel gel based single-tube assays which not only detect Old World Orthopoxviruses, but are also specific for variola virus. These multiplex assays employ three primers; two consensus primers generate an amplicon diagnostic of an Old World Orthopoxvirus and the third primer simultaneously binds to a variola-specific polymorphism and initiates extension of a shorter PCR product to detect the presence of variola virus. Examination of amplified PCR products by agarose gel electrophoresis allows the distinction of one (Orthopoxvirus present) or two (variola virus detected) PCR products. We show here the examination of these assays with DNA samples from an extensive panel including 43 variola and 50 other Orthopoxvirus isolates.

All variola virus DNA was prepared from strains kept at the WHO repository at the CDC, Atlanta, USA. The panel included DNA from 43 isolates obtained from five continents from between 1939 and 1977 (Table 1) . Variola virus isolates were grown on BSC40 cells whereas other Orthopoxviruses (Table 1) were grown on MA104 cells (Pulford et al., 2002) . Archived clinical tissue samples were recovered from liquid nitrogen.

All chemicals were purchased from Sigma Aldrich unless otherwise stated. Assays were initially developed using Orthopoxvirus DNA prepared from partially purified virus. MA104 cells infected with Orthopoxviruses were Dounce homogenised, centrifuged for 15 min at 1000 ×g and the supernatant was layered onto 10 ml of 36% (w/v) sucrose and centrifuged at 30,000 × g. The pellet was resuspended in 100 l 10 mM Tris pH 8.0, 1 mM EDTA containing 2.5 g proteinase K (Boehringer Mannhiem), 140 mM NaCl, 1% SDS and 1% 2-mercaptoethanol and incubated at 55 • C for 30 min. The sample was then phenol:chloroform extracted and the aqueous phase collected, 10 l 3 M NaCl was added and the genomic DNA was precipitated by addition of 2.5 vol. 100% ethanol and centrifugation for 10 min. The DNA pellets were washed with 70% ethanol, air dried, resuspended in 50 l distilled water and stored at +4 • C. All variola virus DNA samples prepared by the CDC were obtained from 0.1 ml of infected cell suspension. Samples were heat inactivated at 55 • C overnight and the samples were checked for sterility in tissue culture before processing with the Aquapure genomic lysis kit (Biorad) according to manufacturer's instructions. DNA prepared from clinical specimens was performed using the Aquapure genomic lysis kit for tissues according to manufacturer's instructions. Final DNA pellets were resuspended in up to 100 l of DNA hydration buffer and incubated overnight at room temperature before storage at 4 • C.

The variola virus specific primers were designed after compiling sequences for orthologs of the vaccinia virus Copenhagen A13L and A36R genes (Pulford et al., 2002) . Primers were designed to interrogate a specific polymorphism and specificity was increased by incorporating an adjacent mis-match base (Fig. 1 C 5 -AAGATTATTGTTGCCTCCTTTGAC-3 Antisense A13L3

S 5 -TGTTTCTGGAGGAGGCĀ gḠ G-3 Antisense A36R1

C 5 -TCTTATCACAGTGACCGTAGTTGC-3 Sense A36R2

C 5 -GTAATGAACGGATTTGACTTGCTAC-3 Antisense A36R3

S 5 -TTTGTTCATTACAATCATTATTTATTAGgC -3 Antisense

Control Templates (CT) A13L3CT 5 -TGTTTCTGGAGGAGGCĀ AḠ TTTAAATTCGGACT-3 A36R3CT 5 -TTTGTTCATTACAATCATTATTTATTAGCC CGCGTGCTTCCAG-3

Design at Primer 3 -end

Primer types include Orthopoxvirus consensus (C) and variola virus specific (S) oligonucleotides. Variola virus specific polymorphisms are shown underlined and mismatch bases introduced into each primer sequence are shown in lower case. et al., 1992) . A third Orthopoxvirus primer upstream of the variola-specific primer was included to generate a multiplex assay (Fig. 1) . The Orthopoxvirus generic product in both multiplex assays is larger than the variola virus specific amplicon.

High quality HPLC purified primer sets were used for multiplex and ARMS assays (Cruachem). Primers were designed with Oligo 6.0 software to have matching melting temperatures with minimum secondary structure and primer-dimer character. Ten micromoles working stocks of each primer were prepared and were added in varying ratios for optimal multiplex assay performance.

Positive control templates (CT) were generated from vaccinia virus IHD-J DNA by PCR using synthetic oligonucleotide primers that contained variola virus specific polymorphisms (Table 2 , underlined). The CT primers were combined with the consensus primer in a PCR and the products were purified using a Qiaquick column and diluted 1/10,000 before they were used as control samples in PCR (see Fig. 2 ).

All PCR reagents were purchased from Roche. Master mixes of reagents were prepared and 25 l volumes were dispensed with 1 l of sample DNA for PCR. Each A13L PCR assay included 200 nM of generic primers and 400 nM of variola-specific primer, whereas A36R PCR assays used 400 nM of generic primer and 200 nM of variola-specific primer. All PCR assays used 320 nmol of dNTPs (Roche), 3 mM MgCl 2 and 0.05 U/l Taq DNA polymerase (Roche). Variola virus PCR assays contained between 0.3 and 2.8 ng of infected cell culture DNA per reaction. 

camelpox virus CP1, (4) cowpox virus Brighton, (5) cowpox virus EP1, (6) cowpox virus Norway, (7) ectromelia virus MP1, (8) monkeypox virus Z1, (9) racoonpox virus VR838, (10) variola virus synthetic control template, (11) water and (M) 100 bp ladder (Roche). Ten microliters of PCR product was loaded on a 1% agarose gel.

All PCR assays were performed with an MJ Research PTC 200. Assays were optimised for specificity using the MJ 96V thermal gradient block and with a panel of Orthopoxvirus DNA or synthetic control templates. Multiplex and ARMS assays with variola virus genomic DNA were performed at the CDC, Atlanta using thermal cycling programs with predicted temperature algorithms. The optimised protocol for both assays was 94 • C 5 min; (94 • C 30 s, 60 • C 45 s, 72 • C 30 s) × 20 cycles; (94 • C 30 s, 60 • C 45 s, 72 • C 30 s + 10 s/cycle) × 10 cycles; 72 • C 10 min; +4 • C.

To evaluate the specificity of the multiplex PCR we tested the A13L and A36R multiplex assays with a small panel of Orthopoxvirus DNA samples (Fig. 2) . Synthetic control templates were used to confirm the functioning of the variola virus specific primer in each multiplex assay (lane 10). The shorter variola-specific amplicon was absent from all the Orthopoxvirus genome samples (lanes 1-9) but was produced by the control template (lane 10) confirm- ing assay specificity. The consensus primers for the A13L and A36R genes produced PCR amplicons for all the Old World (lanes 1-8), but not for the New World (lane 9) Orthopoxviruses. A more extensive survey was then performed to evaluate the reliability of variola virus specific amplification using a larger panel of Orthopoxvirus DNA samples. These cross-reactivity assays are summarised in Table 3 . Multiplex and ARMS assay formats were employed with 7 camelpox, 21 cowpox, 6 ectromelia, 8 monkeypox and 7 vaccinia virus isolates. In all assays performed no variola virus product was generated in the A13L or A36R ARMS or multiplex assay formats. However, the A13L consensus primers proved unreliable at amplifying a generic Orthopoxvirus product with some cowpox virus strains (Table 3) . 

To establish the specificity of the variola-specific primer we then performed some ARMS assays using a small panel of Orthopoxvirus genomes including variola genomic DNA (Fig. 3) . The combination of the A13L1 and 3 primers produced a 416 bp product with variola DNA (lane 1), but no product with any other Orthopoxviruses in this experiment (lanes 2-5). A similar result was obtained with the A36R1 and 3 primers which produced a 374 bp variola-specific product (lane 1), but no product with the other Orthopoxviruses (lanes 2-5).

The ARMS is an imperfect method for identifying SNPs because the absence of an amplified product reveals lit- tle information about the assay performance. Therefore, we tested all variola DNA samples with the multiplex assay format to report the presence of Orthopoxvirus DNA and to confirm if the hypothesised variola-specific SNPs were present. As anticipated, all the variola virus genomes, produced two PCR amplicons, the smaller band represented a variola-specific product and the larger band was an Orthopoxvirus generic amplicon (Fig. 4) . No bands were amplified from mock-infected BSC40 cell DNA in any assay (lane 25). A second panel of 23 more variola isolates was also tested with the A13L3 multiplex assays (Fig. 4, A13L multiplex b). Two PCR products were always amplified from all 43 variola strains tested demonstrating that the polymorphisms exploited by these multiplex assays were conserved.

To evaluate the performance of the multiplex for diagnosis we performed assays using DNA extracted from lesion or scab samples obtained from smallpox and chickenpox skin biopsies. These samples offered a significant test for the variola virus specific multiplex PCR because the DNA was of unknown quality being prepared from archived patient scabs collected during the eradication campaign. Seven separate variola virus isolates (Fig. 5, lanes 1-7) and one chickenpox (lane 8) sample prepared from scabs were compared using the A13L3 multiplex assay only. All seven biopsy samples produced two amplicons. No products were amplified from varicella zoster DNA (lane 8) confirming the suitability of this assay for diagnosis from clinical specimens.

This paper describes an easy method for performing fast, reproducible and specific PCR assays with the minimum of equipment or reagents. To achieve this goal we compared Orthopoxvirus ortholog sequences of the vaccinia virus Copenhagen A13L and the A36R genes using a panel of African and Eurasian viruses. These two genes both code for virus membrane proteins, and although they are present in all Orthopoxviruses so far sequenced, they display remarkable sequence heterogeneity (Pulford et al., 2002) . We exploited this diversity to design some new variola virus specific PCR assays.

The principles established for the ARMS PCR were exploited to design an oligonucleotide capable of specifically priming and extending from variola virus SNP's. The A13L-3 primer utilised two variola unique polymorphisms in close proximity and introduced a mismatch base A → G to generate further instability at the primer 3 -end (Table 2) . This primer produced a characteristic 416 bp amplicon with variola virus DNA (Fig. 3, lane 1) in the ARMS assay. Priming events from Orthopoxvirus genomes other than variola virus were inhibited strongly by this cluster of three non-matched bases at the 3 -end of the A13L3 oligonucleotide primer making it highly specific.

The A36R-3 primer contained a single variola polymorphism and an adjacent base mismatch C → G to create further instability at the 3 -end. This primer worked specifically with variola DNA in an ARMS assay when combined with the A36R-1 primer, and also worked specifically in a multiplex with all variola viruses tested. The consensus primer pair A36R-1 and A36R-2 faithfully produced a larger ∼523 bp amplicon with all Orthopoxviruses tested.

The quantity of shorter variola virus specific amplicons should normally exceed longer PCR products because they are synthesised more efficiently. However, the incorporation of a mis-match base into the variola-specific primers reduces their binding energy and subsequently the efficiency of product amplification (Fig. 4) . The A13L multiplex also revealed minor ∼1100 bp bands on gels with samples containing variola virus DNA only (Fig. 4) . The size of this extra band corresponds to the sum of the consensus and specific products together, and may represent a heteroduplex. The production of potential minor heteroduplex complexes was also observed for other multiplex assays we performed (not shown) which may be the result of the repetitive sequence elements identified in the A13L orthologs (Pulford et al., 2002) . Orthopoxvirus genes frequently contain repetitive sequence elements (Massung et al., 1996) . Consequently, the structural characteristics of this sequence might explain the low abundance of variola virus specific products in the A13L-3 multiplex assays. Knight et al. (1995) described a variola virus specific PCR assay that could differentiate between variola alastrim minor and major isolates based on the size of the amplified product. A recent evaluation of this published diagnostic method with a large panel of other Orthopoxviruses revealed that a subset of cowpox viruses also produce amplicons corresponding exactly with the size described for variola minor strains . We analysed the performance of the ARMS and multiplex A13L and A36R assays with the same cowpox virus subset (Table 3 , cowpox viruses EP-2, OPV89/1, OPV89/5, OPV90/1, OPV91/1 and OPV91/3) and observed that no variola-specific amplicons were produced from these or any other Orthopoxvirus DNA samples.

It is clear that the production of an ever-expanding Orthopoxvirus sequence database (http://www.poxvirus.org/ index.html) has given researchers the opportunity to produce many new assays (Espy et al., 2002) , but the real value of any diagnostic assay can only be provided by the quantity and diversity of the samples tested. The ARMS and multiplex assays described herein were performed and validated with a very large panel of variola virus and Orthopoxvirus strains to confirm that the SNPs probed by these assays are preserved and unique to variola virus genomes.

The clinical presentation of a generalised exanthum is not uncommon. Smallpox was often confused with a range of common skin conditions, and frequently chickenpox and monkeypox have been difficult to differentiate from smallpox at early times in clinical presentation (Fenner et al., 1988; Breman and Henderson, 2002) . The assays described here, were able to differentiate between DNA from smallpox, monkeypox and chickenpox and have demonstrated their reliability and specificity with a large collection of Orthopoxvirus samples, including clinical specimens. The application of these multiplex and ARMS PCR assays offer a simple and inexpensive alternative to the sequencing of amplicons generated from A13L or A36R orthologs for Orthopoxvirus identification.

The genome of camelpox virus

The complete genomic sequence of the modified vaccinia Ankara strain: comparison with other orthopoxviruses

Diagnosis and management of smallpox

A variant of variola virus, characterized by changes in polypeptide and endonuclease profiles

Orthopoxvirus DNA: a comparison of restriction profiles and maps

Detection of smallpox virus DNA by LightCycler PCR

Smallpox and its Eradication, WHO

Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene

Transmission potential of smallpox in contemporary populations

The complete DNA sequence of vaccinia virus

Detection of the hepatitis B virus major pre-core mutation by the amplification refractory mutation system technique

The sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox

Smallpox as a biological weapon: medical and public health management

The potential of 5 nucelase PCR for detecting a single-base polymorphism in Orthopoxvirus

Real-time microchip PCR for detecting single-base differences in viral and human DNA

Polymerase chain reaction identification of smallpox virus

The use of arms PCR and RFLP analysis in identifying genetic profiles of virulent, attenuated or vaccine strains of TGEV and PRCV

Smallpox research activities: U.S. interagency collaboration

Presence of hepatitis B and C viral genomes in US blood donors as detected by polymerase chain reaction amplification

Detection and differentiation of old world orthopoxviruses: restriction fragment length polymorphism of the crmB gene region

Conservation and variation in Orthopoxvirus genome structure

Analysis of the complete genome of smallpox variola major virus strain Bangladesh-1975

Terminal region sequence variations in variola virus DNA

Modeling potential responses to smallpox as a bioterrorist weapon

Amplification of 'variola virus-specific' sequences in German cowpox virus isolates

Sequence alterations within and downstream of the A-type inclusion protein genes allow differentiation of Orthopoxvirus species by polymerase chain reaction

Gene for A-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxviruses

Specific detection of mousepox virus by polymerase chain reaction

Specific detection of monkeypox virus by polymerase chain reaction

Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS)

Orthologs of the vaccinia A13L and A36R virion membrane protein genes display diversity in species of the genus Orthopoxvirus

PCR strategy for identification and differentiation of small pox and other orthopoxviruses

Functional organization of variola major and vaccinia virus genomes

The genomic sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact ORFs for immunomodulatory and host range proteins

Alastrim smallpox variola minor virus genome DNA sequences

Analysis of the monkeypox virus genome

Nucleotide sequence of 42 kbp of vaccinia virus strain WR from near the right inverted terminal repeat

Real-time PCR assay to detect smallpox virus

The authors wish to thank Gudrun Zoeller of the Institute of Microbiology, Munich, for her technical assistance. The authors would also like to acknowledge the help of Dr. Yu Li at the CDC for his advice.