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Journal of Clinical Microbiology, April 2008, p. 1507-1509, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.00158-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Smallpox Virus Resequencing GeneChips Can Also Rapidly Ascertain Species Status for Some Zoonotic Non-Variola Orthopoxviruses

Irshad M. Sulaiman,^* Scott A. Sammons, and Robert M. Wohlhueter

Biotechnology Core Facility Branch, Division of Scientific Resources, National Center for Preparedness, Detection and Control of Infectious Diseases, Coordinating Center for Infection Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

Received 25 January 2008/ Accepted 1 February 2008

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We recently developed a set of seven resequencing GeneChips for the rapid sequencing of Variola virus strains in the WHO Repository of the Centers for Disease Control and Prevention. In this study, we attempted to hybridize these GeneChips with some known non-Variola orthopoxvirus isolates, including monkeypox, cowpox, and vaccinia viruses, for rapid detection.

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We used full genomic sequence information from 24 strains of smallpox virus (Variola virus) to develop a set of seven 30-kb smallpox virus resequencing GeneChips encompassing all genetic variations known at that time and resequenced 14 strains of this virus (3, 7). Some of the smallpox virus resequencing GeneChip sets are being held at the Centers for Disease Control and Prevention (CDC) for rapid characterization, should a future variola virus release ever occur.

Monkeypox virus, cowpox virus, and vaccinia virus are orthopoxviruses; are closely related to smallpox virus; and are recognized to cause zoonotic diseases. Recently, some of the strains of these viruses were documented in humans; a comparative genetic analysis with variola virus strains has led to speculation that these viruses might fill the niche vacated by smallpox virus eradication (3, 5). The present study describes our attempts to hybridize smallpox virus resequencing GeneChips with some non-variola orthopoxvirus isolates, namely, monkeypox virus, cowpox virus, and vaccinia virus, for their rapid identification and addresses the following question: can we garner useful information by hybridizing variola virus-related viruses to GeneChips designed to resequence variola virus variants?

The smallpox virus resequencing GeneChips were manufactured by Affymetrix (Santa Clara, CA). Viral DNA was extracted from infected cells and, in some cases, from purified virions, as described before (3, 5). To amplify a genome, 22 overlapping amplicons were amplified by a previously reported protocol (3, 4, 5, 7). The purified PCR amplicons corresponding to the genome segment covered by each of the seven GeneChips were pooled and hybridized by the manufacturer's protocol (Affymetrix). The hybridized GeneChips were washed, stained, and scanned as described previously (7). The values of scanned raw pixel array data files (.DAT) were integrated into cell intensity files (.CEL) with Affymetrix GeneChip operating software and were used to make base calls and to assign quality scores. The data were analyzed by two implementations, i.e., by use of Affymetrix GeneChip DNA analysis software and by use of the RA tools of the base-calling Abacus algorithm (2). The ClustalX program was used to align multiple sequences (8).

In this study, two strains of monkeypox virus (strains MPXV_RCG_2003_358 and MPXV_ZAI_1979_005) and one strain each of cowpox virus (strain CPXV_GER91_3) and vaccinia virus (strain VACV_ACAM2000) were hybridized with five different sets of smallpox virus resequencing GeneChips. Of these, one set was used to investigate each strain except for the cowpox virus strain, which was hybridized with two sets. Attempts were also made to hybridize a nonorthopoxvirus strain (severe acute respiratory syndrome [SARS]-associated coronavirus strain CDC200301157) with an additional set of smallpox virus GeneChips. The nucleotide sequences generated by the GeneChip sets were integrated and compared with those sequences previously obtained by capillary-based dideoxy sequencing on an ABI 3730 sequencer (3, 5). By using GeneChip hybridization, the data on the complete genome can be obtained within 24 h after hybridization of the purified PCR products.

The smallpox virus resequencing GeneChip set was designed to comprehend 24 major and minor variola virus strains, with the sequence of variola virus strain VARV_CHN48_horn tiled as the major reference or guide sequence; hence, the computational alignment features (insertion or deletions) calculated here are expressed relative to this sequence (7). In addition, a few unique sequences belonging to several non-variola orthopoxvirus strains, including cowpox virus, monkeypox virus, vaccinia virus, ectromelia virus, camelpox virus, and taterapox virus, were also tiled as minor reference sequences in these GeneChips (data not shown).

The call rates observed among the seven-GeneChip set varied for all three strains of orthopoxvirus hybridized. For two strains of monkeypox virus, there was no significant difference in the call rates; these ranged from 28.0% to 74.0%, with an average of 56.8%. The call rates for the VACV_ACAM2000 strain of vaccinia virus ranged from 26.8% to 73.0% across the GeneChip set. Comparable call rates (range, 24.6% to 71.0%; average, 48.6%) were observed in replicate hybridized GeneChip sets (Table 1). The high call rate (96.0%) for hybridization with these GeneChips observed for variola virus in our previous study and reported here for comparison represents a benchmark for a completely homologous sequence (7). The occurrence of lower call rates in non-variola viruses may be due to percent nucleotide identities between major references (variola virus strain VARV_CHN48_horn) tiled in GeneChips with respective non-variola virus genomes. The comparative analysis revealed a lower percentage of nucleotide sequence identity between major reference (variola virus) and non-variola virus genomes (for example, the nucleotide sequence identities were 81.8% with the MPXV_RCG_2003_358 strain of monkeypox virus, 75.7% with the CPXV_GER91_3 strain of cowpox virus, and 81.6% with the VACV_ACAM2000 strain of vaccinia virus). Nevertheless, the smallpox resequencing GeneChip set completely failed to hybridize with the genomic cDNA of a nonorthopoxvirus (SARS-associated coronavirus) isolate (Table 1). The high percentage of nucleotide identities (>90%) observed between the major reference strain and the other 14 strains of variola virus should be the reason for successful hybridization and the higher call rates observed (7). A similar result was also evident when the genomes of two SARS-associated coronavirus strains were hybridized with the Affymetrix SARS-associated coronavirus resequencing GeneChips (6).

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TABLE 1. Call rates observed among hybridized smallpox virus resequencing GeneChips with orthopoxvirus and nonorthopoxvirus strainsa

Given that our experimental design hybridized heterologous species against variola virus-specific sequence probes, a low call rate is to be construed not as a technical failure but as a rough measure of sequence mismatch. The chip-by-chip tabulation of call rates, as shown in Table 1, thus represents an average mismatch across the seven tandem regions of about 29,000 bases of the variola virus sequence, as apparent from GeneChip hybridization. Furthermore, hybridization data provide information not only about the average sequence mismatch but also about the patterns of match and mismatch across the entire genome, most conveniently captured in the Basic Local Alignment Search (BLAST) tool (1). The BLAST analyses of the test DNA used in this study against all orthopoxvirus genomes in the public domain unambiguously identified the test DNA as that of monkeypox virus, cowpox virus, or vaccinia virus.

The species-specific status of the non-variola virus isolates hybridized in this study was further confirmed by examining the hybridization results for their respective internal controls (for vaccinia virus, 22 species-specific minor instruction sets were tiled across the GeneChip set, and these numbers were 26 and 38 for cowpox virus and monkeypox virus, respectively); in all cases these small unique fragments of a genome showed complete concordance.

In summary, the smallpox virus resequencing GeneChip set that we initially developed for the rapid characterization of smallpox virus genomes can also identify close relatives of smallpox virus, namely, monkeypox virus, cowpox virus, and vaccinia virus, at the species level.

 
We thank Inger Damon and her associates, including Linda Stempora and Richard Kline, of CDC's poxvirus program, whose maintenance of the WHO Repository, propagation of viruses in the BCL4 environment, and extraction of their DNA have enabled these studies. We also thank Joseph Esposito and Michael Frace, who have led our team efforts of conventional sequencing of orthopoxvirus genomes and who have thus provided the "gold standard" against which the accuracies of GeneChip resequencing could be measured. We thank Carolyn Black for her critical comments on the manuscript.

The findings and conclusions in this report are ours and do not necessarily represent the views of the funding agency.

 
* Corresponding author. Mailing address: Biotechnology Core Facility Branch, Division of Scientific Resources, National Center for Preparedness, Detection and Control of Infectious Diseases, Coordinating Center for Infection Diseases, Centers for Disease Control and Prevention, Mailstop G-36, 1600 Clifton Road, Atlanta, GA 30333. Phone: (404) 639-1644. Fax: (404) 639-1331. E-mail: ISulaiman{at}cdc.gov

Published ahead of print on 13 February 2008.

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Journal of Clinical Microbiology, April 2008, p. 1507-1509, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.00158-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sulaiman, I. M. Articles by Wohlhueter, R. M. Search for Related Content PubMed PubMed Citation Articles by Sulaiman, I. M. Articles by Wohlhueter, R. M.