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5 Genomic Analysis U nderstanding the biology of variola virus and its genetic relatives is critical for developing countermeasures for smallpox, and genome sequencing is one of the most effective initial steps in achieving an understanding of the biology of any life form. The genome sequence is the “blueprint” that describes the entire suite of biological capabilities of any cellular organism or virus. With the recent rapid growth in genome sequencing capabilities, it has become increasingly clear that genomics (the study of genome sequences and of the functions they encode) can provide unexpected insights into host–pathogen interactions, the evolutionary his- tory of a virus, and evolutionary relationships among viruses. Poxvirus genome sequence analysis can be used to identify potential targets for the development of therapeutics and vaccines, and offers the promise of reveal- ing the molecular events underlying smallpox infection and pathogenesis and the host response. The last 10 years have seen enormous technological advances leading to much more rapid and much less expensive methods for genome sequencing. Today it is possible to sequence the complete genomes of all variola strains in both authorized repositories in less time and at a fraction of the cost required to sequence one variola virus genome in 1999. While Chapter 3 provided an overview of poxvirus genomics, this chapter presents a more detailed review of variola genomics and the progress that has been made in this area since 1999. Since then, a more refined understanding of the evolution and viral population structure of variola virus has emerged from genomic analysis. In addition, as with monkeypox virus strain analysis (Chapter 3), the sequencing of geographically distinct isolates of variola has 

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0 LIVE VARIOLA VIRUS yielded some important clues as to the viral determinants of human disease. This progress and these discoveries, as well as the remaining unanswered questions about the links between viral genetics and disease manifestations, serve as the foundation for the committee’s conclusion and recommenda- tion regarding variola genomics (see Chapter 10). The conclusion of the 1999 IOM committee on this subject provides context for the discussion that follows: genomic sequencing and limited study of variola surface proteins derived from geographically dispersed specimens is an essential foundation for important future work. Such research could be car- ried out now, and could require a delay in the destruction of known stocks, but would not necessitate their indefinite retention. The 1999 committee believed that the sequencing of multiple strains would provide greater understanding of genetic variation and variation in genome structure and content among strains, particularly at the termi- nal ends (where genes associated with pathogenicity and virulence often reside). This chapter reviews work done to date to analyze the genome of variola virus, the additional work on variola genomics needed to support the development of smallpox countermeasures and increase understanding of smallpox infection and pathogenesis and the host response, and the need for live variola virus in this work. SEqUENCE ANALySIS The genome of variola virus (VARV) contains approximately 186 kilo- bases of double-stranded DNA and approximately 200 nonoverlapping open reading frames (ORFs). Each end of the genome is covalently closed, and regions of inverted terminal repeats (ITRs) flank the central coding region. VARV is unique among the poxviruses in that its ITRs do not con- tain ORFs (Massung et al., 1996; Esposito et al., 2006). Work published on the genomics of variola virus since 1999 has been restricted largely to isolates held in the CDC repository. In work published in 2006, full genome sequences were determined for 43 geographically distinct VARV isolates held in the CDC repository. Several other variola genome sequences are also available at present, for a total of at least 49. These strains are not necessarily representative of the extant global variola virus population from the last half of the twentieth century, but were selected because they were isolated from cases of smallpox that occurred in geographically diverse regions of the world for which reasonably reliable epidemiological data and case fatality rates were available.

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 GENOMIC ANALYSIS The analysis of these 43 genome sequences, in combination with the two previously determined full genome sequences, revealed a high degree of conservation of centrally located coding region sequences (mid-CRS) among strains, supporting a role for these ORFs in ensuring the fitness of the virus through its life cycle (Esposito et al., 2006). Furthermore, the ter- minal CRS regions, adjacent to the ITRs, display variation among isolates with differing case fatality rates. Nearly 90 percent of VARV predicted ORFs can be identified in the genomes of other orthopoxviruses, with the remaining VARV ORFs being found as partial forms in other orthopoxvirus genomes (Esposito et al., 2006). Over the entire roughly 186,000 base genome, pairs of variola strains differ by as many as about 700 single nucle- otide polymorphisms (SNPs) and about 90 insertions or deletions (indels), and by as few as a handful of each (overall, among all viruses, there are 1,782 specific SNPs and 4,812 specific indels). Taken together, these data indicate restricted variability in the overall genome sequence and support the notion that the terminal CRS regions contain ORFs important for host interaction and pathogenesis, while the mid-CRS-region ORFs are critical for expressing conserved proteins important to virus replication (Esposito et al., 2006; Moss, 2007). Phylogenetic analysis of these epidemiologically distinct VARV isolates, isolated from patients over a period of 30 years, reveals two primary clades of VARV with distinct clustering based on the geographic region from which the source patients derived. One clade (“A” in Figure 5-1) includes variola major virus isolates from Asia that were associated with clini- cally severe (high case fatality rate) cases of smallpox and variola isolates from east, central, and southern Africa associated with disease of variable severity. The other clade consists of two subclades, one comprising alas- trim minor isolates from South America (“B” in Figure 5-1), which were associated with mild smallpox disease, and the other (“C” in Figure 5-1) comprising variola isolates from west Africa that were associated with intermediate disease severity (Li et al., 2007). The tendency of strains to cluster based on geography has provided clues as to how the virus spread among humans around the world. Some relationships between variola genome sequences and disease severity have been identified, although these associations are only broadly defined at present because the range of case fatality rates within some clades is large. For example, the case fatality rates of isolates from Asian clade C range from <1 percent to 38 percent, compared with 8 percent to 12 percent for west African clade A and 0.8 percent for South American clade B. Furthermore, both viral and host features, such as age and nutritional status, are important in determining clinical outcome. Based on genome sequence comparisons, variola virus is most closely related to camelpox and taterapox viruses, with which it shares approxi-

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 LIVE VARIOLA VIRUS FIgURE 5-1 Variola virus evolutionary relationships based on an alignment of a conserved mid-region of genomic DNA sequences of 45 isolates (from Esposito et al., 2006; Figure 3). The isolates are from smallpox case-patients in west Africa (clade A), South America (clade B), and Asia (clade C); the Asian clade C includes a subgroup of non–west African African variants. Case fatality rates associated with some isolates are indicated in parentheses. SOURCE: Esposito et al., 2006. Reprinted with permission from AAAS. mately 98 percent overall sequence similarity (Esposito et al., 2006). VARV probably arose from an ancestral rodent-associated variola-like virus in Africa between 16,000 and 68,000 years ago (Li et al., 2007). The data also indicate a different evolutionary history for the mid-CRS and terminal CRS regions of the VARV genome among the different clades (Esposito et al., 2006; Li et al., 2007). According to WHO (Lavanchy, 2008; WHO, 2008), 891 isolates (120 strains) were held at the authorized repository at VECTOR until 2008, when it was reported that 200 nonviable and duplicate samples were destroyed. It is not clear whether or to what degree the remaining 691 isolates (120 strains) at VECTOR and the 406 unsequenced isolates (184 unsequenced strains) at CDC offer novel features not found among the at least 48 isolates sequenced thus far (2 sequenced prior to 2000 and

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 GENOMIC ANALYSIS 47 since then; 1 sequenced twice [Bangladesh, 1975]). However, it is rea- sonable to assume that additional diversity would be discovered if these strains were sequenced. In comparison with the significant effort required to determine a com- plete poxvirus genome sequence in 1993, the effort necessary today to sequence all remaining variola isolates would be relatively minor. In fact, if DNA were made available, current next-generation sequencing technol- ogy would enable the determination of complete genome sequences for all remaining variola strains in a total of several weeks by one laboratory, at a low cost. Furthermore, it should be feasible to obtain sufficient DNA from each strain using current whole-genome amplification techniques, obviating the need for in vitro cultivation of these strains for the purpose of genome sequencing. Since fewer African than Asian isolates have been sequenced, obtaining more genome sequence information about these isolates should be a priority. Given these advances, the scientific benefits of sequencing all remaining isolates today vastly outweigh the costs. BEyOND gENOMIC ANALySIS Significant progress has been made in poxvirus genomics. More than 111 poxvirus genome sequences are now available (http://www.poxvirus. org/), and 48 VARV genomes are available for public access in GenBank (http://www.ncbi.nlm.nih.gov/Genbank/). Further, plaque phenotyping and assessment of comet morphology for the sequenced VARV isolates have been completed, deepening understanding of the relationship between the biological properties of VARV isolates and their genome sequences (Olson et al., 2009). Analyzing VARV genome sequences yields insights into viru- lence; greatly improves the reliability of nucleic acid-based detection and diagnostic assays (see Chapter 8); and makes it possible to begin to under- stand better the biology of this virus, facilitating the development of new therapeutics (see Chapter 6). However, meaningful exploitation of genome sequence requires an assessment of functional attributes. Additional work is necessary to under- stand the molecular mechanisms of viral pathogenesis and replication in order to support the development of effective countermeasures and means of detection. Among the possible approaches are targeted assessments of genes or gene products of interest, as well as genome-wide “functional genomics” methodologies, such as analysis of (virus and host) genome-wide transcript and small RNA abundance, profiling of proteins and phosphoproteins, and analysis of protein–DNA binding patterns. Recent advances in computa- tional methods allow identification of gene networks, metabolic pathways, and genetic modules and nodes (Litvin et al., 2009), all of which may reveal novel, critical targets for therapeutic intervention in both virus and host.

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 LIVE VARIOLA VIRUS Additional insights into VARV pathogenesis will come from charac- terization of the VARV proteome, the entirety of the proteins expressed by VARV. Work of this sort has been undertaken with vaccinia virus and other orthopoxvirus proteins (Chung et al., 2006; Resch et al., 2007). However, the ability to extrapolate findings from vaccinia virus to variola virus is unclear, but almost certainly limited. NEED FOR LIVE VARIOLA VIRUS In the past, genome projects have required large amounts of high-quality genomic DNA, which in turn has usually necessitated propagation of the agent to high titer in the laboratory. Today, however, the ability to isolate and amplify DNA from microbes is greatly improved, such that adequate DNA can be generated from a single bacterial cell for full-genome projects (Marcy et al., 2007). In general, live variola virus is not needed for variola genome sequence analysis as long as DNA of adequate quantity and quality is available. The latter need can be met either by cultivation and DNA har- vesting or by DNA amplification methods, the products of which can be saved in the form of genomic clones or amplified DNA. On the other hand, live variola virus would be needed to perform functional studies (such as studying RNA or protein expression or host interactions) for the purpose of understanding pathogenesis so as to identify new targets for therapeutics. REFERENCES Chung, C.S., C. H. Chen, M. Y. Ho, C. Y. Huang, C. L. Liao, and W. Chang. 2006. Vaccinia virus proteome: Identification of proteins in vaccinia virus intracellular mature virion particles. Journal of Virology 80(5):2127–2140. Esposito, J. J., S. A. Sammons, A. M. Frace, J. D. Osborne, M. Olsen-Rasmussen, M. Zhang, D. Govil, I. K. Damon, R. Kline, M. Laker, Y. Li, G. L. Smith, H. Meyer, J. W. Leduc, and R. M. Wohlhueter. 2006. Genome sequence diversity and clues to the evolution of variola (smallpox) virus. Science 313:807–812. Lavanchy, D. 2008. WHO Smallpox program: Live variola virus. Presentation to the Com- mittee, December 18–19, 2008. Li, Y., D. S. Carroll, S. N. Gardner, M. C. Walsh, E. A. Vitalis, and I. K. Damon. 2007. On the origin of smallpox: Correlating variola phylogenics with historical smallpox records. Proceedings of the National Academy of Sciences of the United States of America 104:15787–15792. Litvin, O., H. C. Causton, B. J. Chen, and D. Pe’er. 2009. Modularity and interactions in the genetics of gene expression. Proceedings of the National Academy of Sciences of the United States of America. [Epub ahead of print]. Marcy, Y., C. Ouverney, E. M. Bik, T. Lösekann, N. Ivanova, H. G. Martin, E. Szeto, D. Platt, P. Hugenholtz, D. A. Relman, and S. R. Quake. 2007. Dissecting biological “dark matter” with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. Proceedings of the National Academy of Sciences of the United States of America 104:11889–11894.

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 GENOMIC ANALYSIS Massung, R. F., V. N. Loparev, J. C. Knight, A. V. Totmenin, V. E. Chizhikov, J. M. Parsons, P. F. Safronov, V. V. Gutorov, S. N. Shchelkunov, and J. J. Esposito. 1996. Terminal refion sequence variations in variola virus DNA. Virology 22 (2):291–300. Moss, B. M. 2007. Poxviridae: The viruses and their replication. In Fields’ virology, 5th edi- tion, edited by B. N. Fields, D. M. Knipe, P. M. Howley, and D. E. Griffin. Philadelphia: Lippincott Williams & Wilkins. Pp. 2906–2946. Olson, V.A., K. L. Karem, S. K. Smith, C. M. Hughes, and I. K. Damon. 2009. Smallpox virus plaque phenotypes: genetic, geographical and case fatality relationships. Journal of General Virology 90(Pt 4):792–798. Resch, W., K. K. Hixson, R. J. Moore, M. S. Lipton, and B. Moss. 2007. Protein composition of the vaccinia virus mature virion. Virology 358(1):233–247. WHO (World Health Organization). 2008. WHO Advisory Committee on Variola Virus Research Report of the Tenth Meeting. November 19–20. Geneva, Switzerland: WHO. http://www.who.int/csr/resources/publications/WHO_HSE_EPR_2008_9/en/index.html (accessed March 23, 2009).

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