Live Variola Virus Considerations for Continuing Research (2009) / Chapter Skim
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8 Methods for Detection and Diagnosis
8 Methods for Detection and Diagnosis
Pages 111-122
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From page 111... ...
Recovery of infectious virus from infected persons using tissue culture methods was feasible but was seldom used. Despite the eradication of smallpox, the need remains for robust and safe methods of detection of variola virus and diagnosis of the disease.
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From page 112... ...
Such advances in detection and diagnosis would facilitate forensic investigations to determine the source of variola virus in the event of an intentional release. This chapter reviews the current status of methods to detect variola virus and diagnose smallpox, relevant regulatory requirements, and the need for live variola virus to achieve advances in the development of detection and diagnostic capabilities.
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From page 113... ...
. The hemagglutinin gene has also been used to design primers for detecting all orthopoxviruses for use with a probe that can distinguish variola from other poxviruses by melting curve analysis, and tested on plasmid DNA (Espy et al., 2002)
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From page 114... ...
Multiplex PCR has also been performed using consensus and variola virus-specific primers based on known single nucleotide polymorphisms (SNPs) in A13L and A36R genes that are different in variola and other poxviruses; these SNPs were identified in PCR products from 43 variola strains but none of 50 other orthopoxviruses (Pulford et al., 2004)
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From page 115... ...
Protein-Based Methods Little work has been done to develop direct protein detection methods for variola. At present, these methods depend on developing antibody reagents that bind specifically to variola proteins that are distinct from those made by other orthopoxviruses.
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From page 116... ...
Specificity for geographically unrelated variola isolates would depend on defining a fully conserved and stable epitope or using a mix of monoclonals that would recognize epitopes in several unique variola virus proteins having no homologues or differing substantially from the related proteins in the other poxviruses. Even when well-characterized reagents are available, designing antigen detection methods that demonstrate the presence of viral proteins in patient materials has been challenging for many human pathogens.
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From page 117... ...
Ideally, tools for detecting the presence of variola virus in the environment would need to be rapid, portable, and easily deployable. Because poxvirus genome detection methods require relatively complex equipment and reagents, it would be necessary at present to bring materials suspected of containing variola virus to a laboratory facility.
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From page 118... ...
, including submission of clinical data to support marketing claims. The potential classification of IVDs for variola virus detection has not been established, although it appears likely, given the critical importance of accurate detection methods, that premarket notification including both general and special controls (Class II designation)
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From page 119... ...
One caveat related to variola detection and smallpox diagnosis is that genomic sequencing of enough geographically diverse isolates is necessary to ensure that PCR tests have adequate specificity. PCR and sequencing of the amplicons would be the first step in a forensic analysis of the source of a variola isolate should a reintroduction of the virus occur, and would also be accomplished most effectively if background information were available on the complete genome sequence of as many variola isolates as possible.
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From page 120... ...
2003. Real-time PCR assay to detect smallpox virus.
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2005. Neutralizing monoclonal antibodies cross-react with fusion proteins encoded by 129L of the ectromelia virus and A30L of the variola virus.
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2007. GeneChip resequencing of the smallpox virus genome can identify novel strains: A biodefense appli cation.
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