ola virus have greater potential for variability among different strains than sequences that are common to the orthopoxvirus family. Therefore, additional sequencing of variola virus samples is critically important for development of the nucleic acid detection technology needed to identify variola virus, as opposed to other orthopoxviruses. There is no guarantee that an emerging strain would be represented in the archive, but understanding of the variation would assist in determining the relation of the new strain to known isolates. Moreover, the specific identification of variola virus would be a necessary feature of detection or diagnosis of variola virus infection should the precise source of the infection be unknown. The two variola major isolates that have been sequenced come from India and Bangladesh, within or near the Indian subcontinent, and one variola minor isolate comes from Brazil. The degree of similarity between these variola virus sequences and those of strains of variola from other parts of the world is unknown. Research on this issue—including sequencing of the entire genome or selected genome segments of additional isolates, or extended PCR and restriction fragment length polymorphism (RFLP) assay of entire genome DNA—should move forward as rapidly as possible.

It may be noted that additional sequencing and PCR/RFLP assay of other orthopoxvirus DNA sequences may be important for specificity issues. This is especially true for monkeypox virus DNA. Little is known about the range of DNA sequence variability among isolates of monkeypox virus.

Concerns analogous to those regarding DNA sequence conservation and variability would hold for protein- or antibody-based detection or diagnostic strategies. Some work on validating target sequences for sensitive or specific detection or diagnosis has been done at CDC and at VECTOR. The current state of that work needs to be critically assessed.

Environmental Detection

Environmental detection would involve instrumentation for sampling the environment. For example, a vacuum system with a contained filter trap could be developed for air sampling, and various adsorbents could be prepared for surface sampling. For the most part, any orthopoxvirus could be used in place of variola virus to devise suitable sampling techniques, assuming similar biophysical characteristics and stability (as appears to be the case). However, variola virus or possibly another live orthopoxvirus expressing variola virus surface protein(s) or containing variola virus DNA might be essential for final testing of a detection technology and strategy. For example, a filter coated with a variola-specific monoclonal antibody might trap variola virus. Identification of the virus might then rely on PCR for DNA testing or a second antibody for antigen testing. A solely protein-based detection assay, such as those using monoclonal antibodies in antigen capture, would be relatively insensitive, but might be faster



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