Antigenic subtyping has been applied for many years to classify viral infections and outbreaks. Elaborate networks of surveillance teams, for example, are monitoring the antigenic characteristics and epidemiology of influenza virus strains isolated from infected patients, to determine the best vaccine cocktail to use in the coming year. The advent of rapid sequencing techniques has opened the door to obtaining detailed fingerprints of viruses that could provide important clues as to their source. In general, a selected region of the viral genome is sequenced from numerous isolates and subjected to comparative phylogenetic analysis. Despite the increasing desire to rely on molecular analyses for all the answers, one should not overlook the fact that phenotypic features of the virus (tropisms, cytopathic effects, etc.) may provide important clues to facilitate identification.
As with other microbial species, the analysis of viral genomic segments can provide important clues as to the relatedness and origins of infections. Due to their small size and high rate of evolution, several considerations should be kept in mind when applying comparative molecular forensic analyses to viruses. First, for statistical reasons, it is usually advisable to look at as large a number of regions with variable sequence content as possible in the viruses that are isolated. Cost constraints, however, often make this impractical. Many viral genes, especially in RNA viruses, contain regions under rapid evolution and others that are under significant evolutionary constraint. In choosing a region of the viral genome to focus the analysis upon, it is important to consider that only rapidly evolving regions will provide enough useful variability when comparing agents from recently acquired infections. Second, if possible, it is extremely useful to include in the analysis a significant number of control viruses isolated from the surrounding population. This will allow for a full consideration of the background viruses in the local environment, and allow for a stronger statistical argument for relatedness to a predicted infection source. Finally, the overall strength of the argument for relatedness between two viruses requires detailed phylogenetic and statistical analyses that consider all alternative hypotheses. It will be important to get as close to "100%" certainty when one is performing the analysis for use in a legal rather than scientific context.
The tracking of HIV infections provides excellent examples of the successful application of molecular forensics to identify the source of a viral infection. In 1990, an HIV-positive dentist in Florida was suspected to be the source of HIV infection in six patients with no known risk factors. The sequencing of the env gene from viruses isolated from the doctor and patients strongly corroborated the epidemiological data that suggested the transmission route.1 In more recent cases, polymerase chain reaction (PCR) amplification of the HIV genes followed by phylogenetic analyses have been used to suggest HIV transmission from a surgeon to a patient2 and from a nurse to a patient.3 Molecular forensics has been applied to other viral infections in addition to HIV The nosocomial spread of specific strains of hepatitis C virus infections in hemodialysis units, for example, has been documented in several instances.4,5
The examples above involve molecular epidemiology of conventional viral pathogens. In order to rapidly identify viral agents involved in biocrimes, more work is needed to develop supportive resources. There is a clear need to establish an extensive sequence database of possible species. The identification of unique patterns and signatures beforehand will greatly facilitate the elabora tion of the strain and perhaps the source of the suspect agent. In the case of recombinant biowarfare agents, the bioengineered features along with the strain background should give reasonable clues to the source.
In closing, the goal of this chapter is to provide a background in the fundamentals of human virology and provide an overview of the utility and issues surrounding the use of molecular forensics and epidemiology to the world of virology. The rapid evolution of genomic technologies should continue to expand the capacity and impact of this exciting field. I hope that this chapter can be used as a stepping-stone to appreciate and give perspective to these advances.
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