Viral Forensics the Schmidt Case

The value of phylogenetic analyses in forensics is well illustrated by the recent and curious criminal case of the State of Louisiana versus Richard J. Schmidt.50 The uncontested facts in this case are that a gastroenterologist from Lafayette, Louisiana broke into the home of his former mistress and office nurse late at night on August 4, 1994, and that he argued with her and gave her an intramuscular injection. He claimed it was a vitamin B shot. She claimed it was HIV She had begun feeling ill several months after the injection, and a blood test in January 1995 revealed that she had become infected with HIV She was a periodic blood donor, and based on tests of those previous blood donations, had a clear record without prior infection. She did not engage in any behaviors placing her at high risk for infection, and her sexual contacts over the previous nine years all tested negative for HIV He was a community leader and Vietnam War veteran. Schmidt admitted to having a long-term affair with her, but maintained the infection was not his doing.

She went to the District Attorney's office to file charges on learning that she was HIV-positive. Moving quickly, the DA's detectives obtained a search warrant and proceeded to the accused physician's office, where they seized his record books for blood samples drawn from patients and a vial of blood sitting in the refrigerator in a back room in his office. This was unusual; patients' blood samples were sent to the lab soon after being drawn, and none were routinely stored there. The physician claimed that this sample, drawn from one of his HIV-positive patients, was for his own use and research. Was the physician telling the truth? Might this blood sample link the physician to his nurse assistant and former mistress' infection? The next logical step in the investigation would clearly be phylogenetic analyses. Phylogenetic analyses of viral DNAs showing little or no relationship between HIV lineages from the nurse and the alleged source (the blood vial seized from the physicians office) could help demonstrate the physician's innocence, whereas a close, sister relationship among those lineages, in the context of an epidemiological sampling of HIV, would be consistent with the physician's alleged role in transmission.

Are phylogenetic analyses better than other, more routine methods of forensic analyses? In some instances, yes. Analyses based on similarity alone, such as comparison of genetic fingerprinting data in which restriction fragment patterns for hypervariable DNA sequences are compared, are subject to greater bias from similarity due to chance, known as homoplasy, rather than similarity due to common ancestry, known as homology. By contrast, phylogenetic analyses attempt, explicitly, to show the pattern of common descent among samples analyzed, rather than simple similarity. Phylogenetic analyses have the potential to show homoplasious similarity for what it is, convergence, and not to be misled, when the desired information is evolutionary relationships.

Portions of the HIV env gp120 gene and the RT genes were specifically chosen to be sequenced in an attempt to maximize the phylogenetic information in the dataset. The env gene evolves relatively quickly, being selected upon by hosts' immune systems, and is capable of recovering relationships among recently diverged HIV lineages. The RT gene evolves more slowly, due to greater functional constraints, and can provide insight into relatively older divergences. Using both sequences thus provides a broader range of evolutionary rates than either does alone.

Initial phylogenetic analyses were conducted using maximum parsimony with the software program PAUP,51 and final analyses were conducted using an explicit model for sequence evolution seeking to account for heterogeneity in rates of change across nucleotide sites and across virus lineages. This was done using the maximum-likelihood optimality criteria in a bayesian context with the program MrBayes.52 All analyses of the HIV env gp120 sequences from the nurse, the alleged source, and the epidemiological sampling of HIV patients were congruent in showing the HIV sequences from the victim to form a single monophyletic clade, in showing the alleged source to form a single mono-phyletic clade, and in showing those two clades to be closest relatives (sister taxa) relative to the epidemiological sample. This is consistent with the accusation that the physician used the blood sample from one of his patients to infect the nurse, although this rapidly evolving sequence provides no information regarding the direction of infection.

The more slowly evolving RT sequences also indicate their close evolutionary relationship, but with an additional and valuable piece of information. Based on RT sequences, viruses from the victim arose from within the clade of viruses from the alleged source. That is, the alleged source viruses are para-phyletic (incomplete in this case) unless the victim's viruses are included and nested within that group. This analyses does provide more direct evidence about the direction of infection, with the clear implication that viruses from the alleged source were used to infect the victim. Viral lineages from the alleged source diverged prior to divergences among the victim's viruses. This difference from the tree topologies based on env sequences can be traced to the slower rate of RT sequence change, with longer associated coalescence times for gene lineages, showing an earlier set of divergence events, as expected.

The phylogenetic analyses and rationale above are mainstream methods among evolutionary biologists; however, phylogenetic thinking and explicit use of evolutionary trees to track genealogy and transmission of virus or bacterial strains between individuals is not yet common in the U.S. courts. The case of the State of Louisiana versus Richard J. Schmidt set a precedent in this area. A pre-trial admissibility hearing was held regarding the proposed use of phylogenetic analyses in the criminal trial accusing Schmidt of attempted murder. Despite the efforts of the defense to block their admissibility, based on arguments that the viruses were evolving too rapidly to allow tracing of their shared ancestry (they were not), the judge ruled that phylogenetic analyses did meet judicial standards of admissibility, being subject to empirical testing, published in peer reviewed sources, and generally accepted within the scientific community.

Though not a panacea, phylogenetic analyses will prove useful in a range of forensic investigations. As with other molecular forensic approaches they can be particularly effective in demonstrating the innocence of accused individuals. They can be also be useful in tracing sources for any transfer of infectious materials whether viral, bacterial, or protozoan, involving accidental contamination or intentional infection in personal crimes or acts of terrorism. However, these applications are potentially limited by rates of sequence change, which must be sufficiently fast to provide a record of phylogenetic relatedness, but slow enough to preserve sufficient phylogenetic signal, prior to its being overwritten with multiple substitutions at individual sites. Application of phylogenetic analyses can also be complicated by the propensity of some viruses to recombine.

The defendant was found guilty of attempted murder and sentenced to 50 years in prison, the maximum allowable under the law. In this particular case, tried by jury, the phylogenetic evidence was consistent with the prosecution's case; however, there was other evidence that the jury may have found even more compelling. This included the physician having hidden the notebooks of his blood sampling and having a history of threats against the victim as she tried to end their affair. On March 4, 2002 the U.S. Supreme Court rejected an appeal of the verdict, thus establishing precedent for use of phylogenetic analyses in U.S. courts of law.

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