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Discovering Novel Pathogens

Next-generation sequencing uncovers disease-causing microbes.
February 13, 2008

The next-generation sequencing technology that was harnessed to assemble the entire sequence of James Watson’s genome has been put to a new and potentially life-saving use: identifying novel pathogens. After several other identification techniques failed, the new sequencing approach was used to discover a never-before-seen virus that was likely responsible for the deaths of three transplant patients who received organs from the same donor.

Mystery microbes: A next-generation sequencing technique allowed researchers to identify the virus that likely killed three transplant patients who received organs from the same donor. Because the technique is “unbiased,” it could pick up the virus even though it was highly dissimilar at the nucleotide level to its nearest viral kin, lymphocytic choriomeningitis virus (LCMV). In the top panel, cells infected with the new virus have been stained using antibodies against LCMV and its relatives. In the bottom panel, individual virus particles (denoted with arrows) are revealed by electron microscopy.

The technique, called unbiased high-throughput pyrosequencing, or 454 sequencing, was developed by 454 Life Sciences, owned by Roche. This is the first time it was used to probe for the cause of an infectious-disease outbreak in humans, and experts say that it could ultimately usher in a new era in discovering and testing for agents of infectious disease.

“This is going to begin to allow us to understand the etiology of infections that had previously gone undiagnosed,” says Richard Whitley, professor of medicine at the University of Alabama at Birmingham, who was not involved with the research.

Last spring, several weeks after receiving organs from a single donor, three Australian transplant patients became ill with fever and encephalitis; within six weeks of the operation, all three had died. When traditional methods failed to identify the cause of the patients’ deaths, the Victorian Infectious Disease Reference Laboratory turned to W. Ian Lipkin, director of the Laboratory for Immunopathogenesis and Infectious Diseases at Columbia University’s Mailman School of Public Health, for assistance.

To find the mystery pathogen responsible for the deaths, Lipkin’s team extracted RNA from the tissues of two of the patients and prepared the sample by treating it with an enzyme that removed all traces of human DNA; this enriched the sample for viral sequences. The researchers then amplified the RNA into millions of copies of the corresponding DNA using a reverse transcriptase polymerase chain reaction (PCR). Usually, PCR requires some advance knowledge of the sequence in question because it relies on molecular primers that match the string of code to be amplified. But 454 sequencing avoids that problem by using a large number of random primers.

The resulting strands of DNA were sequenced using pyrosequencing, which determines the sequence of a piece of DNA by adding new complementary nucleotides one by one in a reaction that gives off a burst of light. Pyrosequencing allows for fast, simultaneous analysis of hundreds of thousands of DNA fragments. Although traditional pyrosequencing generally produces relatively short chunks of sequence compared with earlier sequencing techniques, 454 Life Sciences has improved upon the technology such that longer reads are possible.

When 454 Life Sciences used this technique to sequence James Watson’s genome, its approach was nearly identical. Lipkin’s modification was to eliminate human DNA so that only the mystery pathogen’s genetic material would remain.

Once the sequences were generated, Lipkin used computational techniques developed in his laboratory to filter out any remaining human sequences (which sometimes linger due to the presence of human RNA) and to piece together the many sequence fragments into longer strings. Of the more than 100,000 sequences initially produced, a mere 14 matched viral proteins in a database of all known microbes’ sequences.

“If we had used a different sequencing strategy–one that gives you shorter reads–or if we had not used the sample preparation to enrich [for viral sequences], we would never have captured those,” says Lipkin.

The virus from the patients’ tissues was most closely related to a pathogen called lymphocytic choriomeningitis virus (LCMV), which is known to cause meningitis in humans. While LCMV has been implicated in transplant-associated illness before, the sequence of the new virus was different enough that existing methods could not have detected its presence. The results of the analysis were published online last week in the New England Journal of Medicine (NEJM).

Once it had characterized the LCMV-like virus, the group was able to design probes to test specifically for its presence. The group found evidence of the virus in several tissue samples from all three transplant recipients.

Unbiased high-throughput pyrosequencing has become a critical tool in Lipkin’s lab, which is a member of the World Health Organization and helps train and equip public-health workers around the world. Lipkin has successfully used the technique to identify 20 viruses to date, including the Israel acute paralysis virus thought to be responsible for colony collapse disorder in bees. “There are all sorts of things that we’ve been able to identify using this approach,” says Lipkin. “It’s really quite powerful.”

Because the sequencing technique is not biased toward known organisms, it is ideally poised to track down previously unknown pathogens. “We’re finding the needle in the haystack, even without knowing what the needle looks like a priori,” says Michael Egholm, vice president of research and development at 454 Life Sciences and a coauthor of the NEJM report.

“There’s an enormous amount of uncharted territory in microbiology,” says Lipkin. As many as 40 percent of cases of central nervous system disease cannot be traced back to a specific culprit. For respiratory illness, the figure is 30 to 60 percent. In the United States alone, 5,000 deaths each year result from unidentified food-borne infections. “The advent of molecular tools like the one we’ve described here will be important in identifying the pathogenesis of a wide variety of diseases, acute and chronic,” says Lipkin.

According to Whitley, understanding the microorganisms that cause these diseases could lead to more effective treatments.

As powerful as 454 sequencing is for discovering new pathogens, it is not fast or cost efficient enough for use in routine screening of transplant tissue. But microbes discovered using this technique could be incorporated into existing screening techniques. “As we do more and more transplantation medicine,” says Lipkin, “it’s going to become critical that we find faster, more efficient, less expensive ways to screen to ensure safety.”

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