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CDC Researchers Use Sequencing, Phylogenetics to Identify Novel Virus

NEW YORK (GenomeWeb) – Researchers from the US Centers for Disease Control and Prevention have identified a novel virus linked to a man's 2014 death following multiple tick bites.

As the CDC's Erin Staples and her colleagues reported in Emerging Infectious Diseases, they used next-generation sequencing combined with phylogenetic analysis to trace the unknown virus  — dubbed Bourbon virus for the county in Kansas where the patient lived — to the genus Thogotovirus. This is, the researchers noted, the first time a virus belonging to this group has been associated with disease in humans, though there have been seven symptomatic Thogotovirus infections reported.

"The discovery of Bourbon virus, in addition to recent discoveries of tick-associated Heartland and severe fever with thrombocytopenia syndrome viruses, suggests that the public health burden of these pathogens has been underestimated," Staples and her colleagues wrote. "As nonselective molecular methods of pathogen identification (i.e., [next-generation] sequencing) become more widely used, ideally in combination with classical microbiologic techniques, it is anticipated that similar discoveries will be made in the future."

The patient, a man older that 50 years, came down with nausea, weakness, and diarrhea a few days after being bitten by ticks while working outdoors. His symptoms worsened, and he was treated with doxycycline for a presumed tick-borne illness, though his health continued to deteriorate. The patient died 11 days after becoming ill.

Samples from the patient, the researchers noted, were tested for Rocky Mountain spotted fever, Lyme disease, and ehrlichiosis, and came back negative as did tests for hepatitis B and C viruses, West Nile virus, HIV, and a number of other viruses.

However, testing for antibodies against Heartland virus, a virus first described in 2012, using plaque reduction neutralization indicated the presence of another virus.

To identify this unknown virus, Staples and her colleagues performed next-generation sequencing on cell culture supernatants using Thermo Fisher Scientific's Ion Torrent PGM platform. These viral bits had about 70 percent identify in common with Dhori virus, a tick-borne Orthomyxoviridae. Using real-time RT-PCR, the researchers verified the presence of this novel virus in blood and serum samples. The high levels of this virus in the patient's samples suggested that it was behind his illness and death.

The investigators constructed three phylogenies using a neighbor-joining approach with 2,000 bootstrap replicates that placed this novel virus near the Dhori and Batken viruses, in all three trees. The branch lengths, the researchers noted, were quite long, indicating a distant evolutionary relationship. While the Dhori and Batken viruses have been found in tick species, these viruses have only been observed in the Eastern Hemisphere.

The investigators are also searching for additional Bourbon virus infection cases and are examining its potential distribution, vectors, and reservoirs.

"These data will be critical to further characterize the epidemiology and illness caused by Bourbon virus and to implement potential prevention and control measures," they said.