The study began when Jesse Bloom, a biologist who studies flu viruses at the Fred Hutchinson Cancer Research Center, went downstairs for coffee and ran into Steven Pergam, a doctor at the same center. Bloom was interested in studying long-term flu infections, and Pergam mentioned that they happened to have a a freezer of snot samples from long-term flu patients. A decade ago, researchers at Fred Hutch had run a clinical trial on flu in cancer patients with bone-marrow transplants. The samples had been sitting in storage the whole time.
“We realized now that we have these powerful deep sequencing methods, we might be able to to repurpose these samples,” says Katherine Xue, a graduate student in Bloom’s lab. Deep sequencing is a technique for identifying rare mutations. Since the flu virus is evolving all the time, there are many different mutations among the millions of viruses in any single patient. If you go through the sequence only a few times in the sample, you’re only going to pick up the average virus. If you go through it at least 200 times, like Xue and her colleagues did, you’re going to see those rare mutations.
At first, the team didn’t know what to expect. The four patients in their study obviously didn’t have typical medical histories—given the cancer, bone-marrow transplant, and later use of the antiviral medication Tamiflu to combat their flu infections. But the team started to see some of the same mutations in the patients. The fact that these samples were old turned out to be fortuitous: The researchers could compare the mutations in these patients to flu variants that circulated years later. And again, they found similarities. For example, a particular mutation in three patients that altered the virus’s outer protein shell would become common in H3N2 viruses around the world by 2015.
To be clear, this is not because Seattle is some flu epicenter and these patients spread their H3N2 virus to the rest of the world. Rather, it suggests evolutionary pressures for the viruses in these individuals are the same pressures that apply on a global scale.
One way to think about this, says Katia Koelle, a virologist at Duke who was not involved with the research, is that the human immune system is always out to get flu viruses. The immune system makes antibodies that stick to the lollipop-shaped proteins jutting out of the virus’s shell. This neutralizes the virus. But if the very tip of the lollipop protein gets mutated, then the virus can escape the immune system. So that explains why viruses in these patients and around the world are acquiring mutations in the same places. Other molecular constraints make it so that it’s also often the same mutation in the same place.
Koelle’s lab has deep sequenced flu viruses in healthy people, who are sick for only a few days. The virus evolves in them, too. But, she says, “it’s hard for a new variant to get to high frequency in a short amount of time.” In other words, a beneficial new mutation may arise, but it may not have time to that variant to become common enough to spread another person before your immune system clears the infection.