ABOVE: Specimens in the basement of the Berlin Museum of Medical History of the Charité NAVENA WIDULIN, BERLIN

As the world’s most infamous flu pandemic (often referred to as the Spanish flu) raged from 1918–1920, scientists had very few tools available to help them combat or understand the disease. Researchers didn’t even know that a virus was responsible for the disease until the causal agent was finally isolated in a lab in 1930. In the years and decades that followed, improving technology has allowed researchers to look back and learn more about the often-fatal pathogen, but questions remain about the pandemic’s course, especially regarding how and why the virus changed over time.

Research published today (May 10) in Nature Communications fills in some of the gaps in that body of knowledge: researchers managed to extract viral genomes from tissue samples of people who caught the 1918 pandemic flu in different years to show how the virus mutated over time to adapt to the human immune system. They conclude that the virus may have evolved into the pathogen that circulated as a seasonal flu after the pandemic ended.

“The Spanish influenza from 1918 is still a big mystery and riddle because there are so many questions [about] what really happened at the time,” study coauthor Thorsten Wolff, a respiratory virus researcher at the Robert Koch Institute in Germany, told reporters at a press conference.

Studies of the nature and evolution of the 1918 pandemic virus have been limited by two major barriers: very few samples of the virus exist, and those that do are locked away in preserved, century-old specimens. Extracting them, experts tell The Scientist, is no small feat.

Colorized transmission electron microscope of the virus that caused the 1918 flu pandemic
Colorized transmission electron microscope image of the 1918 H1N1 flu virus

“When we started this work, there were only 18 specimens. . . . There was no genome-wide information about the early [stage] of the pandemic,” Sébastien Calvignac-Spencer, also of the Robert Koch Institute, said at the press conference. “Any new genome . . . can really add to our knowledge.” But, he added, “these specimens had and still have a terrible reputation” of being difficult to work with.

Working with 13 formalin-fixed lung samples that were collected from people who died in Berlin between 1913 and 1920 of the flu or other bronchial diseases, Wolff, Calvignac-Spencer, and their colleagues only managed to extract one complete and two partial genomes. But these included the first-ever genomes of the virus from before the pandemic’s initial 1918 peak, granting new insight into the genetic changes that the virus underwent as it first adapted to humans.

See “The Hunt for a Pandemic’s Origins

As the researchers compared the newly sequenced genomes to two previously sequenced genomes of the notorious flu virus from 1918, the researchers noticed that different mutations in the gene for the polymerase complex—the enzyme that mediates viral replication and is thought to be a major factor in the virus’s outsize pathogenicity—were prevalent in different years, allowing them to plot out a rudimentary history of the flu’s evolution over the course of its multiple waves.

“I think the coolest part of this paper was that they were able to pull these sequences out of these formaldehyde-fixed tissues,” Emily Bruce, a virologist at the Larner College of Medicine at the University of Vermont who didn’t work on the study, tells The Scientist, “and the fact that they found specific point mutations in the polymerase that they think contributed to changes in virulence as the pandemic progressed.”

The team was able to reconstitute that polymerase complex and link those mutations and others in the underlying gene to specific phenotypic changes that they say were adaptive responses. For example, the team identified multiple genetic alterations that, in some cases, nearly doubled the enzyme’s activity. By connecting the prevalence of mutations in a given specimen to the month and year of the person’s death, the researchers saw that mutations that increased disease severity coincided with the peaks of the pandemic, suggesting that the pandemic worsened after the virus adapted to its human hosts.

The team “found that there was a change in activity related to adaptive processes,” Wolff said at the press conference, indicating that “the virus is trying to optimize replication . . . in the human population.”

The “ability to take these archived medical samples and get this really interesting genomic data from them to help answer some really critical questions about the Spanish flu pandemic” was the “real takeaway from this paper,” says Emma Loveday, an influenza researcher at Montana State University who wasn’t involved in the study.

The researchers also suggest that the seasonal H1N1 flu virus that began circulating after the 1918 pandemic ended (and continued to circulate until supplanted by the 1957 H2N2 pandemic flu) is the direct descendant of the pandemic virus.

See “Plenty of Evidence for Recombination in SARS-CoV-2

Scientists have linked the 1918 pandemic flu to the subsequent seasonal flu before, but via different mechanisms. In the past, researchers have proposed that the seasonal H1N1 virus that emerged after the pandemic did so as the result of reassortment, the shuffling of genes among multiple viruses infecting the same host cell, meaning that the virus may have become less lethal after swapping genes with other flu viruses. The authors write in the new paper that their results don’t debunk the reassortment hypothesis; they simply show that H1N1 could have descended directly from the pandemic flu, no reassortment necessary.

A poster from 1918 with written instructions for how to prevent the flu from spreading.
A public health poster published during the 1918 pandemic with advice for stopping the spread of the flu

Loveday tells The Scientist that the idea that the seasonal H1N1 virus that followed the 1918 pandemic stemmed from the pandemic virus is well established, but the underlying mechanism was less clear.

Wolff and Calvignac-Spencer say that going forward, their goal is to extract and analyze genomes from more specimens so that they can flesh out what they describe as preliminary findings and answer more questions about the 1918 pandemic virus. Because there are no sequences from the 15 or so years’ worth of flu seasons following the pandemic, fully charting the evolution of the virus for now remains impossible.

“It is actually very, very difficult to [find] such specimens,” Calvignac-Spencer says. “We were crazy lucky to find a handful of those in the [Berlin Museum of Medical History] just around the corner.”

Bruce says that, hypothetically, it would be interesting to see what scientists could learn if they reconstituted the virus at different evolutionary stages, recreating the various mutations that occurred over time and testing their phenotypic effects in tissue cultures and live animals. “What do [the various genetic changes over time] mean in the virus’s ability to transmit or cause disease? We know the polymerase is an important determinant in virulence, but we don’t know exactly how,” she says.

But she concedes that doing so would require working in facilities with a higher biosafety level than the current study because the researchers only looked at the polymerase, and that there would be more regulatory and logistical hurdles to clear in order to study viable virus.

Bruce notes that the findings presage humanity’s current experience with COVID-19, and that they may help in understanding future pandemics as well. “What [the authors] are saying is these mutations may represent hallmarks of early adaptions to humans,” she says. The study shows “that this is the normal thing that’s going to happen: in a pandemic, you’re going to have waves,” in part because changes in the viral sequence are selected for.

“Today we still have very poor information on the genetic sequences over the course of the [1918] pandemic,” she adds. The data that do exist can help “us understand things that were maybe surprising to us in 2020.”