The recipient of the 2018 Crow Award reveals details of flu evolution at the smallest —and largest—scales.

For many viral diseases, a vaccine can provide lifelong protection. But for flu, you need a new shot every year. The influenza virus evolves so fast it presents a constantly moving target for both our immune systems and public health authorities, fueling epidemics like the particularly bad season we just endured. With over 30,000 people hospitalized in the United States alone this season, the flu provides a dramatic reminder of the importance of understanding evolutionary dynamics.

Katherine Xue, a graduate student at the University of Washington, is revealing the mechanics of influenza evolution on scales ranging from an individual person up to the entire planet. Xue was awarded the 2018 James F. Crow Award for Early Career Researchers for her doctoral work on the subject after a presentation at the Population, Evolutionary, and Quantitative Genetics Conference in May.

In a special session of talks by Crow Award finalists, Xue spoke about using deep sequencing to examine diversity in flu virus populations.

“Up until recently, we were only able to look at the average genetic identity across the millions or billions of flu viruses in a single infection,” says Xue. “We’ve used deep sequencing to show that within a single infection there are fast evolutionary dynamics that have been invisible to previous technologies.”

Xue approaches clinical topics with the conceptual tools developed by evolutionary and population geneticists. Linking ideas across fields is characteristic of Xue, says her mentor Jesse Bloom (Fred Hutchinson Cancer Research Center / University of Washington), whether it’s between medicine and evolutionary theory or between science and the humanities.

“What makes Katherine stand out is her ability to think about big scientific concepts and connect ideas,” says Bloom. “She’ll see and connect ideas in ways that I can’t.”

Viral cooperation

When Xue first rotated in Bloom’s lab, she was working on a molecular virology project about how a particular viral protein binds to a cell. In the course of examining sequence databases, she noticed the mutation she was studying was often ambiguously annotated.

Inspired by this hint of population diversity, she wondered whether the two viral variants might interact with each other. She was able to establish that the mutation tended to occur alongside the wild type version within a population; the mutation was deleterious to virus reproduction on its own but beneficial when mixed with wild-type. This example of cooperation suggests that interactions between different variants within flu populations can be important factors in virus evolution.

A glimpse of evolution in action

But can evolution be detected within the virus population of a single individual? Xue was drawn to the question of how global flu evolution traces back to the founding infections in which each mutation must first arise.

“I was intrigued because it was hard to imagine how this works,” says Xue. “Flu infections are very short; there’s not a lot of time for a new mutation to reach frequencies large enough to ensure it makes it over to the next infected person.” In the language of population geneticists, flu populations are repeatedly subjected to extreme bottlenecks. But observing such rapid evolution in action is extremely challenging.

Xue and her colleagues in the Bloom lab used a unique approach to get around this problem. They partnered with clinicians Michael Boeckh and Steve Pergam at the Fred Hutchinson Cancer Research Center, who had collected samples from four immunocompromised patients over the course of their months-long flu infections. Deep sequencing these samples gave them an in-depth view of a process that would normally be finished within days in a person with healthy immune defenses.

“I initially had doubts that this project would show us anything interesting or be worth doing,” says Bloom. “But Katherine is very independent and persistent, and she kept going despite my occasional words of discouragement.”

The results were dramatic. Over the span of about two months, there was a substantial amount of flu evolution within each patient. Mutations arose regularly, fluctuated in frequency, and even became fixed in the population in a few cases. They also saw evidence that some of these changes are due to selection. The same mutations would often arise independently and then rise to substantial frequencies in multiple patients, suggesting these particular changes were adaptive.

Remarkably, the mutations that arose repeatedly in different patients were sometimes the same mutations that spread through the global flu population within the next decade. The immunocompromised patients seemed to be microcosms of global evolutionary patterns. “We were astonished,” said Xue.

Most of these recurring mutations affect the part of the flu haemagglutinin protein that is most recognized by the host immune system, so the team hypothesizes that the changes help the flu escape host defenses.

These results raise many questions about how evolutionary dynamics interact across scales. How far do the conclusions generalize? Where and when do natural selection and genetic drift act? How do normal week-long infections generate enough diversity to fuel rapid global evolution? Could understanding these processes translate to better flu season predictions? Xue’s graduate research continues to explore flu evolution with these questions in mind.

Connecting ideas

The scientific big picture is never far from Xue’s mind, it seems. Alongside her thesis research, Xue is pursuing a certificate in science and technology studies, with a capstone project on the history of flu research. “I have really loved being part of the history, philosophy, and sociology of science community here,” she says. “It’s given me a lot of perspective that has been really enriching.”

A few years ago Xue helped start the UW Genomics Salon, which is a group of students and postdocs who take part in freeform discussions about the intersections of science and society. These discussions touch on policy, advocacy, communication, education, representation, law, art, and a host of other topics.

Bloom thinks Xue’s broad interests are yet another reflection of her creativity and ability to link ideas across fields. Before graduate school, she spent a few years working as a science writer for the Harvard Magazine. “She’s an exceptionally good science communicator and very dedicated to creating connections between science and other fields. It’s pretty awesome to have someone like that around!”

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Watch #PEQG18 presentations from all the other  outstanding finalists for the Crow Award here.

Guest post by Nina Wolff pays tribute to long-standing GSA member Margaret Lieb.

Margaret (Peggy) Lieb died on March 8, 2018 in South Pasadena, California at the age of 94. After attending schools in New Rochelle, NY, she graduated magna cum laude from Smith College, and subsequently studied with  H.J. Muller at Indiana University and with Francis Ryan at Columbia University, where she received her PhD degree. Following postdoctoral studies at Caltech in the laboratory of Max Delbruck, and in Paris at the Pasteur and Radium Institutes, Lieb taught at Brandeis University and then moved to the Medical School of the University of Southern California where she continued her research and teaching for 45 years. After her retirement, she continued to be active as an Emerita member of the faculty, and as a garden docent at the Huntington Museum and Botanical Garden.

While at Caltech, Lieb published one of the first studies of phage lambda, and subsequently isolated and characterized a large number of mutations in the repressor gene of the phage. Her studies of lysogenization indicated that the active repressor was a dimer, a conclusion later confirmed by biochemical studies in other laboratories. While mapping mutations in the lambda repressor gene, she observed that excess recombination (negative interference) was associated with mutations arising from the deamination of 5-methylcytosine. This led to the identification of a novel mismatch repair gene (vsr) in E. coli – a gene that is adjacent to the gene for cytosine methylase. The Vsr function reduces the probability of mutations that occur due to spontaneous deamination of 5meC. Although genes related to vsr appear to be limited to bacteria, the search for genes like vsr in eukaryotes, where 5-methycytosine has important regulatory functions, has led others to the discovery of additional specific repair activities in higher organisms.

In 1972-1973, Lieb served as Program Directory of the Genetic Biology program of the National Science Foundation. She was elected Chairman of the Virology Division of the ASM in 1975, and served on the editorial boards of Journal of Virology and GENE. She was a Fellow of the American Association for the Advancement of Science (AAAS).

Peggy Lieb maintained an active interest in the research of her colleagues, and will also be missed by the students and post-doctoral fellows who spent time in her lab. Her high standards of performance in the classroom and in the lab were challenging and also appreciated by those who knew her.

Sequencing a genome is not as simple as reading a book. All those neatly lined up letters are the final product of a complex process made up of many intricate steps that can—and do—go wrong. In a report published in G3: Genes|Genomes|Genetics, Peccoud et al. put their painful sequencing experiences to good use providing new insights into a common sequencing problem: artificial chimeras.

Sequencing typically requires cutting up genetic material into fragments. These fragments are then amplified by PCR, and these amplified fragments are then sequenced. The end result is millions of short sequences, called reads. These reads can then be aligned to a reference sequence to identify changes like recombination and mutations.

The authors of the G3 study originally set out to identify recombination events between dengue virus and its host mosquito. They sequenced RNA from virus-infected mosquito cells, and they added pillbug RNA to a separate batch to serve as a control. Unexpectedly, the authors found virus-mosquito and virus-pillbug recombinant reads at similar frequencies. Since the virus RNA had never been in contact with the pillbug RNA before the sequencing procedure, they concluded that most, if not all, of these recombination events must have happened during the amplification or sequencing steps.

False-positives are always disappointing, but instead of giving up, the authors used their data and data from previous studies to better understand how the artificial reads occurred, as well as to learn how to better filter them.

This investigation revealed certain characteristics that are shared by both real and fake recombinant reads, including microhomology around the recombination junction. Crucially, they found that biologically-generated recombination almost always joins sequences in the same orientation, whereas artificial recombinant reads are often joined in opposite directions. The authors explain that this is likely due to template switching during the PCR step of sequencing.

Knowing the traits of false-positive reads may allow researchers to more carefully filter their data in future studies, ensuring they get the most accurate information possible—and knowing that what appears to be a dead end can still yield useful insights may help graduate students sleep better at night.

CITATION

A Survey of Virus Recombination Uncovers Canonical Features of Artificial Chimeras Generated During Deep Sequencing Library Preparation

In the early 1940s, many biologists doubted bacteria had genes. After all, they seemed to play by their own genetic rules: they appeared to lack chromosomes, meiosis, mitosis, sex, and all the other trappings of Mendelian inheritance. They even seemed to show a kind of Lamarckian inheritance, in which an individual could pass on traits acquired during its lifetime.

Then, in 1943, Salvador Luria and Max Delbrück published an article in GENETICS that marked the birth of bacterial genetics, revealing that this apparently Lamarckian inheritance was in fact a case of random mutation. Luria and Delbrück’s work wasn’t just a boon for bacterial genetics. Brought together by biophysics and war, the duo’s brief decade of collaboration also fostered the emergence of molecular biology by developing the viruses that infect bacteria into a streamlined genetic model.

In the February issue of GENETICS, Andrew Murray introduces the 1943 paper as one of the journal’s 100th anniversary Classics.

The roots of Luria and Delbrück’s partnership traced back to a paper published just as war was brewing in Europe in 1935. Luria was in Italy, a newly qualified doctor who was bored with medicine; Delbrück was in Germany, a young physicist who had grown bored with physics. Delbrück had collaborated with some biologists from a discussion group—his “little club”— contributing a quantum mechanical model of the gene to a paper on mutations and gene structure. Luria read the paper a few years later when he was in Rome completing a specialty in radiology and dabbling in physics under soon-to-be-Nobel-Prize-winner Enrico Fermi’s wing. Luria’s imagination was sparked by the paper’s proposal that the abstract concept of a gene, which was then still hazy with mystery, could be investigated as a concrete “collection of atoms.” It seemed, he would later say, “to open the way to the Holy Grail of biophysics.”

Luria wondered how to test the new ideas and stumbled on a possibility. He met a microbiologist on a stalled trolley car who turned out to be measuring bacteria in the Tiber River using bacteriophages—viruses that infect bacteria. Luria became intrigued by the possibilities of probing gene structure using the phage as a stripped-back biological system.

Bacteriophages (white polygonal shapes) attached to a bacterial cell, ready to transfer their genome into the bacterium. By Dr Graham Beards [CC BY-SA 3.0] via Wikimedia Commons.

“I didn’t make much progress in reading these forbidding-looking papers; every genotype was about a mile long, terrible, and I just didn’t get any grasp of it.”

–Max Delbrück, 1978

Then he began talking to Emory Ellis, a postdoc in the department at Caltech who had isolated some bacteriophage from Los Angeles sewage; Delbrück realized that he had found the system he had been searching for:

“I was absolutely overwhelmed that there were such very simple procedures with which you could visualize individual virus particles; […] This seemed to me just beyond my wildest dreams of doing simple experiments on something like atoms in biology.”

–Max Delbrück, 1978

Back in Italy, Luria was encouraged to hear of Delbrück’s phage epiphany and, in 1938, hoped to use a government fellowship to travel to the US to work with the displaced physicist. But just as soon as he was awarded the money, Italian dictator Benito Mussolini announced a series of Nazi-influenced race laws. Luria’s fellowship was unceremoniously withdrawn because he was Jewish. With no funding to work with Delbrück, he sought shelter and grant money at Marie Curie’s Radium Institute in Paris, where he bombarded bacteriophage with radiation to study their molecular make up.

Then, on June 13, 1940, as the German Army advanced on Paris, Luria and his friends were forced to flee the city on bicycles. After making his way nearly 500 miles to Marseilles and the US embassy (via a combination of bike and freight train), Luria took a ship to New York. Once the US, he quickly obtained funding to restart his investigations and at a conference in December, he finally met the one other person in the world who thought phage was the key to understanding the gene: Delbrück. They immediately agreed to join forces.

French refugees, 19 June 1940. After the Nazi invasion, millions of people fled South. Photo: Bundesarchiv, Bild 146-1971-083-01 / Tritschler / CC-BY-SA 3.0 via Wikimedia Commons

Among the first problems the pair tackled was the emergence of resistance in bacteria. When a culture is grown from a single cell and then exposed to phage, the infected cells die. But if left for a few hours or days, virus-resistant cells start growing and the culture revives itself. Many biologists held the view that these survivors were the descendants of cells that had developed resistance to the phage as a result of some direct interaction between the virus and bacteria.

But others saw no reason to believe that bacteria operated under entirely new rules of heredity. Just as for plants and animals, they argued that resistance mutations arise at random, and the virus acts as selective force that enriches the population for pre-existing cells that happen to be resistant.

At first, Luria and Delbrück struggled to come up with experiments to put this disagreement to rest. Luria tried measuring the proportion of virus resistant cells in growing bacterial cultures, but he seemed to get different results every day. Then, he had a chance encounter with a colleague at a slot machine:

“Not a gambler myself, I was teasing him about his inevitable losses, when he suddenly hit a jackpot, about three dollars in dimes, gave me a dirty look, and walked away. Right then I began giving some thought to the actual numerology of slot machines; in so doing it dawned on me that slot machines and bacterial mutations have something to teach each other.”

-Salvador Luria 1984

Luria realized he could distinguish between the acquired resistance and the random mutation hypothesis using statistics. If resistance were caused by exposure to the virus, it should arise only after the virus was added, at a relatively consistent proportion. But if resistance emerged by mutation at random times, then the proportion of resistant cells would be much more variable, depending on how early the mutation arose. Delbrück calculated the statistical distribution they would expect from random mutations and, to their joy, Luria’s experimental data matched it. They had shown that bacteria had genes that mutate at random, providing fodder for natural selection. Their approach also provided a quantitative approach for studying bacterial mutation rates.

The Luria–Delbrück experiment. (A) If resistance is induced by the presence of the phage in the final, assay plate, independent cultures should yield roughly similar numbers of resistant colonies. (B) If resistance mutations arise spontaneously during the cell divisions prior to plating, the number of resistant colonies will depend on how early in the culture the mutation arose. Image: Madprime via Wikimedia commons

The fruit of this community-building was a transformative molecular understanding of heredity within a remarkably short period. Luria and Delbrück’s phage epiphanies turned out to be science jackpots.

CITATIONS

Bertani, G. (1992). Salvador Edward Luria (1912-1991). Genetics, 131(1), 1. http://www.genetics.org/content/131/1/1

Fischer, E. P. (2007). Max Delbrück. Genetics, 177(2), 673-676. http://www.genetics.org/content/177/2/673

Luria, S. E., & Delbrück, M. (1943). Mutations of bacteria from virus sensitivity to virus resistance. Genetics, 28(6), 491. http://www.genetics.org/content/28/6/491

Murray, A. (2016). Salvador Luria and Max Delbrück on Random Mutation and Fluctuation Tests Genetics 202(2) 367-368; DOI:10.1534/genetics.115.186163  http://www.genetics.org/content/202/2/367