Amazing Science

Mitochondria are critical organelles that form networks within our cells, generate energy dynamically, contribute to diverse cell and organ function, and produce a variety of critical signaling molecules, such as cortisol. This intracellular microbiome can differ between cells, tissues, and organs. Mitochondria can change with disease, age, and in response to the environment. Single nucleotide variants in the circular genomes of human mitochondrial DNA are associated with many different life-threatening diseases. Mitochondrial DNA base editing tools have established novel disease models and represent a new possibility toward personalized gene therapies for the treatment of mtDNA-based disorders.

Climate change is shifting the ranges of many disease-carrying species like ticks and mosquitoes. Scientists warn that the U.S. is underprepared for a potentially devastating surge in infections. In the summer and fall of 2021, West Nile virus spread rapidly through Arizona’s Maricopa County and other areas of the state. The outbreak, with more than 1,700 cases reported and 127 deaths. was the largest in the United States since the mosquito-borne virus first emerged in this country in 1999. But with the nation facing a far larger public health crisis with the Covid-19 pandemic, it went almost unnoticed. Even before Covid-19 arrived, the public health response to diseases transmitted to humans by vectors like fleas, ticks and mosquitoes — including West Nile, Zika, dengue fever, Lyme disease, and others — was muted, perhaps because the number of reported cases has been relatively low, and the public largely unaware of the health risks such diseases pose. With climate change accelerating, however, shifting the ranges of many disease-carrying species and sharply increasing infections, scientists and others warn that the nation’s public officials, as well as hospitals and doctors, are underprepared for a potentially devastating surge in infections. Research on vector-borne diseases and disease surveillance, they note, are underfunded by federal and local governments, leaving the country vulnerable to outbreaks.

“Without sustained funding in local vector control and surveillance, it ends up stymieing that response of looking for the threats before they become really huge causes for concern for local public health,” said Chelsea Gridley-Smith, director of environmental health for the National Association of County and City Health Officials (NACCHO). In the United States, cases of 17 different vector-borne diseases have been reported to the Centers for Disease Control and Prevention, and nine pathogens new to the country have been identified since 2004, according to a 2020 report by the agency, which noted that the data for 2019 and 2020 might be incomplete due to underreporting during the Covid-19 pandemic. Reported cases of vector-borne diseases more than doubled from 2004-2019, to more than 800,000 cases. But those figures are almost certainly an undercount, CDC officials said in a presentation to Congress last year. Only 2% to 3% of West Nile cases and about 10% of Lyme disease cases are reported, said Lyle Petersen, the director of the CDC’s Division of Vector-Borne Diseases in Fort Collins, Colo. Overall, cases of vector-borne diseases are probably underreported by 10-fold to 80-fold, according to Benjamin Beard, the CDC division’s deputy director.

Petersen noted that addressing vector-borne disease involves formidable challenges, including a lack of vaccines for diseases found in the continental United States; the difficulty in diagnosing some diseases in their early stages; and the sheer number of emerging pathogens. Tick-borne diseases comprise the largest share of vector-borne diseases by far — over 80% of reported cases are caused by ticks. Longer summers, rising temperatures, and the expanding ranges of tick species such as Ixodes scapularis, the black-legged tick, and Amblyomma americanum, the lone star tick, are leading to an increased chance of human exposure to pathogens over a larger geographic area. The range of Ixodes scapularis, a tick that transmits Lyme and other diseases, expanded greatly over two decades, with the number of counties with established populations more than doubling from 1996 to 2015.

Similarly, milder year-round temperatures mean that some mosquitoes may overwinter or emerge earlier in the spring. In the case of West Nile, this affects not just the mosquitoes carrying the virus but the virus itself, which replicates faster in warm temperatures. “So the mosquitoes actually are more infectious to people when they bite them,” Beard said. Nelson Nicolasora, medical director for the infectious disease program at Banner University Medical Center in Phoenix, said that while the 2021 West Nile virus outbreak was “nothing like” the Covid-19 pandemic, the illness was “life-changing” for people who suffered debilitating neurological disease. West Nile usually causes mild, flu-like symptoms, but about 1 in 150 people who are infected will develop severe neuroinvasive disease. “It can be devastating,” Nicolasora said. Two of his patients died during the 2021 outbreak, he said, and others faced serious short-term and longer-term effects: Some required a ventilator to breathe, or rehabilitation to regain the ability to walk. Irene Ruberto, vector-borne and zoonotic disease program manager at the Arizona Department of Health Services, said that even though public health officials in the state were aware of the cyclical nature of West Nile virus infections from year to year, they had no idea the infection rate would be so high in 2021. It’s difficult to predict how many infections will occur in a given year, Ruberto said, because many factors are involved, including mosquito density, local environments, and the climate.

“We do know that birds play a role,” acting as an amplifying host for the virus, Ruberto said, which adds complexity to understanding virus transmission. While West Nile is transmitted to humans by mosquitoes, mosquitoes get the virus through biting an infected wild bird. And different species of birds vary in their ability to transmit the virus once they’re infected. Ruberto said the state health department in Arizona doesn’t on its own have the funding or the capacity to analyze the 2021 outbreak to understand the factors that drove it. Instead, she said, the department is working with the CDC and universities to study the 2021 data and develop a model to predict future outbreaks. However, Ruberto said she’s even more concerned about the emergence in her state of another vector-borne disease: dengue fever. In 2022, two locally transmitted cases of dengue were discovered in Arizona, the first appearance of the disease in the state in modern times. Though dengue — known colloquially as “breakbone fever” because of the severe joint pain and muscle spasms it can cause — is also transmitted by mosquitoes, it differs from West Nile in an important way: The virus can be spread from one infected person to another person through a mosquito bite...

Corals, sturgeon and other aquatic creatures harbor signs of infection by influenza and its distant relatives. The influenza virus might actually have started in fish. Researchers trawling genetic databases have discovered a distant relative of influenza viruses — which are responsible for seasonal flu, not to mention the avian flu roiling the globe — in sturgeon [1]. The authors also found that the wider virus family that includes influenza probably originated hundreds of millions of years ago in primordial aquatic animals that evolved well before the first fish. Viruses in this group seem to be especially adept at jumping between hosts, says Mary Petrone, a virologist at the University of Sydney, Australia, who co-authored the preprint describing the findings. Knowing about ancient host jumps could help scientists identify viruses with the potential to spark new human epidemics. The study was posted on 16 Feburary 2023 to the preprint server bioRxiv and has not yet been peer reviewed.

Influenza’s origins story

Like many virologists, Petrone spent the first couple years of the pandemic intensively studying SARS-CoV-2. But when she moved to Australia to do postdoctoral research, Petrone wanted to steer clear of human infections and spend time in one of the country’s most famous ecosystems. “After working on COVID for two years, I thought going to coral reefs to do fieldwork sounded really good,” she says. Corals are part of a phylum called Cnidaria, whose ancestors branched off from other animals around 600 million years ago. Petrone hoped that studying corals could reveal the deeper history of viruses that infect animals — particularly those with RNA genomes. This viral group includes numerous human and animal pathogens. Petrone’s first call was not to a diving shop but to Zoe Richards, a coral-reef researcher at Curtin University in Bentley, Australia, who provided samples of two coral species collected off the coast of Western Australia. Analysis of RNA collected from the corals found evidence of infection with viruses that belong to a grouping called Articulavirales, which includes influenza’s family of viruses and a group called Quaranjaviruses. The latter group’s members circulate in ticks and occasionally spill over into humans, birds and other vertebrates. The new analysis suggests that coral-infecting viruses are part of an ancient viral family that probably emerged around 600 million years ago, and later gave rise to other members of Articulavirales, including influenza and Quaranjaviruses.

Secrets of the hagfish

The discovery got Petrone wondering whether influenza viruses might also have been born at sea. There was already some evidence for this. In 2018, researchers identified a distant relative of influenza in hagfish [2]. These slimy, jawless creatures descended from an early lineage of vertebrates, and the study’s authors hinted that influenza evolved alongside vertebrates. Searching genetic databases, Petrone found influenza-related RNA sequences in samples from Siberian sturgeon (Acipenser baerii). Sturgeon are jawed vertebrates, more closely related to humans than hagfish are. But the sturgeon virus had branched off from the main influenza family tree before any other known influenza virus, including the hagfish virus. The discovery of the two early lineages of influenza suggest that influenza probably infected aquatic animals, including fish, before moving onto land, says Petrone. But it’s not clear whether influenza moved onto land with early terrestrial vertebrates, or jumped from sea to land more recently.

To determine this, researchers will need to look for relatives of influenza in more animals and gain a better understand how the virus spreads between host species, researchers say.

Born at sea

Jie Cui, an evolutionary virologist at the Pasteur Institute of Shanghai in China, agrees that influenza and its wider family probably emerged from the sea. In 2021, his team analysed deep-sea lobster genomes and identified viruses that are part of influenza’s wider group [3]. “There is great untapped viral diversity in aquatic environments,” he says. Robert Gifford, an evolutionary virologist at the University of Glasgow, UK, says it would be surprising to find a major group of viruses that didn’t arise in aquatic environments because of the ancient nature of marine life. “The study provides compelling evidence that influenza viruses have an aquatic origin.” Identifying ancient host jumps could also help researchers gauge the risk that certain viruses pose to humans, researchers say. Petrone’s team found signs that Quaranjaviruses that infect ticks might have jumped to the creatures after first circulating in crustaceans. Uncovering such jumps shows that the study of aquatic viruses “can help us to better understand the historic emergence and evolution of viruses with zoonotic potential”, adds Chantal Vogels, an arbovirologist at the Yale School of Public Health in New Haven, Connecticut. Gifford agrees that studies such as Petrone’s could help to identify viruses that have the capacity to spark epidemics in humans and other animals. But he cautions that conclusions about ancient host jumps can change as more of the viral family tree gets filled in, reshuffling relationships.

Cited research available in bioRxiv (Feb. 16, 2023):

https://doi.org/10.1101/2023.02.15.528772

In a new paper published in the journal Nature Astronomy, astronomers with Breakthrough Listen Initiative — the largest ever scientific research program aimed at finding evidence of alien civilizations — present a new machine learning-based method that they apply to more than 480 hours of data from the Robert C. Byrd Green Bank Telescope, observing 820 nearby stars. The method analyzed 115 million snippets of data, from which it identified around 3 million signals of interest. The authors then inspected the 20,515 signals and they identified 8 previously undetected signals of interest, although follow-up observations of these targets have not re-detected them.

“The key issue with any techno-signature search is looking through this huge haystack of signals to find the needle that might be a transmission from an alien world,” said Dr. Steve Croft, an astrophysicist at the University of California, Berkeley and a member of the Breakthrough Listen team. “The vast majority of the signals detected by our telescopes originate from our own technology — GPS satellites, mobile phones, and the like. Our algorithm gives us a more effective way to filter the haystack and find signals that have the characteristics we expect from techno-signatures.”

Classical techno-signature algorithms compare scans where the telescope is pointed at a target point on the sky with scans where the telescope moves to a nearby position, in order to identify signals that may be coming from only that specific point.

These techniques are highly effective. For example, they can successfully identify the Voyager 1 space probe, at a distance of 20 billion km, in observations with the Green Bank Telescope. But all of these algorithms struggle in crowded regions of the radio spectrum, where the challenge is akin to listening for a whisper in a crowded room.

The process developed by the team inserts simulated signals into real data, and trains an artificial intelligence algorithm known as an auto-encoder to learn their fundamental properties. The output from this process is fed into a second algorithm known as a random forest classifier, which learns to distinguish the candidate signals from the noisy background. “In 2021, our classical algorithms uncovered a signal of interest, denoted BLC1, in data from the Parkes telescope,” said Breakthrough Listen’s principal investigator Dr. Andrew Siemion, an astronomer at the University of California, Berkeley.

A 53-year-old man in Germany has become the third person with HIV to be declared cleared of the virus after a procedure that replaced his bone marrow cells with HIV-resistant stem cells from a donor. For years, antiretroviral therapy (ART) has been given to people with HIV with the aim of lowering the virus to almost undetectable levels and preventing it from being transmitted to other people. But the immune system keeps the virus locked up in reservoirs in the body, and if an individual stops taking ART the virus can begin replicating and spreading. A true cure would eliminate this reservoir, and this is what seems to have happened for the latest patient, whose name has not been released. The man, who is being referred to as the ‘Düsseldorf patient’, stopped taking ART in 2018 and has remained HIV-free since. But the risks associated with the procedure mean it is unlikely to be widely used in its current form.

The stem-cell technique involved was first used to treat Timothy Ray Brown, often referred to as the Berlin patient. In 2007, he had a bone marrow transplant, in which those cells were destroyed and replaced with stem cells from a healthy donor, to treat acute myeloid leukemia. The team treating Brown selected a donor with a genetic mutation called CCR5Δ32/Δ32, which prevents the CCR5 cell-surface protein from being expressed on the cell surface. HIV uses that protein to enter immune cells, so the mutation makes the cells effectively resistant to the virus. After the procedure, Brown was able to stop taking ART and remained HIV-free until his death in 2020. In 2019, researchers revealed that the same procedure seemed to have cured the London patient, Adam Castillejo. And, in 2022, scientists announced that they thought a New York patient who had remained HIV-free for 14 months might also be cured, although researchers cautioned that it was too early to be certain. Ravindra Gupta, a microbiologist at the University of Cambridge, UK, who led the team that treated Castillejo, says the latest study “cements the fact that CCR5 is the most tractable target for achieving a cure right now”.

An Australian company resurrects flesh of lost species to demonstrate potential of meat grown from cells as food source. Recently, they created a mammoth meatball, resurrecting the flesh of the long-extinct animals. The project aims to demonstrate the potential of meat grown from cells, without the slaughter of animals, and to highlight the link between large-scale livestock production and the destruction of wildlife and the climate crisis. The mammoth meatball was produced by Vow, an Australian company, which is taking a different approach to cultured meat. There are scores of companies working on replacements for conventional meat, such as chicken, pork and beef.

But Vow is aiming to mix and match cells from unconventional species to create new kinds of meat. The company has already investigated the potential of more than 50 species, including alpaca, buffalo, crocodile, kangaroo, peacocks and different types of fish. The first cultivated meat to be sold to diners will be Japanese quail, which the company expects will be in restaurants in Singapore this year. “We have a behaviour change problem when it comes to meat consumption,” said George Peppou, CEO of Vow . “The goal is to transition a few billion meat eaters away from eating [conventional] animal protein to eating things that can be produced in electrified systems.

“And we believe the best way to do that is to invent meat. We look for cells that are easy to grow, really tasty and nutritious, and then mix and match those cells to create really tasty meat.” Tim Noakesmith, who cofounded Vow with Peppou, said: “We chose the woolly mammoth because it’s a symbol of diversity loss and a symbol of climate change.”

The creature is thought to have been driven to extinction by hunting by humans and the warming of the world after the last ice age. The initial idea was from Bas Korsten at creative agency Wunderman Thompson: “Our aim is to start a conversation about how we eat, and what the future alternatives can look and taste like. Cultured meat is meat, but not as we know it.” Plant-based alternatives to meat are now common but cultured meat replicates the taste of conventional meat.

Cultivated meat – chicken from Good Meat – is currently only sold to consumers in Singapore, but two companies have now passed an approval process in the US. In 2018, another company used DNA from an extinct animal to create gummy bears made from gelatine from a mastodon, another elephant-like animal.

Vow worked with Prof Ernst Wolvetang, at the Australian Institute for Bioengineering at the University of Queensland, to create the mammoth muscle protein. His team took the DNA sequence for mammoth myoglobin, a key muscle protein in giving meat its flavor, and filled in the few gaps using elephant DNA. This sequence was placed in myoblast stem cells from a sheep, which replicated to grow to the 20bn cells subsequently used by the company to grow the mammoth meat. “It was ridiculously easy and fast,” said Wolvetang. “We did this in a couple of weeks. Initially, the idea was to produce dodo meat, he said, but the DNA sequences needed do notvyet exist!"

A decade after a landmark report on Americans' shorter lives, the problem has only gotten worse. Unlike other wealthy nations, U.S. life expectancy has not bounced back from the pandemic. Just before Christmas, federal health officials confirmed life expectancy in America had dropped for a nearly unprecedented second year in a row – down to 76 years. While countries all over the world saw life expectancy rebound during the second year of the pandemic after the arrival of vaccines, the U.S. did not. Then, last week, more bad news: Maternal mortality in the U.S. reached a high in 2021. Also, a paper in the Journal of the American Medical Association found rising mortality rates among U.S. children and adolescents. "This is the first time in my career that I've ever seen [an increase in pediatric mortality] – it's always been declining in the United States for as long as I can remember," says the JAMA paper's lead author Steven Woolf, director emeritus of the Center on Society and Health at Virginia Commonwealth University. "Now, it's increasing at a magnitude that has not occurred at least for half a century." Across the lifespan, and across every demographic group, Americans die at younger ages than their counterparts in other wealthy nations. How could this happen? In a country that prides itself on scientific excellence and innovation, and spends an incredible amount of money on health care, the population keeps dying at younger and younger ages.

An unheard alarm

One group of people are not surprised at all: Woolf and the other researchers involved in a landmark, 400-page study ten years ago with a name that says it all: "Shorter Lives, Poorer Health." The research by a panel convened by the National Academy of Sciences and funded by the National Institutes of Health compared U.S. health and death with other developed countries. The results showed – convincingly – that the U.S. was stalling on health advances in the population while other countries raced ahead. The authors tried to sound an alarm, but found few in the public or government or private sectors were willing to listen. In the years since, the trends have worsened. American life expectancy is lower than that of Cuba, Lebanon, and Czechia.

Ten years later, here's a look back at what that eye-popping study found, and why the researchers involved believe it's not too late to turn the trends around.

Beyond bad habits

Americans are used to hearing about how their poor diets and sedentary lifestyles make their health bad. It can seem easy to brush that off as another scold about eating more vegetables and getting more exercise. But the picture painted in the "Shorter Lives" report could shock even those who feel like they know the story. "American children are less likely to live to age 5 than children in other high-income countries," the authors write on the second page. It goes on: "Even Americans with healthy behaviors, for example, those who are not obese or do not smoke, appear to have higher disease rates than their peers in other countries." The researchers catalog what they call the "U.S. health disadvantage" – the fact that living in America is worse for your health and makes you more likely to die younger than if you lived in another rich country like the U.K., Switzerland or Japan. "We went into this with an open mind as to why it is that the U.S. had a shorter life expectancy than people in other countries," says Woolf, who chaired the committee that produced the report. After looking across different age and racial and economic and geographic groups, he says, "what we found was that this problem existed in almost every category we looked at." That's why, says Eileen Crimmins, professor of gerontology at the University of Southern California who was also on the panel that produced the report, they made a deliberate choice to focus on the health of the U.S. population as a whole. "That was a decision – not to emphasize the differences in our population, because there is data that actually shows that even the top proportion of the U.S. population does worse than the top proportion of other populations," she explains. "We were trying to just say – look, this is an American problem."

Digging into the 'why'

The researchers were charged with documenting how Americans have more diseases and die younger and to explore the reasons why. "We were very systematic and thorough about how we thought about this," says Woolf. The panel looked at American life and death in terms of the public health and medical care system, individual behaviors like diet and tobacco use, social factors like poverty and inequality, the physical environment, and public policies and values. "In every one of those five buckets, we found problems that distinguish the United States from other countries." Yes, Americans eat more calories and lack universal access to health care. But there's also higher child poverty, racial segregation, social isolation, and more. Even the way cities are designed makes access to good food more difficult. "Everybody has a pet thing they worry about and say, 'it's oral health' or 'it's suicides' – everyone has something that they're legitimately interested in and want to see more attention to," says John Haaga, who was the director of the Division of Behavioral and Social Research at the National Institute on Aging at NIH, before he retired. "The great value of an exercise like this one was to step back and say, 'OK, all of these things are going on, but which of them best account for these long-term population level trends that we're seeing?' " The answer is varied. A big part of the difference between life and death in the U.S. and its peer countries is people dying or being killed before age 50. The "Shorter Lives" report specifically points to factors like teen pregnancy, drug overdoses, HIV, fatal car crashes, injuries, and violence.

"Two years difference in life expectancy probably comes from the fact that firearms are so available in the United States," Crimmins says. "There's the opioid epidemic, which is clearly ours – that was our drug companies and other countries didn't have that because those drugs were more controlled. Some of the difference comes from the fact that we are more likely to drive more miles. We have more cars," and ultimately, more fatal crashes. "When we were doing it, we were joking we should call it 'Live free and die,' based on the New Hampshire slogan, ['Live free or die']," Crimmins says. "The National Academy of Sciences said, 'That's outrageous, that's too provocative.' " There are some things Americans get right, according to the "Shorter Lives" report: "The United States has higher survival after age 75 than do peer countries, and it has higher rates of cancer screening and survival, better control of blood pressure and cholesterol levels, lower stroke mortality, lower rates of current smoking, and higher average household income." But those achievements, it's clear, aren't enough to offset the other problems that befall many Americans at younger ages. All of this costs the country tremendously. Not only do families lose loved ones too soon, but having a sicker population costs the country as much as $100 billion every year in extra health care costs. "Behind the statistics detailed in this report are the faces of young people – infants, children, and adolescents – who are unwell and dying early because conditions in this country are not as favorable as those in other countries," the paper's authors wrote....

Researchers found associations between certain viral illnesses and the risk of Alzheimer’s and other neurodegenerative diseases.  Neurodegenerative diseases can damage different parts of the nervous system, including the brain. This may lead to problems with thinking, memory, and/or movement. Examples include Alzheimer’s disease (AD), multiple sclerosis (MS), and Parkinson’s disease (PD). These diseases tend to happen late in life. There are few effective treatments. Previous findings have suggested that viruses may play a role in certain neurodegenerative diseases. 

A recent study found a link between Epstein-Barr virus infection and the risk of MS. There are also concerns about cognitive impacts from SARS-CoV-2, the virus that causes COVID-19. A research team led by Drs. Mike Nalls, Kristin Levine, and Hampton Leonard of NIH's Center for Alzheimer’s and Related Dementias examined links between viruses and neurodegenerative disease more generally. To do so, they analyzed data from the FinnGen project. This is a repository of biomedical data, or biobank, from more than 300,000 people in Finland. The team searched the biobank for people who had been diagnosed with one of six different conditions: AD, amyotrophic lateral sclerosis (ALS), generalized dementia, vascular dementia, PD, and MS. They then checked how many had been hospitalized for a viral illness before. To confirm their findings, they looked for the same associations in the UK Biobank, which contains data from almost 500,000 people in the United Kingdom. Results appeared in Neuron on January 19, 2023.

The researchers found 45 associations between viruses and neurodegenerative diseases in FinnGen. Of these, 22 also appeared in the UK Biobank. The strongest association was between viral encephalitis—brain inflammation caused by a virus—and AD. A person with viral encephalitis in the FinnGen database was 30 times as likely to be diagnosed with AD as someone without encephalitis. Results were similar in the UK Biobank; people with viral encephalitis were 22 times as likely to develop AD as those without. The team also found, in FinnGen, the association between Epstein-Barr virus and MS that was described before. The association wasn’t seen in the UK Biobank, but this may reflect how the different biobanks use hospital diagnostic codes; Epstein-Barr viruses are common and so often not noted. Influenza with pneumonia was associated with all the neurodegenerative diseases except MS. The researchers only included cases of influenza severe enough to need hospitalization in the study.

Thus, these associations only apply to the most severe cases of influenza. FinnGen contains data on the same people over time. The team used this to examine how the associations depended on the time since infection. They found that some viral infections were associated with increased risk of neurodegenerative disease as much as 15 years later. The researchers note that vaccines exist for some of the viruses they identified. These include influenza, varicella-zoster (which causes chickenpox and shingles), and certain pneumonia-causing viruses. Vaccination might thus reduce some of the risk of the conditions they examined. “The results of this study provide researchers with several new critical pieces of the neurodegenerative disorder puzzle,” Nalls says. “In the future, we plan to use the latest data science tools to not only find more pieces but also help researchers understand how those pieces, including genes and other risk factors, fit together.”

A study employing CRISPR/Cas9 to explore the evolutionary beginnings of some giant viruses finds evidence that their large genomes arose from gene duplications.  Most viruses are small and carry minimal genomes. Even one of the largest small viruses, Vaccinia, measures merely one-fiftieth the size of a pollen grain and contains only 270 genes.

Giant viruses flout these rules. With sizes that rival small bacteria and genomes that contain thousands of genes, their complexity emulates that of cellular life. How these viruses came to be so large has been the subject of much debate. Now, scientists are finally poised to unravel the mystery of their evolutionary origins, thanks to a suite of CRISPR/Cas9-based tools described in a Nature Communications paper from January.

“It was by chance that we encountered the first giant virus,” says Chantal Abergel, a virologist at Aix-Marseille University in France. “It was Mimivirus, and it was actually mistaken for a bacterium.” In the 20 years since that discovery, virologists have prioritized exploring the diversity of giant viruses. Now that they’ve found a fair few, the focus has shifted towards studying their evolution in more detail with molecular biology techniques.

Evolutionary biologists have grappled over two possible origins of giant viruses. One possibility is that they were once cellular organisms that shrunk physically and genetically over time. But most virologists now suspect giant viruses grew out of much smaller ones—though the evidence supporting either hypothesis is scant.

To begin addressing this origin question, Abergel decided to examine how the essential genes in the Pandoravirus genome are distributed. In cellular organisms, essential genes are scattered throughout the genome—so if giant viruses are essentially reduced cells, one would expect a similar pattern. Alternatively, if the genes are clumped, that could indicate the viruses’ large genomes started out in a more compact form. One way to locate a virus’s essential genes is to knock out genes one at a time to find the ones that are needed for virus production. But to do that with a giant virus, Abergel needed a gene-editing system that worked in members of the group. With the help of Hugo Bisio, a postdoctoral researcher in Abergel’s lab, and colleagues at Aix–Marseille University, Abergel used a CRISPR/Cas9-based gene-editing system to modify the genome of the amoeba Acanthamoeba castellanii and the giant virus Pandoravirus neocaledonia, which infects it.

The CRISPR/Cas9 system was designed to delete specific genes and consists of two guide RNAs and a Cas9 scission enzyme. Similar to other CRISPR/Cas9 systems, each guide RNA contains 17 to 20 bases designed to bind to one specific location on the genome of the giant virus or the amoeba, allowing the Cas9 scission enzyme to cut the genome at that site. The amoeba A. castellanii contains 25 copies of each chromosome, making it difficult to design an efficient CRISPR/Cas9 system that could delete each gene copy. To overcome this issue, the researchers modified their CRISPR/Cas9 system to generate a chain reaction. Each time DNA was cut to remove a gene, a DNA segment encoding the Cas9 enzyme and the guide RNAs responsible for the cut would take the place of the missing gene in the genome. This allowed gene deletions to repeat and propagate until all copies were removed.

Once they optimized their CRISPR/Cas9 system, the team deleted each gene separately from the Pandoravirus genome and measured the resulting change in virus production, in order to determine how important each gene is to the virus’s lifecycle. They found that essential genes clustered together at one end of the genome and were segregated from nonessential genes at the other end. This level of gene orderliness has not been seen in viruses, according to Bisio. Even bacterial genomes aren’t quite so tidy: While they do group genes with linked functions together into gene clusters known as operons, these tend to be dispersed throughout the genome rather than grouped all together in one spot. Bisio says the cluster of essential genes may echo a smaller “core genome” of an ancient virus. This genome could have become elongated through multiple rounds of gene duplication that were biased in one direction to produce an additional set of spare nonessential genes. This could explain how modern-day giant viruses came to possess thousands of genes. “Our data indicate that complex viruses arose from smaller and simpler ones,” Bisio tells The Scientist in an email—noting that it will take further research to determine whether that’s true of all giant viruses or just Pandoravirus. Other studies found that some genes in giant viruses were usurped from their amoeba hosts, suggesting gene exchange is another way giant viruses increased in size. The team then set their sights on one of the many evolutionary mysteries of Pandoravirus: its lack of a capsid. Small viruses package their genomes into capsids made of viral proteins. While some giant viruses, such as Mimivirus, continue this tradition, others, including Pandoravirus, do not. If giant viruses did indeed evolve from smaller ones, there could be traces of capsid proteins hiding in their genomes. So, the researchers set out to study the function of potential capsid protein remnants in a close cousin of Pandoravirus, the smaller Mollivirus, which can also infect A. castellanii.

Researchers have suspected that a Mollivirus protein called ml_347 evolved from a capsid gene based on its gene’s sequence and predicted 3D shape. So, the team investigated its function by deleting the gene using their CRISPR/Cas9 system. They found that the gene is important for Mollivirus assembly, which the authors say is intriguing given its possible capsid ancestry. It’s possible that, as capsids were lost in giant virus evolution, obsolete capsid genes were adapted for new assembly functions. Frederik Schulz, an evolutionary biologist with the DOE Joint Genome Institute in California who wasn’t involved with the study but who has worked with Chantal Abergel in the past, tells The Scientist that the findings align with recent discoveries. “There was a debate for a long time [about] how giant virus[es] evolved,” he says. “The working hypothesis in the previous years was that they evolved from smaller viruses, and that’s exactly what Chantal and her team could show and confirm using their CRISPR/Cas9 gene-editing approach.” Schulz notes that it will be exciting to see the CRISPR/Cas9 technology introduced into other host species, such as algae, which would allow researchers to expand research into a greater variety of giant viruses. He also points out that the system only works for viruses that replicate in a host cell’s nucleus, while most giant viruses replicate in cytoplasmic structures called viral factories, which the Cas9 enzyme and guide RNAs can’t penetrate. Still, Bisio says there’s much left to discover in Pandoravirus. “[This CRISPR/Cas9 technology is] a goldmine to find new functions,” he says—one that he and his colleagues are eager to employ to tease apart what all the virus’s genes do. 

Cited research published in Nat. Comm. (Jan. 26, 2023):

https://doi.org/10.1038/s41467-023-36145-4