NIAID Scientists Explore Pathways of Viral Evolution

Viruses are known to evolve quickly, in part because one generation of viral growth occurs in just minutes—an instant compared to the decades taken for a human generation to occur. This evolution happens at a vast scale, too. There are more viruses on the planet than stars in the universe. That’s trillions of trillions of viruses, all evolving at breakneck speed—and yet, researchers have found that viruses often evolve to arrive at similar genetic features. NIAID scientists recently developed new tools to understand this process in an important group of viruses called enteroviruses, which are responsible for several dangerous diseases, including polio; hand, foot, and mouth disease (HFMD); and acute flaccid myelitis (AFM). Their findings have implications for the evolution of other viral pathogens, as well.

The enterovirus that causes polio, Poliovirus, may be the most well-known. It infects the nervous system, leading to paralysis and death in severe cases. Although vaccination against polio has resulted in eradication of the virus from many parts of the world, the virus continues to emerge in some places, posing a threat to public health. HFMD is caused by different enteroviruses from a group often referred to as non-polio enteroviruses, including enterovirus A71 (EV-A71) and coxsackie virus A6 (CV-A6). HFMD is very contagious and often results in a fever, sore throat, and rash, with symptoms more commonly occurring in children than adults. Although uncommon, HFMD can result in serious complications, and so large outbreaks can raise public health concerns. In rare cases, non-polio enteroviruses such as EV-A71 and EV-D68 can also lead to AFM, a dangerous disorder of the nervous system that can result in permanent impairment. Despite the risks they pose, many enteroviruses and the diseases they cause are not well understood. It’s important for scientists to uncover how enteroviruses grow and evolve so they can develop new countermeasures against these diseases.

Patrick Dolan, Ph.D., chief of the Quantitative Virology and Evolution Unit in the Division of Intramural Research, is investigating aspects of enterovirus evolution. His research group developed a new method called SEARCHLIGHT (short for scRNA-seq–enabled acquisition of mRNA and consensus haplotypes linking individual genotypes and host transcriptomes). The method homes in on individual viruses within a rapidly evolving viral population. Using cutting-edge technology, the scientists examined the communities of viruses in single cells during viral infection. This method allows for the exploration of the concept of “viral quasi-species”—a term coined by scientists to describe how populations of viruses rapidly evolve when faced with new environmental pressures. The researchers observed how, in specific conditions, viral populations explore their “genetic neighborhood”—the possible ways they could adapt—through mutation to find more successful genetic strategies. In addition, they observed how EV-D68 and EV-A71, two distantly related members of the enterovirus family, followed common routes of evolution under similar conditions, suggesting that the routes viruses travel to explore their genetic neighborhood are shaped by common features of the viruses. Insights from this research may allow researchers to predict how new viral variants emerge and develop strategies to counter them.

To further explore features of enterovirus evolution, Dr. Dolan’s team used an innovative method to map all the sites of the genome known to foster insertions and deletions, or InDels. InDels are major evolutionary events in which short genetic sequences can be removed or added, as opposed to single substitutions in the genetic sequence, which are minor events that generally have more modest effects on gene function. The team examined tens of thousands of mutations in EV-A71, finding that InDels were only tolerated at specific hotspots, many of which appear to be critical for adaptions in viral function and immune evasion. For instance, the researchers observed many InDels in the regions of the genome dedicated to forming the viral capsid—the outer shell of the virus that interacts with the body’s machinery to enter cells, as well as triggering an immune response. This suggests that InDels in this region provide a mechanism for the virus to adapt new ways to infect cells and trick the immune system, which are critical functions for viral propagation. Based on these findings, InDels contribute significantly to the evolution and diversification of viruses. This new map of InDel sites may provide novel avenues for investigation of viral functions and interventions against them.

These studies demonstrate the power of advanced genetic technology to explore important questions in enterovirus evolution. Because enteroviruses have genetic similarities to many other viruses, this research has further implications towards discovering mechanisms of adaptation in a range of pathogens. A deepened understanding of viral evolution will provide researchers with new ways to develop targets for vaccines, therapeutics, and other countermeasures. Additionally, this research highlights the importance of investing in basic research to into how viruses adapt and function, fostering a body of knowledge researchers can draw upon for the development of interventions against infectious diseases. 

References:

• N Dábilla N, PT Dolan. Structure and dynamics of enterovirus genotype networks. Science Advances DOI: 10.1126/sciadv.ado1693 (2024).
• W Bakhache et al. Deep mutation, insertion and deletion scanning across the Enterovirus A proteome reveals constraints shaping viral evolution. Nature Microbiology DOI: 10.1038/s41564-024-01871-y (2024).

NIAID-led scientists’ discovery of a hidden gene variant that causes some cases of a devastating inherited disease will enable earlier diagnosis of the disorder in people with the variant, facilitating earlier medical care that may prolong their lives. The researchers are working on a treatment for this unusual form of the rare autoimmune disease, known as APECED, and have traced its evolutionary origins. The findings are published in the journal Science Translational Medicine. 

APECED—short for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy—causes multi-organ dysfunction, usually beginning in childhood, and can kill up to 30% of people with the syndrome. If diagnosed early and treated by a multidisciplinary healthcare team, however, people with APECED can survive into adulthood. Scientists in NIAID’s Laboratory of Clinical Immunology and Microbiology (LCIM) have developed a world-class APECED diagnostic and treatment program, currently caring for more than 100 patients as part of an observational study and serving as a resource for clinicians across the globe.

APECED is caused by mutations in a gene called AIRE, which provides instructions for making a protein that keeps the immune system’s T cells from attacking the body’s tissues and organs. These genetic mutations reduce or eliminate the protein’s normal function, leading to autoimmunity. 

Most people with APECED are diagnosed based on their clinical signs and symptoms as well as on genetic testing that confirms they have a disease-causing mutation in the AIRE gene. However, as the LCIM team studied people who came to NIH with APECED, they found 17 study participants with clinical signs and symptoms of the disease but no detectable mutations in AIRE. These participants shared two notable characteristics. The families of 15 of the 17 participants were wholly or partly from Puerto Rico, a relatively small, self-contained geographic area, suggesting that the individuals’ disease might have the same genetic cause. In addition, all 17 participants had the same harmless mutation to a single building block, or nucleotide, in both copies of their AIRE gene (one inherited from each parent). This suggested they all might have a similar stretch of genetic material in or around AIRE. These clues led the researchers to start hunting for a unique genetic mechanism that could be causing APECED in the group. 

The Quest for a Genetic Cause

Using technologies called whole-exome sequencing and whole-genome sequencing, the scientists determined the order of all the nucleotides in the DNA of each study participant. By examining and comparing these genetic sequences, the researchers discovered that the 17 participants had the same mutation to a single nucleotide located in a different part of the AIRE gene than the mutations commonly known to cause APECED. APECED-causing mutations usually occur in parts of the AIRE gene called “exons,” which contain the DNA code for the protein. The mutations also sometimes occur at either end of the large, non-coding sections of AIRE called “introns,” which are located in-between the exons. The newly discovered mutation was in the middle of an AIRE intron rather than at either end, so how it caused disease was initially unclear.

To solve this puzzle, the researchers examined what happens when the version of AIRE with this mid-intron mutation gets transcribed into mature messenger RNA (mRNA), the protein precursor. Normally, a molecule called a spliceosome detects the boundaries between introns and exons, cuts out the exons, and “pastes” them together in order. The scientists discovered that the mid-intron AIRE mutation fools the spliceosome into “thinking” that part of the intron is an exon, leading it to cut and paste part of the intron—extraneous genetic material—into the mature mRNA. This gives cells instructions to make an AIRE protein with an incorrect amino-acid sequence at one end. The researchers predicted and then showed that this protein can’t function normally, confirming that the mid-intron AIRE mutation causes APECED in the 17 study participants who previously lacked a genetic diagnosis. 

The scientists anticipate that the newly discovered AIRE variant will be added to genetic screening panels given to people who doctors suspect have APECED or who have a family history of the disease. This could facilitate earlier diagnosis and treatment of people with the mid-intron AIRE mutation, potentially prolonging their lives. It will also enable these individuals to receive genetic counseling to inform their family planning decisions. According to the researchers, the new findings also suggest that there may be other undiscovered, mid-intron mutations that cause APECED or other inherited diseases.

A Potential Treatment in the Making

Now NIAID LCIM scientists are working on a treatment for APECED caused by the mid-intron mutation. They engineered five different strings of nucleic acids, known as antisense oligonucleotides (ASOs), designed to hide the mutation from the spliceosome. Laboratory testing in cells with the mid-intron AIRE mutation showed that one ASO worked. Unable to “see” the mutation, the spliceosome cut out the correct AIRE exons and pasted them together to make mature mRNA that could be translated into a normal AIRE protein. Next, the researchers will test this mutation-masking tool in a mouse model of APECED with this specific mid-intron mutation. They expect results in two to three years. 

ASOs are an emerging form of treatment for rare genetic diseases, sometimes custom-made for just one person.

Origins of the Mutation

Through genetic and statistical analyses, the researchers estimated that the mid-intron mutation first occurred about 450 years ago. This timing coincides with when the first Europeans colonized Puerto Rico, hailing from the Cádiz province of Spain. Notably, one of the two study participants who did not have Puerto Rican ancestry also was from Cádiz and had the same set of DNA variants on one of his chromosomes as the participants with Puerto Rican ancestry. According to the researchers, these findings suggest that one or a few early Spanish colonizers of Puerto Rico carried the mid-intron AIRE mutation, and it eventually became a major cause of APECED in the Puerto Rican population. Further studies are needed to determine the prevalence of this cause of APECED among Puerto Ricans and other populations with Spanish ancestry.    

By contrast, one member of the study cohort had no known Puerto Rican or Spanish ancestry and did not share the same set of DNA variants as the other 16 participants. The investigators say this suggests that the mid-intron AIRE mutation also emerged independently in North America and will likely be found in additional Americans with APECED who do not have Puerto Rican or Spanish ancestry.

Note: APECED is also known as APS-1, short for autoimmune polyglandular syndrome type 1. 

References 

S Ochoa et al. A deep intronic splice-altering AIRE variant causes APECED syndrome through antisense oligonucleotide-targetable pseudoexon inclusion. Science Translational Medicine DOI: 10.1126/scitranslmed.adk0845 (2024).

D Karishma et al. Antisense oligonucleotides: an emerging area in drug discovery and development. Journal of Clinical Medicine DOI: 10.3390/jcm9062004 (2020).

F Collins. One little girl’s story highlights the promise of precision medicine. NIH Director’s Blog. https://directorsblog.nih.gov/tag/milasen/ Oct. 23, 2019. Accessed Oct. 30, 2024.

Findings From NIH-Funded Study Could Facilitate Early Treatment of Neonatal Sepsis

Scientists have identified a four-gene signature detectable in newborns’ blood at birth that predicts before symptom onset whether a baby will develop sepsis during the first week of life, according to a study co-funded by the National Institutes of Health’s National Institute of Allergy and Infectious Diseases (NIAID). Sepsis is a potentially life-threatening condition that arises when the body's response to infection injures its own tissues and organs. Using the newly discovered genetic signature to identify newborns who will develop sepsis could facilitate early treatment and obviate the need to give antibiotics to all newborns with suspected sepsis but lacking a definitive diagnosis. The findings were published today in the journal eBioMedicine. 

Two to 3% of newborns globally develop sepsis, and 17.6% of those babies die. The signs of sepsis in newborns—such as irritability, poor feeding and respiratory distress—are common to many illnesses. Consequently, clinicians sometimes misdiagnose newborn sepsis or suspect it too late, leading to death. If a clinician does suspect that a newborn has sepsis, they give the baby antibiotics pending confirmatory laboratory diagnosis of infection. The most common diagnostic technique takes several days, however, and is often inconclusive. As a result, clinicians often must decide between stopping antibiotics early and risking under-treatment, or giving antibiotics based only on a clinical diagnosis and risking serious side effects and development of antimicrobial resistance.

The NIAID-supported study aimed to find a way to accurately predict sepsis in newborns so it can be diagnosed and treated early while avoiding unnecessary antibiotic use. The researchers conducted their study in a subset of 720 initially healthy, full-term newborns who were enrolled in a larger clinical trial at two community health centers in The Gambia, West Africa. Blood was collected from all babies at birth.

Thirty-three infants were hospitalized within the first month of life for clinical signs suggestive of sepsis. Of those, 21 babies were diagnosed with sepsis, including 15 within the first week of life, which is considered early-onset sepsis. Twelve babies were diagnosed with non-septic localized infections. The researchers matched these 33 babies with 33 healthy controls and analyzed their blood to identify genes that were comparatively more active or less active at birth in each of the four groups. Using machine learning methods, the researchers detected four genes that were comparatively more active at birth only in those newborns who developed early-onset sepsis. The four-gene signature was 92.5% accurate at predicting at birth which of the 66 infants would develop early-onset sepsis. 

The researchers tested the predictive accuracy of this gene signature in a different group of 12 infants whose blood had been collected soon after birth. Half had developed early-onset sepsis, while the other half had remained healthy. The four-gene signature predicted sepsis with 83% accuracy in this group. Further research is needed to determine how well the gene signature predicts early-onset sepsis in much larger groups of newborns.

The study was led by Robert E. W. Hancock, Ph.D.; Tobias R. Kollmann, M.D., Ph.D.; Beate Kampmann, M.D., Ph.D.; and Amy H. Lee, Ph.D. NIAID co-funded the study through its Human Immunology Project Consortium (HIPC) and Immune Development in Early Life program. 

The larger study that enrolled the 720 newborns was called Systems Biology to Identify Biomarkers of Neonatal Vaccine Immunogenicity, sponsored by Boston Children's Hospital and funded by NIAID through the HIPC. More information is available in ClinicalTrials.gov at study identifier NCT03246230.

Reference: An et al. Predictive gene expression signature diagnoses neonatal sepsis before clinical presentation. eBioMedicine DOI: 10.1016/j.ebiom.2024.105411 (2024).