A new imaging technique for studying the structure of a childhood disease, respiratory syncytial virus (RSV), could provide scientists with the information they need to develop new antiviral drugs and perhaps even a vaccine to prevent severe infections.
By the time they’re two years old, most children have had RSV and suffered symptoms no worse than a bad cold. But for some children, especially premature babies and those with underlying health conditions, it can lead to pneumonia and bronchitis—which can require hospitalization and have long-term consequences.
The technique could clarify how RSV enters cells, how it replicates, how many genomes it inserts into its hosts—and perhaps why certain lung cells escape relatively unscathed.
“We want to develop tools that would allow us to get at how the virus really works,” says Philip Santangelo, an associate professor of biomedical engineering at Georgia Institute of Technology (Georgia Tech) and Emory University. “We really need to be able to follow the infection in a single living cell without affecting how the virus infects its hosts, and this technology should allow us to do that.”
While RSV will be the first target for the work, the researchers believe the imaging technique could be used to study other RNA viruses, including influenza and Ebola.
“We’ve shown that we can tag the genome using our probes,” says Santangelo. “What we’ve learned from this is that the genome does get incorporated into the virion, and that the virus particles created are infectious. We were able to characterize some aspects of the virus particle itself at super-resolution, down to 20 nanometers, using direct stochastic optical reconstruction microscopy (dSTORM) imaging.”
What is RSV doing?
RSV can be difficult to study. For one thing, the infectious particle can take different forms, ranging from 10-micron filaments to ordinary spheres. The virus can insert more than one genome into the host cells and the RNA orientation and structure are disordered, which makes it difficult to characterize.
For the study, published in ACS Nano, researchers used a probe technology that quickly attaches to RNA within cells. The probe uses multiple fluorophores to indicate the presence of the viral RNA, allowing them to see where it goes in host cells—and to watch as infectious particles leave the cells to spread the infection.
“Being able to see the genome and the progeny RNA that comes from the genome with the probes we use really give us much more insight into the replication cycle,” Santangelo says. “This gives us much more information about what the virus is really doing. If we can visualize the entry, assembly and replication of the virus, that would allow us to decide what to go after to fight the virus.”
The research depended on a new method for labeling RNA viruses using multiply-labeled tetravalent RNA imaging probes (MTRIPS). The probes consist of a chimeric combination of DNA and RNA oligonucleotide labeled internally with fluorophores tetravelently complexed to neutravidin. The chimeric combination was used to help the probes evade cellular defenses.
“There are lots of sensors in the cell that look for foreign RNA and foreign DNA, but to the cell, this probe doesn’t look like anything,” Santangelo says. “The cell doesn’t see the nucleic acid as foreign.”
A tight bind
Introduced into cells, the probes quickly diffuse through a cell infected with RSV and bind to the virus’s RNA. Though binding tightly, the probe doesn’t affect the normal activities of the virus and allows researchers to follow the activity for days using standard microscopy techniques. The MTRIPS can be used to complement other probe technology, such as GFP and gold nanoparticles.
Work done by graduate student Eric Alonas to concentrate the virus was essential to the project, Santangelo says. The concentration had to be done without adversely affecting the infectivity of the virus, which would have impacted its ability to enter host cells.
“It took quite a bit of work to get the right techniques to concentrate the RSV,” he says. “Now we can make lots of infectious virus that’s labeled and can be stored so we can use it when we want to.”
To study the infection’s progress in individual cells, the researchers faced another challenge: living cells move around, and following them complicates the research. To address that movement, the laboratory of Thomas Barker used micro-patterned fibronectin on glass to create 50-micron “islands” that contained the cells during the study.
Why some, but not others?
Among the mysteries that the researchers would like to tackle is why certain lung cells are severely infected—while others appear to escape ill effects.
“If you look at a field of cells, you see huge differences from cell to cell, and that is something that’s not understood at all,” Santangelo says. “If we can figure out why some cells are exploding with virus while others are not, perhaps we can figure out a way to help the bad ones look more like the good ones.”
One of the challenges of studying RSV is maintaining its activity in the laboratory setting—a problem parents of young children don’t share.
“When you handle this virus in the lab, you have to always be careful about it losing infectivity,” Santangelo says. “But if you take a room full of children who have not been infected and let one infected child into the room, 15 minutes later all of the children will be infected.”
Researchers from Emory University and Vanderbilt University contributed to the research, which was supported by the National Institutes of Health’s National Institute of General Medical Sciences.
Source: Georgia Tech