When asymptomatic viral infections turn deadly: Lessons from flies

Fruit flies

When Dr. Jonathan Kagan’s student came to him complaining of dying fruit flies, the two were unaware that their research was about to take an unexpected turn. Their goal in establishing Drosophila lines had been to study virus-host interactions. It was quickly subverted when the flies died on exposure to carbon dioxide, used when transferring flies between vials.

This was surprising on two fronts. First, carbon dioxide is routinely used to anesthetize the flies, with no ill effects. Second, the uninfected flies did not die. The virus used to infect the flies, called vesicular stomatitis virus (VSV), normally does not cause symptoms, even with the virus making several thousand copies of itself.

Digging through decades-old research, some of it in French, much of it only available as abstracts, Kagan and his student, Jonathan Chow, found similar observations of infected flies dying under carbon dioxide had been made before. Kagan’s group decided to dig deeper and found surprising results: the carbon dioxide and the virus had a synergistic effect on the fruit fly nervous system.

“When the virus gets into the brain, the carbon dioxide will cause what we call neuro-trauma,” says Kagan, a scientist in Boston Children’s Hospital’s Division of Gastroenterology, Hepatology and Nutrition. Kagan thinks this finding, reported last month in Cell Host & Microbe, could ultimately have broad implications, from mosquito control to Alzheimer’s disease in humans.

A double hit

Kagan and his colleagues set out to understand the reason for their serendipitous discovery. First, they collaborated with astrophysicists at Columbia University who have developed algorithms that can track miniscule movements of flies. The FlyWalker software can analyze brain function indirectly by measuring the motion of the flies’ legs, walking speed, coordination and other factors. Flies infected with VSV harbored massive amounts of virus but displayed no symptoms of it. But, in the presence of carbon dioxide, the infected flies were unable to stand upright due to an acute paralysis that ultimately led to death.

Image of fruit fly in FlyWalker software
Fruit fly movement recorded by the FlyWalker program.
Annotation: Left (L), Right (H), Front (F), Middle (M) and Hind (H) legs.

Kagan’s team found that infected mosquitoes suffered the same fate – death on exposure to carbon dioxide. But to answer how VSV was disrupting the flies’ movements, they turned back to Drosophilae, a well-understood model system that is easy to manipulate at the genetic level.

VSV synthesizes only five proteins, so the team set out to find out which protein was making the flies more susceptible to carbon dioxide — without actually infecting them with the virus. They developed five different lines of flies, each capable of synthesizing one viral protein. “We made transgenic flies that expressed each of the viral genes, and then asked, ‘do any of them reproduce the infectious sensitivity [to carbon dioxide]?’” says Kagan.

None of the flies were infected with VSV. However, the group of flies producing the VSV-glycoprotein (VSV-G) was as prone to death on exposure to carbon dioxide as flies infected with the whole virus. In these flies, fusion of the neurons and glial cells was apparently the cause of death, since neither cell type was able to function properly.

Learning from flies

Latent or asymptomatic viral infections can make humans vulnerable to disease in the presence of a secondary stress. One example of this is during chronic stage hepatitis C virus infection. Alcohol intake in infected patients is known to promote viral replication while diminishing the immune response, exacerbating liver damage. Conversely, there are also cases in which a latent virus can provide a protective function. For example, the herpes virus in its latent stage can provide immunity to certain bacterial infections. Thus, even with no observable symptoms of viral infection, the presence of a secondary agent intensifies any changes made by the virus.

“Our study might suggest an example of how a single viral protein can cause predispositions to disease as opposed to active tissue destruction induced by the virus,” says Kagan. In humans, singular viral proteins are produced during the latent stage of an Epstein-Barr viral infection even in the absence of active viral replication. More precise therapeutics targeting the viral protein may be required in these cases. Thus, therapy to kill the virus may not eliminate the viral proteins that could cause problems in the presence of environmental stresses.

In the future, fruit flies might present a way to identify key molecules in the pathways that lead to chronic neurological ailments, as well as protective factors. Diseases like Alzheimer’s develop over long periods of time, making cause-and-effect studies a lengthy procedure. Not so in flies. “We’ve discovered the fastest neuro-trauma that can be elicited by a viral infection ever,” says Kagan.

Another potential application of this study’s findings is one Kagan describes as a “fantasy.” He envisions leveraging secondary stresses to control virus-carrying mosquitoes. He hopes that one day, researchers will be able to use such tactics to curb insect-borne disease.