Scientists at Rutgers University in New Brunswick have discovered a virus that caused a nationwide die-off of superworms, which are used as food for birds, reptiles and other domestic animals and, increasingly, as an alternative source of protein for humans. In doing so, they have developed a new method for searching for and identifying emerging viruses and pathogens in humans, plants and animals.

Using crushed beetle carcasses that form a pulp and an electron microscope cooled with liquid nitrogen, the scientists report in cell that they discovered something they called Zophobas morio Black Wasting Virus.

The name derives from the virus’s lethal effect on a species of darkling beetle native to the subtropics, Zophobas morio, particularly in the insect’s immature larval stage when it hatches from its eggs as large, brown superworms. This species was called the “superworm” because its larvae, about 5 cm long, are larger than any others reared for food.

The protein-rich larvae of Z. morio, a staple diet for captive, often exotic reptiles, birds, fish and amphibians around the world, began mysteriously dying in 2019, puzzling pet food suppliers and pet owners.

Jason Kaelber, author of the study and associate research professor at the Institute for Quantitative Biomedicine (IQB) at Rutgers-New Brunswick, collaborated with Judit Penzes, the study’s first author and a postdoctoral fellow at the IQB.

“Judit wanted to find out why beetle farmers were losing all their superworm colonies to a deadly disease, and I wanted to find ways to discover new viruses that didn’t rely on DNA or RNA sequencing,” Kaelber said. “We eventually discovered the virus that was sweeping the country and killing superworms.”

The scientific investigation began over a year ago when Penzes, a molecular virologist, was contacted by owners of bug farms whose superworms were mysteriously dying out at an alarming rate. Penzes was already well known in the industry because she had previously isolated a virus that killed crickets, another popular pet food.

She started collecting superworms from pet stores in New Jersey. “When I went to a pet store, I would immediately go to the feeder insect section, open the containers and look at the worms,” ​​she said. “They were all infected. I told the store owners what I saw, that I was researching this virus, and asked if I could have the container. They immediately agreed. They told me to take as many as I needed.”

She returned to her lab, took a Magic Bullet blender, threw the worm carcasses in and blended them at high speed. This created a slurry of bug juice, which she processed through a virus purification process that separated the virus based on its density. In the final step, she shined a fluorescent light on the centrifuge tube and the virus glowed blue.

“When I saw it, I said, ‘I got you,'” Penzes said. “That’s when I knew it was actually a virus.”

Penzes then examined the virus together with his colleague Kaelber, who is also an electron microscopist, using a cryo-electron microscope, which provides a three-dimensional view of the virus, including its interior.

“You take a virus, a protein, a cell, etc. and freeze it so fast that the water freezes without turning into ice crystals,” Kaelber said. “We can actually figure out the amino acid sequence of the protein without analyzing the DNA and just by looking at that 3D structure because we have such sharp resolution.”

They compared the protein’s structure to all known proteins using Rutgers University’s Protein Data Bank database and found that it is similar to, but not identical to, a cockroach virus and that it belongs to a family of animal viruses known as parvoviruses.

“It is new and different from anything that has been sequenced or imaged before,” Penzes said.

The scientists are also grateful to superworm farmers across the country who voluntarily sent samples when the study became known. “The farmers’ eagerness to help us research the virus contributed enormously to getting this study published,” Penzes said.

According to Kaelber, the project provides a “proof of concept” that cryo-electron microscopy can be used to directly discover and characterize new pathogens.

“If there is ever a really significant outbreak in the future, we will want to use every tool at our disposal to see what we can find,” Kaelber said. “We want to make diagnostic cryo-electron microscopy routine so that when we have unknown infectious diseases, we have many opportunities to identify the pathogen on the same day.”

Cryo-electron microscopy has gained popularity in recent years and has become a more common method for 3D analysis of known samples, but the Rutgers work marks the first time the method has been used on an unknown pathogen.

After discovering the virus, researchers tested a way to protect the Z. morio beetles from the disease by injecting them with a closely related virus from a different species that does not cause symptoms. They are currently developing a vaccine based on this work.

“The discovery is important for two reasons,” Kaelber said. “First, beetle breeders can use this information to protect their colonies and understand what measures will be effective or ineffective in controlling the epidemic. Second, the beetle epidemic was a real-world test of the technology, which we hope can be useful for quickly investigating future outbreaks in humans, plants or animals.”

Scientists Martin Holm of the Rutgers Institute for Quantitative Biomedicine and Samantha Yost of REGENXBIO Inc. in Rockville, Maryland, are also authors of the study.