Draining dengue: Alexander Franz uses genetically engineered mosquitoes to fight arboviruses

By Lauren Puckett

Alexander Franz stands before a row of perforated metal boxes. He surveys each one, peering at the contents, before stepping forward and taking a box into his arms.

In it is a swarm of mosquitoes – over 400 of them.

At the sudden movement, they swell like a black gust of wind. They land on the edges of the metal screens that block their escape.

These are Aedes aegypti mosquitoes, the most frequent carriers of dangerous viruses such as dengue fever and Zika.

Franz smiles and continues the conversation; he’s comfortable being in a room filled with bugs. As an assistant professor of veterinary pathobiology at MU, Franz handles a box of mosquitoes the way a carpenter handles a hammer. It’s just another tool of the trade.

It’s from this mosquito laboratory that Franz and his colleagues attempt to genetically modify the insects. Their goal is to halt the transmission of mosquito-borne illnesses throughout the bugs’ bodies.

Alexander Franz handles a box of live mosquitoes. Photo by Lauren Puckett.

Alexander Franz handles a box of live mosquitoes. Photo by Lauren Puckett.

With the recent media attention on the Zika virus and its connection to the birth defect microcephaly, mosquito researchers such as Franz are getting a spotlight cast over their work. Together, they’re searching for weapons against the viruses causing death and defect across the world.  

Franz’s lab staff targets the dengue virus. They fight the disease by snipping and stitching pieces of the virus strain into a gene “cassette,” like a DNA package, and injecting this “cassette” into the mosquito, This makes the insect transgenic – containing DNA designed in a lab.

A dangerous call to action

Franz’s work targets the dengue virus because he views it as one of the most medically important diseases to date. Franz feels, compared to dengue, the hype over Zika is relatively overblown.

“If you ask most people in Brazil, most of them say [one of their] biggest problems is dengue,” Franz said.

Dengue virus is a leading cause of death in the tropics and subtropics, according to the U.S Centers for Disease Control. Dengue is the most common mosquito-borne virus; 40 percent of the world’s population lives in areas where dengue is a danger. The CDC estimates as many as 400 million people are infected yearly, particularly in Africa, the southern areas of Asia, and the lower Americas. And, as of now, there is no vaccine or specific medicine available to treat dengue, according to the World Health Organization.

Around 5 percent of individuals who contract dengue will become severely ill, according to Dr. William Roland, a physician and professor of clinical medicine in the infectious disease division at the MU School of Medicine. This “severe dengue” can be life-threatening.

In 2015, 2.35 million cases of dengue were reported in the Americas, and 10,200 of these cases were diagnosed as severe dengue, according to WHO data. This led to 1,181 deaths.

Typical dengue symptoms are flu-like, such as fevers and headaches, Roland wrote in an email, but sometimes the symptoms cause internal bleeding. This can lead to cardiac arrest and death, Franz said. When the virus spreads as quickly as dengue does, it can be dangerous to an entire community.

Franz uses genetically modified mosquitoes to fight dengue fever. He's been at MU for about three years now. Photo by Lauren Puckett.

Franz uses genetically modified mosquitoes to fight dengue fever. He’s been at MU for about three years now. Photo by Lauren Puckett.

Franz came to MU around 3 years ago, he said, because he wanted to focus specifically on dengue. Originally from Berlin, Franz moved to the U.S in 2000 to work with mosquito-borne viruses at Colorado State University before coming to MU.

While no cases of the dengue virus have been confirmed in Columbia, according to Roland, Franz believes studying dengue is vital regardless of who and where it’s hurting. It’s a matter of global importance.

The cutting room

Franz and his fellow researchers can retrieve strains of dengue, and even a strain of Zika virus, at the MU Laboratory of Infectious Disease Research, a Level 3 biocontainment facility. Because the viruses are a public health risk, the facility is at BSL-3, meaning the lab “works with microbial agents that can cause severe and oftentimes treatable diseases and require additional precautions over BSL-2,” according to the MU LIDR website

After receiving a virus from the MU LIDR, Franz and his team can prepare the strain for their research.

Velmurugan Balaraman, a postdoctoral researcher working in Franz’s lab, is an experienced molecular biologist and virologist. He spends time doing the nitty-gritty work of creating transgenic mosquitoes. It’s a complicated scientific process, but it’s a bit like giving mosquitoes superpowers. If an Aedes aegypti mosquito is Clark Kent, then a molecularly cloned piece of the dengue-2 virus is a sunbeam, blessing him with the strength of Superman.

First, Balaraman identifies an area of the dengue virus RNA genome* that doesn’t mutate. He then copies the area, cloning it into an artificial DNA construct, called a plasmid, that acts as a gene carrier. When the plasmid is inserted into the mosquito’s eggs, the gene “cassette” is inserted into the mosquito’s genome.* It’s like giving Superman a dose of energy from the sun. The mosquito gains amazing strength against dengue fever, like Superman gaining super strength. When Balaraman adds a new gene cassette to the mosquito’s genes, the mosquito’s body develops resistance and the infecting dengue virus essentially loses its power; it struggles to replicate or spread. When the insect takes a blood meal, an antiviral response is triggered and the dengue virus struggles to replicate or spread.*

Velmurugan Balaraman uses RNA interference to genetically edit mosquitoes. Photo by Lauren Puckett.

Velmurugan Balaraman uses RNA interference to genetically modify mosquitoes. Photo by Lauren Puckett.

And much like Kryptonite glows green in the Superman films, transgenic mosquitoes are given a “marker” that makes their eyes glow under a fluorescent microscope. Markers are put into transgenic mosquitoes so they can be visually identified from non-transgenic mosquitoes.

From there, Balaraman said the researchers cross and breed these mosquitoes. Transgenic resistance is a trait that can pass from parent to offspring.

“Our specific goal is population replacement,” Balaraman said. “We want a pool that is resistant to the virus.”

In other words, Franz, Balaraman and the rest of their team want a mosquito revolution. They’re seeking to create an entire population of transgenic mosquitoes resistant to the dengue-2 serotype, and then to craft a family of mosquitoes resistant to all four possible dengue serotypes. If they can achieve this, they could have a real chance at stopping the spread of dengue.

The research is not yet ready to test in the field, but Balaraman said the project is rewarding.

“No one is breathing over my shoulder,” Balaraman said, laughing. “I can say what I feel. I like being in this. Genetic manipulation is exciting to me.”

Life in the lab

In the lab, a woman sits in a stool and presses her eyes against a microscope. Using a little scoop, she delivers squirming baby mosquitoes from pan to petri dish, one after another, until she looks into the microscope again.

She sees that some of the offspring have glowing eyes, the implanted sign that a mosquito is transgenic.

The lab technician, Jingyi Lin, considers herself a mosquito caretaker. She watches over at least 15 different populations of transgenic mosquitoes in the lab, populations that she has to “regrow,” or repopulate, every three months.

Lin said she can screen as many as 10,000 larvae in a week.

It’s a learning process for her, she said. But now she’s caring for mosquitoes and growing cells and working with a violent virus, learning under the guidance of Franz.

Asher Kantor tries to understand how the dengue virus penetrates the midgut barrier of a mosquito. Photo by Lauren Puckett.

Asher Kantor tries to understand how the dengue virus penetrates the midgut barrier of a mosquito. Photo by Lauren Puckett.

“Franz is an easy-going person, but he’s hard-working too,” Lin said. “He works day and night and sometimes he has so many different ideas.”

One of Franz’s ideas is to learn more about virus infection of mosquito tissues, figuring out how mosquito-borne viruses pass through the insect’s body. He’s assigned a couple of researchers, including Asher Kantor, a graduate student at MU, to dissect the creatures and hone in on one area in particular: the midgut.

When an Aedes aegypti mosquito gets infected with a virus, the virus enters and replicates in its midgut system before spreading to its salivary glands, Franz said. As a mosquito chomps on a human host, the virus spreads from the mosquito’s saliva to the human’s bloodstream.

The midgut in an Aedes aegypti mosquito a bit like a sack that holds the organs in shape. When the mosquito takes a gulp of blood rich with dengue-2, the virus sits and stews in this sack. Ordinarily, the barrier around the gut, like a membrane, isn’t easily penetrable. But somehow, the virus is able to cross the barrier of the gut and spread to the mosquito’s salivary glands. Franz says there must be some kind of biochemical pathway that allows this to happen.  

To figure this out, he’s handed the reins to Kantor. Kantor studies a little something called matrix metalloproteinases, a group of proteins that may allow the dengue virus to break through the midgut membrane.

“This mechanism is a topic where no one has a firm idea of what actually occurs,” Kantor said. “The more you know about [how the virus spreads], the better you can combat it.”

Like Lin and Balaraman, Kantor appreciates the autonomy Franz allows his lab staff. Franz believes in attacking the subject of virus transmission from more than one angle, allowing Lin and Kantor to continue research even when, at this point, it’s mainly information-gathering.

Franz “gives you a good idea of what he wants to occur, and then he gives you independence,” Kantor said.

Making progress

With the recent panic over the Zika virus, genetically modified mosquitoes are being heralded as a possible “solution” to the virus problem. But the research is slow-moving, and Franz said he’s concerned the “hysteria,” as he called it, over Zika will overshadow the greater danger that is dengue.

Franz and his team of researchers are trying to fight a way to stop dengue fever from spreading. From left to right: Velmurugan Balaraman, Asher Kantor, Jingyi Lin, Alexander Franz and Shengzhang Dong. Photo by Lauren Puckett.

Franz and his team of researchers are developing a way to stop dengue fever from spreading. From left to right: Velmurugan Balaraman, Asher Kantor, Jingyi Lin, Alexander Franz and Shengzhang Dong. Photo by Lauren Puckett.

“Zika has not been reportedly fatal,” Franz said. “But dengue has.”

And according to a March 17 article from Wired Magazine, dengue is likely to show up along the U.S Gulf Coast soon.

While the research isn’t yet field-ready, it’s not without reward. Franz’s work with mosquitos has been published in multiple research journals, including PLoS One and Insect Molecular Biology, for which he won an award for best paper in 2014. His lab’s research is currently supported by two National Institutes of Health grants.

“Our system is different,” Franz said. “What we’re trying to do really works.”

*Changes in word choice have been made for clarification.


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