Defeating Venezuelan equine virus with a decoy

A decoy molecule could help to contain outbreaks of Venezuelan equine encephalitis infection

November25th,2020
Mel J. Yeates

ST. LOUIS—Researchers at Washington University School of Medicine in St. Louis have identified a molecule that protects mice from brain infections caused by Venezuelan equine encephalitis virus (VEEV), a mosquito-borne virus that causes fast-spreading, deadly outbreaks in Mexico, Central America and northern South America.

Public health officials have struggled to contain VEEV outbreaks, since there are currently no effective drugs or vaccines. As a potential drug, the molecule — which has been described in a paper published in Nature — could serve as a much needed tool to control the virus.

“This virus can infect many species of wild mammals, and every few years it jumps from animals to humans via mosquitoes and causes thousands of infections and many deaths,” said senior author Michael S. Diamond, M.D., Ph.D., the Herbert S. Gasser Professor of Medicine and a professor of molecular microbiology, pathology and immunology. “There’s concern that with global warming and population growth, we’ll get more outbreaks.”

The VEEV virus homes in on neurons. People experiences symptoms like headache, muscle pain, fatigue, vomiting, nausea, diarrhea, sore throat and fever within a week. In the most serious cases, the virus gets past the blood-brain barrier, causing encephalitis, which can be fatal in up to a quarter of patients.

Diamond and colleagues — including first authors Hongming Ma, Ph.D., an instructor in medicine, and Arthur S. Kim, Ph.D., a postdoctoral researcher — began by searching for the protein “handle” on the surface of animal cells that the virus attaches to and uses to get inside cells. The researchers theorized that a drug that stops the virus from grabbing that handle could stymie infection and prevent disease.

But first, the researchers had to make a form of the virus they could easily work with. During the Cold War, the U.S. and the Soviet Union attempted to weaponize the virus — and it’s still classified as a select agent, so only high-security labs are allowed to work with it. 

Instead, the researchers and their colleagues took Sindbis virus — a related virus that causes mild fever and rash — and swapped out some of its genes for some from VEEV. The resulting hybrid virus, Sindbis-VEEV, infects cells like authentic VEEV but is unable to cause severe disease.

Using genome-wide CRISPR screening, researchers deleted genes in mouse neuronal cells until they found one called LDLRAD3, the absence of which kept Sindbis-VEEV from infecting cells. LDLRAD3 codes for a little-studied surface protein. Further experiments verified its importance — adding LDLRAD3 back to neuronal cells restored the virus’s ability to infect cells. The human LDLRAD3 gene is nearly identical to its mouse equivalent, and knocking out the human gene also reduced infection in multiple cell lines. 

When the researchers added LDLRAD3 to a different cell type that is normally resistant to infection, the virus was able to infect the cell. Co-author William Klimstra, Ph.D., at the University of Pittsburgh, separately replicated the findings using authentic, highly virulent VEEV.

LDLRAD3 isn’t the only way the virus gets inside cells; a small amount of virus is able to infect cells lacking the protein. But it’s clearly the primary way in. The researchers created a decoy handle using a piece of the LDLRAD3 protein. Any virus particles that mistakenly latch onto the decoy handle would fail to infect cells and instead would get destroyed by the immune system.

“Available transcriptomics data suggest that LDLRAD3 expression occurs in neurons of the brain, which is a site of VEEV infection and pathogenesis,” notes that article. “Because LDLRAD3 mRNA has also been reported to be expressed in epithelial, muscle and myeloid cells15, it could have additional roles in VEEV tropism.”

To test the decoy in a living animal, the researchers injected mice with authentic, virulent VEEV in two ways: under the skin to mimic a mosquito bite, or directly into the brain. They gave the mice either the decoy handle or a placebo for comparison, either six hours before or 24 hours after infection. 

In the experiments, all of the mice that received the placebo died within a week. In most cases, all of the mice that received the decoy molecule survived — although in the most stringent experiment, in which the virus was injected into the brain, two of the 10 mice died despite receiving the decoy handle.

“Low levels of residual VEEV infection were observed in the absence of LDLRAD3 expression in N2a or SH-SY5Y cells, which suggests that additional factors might contribute to cell entry,” the article explains. “Whether this is due to interaction with laminin-binding proteins or other host factors is undetermined. Mosquitoes—a natural host for VEEV—lack an apparent LDLRAD3 orthologue and thus must have separate entry receptors. Moreover, as EEEV and WEEV do not require LDLRAD3 for infection, additional receptors for this virus family probably exist.”

An antiviral drug based on a human — rather than a viral — protein has a major advantage:  it’s unlikely that the virus could evolve resistance to it. The researchers noted that any mutation that enabled the virus to avoid the decoy handle would probably make it unable to attach to cells, as well. 

“In an outbreak situation, you may be able to use a drug like this as a countermeasure to prevent transmission and further spread,” Diamond pointed out.

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