Identifying a gene that helps cells resist West Nile and Zika viruses with CRISPR

Zika Vaccine

UT Southwestern researchers today report the first use of CRISPR genome-wide screening to identify a gene that helps cells resist flavivirus infection. That nasty class of pathogens includes West Nile virus, dengue fever, Zika virus, and yellow fever.

In a study published in Nature Microbiology, the team led by Dr. John Schoggins, Assistant Professor of Microbiology, used the cutting-edge CRISPR technology to identify the IFI6 gene as a potent antiviral gene targeting flaviviruses. The researchers then used traditional cell culture studies to confirm the gene’s role in protecting against infection by Zika, West Nile, dengue, and yellow fever viruses.

“Other studies have used CRISPR genetic screens to identify cellular genes that are required for flavivirus infection. Our study is the first to use this technology to identify cellular genes that inhibit infection,” said Dr. Schoggins, a Nancy Cain and Jeffrey A. Marcus Scholar in Medical Research, in Honor of Dr. Bill S. Vowell, and a Clayton Foundation Investigator.

“In mammals, cells naturally defend against viral infection through interferon, a molecule that sets off a warning system that a virus has been detected and that the cells need to engage their viral defense systems. The cells do this by activating hundreds of interferon-stimulated genes,” he said. “Flaviviruses cause substantial human disease, and interferon is involved in the body’s innate immune response to these viruses.”

Dr. Schoggins said the team used recently developed genome-wide CRISPR screening technology to identify which of the interferon-induced genes played a major role in suppressing flavivirus infection. He praised the work of graduate student and lead author Blake Richardson and of co-author Dr. Maikke Ohlson, a senior research scientist in his laboratory. “Blake performed all the phenotypic and mechanistic work on how IFI6 inhibits flaviviruses and Dr. Ohlson performed the CRISPR screen that allowed us to uncover IFI6 as a potent suppressor of flavivirus infection,” he said.

“In the CRISPR screen, we used human liver cells and knocked out every gene in the genome – about 19,000 genes – one at a time. We then stimulated the cells with interferon, knowing that this stimulation would normally allow the cells to resist viral infection. For the cells that did not resist infection – because they were missing a gene due to the CRISPR knockout – we used next-generation sequencing to figure out the identity of the relevant genes,” he said.

Dr. Schoggins explained that the CRISPR gene-editing technology made such a study extremely efficient, uncovering the prominent flavivirus-inhibiting role of IFI6.

“The brilliance of the technology is that all of these CRISPR-targeted cells are pooled together in just a few big cell culture dishes. The cells are also bar-coded so you know which gene is missing from each cell when you observe how they respond to the addition of interferon,” he said.

“The technology is super cool,” he added.

In cell culture studies, the IFI6 gene – apparently working via its protein product, also called IFI6 – inhibited yellow fever, a flavivirus known to infect the liver. Cells with a working IFI6 gene also inhibited dengue, Zika, and West Nile viruses, he said. The researchers confirmed those results in liver cells by repeating the experiment in kidney and skin cell lines and in neurons.

Future work will entail drilling down into the molecular mechanism of the IFI6 protein, with the hope that this knowledge may provide a foundation for developing therapies that could target flavivirus infection.

Sporadic West Nile cases have been reported this summer in the United States, he said, adding, “Zika has waned, but I think people still remember it quite well; dengue is an ongoing problem in tropical climates, and there’s currently an outbreak of yellow fever virus in Brazil.”

In earlier studies, Dr. Schoggins and other researchers identified other possible flavivirus resistance proteins using non-CRISPR screening techniques, but the CRISPR technology makes IFI6 appear to play a more prominent role, he said.

Questions still to be answered include whether CRISPR screens in other cell types would give different results. This study focused on liver cells because it began as an investigation of yellow fever, which is known to attack the liver. Zika is known to affect cells in the brain but CRISPR genetic screening in neurons presents logistical challenges, he explained.

Learn more: CRISPR screen identifies gene that helps cells resist West Nile, Zika viruses

 

 

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A new pesticide-free way to limit mosquito populations in some areas and reduce the spread of the West Nile virus.

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Researchers at the University of Waterloo may have discovered a new, pesticide-free way to limit mosquito populations in some areas and reduce the spread of the West Nile virus.

The study by Waterloo researcher Brad Fedy discovered that introducing hungry minnows into bodies of water where mosquitoes breed results in the minnows feeding on mosquito larvae, which dramatically decreases the number of adult mosquitoes capable of carrying the disease.

“The best strategies to limit mosquitoes start at the larval stage. Unfortunately, in North America, control efforts are largely limited to larvicides, which require a repeated application and have potentially negative ecological impacts,” says Fedy. “Addressing the problem with minnows provides many benefits in that it is low-maintenance, cost-effective, better for the environment in many cases, and our health.”

The study took place over three years and introduced minnows into ten treatment reservoirs. Researchers monitored an additional six non-treated reservoirs.

Treatment ponds demonstrated suppressed levels of mosquito larva over each season compared to controls with a model-predicted 114 per cent decrease in larva density within treatment ponds.

“There are many potential advantages to using indigenous fish species as an alternative for larval control including lowered environmental impact, decreased costs regarding time and financial inputs, and the potential for the establishment of self-sustaining fish populations,” said Fedy. “This isn’t a complete solution to the dangers of West Nile, but it should be considered as part of any plan to protect the health of vulnerable populations.”

Fedy and his team discovered the method while researching sage grouse populations in the intermountain west. Sage grouse populations suffer adverse impacts from mosquitoes transferring viruses like West Nile and investigated ways to mitigate those negative impacts. What they discovered could also improve human health.

Learn more: Study finds a pesticide-free way to combat mosquitos and West Nile

 

 

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Using tobacco to thwart West Nile virus

ASU researchers Qiang “Shawn ” Chen and Huafang “Lily ” Lai infiltrate a tobacco plant to produce monoclonal antibodies against West Nile virus.

A new generation of potentially safer and more cost-effective therapeutics against West Nile virus, and other pathogens

An international research group led by Arizona State University professor Qiang “Shawn” Chen has developed a new generation of potentially safer and more cost-effective therapeutics against West Nile virus, and other pathogens.

The therapeutics, known as monoclonal antibodies (MAbs) and their derivatives, were shown to neutralize and protect mice against a lethal dose challenge of West Nile virus—even as late as 4 days after the initial infection.

“The overarching goal of our research is to create an innovative, yet sustainable and accessible, low cost solution to combat the global threat of West Nile virus,” said Chen, a researcher at Arizona State University’s Biodesign Institute and professor in the Department of TEIM.

West Nile virus is spread by infected mosquitoes, and targets the central nervous system. It can be a serious, life-altering and even fatal disease and currently, there is no cure or drug treatment against West Nile virus, which has been widely spread across the U.S., Canada, Latin America and the Caribbean.

“The goal of this latest research was twofold,” said Chen. “First, we wanted to show proof-of-concept, demonstrating that tobacco plants can be used to manufacture large and complex MAb-based therapeutics. Secondly, we’ve wanted to improve the delivery of the therapeutic into the brain to combat West Nile virus at the place where it does the greatest harm.”

The study appears in the March 27 online edition of PLOS ONE. Along with Chen, the research team included Junyun He, Huafang “Lily” Lai, Michael Engle, Sergey Gorlatov, Clemens Gruber, Herta Steinkellner and long-time Washington University collaborator Michael S. Diamond.

Chen’s group has been a pioneer in producing MAbs as therapeutic candidates in plants, including tobacco and lettuce plants. A couple of years ago, his team demonstrated that their first candidate, pHu-E16, could neutralize West Nile infection and protect mice from exposure. MAbs target proteins found on the surface of West Nile virus.

However, this antibody was not able to accumulate at high levels in the brain.

One approach to tackle this challenge is to program into the therapeutic antibodies the capability of binding to receptors that can help the MAbs to cross into the brain. Chen wanted to use this strategy to produce a more effective way to combat West Nile virus.

In the new study, they improved upon their pHu-E16 design, making half a dozen new variants that could, for the first time, lead to the development of MAbs that effectively target the brain and neutralize West Nile virus.

Mice were infected with a lethal dose of West Nile virus, and increasing amounts of a MAb therapeutic were delivered as a single dose the same day of infection. In another experiment, Chen’s team tested whether the therapeutic, called Tetra pHu-E16, could be effective after infection. In this case, the therapeutic was administered 4 days after West Nile virus infection, when the virus has already spread to the brain. In each case, they protected up to 90 percent of the mice from lethal infection.

This is the first instance of such an effect and makes possible neutralizing West Nile virus even after infection by a tetravalent MAb. The tetravalent MAbs design will offer the researchers greater flexibility toward selection of disease, tissue and antigen targets.

For Chen, this also gives promise to his team developing a plant-based system to dramatically reduce the costs of commercial manufacturing of MAbs.

“This study is a major step forward for plant-based MAbs, and also demonstrates for the first time the capacity of plants to express and assemble large, complex and functional tetravalent MAb complexes,” said Chen.

MAbs are a hot and highly competitive research field, having been shown to effectively target cancer, autoimmune and inflammatory diseases. Now a $60 billion market for the biotechnology and pharmaceutical sectors, growth of the market has been hampered by high development costs of producing these in animal cell systems, which when factoring in a long period for manufacturing, R&D and clinical trials, may reach around $1 billion per each therapeutic candidate.

Therapeutic MAbs are typically made in animal host cells and assembled into Y-shaped complexes. Until now, tetravalent MAbs had never been made in a plant system before. To make the potential therapeutics, the group is able to use young tobacco plants and a protein expression system to make and harvest the proteins in the leaves.

For the study, MAbs were rapidly produced in tobacco plants in as little as ten days, giving promise to change the image of scourged product that causes lung cancer into a manufacturing system for societal benefits against infectious diseases.

“It is our hope that these results may usher in new age of cost-effective, MAbs therapeutics against WNV and other neurological diseases,” said Chen. “Our next step is to move this forward with the development of bifunctional MAbs that can target to the brain with the ultimate goal of entering human clinical trials.”

Read more . . .

 

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