Neuroinflammation and Infection: Molecular Mechanisms Associated with Dysfunction of Neurovascular Unit

A Model Virus Capsid Assembles On And Buds Through A Lipid Bilayer Membrane

Assembly and budding of a virus from a membrane microdomain.

Viruses have an outer protein shell called a capsid which surrounds the viral nucleic acid. Enveloped viruses, such as HIV, have an additional layer comprising a lipid bilayer membrane which surrounds the capsid. For some enveloped viruses, the protein capsid assembles on the lipid membrane, driving deformation and eventually budding of the enveloped capsid.

The video shows an animation of a computer simulation trajectory, in which a model capsid (tan) assembles within and buds through a domain (red) within a lipid bilayer membrane (blue). Such simulations enable examining how changing membrane properties affect viral budding, and may shed light on why many viruses preferentially assemble from membrane microdomains such as lipid rafts.

The Culprit Of A Mysterious Superworm Epidemic Finally Identified

In March 2022, Judit Pénzes, a virologist at Rutgers University, was contacted by a farmer in Utah who was worried about the wide-scale die-offs happening in his superworm populations. Superworms, the larvae of the Zophobas morio beetle, are a protein-rich food source for captive reptiles, birds, and amphibians, and in the face of a growing world population, a potential alternative protein source for humans.1 They also chow down on polystyrene, offering an innovative solution to humanity's increasingly dire plastic waste problem.2 But for the last few years, these small but mighty larvae have been threatened by a deadly disease sweeping across the nation.

Judit Pénzes uses advanced microscopy techniques, including cryo-EM, to study viruses.

Judit Pénzes

The frustrated farmer's once-healthy superworms were exhibiting distressed, uncontrollable wiggling and blackening followed by stiffening, liquefaction, and death. He wasn't the only one who had observed these bizarre deaths—for years, insect-rearing facilities throughout the United States had been struggling to determine the cause of this superworm apocalypse. "I went down the rabbit hole to try and figure out what's going on," said Pénzes, who started browsing insect forums and social media posts on the topic dating back to 2019. People put forth humidity, temperature, and even a fungus in the superworms' food as a potential culprit, but Pénzes had another agent in mind. "No one really thought of it being a virus, which was strange at the time because to me, who has seen several viral infections taking place in farmed insects, that was my absolute first guess," she recalled.

Now, with the help of cryogenic electron microscopy (cryo-EM), Pénzes and her colleagues identified the cause of the ongoing agricultural pandemic: a parvovirus that they named the Z. Morio black wasting virus (ZmBWV).3 In characterizing the virus, the researchers identified a prophylactic approach to protect superworm populations. They published their findings in Cell.

"Apart from the cool detective story that it was, there was also lots of very exciting structural biology in there," said Joost Snijder, a structural virologist at Utrecht University who was not involved in the study.

Insect-borne viruses that plague humans, like dengue, are the focus of much research, but over the years, Pénzes has established herself as an expert in the lesser-known viruses that primarily harm the insects themselves. Before joining virologist Jason Kaelber's team at Rutgers University, she worked as a postdoctoral researcher in an insect diagnostic lab at the Armand-Frappier Health Biotechnology Research Center. "When insect farms had problems with their insects dying from unexplainable reasons, then they would reach out to us and would send samples," said Pénzes. "This farmer got my information, that I am the person to contact if you have an insect problem." She agreed to look at the farmer's superworms. "A week later, we found about four pounds of very dead, oozing, stinky, superworm larvae in our mailroom," said Pénzes.

Although she had a hunch that a virus might be to blame for the mass die-offs, she didn't know where to start. "Beetle viruses are really under-investigated," said Pénzes. So, she ground up the dead worms and ran the superworm slurry through a sucrose cushion, a density gradient that separates the liquid contents. Piled up in the lowest rungs of the gradient were her first clues: evidence of viral capsids.

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"A week later, these samples were already in our cryoelectron microscope, and that's when we first got the first glimpse of what this virus actually looks like and what kind of virus it may be," said Pénzes.

Using cryo-EM, the researchers captured a near-atomic-resolution three-dimensional structure of ZmBWV's capsid protein and its genome contents. After querying the Protein Data Bank for known proteins with similar structures, it became clear that they were looking at a virus belonging to the Densovirinae subfamily of the Parvoviridae.

"To infer at the genus level what kind of pathogen you're looking at, this is very impressive to get this just from the EM maps," said Snijder.

Pénzes and her team identified the culprit of a disease afflicting superworms that causes distressed, uncontrollable wiggling and blackening followed by stiffening, liquefaction, and death. They called it the Z. Morio black wasting virus.

Judit Pénzes

Pénzes and her team took a somewhat unusual approach by using cryo-EM as the primary diagnostic tool; sequencing-based techniques are still the most common approach for detecting pathogens.4 Although cryo-EM infrastructure is harder to come by and poses technical constraints, Pénzes noted that EM can help scientists quickly identify a virus at the genus level and, unlike metagenomics, it is not reliant on reference databases that are restricted to known sequences. "It's really cool that you can get that far from the cryo-EM maps, and for an EM expert like Jason that was a logical first thing to do, though, I think in most virology labs, epidemiological labs and most public health institutions, next-generation sequencing will, for a very long time, still remain the go to tool," said Snijder.

To make sure they correctly identified the culprit behind the epidemic, the researchers collected additional samples from other superworm breeders and local pet stores. "Whenever I went to a pet store, I always opened up the superworm containers to see whether they had infected ones," said Pénzes. The symptomatic superworm larvae from these sites tested positive on a diagnostic polymerase chain reaction (PCR) test that they developed for ZmBWV. Additionally, healthy larvae that they infected with the newly identified virus went on to develop the deadly disease.

When Pénzes visited a superworm farm, she noticed that a handful of the breeder's mealworms—a closely-related species of darkling beetle—had died in a manner that looked suspiciously similar to the virus-stricken superworms. A curious Pénzes returned to the laboratory with a sampling of asymptomatic mealworms and tested them for the virus. "To my great surprise, I found the virus at the very same yield as we find them in the blackened superworm carcasses," said Pénzes. However, unlike the superworms, the mealworms experienced a much lower mortality rate, suggestive of species-specific susceptibilities to the same virus. Further experimentation revealed the presence of ZmBWV-like viruses, leaving Pénzes to wonder whether the mealworms also harbored non-pathogenic variants.

Pénzes isolated and purified the viral variants identified in mealworms and introduced them to healthy superworms. "The superworms didn't get sick from it either, so it was clear that we found the non-virulent variant," she said. Furthermore, superworm larvae treated with a non-virulent strain were protected from subsequent exposure to the pathogenic variant, highlighting a potential prophylactic approach for preventing the development of ZmBWV-induced illness.

Pénzes and her team are currently developing a vaccination strategy to deliver non-virulent strains of ZmBWV to beetle larvae. Like many human vaccines, their prophylactic approach wouldn't necessarily prevent infection, but it would reduce mortality, improving not only the lives of the superworms, but the farmers, too.

Disclosure of Conflicts of Interest: Judit Pénzes and her colleagues have submitted a preliminary patent for developing non-virulent ZmBWV strains into a superworm vaccine.

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Viral Vector Efficiency Makes Advanced Therapies Work For Everyone

Viruses make excellent vectors because they can infect hard-to-reach cells and predictably modify gene expression.Credit: Tumeggy/ Science Photo Library Getty Images

Cell and gene therapies are changing lives. They engineer patients' cells or DNA to create personalized therapies, unlocking long-lasting or even curative treatments for diseases with high unmet needs, from cancer to rare genetic disorders.

Currently, these treatments are complex and costly to produce. This translates to high prices, and limits access for patients. "Scientists in academia and industry are working diligently to reduce the cost of these treatments and make access more equitable," says Amitabha Deb, senior vice president of process and analytical development at viral vector manufacturer iVexSol, which uses its lentiviral vector technology to reduce the time and expense involved in cell and gene therapy development.

At the heart of gene therapy is the process of reprogramming cells, using genetic instructions delivered by vehicles called viral vectors. Viruses, such as adeno-associated virus and lentivirus, are very efficient vectors because they can infect hard-to-reach cells and predictably modify gene expression. Producing viral vectors more efficiently is central to accelerating the manufacturing process, making the final product more affordable to patients and health systems.

Cost-effective and reliable vectors

With any new therapy, winning approval is only part of the battle. Once a treatment starts scaling up for clinical use, any gap in vector production could bring costly delays, both for developers expecting a return on their investment, and for patients waiting to benefit from an effective new treatment. Investing in early process development is crucial.

A potential bottleneck is transfection, where genetic material is introduced into the host cells, leading to the production of viruses. This stage is enhanced by using the right transfection reagent to optimize the introduction of genetic material, along with other production parameters, to ensure process scalability, reliability and cost-effectiveness. This improves the odds of achieving high physical and functional titres, and increases the percentage of viral capsids that are filled with therapeutic cargo. "In producing any viral vector, the transfection reagent needs to guarantee both high titre and infectivity, as well as quality, to satisfy the needs of a particular disease indication," says Deb. "It must also be suitable for future GMP production."

Working with expert partners helps reduce process development time and accelerate the journey towards the clinic. Reagent kits can make the transfection process reliable, scalable, cost-effective and standardized — key considerations for viral vector manufacturing. "Without a partner with a deep understanding of the process and product, efforts to reduce costs will be in vain," says Deb.

iVexSol has a strong relationship with US-based Mirus Bio, which has more than 20 years of experience developing technologies for cell and gene therapy delivery. Mirus offers solutions to increase process efficiency for viral vector production, and a unique reagent formulation that aims to boost virus quality and functional titres compared to traditional polymer-based options. "We have tested many transfection reagents and Mirus gives us the best results, yielding more infectious viral particles," says Deb.

Finding the optimal approach to viral vector production can make the difference between success and failure for a new therapy. Efficient production will be reflected in the quality and reach of the final product. "The most important thing is to reduce costs so ultimately your medicines will cost less," says Deb. "Yes, these medicines have unprecedented clinical outcomes, but they need to be accessible to the broadest patient population."

To find out more about Mirus Bio, and how our transfection expertise can benefit your cell or gene therapy programme, visit us here.

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