The COVID-19 pandemic has shown us that we have a lot to learn about viral pathogens and their pathologies. But examination of viruses using epifluorescence and transmission electron microscopy and other traditional methodologies have drawbacks: sample preparation is cumbersome, high-throughput analyses are not possible, and it is not possible to discriminate between infectious virions and their noninfectious counterparts. With flow virometry, researchers have a better option for studying viruses. They can now resolve the size and shape of individual viruses, enumerate live viruses, and discriminate virus particles from their environment. Thus, flow virometry offers researchers a new tool to study viruses to help determine their infectivity profiles, develop identification tools, and create the best vaccine candidates.
A new generation of flow cytometry
Flow cytometry is a potent tool for the rapid multi-parametric analysis of distinct cells or particles in a solution. One or more lasers are used for producing fluorescent and scattered light signals that are then detected using different types of detectors. This application has multiple uses in cancer biology, infectious disease monitoring, immunology, molecular biology, and more recently in viral studies.
Using flow cytometry to study viruses has been challenging, especially since the resolution limit of most flow cytometers is 300–500 nanometers (nm), and the size of most viruses falls below this threshold. Now, a new generation of advanced cytometers with better resolution and enhanced fluorescent labeling, viral capture, and size sorting techniques have all helped overcome these challenges.
In 2013, researchers at the National Institutes of Health in Bethesda, Maryland, coined the term “flow virometry” for the study of viruses using advanced flow cytometry. Flow virometry makes it possible to identify and analyze individual viral particles and viral surfaces, as well as sort viruses based on their surface protein content, lipid composition, genome, or size.
This article will discuss the equipment and procedures needed for flow virometry, as well as applications, challenges, and future uses for this technology.
Equipment and procedures
When compared to standard flow cytometers, advanced instruments have several technical improvements, which include:
- Reduced wide-angle FSC (forward light scatter, which allows for size discrimination) detection, which monitors light between 15° and 70° angles and blocks light in the 0° to 15° range to reduce background noise, crucial for detecting light refracted by smaller objects such as viruses.
- Lasers with wattage up to 300mW compared to the 10–20mW for standard equipment. This is necessary for viruses and other small objects that refract light poorly.
- High-performance photomultiplier tubes/digital focusing systems that are superior to the photodiode detectors in routine flow cytometers.
Because of the number of advanced instruments and different protocols that are now available, researchers need to carefully consider what type of equipment, calibration methodology, virus preparation protocol, and staining technique will help them answer the question at hand.
Viruses can be obtained from a laboratory culture or from the natural environment and diluted in a suitable medium such as 5 percent sucrose-NTE buffer, TRIS-EDTA, or 0.1–1 percent paraformaldehyde (PFA). EDTA buffers prevent the formation of viral aggregates and are better suited for viral sample preparation.
Different protocols are available for staining/labeling viruses. With the use of nucleophilic dyes such as Syto 11 or 13, Styryl-TO, TOTO-1, or LDS 751, researchers can differentiate viruses.
Scaffold labeling is another type of viral staining wherein microspheres/nanoparticles that are preloaded with antibodies targeting specific viral surface antigens are first used to isolate the virus of choice. The bound viruses are then differentiated using antiviral antibodies conjugated with fluorescent stains.
In fluorescent magnetic labeling, magnetic nanoparticles are coupled with antibodies that target specific viral surface antigens and bind these viruses. The attached viruses are then labeled with another antibody specific to a different viral epitope. Thus, non-attached particles in the preparation are excluded. Samples are then purified via magnetic column precipitation.
One of the earliest uses of flow virometry was to enumerate live virus particles during the production of viral vaccines. Since then, flow virometry has been used for the detection and characterization of viruses. Some of the more interesting applications of flow virometry include the following:
Vaccine quality control
By using fluorescent-labeled vaccinia (a poxvirus) virions that were characterized using flow virometry, Tang et al. showed that there was an unexpected level of heterogeneity in viral particle size and ratios of noninfectious to infectious viruses. It was also seen that vaccinia virus tended to aggregate with time in the vaccine preparations. Thus, it became difficult to discriminate between infectious viral particles and noninfectious particles.
This study demonstrated the usefulness of flow virometry in the formulation and preservation of viral vaccines and the assessment of vaccine quality.
Quantification of host proteins on the surface of HIV-1
By using a simple overnight staining protocol and flow virometry, Burnie et al. recently showed that using calibrated flow virometry delivered a highly sensitive, high-throughput characterization of the HIV envelope. This is more efficient when compared to previously reported techniques.
Ultrasensitive flow virometry to enumerate intact viral particles
Niu et al. have reported the use of ultrasensitive flow virometry for detecting the titer of individual viruses, including those as small as 27 nm. The researchers stained the viral genome with SYTO 82 and used a novel in-house nano-flow cytometer for the analysis. Using bacteriophage T7 as the model system, they discriminated intact virions from naked viral genomes and empty capsids.
This protocol will be a valuable tool for virus detection when manufacturing gene delivery systems, phage cocktail therapeutics, and vaccines.
Flow virometry for process monitoring of live virus vaccines
Ricci et al. successfully applied a high-throughput flow virometry assay to identify conformational changes and defective viral particles in live virus vaccines (LVV).
Process analytical tools such as Raman probes and flow virometry, as seen in this case, help monitor virus particles during critical stages of LVV manufacturing. (Raman probes are devices in which fibers are used to deliver an excitation laser beam to a sample and then collect the signal.) They help forestall costly batch failures and provide greater control over the process.
The capacity to discriminate viral particles from other similar sized particles such as exosomes and microvesicles is a challenge. This is because exosomes, microvesicles, and viral particles are all produced via a similar mechanism within infected cells.
Identification becomes even more difficult when you consider that exosomes and microvesicles may contain viral components or may be modulated by viruses—viral microRNAs, proteins, or even entire infectious viral particles can be incorporated within exosomes and microvesicles, affecting the detection of these particles. Also, viruses can themselves integrate host proteins. when they bud from the host cell membranes.
With technological improvements, which include better immunoisolation and gating analysis with specific markers, the above challenges can be overcome.
Another area of concern for researchers using flow virometry is the ability to precisely measure small particles. It has been shown that commercial beads are unsuitable for this purpose because of the differences in their refractive indices. With the use of well-characterized rigid non-enveloped viruses that possess uniform sizes, this challenge can be overcome.
Creating the best vaccine candidates
The COVID-19 pandemic has shown us that viral pathogens pose a threat. With the use of flow virometry, researchers can identify and analyze individual viral particles and viral surfaces, as well as sort them based on their surface protein content, lipid composition, genome, or size. Identifying and sorting viruses will help determine their infectivity profiles, develop identification tools, and create the best vaccine candidates.