To unravel the complexity of the viral life cycle, researchers must rely on model and assay systems to efficiently explore mechanisms that drive infection, replication, and transmission.
While viral surveillance and host genomics studies help us understand viral evolution and identify potential biologic factors impacting infection, researchers need a way to confirm these observations and more deeply explore biologic activities. Virology researchers have long relied on the development of model systems to quickly recreate aspects of real-world infection in a more controlled environment. With an effective model, researchers can develop biologic assays (or bioassays) that can rapidly identify key biologic pathways, investigate specific viral/host factors, and screen drug candidates.
When building bioassays for basic, translational, and pre-clinical research, researchers often use a combination of in vitro and in vivo bioassays, using cellular or animal models respectively. In this blog, we will discuss some details and use cases for bioassay models.
Building and using cell-based assays in virology research
In modern research labs, the majority of basic, translational, and pre-clinical research relies on cellular systems to study specific biologic activities and molecular interactions. [1-3] Specifically, cell-based assays play a key role in studying viral infection and viral inhibition.  While cultured cells don’t perfectly recapitulate target viral hosts, they represent a major step over biochemical assays that don’t use any living components.
By using living cells, researchers can determine necessary and sufficient factors required for effective viral replication. Using cells, researchers can collect massive amounts of data and perform many studies quickly, without needing to wait weeks or months just to breed and raise animals. Additionally, cellular assays eliminate many variables found in an animal host that can obstruct informative investigations on essential viral biology.
The development of cell-based assays starts with investigations into the susceptibility of existing cell lines to specific viruses. These provide a backdrop for building out more complex assays, since basic viral replication indicates that all factors sufficient for infection are present. By comparing cells that are and are not susceptible to viral challenge, researchers can also identify important host-factors, conditions, and mechanisms required by a virus. In one such study, a team led by researchers at the University of Hong Kong studied SARS-CoV-2 infectivity in over 20 cell types (including those isolated from humans) to determine susceptibility, species tropism, replication kinetics, and cellular damage.  These researchers then cross-compared this data with findings for SARS-CoV. Their results showed that SARS-CoV-2 replicated well in five different human cell lines. Like SARS-CoV, SARS-CoV-2 replicated well in Caco2 (human intestinal) cells, but outperformed SARS-CoV in Calu-3 (human pulmonary) cells.
Researchers can also explore studies that attempt to rescue or reduce viral susceptibility, either for basic research or for building a better cell model for subsequent assays. For example, researchers found that A549 (human lung alveolar) cells were not permissive to viral infection but that exogenous expression of ACE2 in A549 enabled SARS-CoV-2 replication.  This indicated that increased receptor abundance was required for replication in these cells, given the low natural expression of ACE2 in A549 cells.
Bioassay cell line development
Once the base susceptibility level is understood, researchers often choose to manipulate their cell lines of choice to increase susceptibility, individually study specific factors, create reporter systems, and beyond.
Users often develop their cell lines into better infectivity models, simply because existing cell lines are not permissive enough to replicate natural transmission conditions sufficiently. For example, you may want to study SARS-CoV-2 replication in human lung alveolar cells using a continuous cell line, like A549. To do so, you would instead use an A549 cell line that expresses additional exogenous ACE2 in your cell-based assays.
In other cases, researchers may want to add a specific receptor not normally expressed and/or remove an existing one. For example, researchers from the University of Melborne, WHO, and the Westmead Millennium Institute, studied whether influenza could infect Chinese hamster ovary (CHO) cells independent of their primary receptor—sialic acid—by instead using human immune receptors that bind the virus.  To investigate this, researchers used CHO Lec2 cells, which don’t express sialic acid on their cell surfaces. Going further, they engineered these CHO Lec2 cell to express human DC-SIGN and L-SIGN proteins. Influenza viruses naturally bearing DC-SIGN or L-SIGN receptors could infect these CHO cells, independent of sialic acid. With stable cell lines, these researchers could then apply these same cells to subsequent studies and viruses.
Additionally, reporter cell line generation and use can massively increase your assay’s throughput. In these cases, researchers often incorporate some marker of viral gene expression, viral enzyme activity, or host response to study infection. As one interesting example, researchers at the University of Heidelberg built a viral infection cell-based assay, by cloning an endoplasmic reticulum (ER)-anchored green fluorescent protein (GFP) into Huh7 and A549-ACE2 cells.  These ER-anchored GFP include a protease cut site recognized by viral proteases, like those found in SARS-CoV-2 and dengue virus. In their study, when viruses infected these cells and expressed their proteases, they cut the reporter site. This led to the translocation of GFP from the ER to the nucleus, due to the inclusion of a nuclear localization signal (NLS) on the GFP construct. By tracking the location of the GFP marker, they could measure viral infection.
Regardless of what genetic changes are needed for your assay, transfecting and/or genome engineering a cell line represents a key step in the assay development process. Transfection allows you to incorporate new genes from a construct into your target cell. However, it’s important to note that there is significant cell-specific variability with respect to what transfection strategies are most efficient.  Alternatively, you may want to make specific edits and mutations in a given cell line’s genome. While a number of genomic editing tools exist, CRISPR is arguably the most important and widely adopted approach. 
Don’t forget to confirm your genetic changes using DNA sequencing! Ensuring your exact cellular changes have been made without off-target effects is crucial to ensuring your bioassays provide valuable and interpretable data.
Taking the next step: animal model development
As useful and important as cell-based assays are, researchers must often move to animal studies to collect additional biologic evidence and confirm observations found during in vitro studies. Common animal models in virology include rodents, rabbits, dogs, horses, ferrets, and non-human primates.  While animal models are not perfect replications of human biology, they do represent a significant increase in biologic complexity and are key to exploring translational potential of cellular observations.
That said, researchers often must make their animal models more “human-like” to improve the relevancy of their assays. For example, research efforts have sought to make mice a better model for infection of MERS-CoV by engineering mice to express human versions of its receptor (DPP4), since the virus does not bind mouse DPP4. 
Like cell lines, researchers must often manipulate animal models for virology studies, either to increase viral replication or to mimic specific disease states. While several gene editing approaches exist for animal molecules, CRISPR has become a marquee method for generating transgenic animals. 
Your virology assay model: from basic research to drug discovery
Biological models play essential roles in modern research. While a lot of basic research can be done with both in vitro and in vivo assays, they also play a central role in translational and pre-clinical settings, like for drug discovery and development. As you embark on the development of your assay, don’t forget that assays benefit from consistent conditions.  Be sure to carefully annotate your assay’s development process, so you can ultimately build effective standard operating procedures down the line.
If you want to start making your own biologic model for your research, Thermo Fisher Scientific has reagents, kits, and services to support your work. For more information and details on relevant research tools from Thermo Fisher Scientific, check out our Virology Resource Page.
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