Characterizing Viral Fitness

Whether it’s assessing the degree to which a virus has been attenuated, comparing the cytolytic properties of two different field isolates, or probing the function of a viral gene, fitness comparisons are integral to both vaccine development and basic virology research. “Fitness” often refers to how efficiently a virus is able to pass through its entire life cycle – spanning everything from attachment, penetration, uncoating, replication, assembly, to virion release.

The traditional method used to measure viral fitness is the labor-intensive plaque assay. Other techniques such as PCR, ELISA, and next generation sequencing allow for higher throughput, but are only used to estimate viral load and do not quantify infectious virus.

Real-time cell analysis using the xCELLigence RTCA system offers automatic and rapid results with high reproducibility. The assay detects changes in the host cell throughout the continuum of a virus-induced CPE and provides quantitative kinetics of the infectious viral activity.

Vaccine & Virology Handbook
Download the Vaccine & Virology Handbook to discover a more accurate method to characterize viral activity. Applications include viral titer determination, detection and quantification of neutralizing antibodies, studying anti-viral drugs, testing virucides, oncolytic viruses, and assessing virus quality or fitness.
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Traditional (or Conventional) methods vs xCELLigence RTCA

Plaque AssaysxCELLigence RTCA Viral Fitness Assay
Counting of number of plaques formed by a virus at varying dilutions to obtain a low multiplicity of infection (MOI). Concentration of virus (titer) is calculated as plaque forming units (PFU) per unit of volume. Direct, real time, and quantitative kinetics for the entire virus life cycle. Cells are plated, infected with virus, and xCELLigence RTCA automatically monitors viral fitness in real time.
Viral plaque formation can take days to weeks to be detectable.Quantitative monitoring of both fast (hours) and slow (days to weeks) CPE. 
Plaque assay provides no information about the onset of CPE or the kinetics of virus-mediated cytotoxicity. xCELLigence provides assessment and quantification of the full virus life cycle.
The manual counting of plaques by visual inspection can be highly subjective and inconsistent since plaque formation rates and sizes can vary dramatically. Measurements are automatically recorded at a user defined frequency and are plotted by the xCELLigence software as Cell Index (CI). Accurate, precise, and highly reproducible method with less manual labor.

 

Example Data: Evaluating Viral Hemorrhagic Septicemia Virus (VHSV) Fitness

Figure adapted from Virology, volume 476, Kim, S. H. et al. “Specific Nucleotides at the 3’-Terminal Promoter of Viral Hemorrhagic Septicemia Virus are Important for Virulence In Vitro and In Vivo,” pages 226–32. Copyright 2015, with permission from Elsevier.

The 3’ terminal sequence of the VHSV genome has an impact on both viral fitness and RNA levels. (A) Predicted secondary structure of the 3’ terminus of the VHSV genome.  (B) Total VHSV positive strand RNA levels two hours post infection.  (C) Quantifying the relative fitness of VHSV mutants using xCELLigence.

Viral hemorrhagic septicemia virus (VHSV) has a negative strand RNA genome that contains a highly conserved nucleotide sequence at its 3’ terminus which is predicted to form a hairpin structure (Figure A).  Øystein Evensen and colleagues at Norwegian University of Life Sciences sought to understand whether this sequence has an effect on VHSV replication and transcription in fish epithelial cells.  Working with a panel of VHSV mutants, total positive strand RNA levels were quantified using qPCR two days post-infection.

As seen in Figure B, whereas the U8C and A7C-U8A mutants had no discernable impact on viral positive strand RNA levels, the A4G-G5A mutant reduced viral RNA levels significantly.  xCELLigence was then used to assess the relative virulence of these different mutants (Figure C; virus was added to cells at the 72 hour time point).  Consistent with its reduced RNA levels, the A4G-G5A mutant (green curve) required a much longer time than WT virus (black curve) to effect complete killing of target cells.  Interestingly, the A7C-U8A mutant (blue curve) also showed a delayed onset of CPE even though its RNA levels were equivalent to WT.  Moreover, the U8C mutant (red curve) was actually more efficient than WT at killing target cells, despite having similar levels of RNA.  This example from VHSV highlights the fact that biomarker (such as RNA) quantifications do not always provide the whole truth of virulence and can be misleading when it comes to evaluating viral fitness, and demonstrates the utility of a functional assay that tracks the entire lifecycle of the virus.

 

Example Data: Evaluating Bluetongue Virus Fitness

Figure reprinted from Veterinary Microbiology, volume 171 (1-2), Coetzee, P. et al. “Viral Replication Kinetics and In Vitro Cytopathogenicity of Parental and Reassortant Strains of Bluetongue Virus Serotype 1, 6 and 8,” pages 53–65. Copyright 2014, with permission from Elsevier.

Reassortment of the bluetongue virus genome gives rise to differences in cytopathogenicity. Vero cells were grown to confluence and then infected (at 23 hours) with identical MOIs of either parental strains or genetic reassortant strains.  Data adapted from reference 2.

Another example of xCELLigence being used to quantitatively evaluate viral fitness involves bluetongue virus, which causes an economically important haemorrhagic disease in both wild and domestic cows, sheep, and goats.  Bluetongue virus has a dsRNA genome that is arranged in 10 different linear segments.  Bluetongue virus displays substantial genetic reassortment over time due to the nature of this genome architecture and the ability of multiple strains of the virus to infect a cell at the same time,.  An important question is how this genome rearrangement affects viral virulence.  To address this, Estelle Venter and colleagues at University of Pretoria generated variants of the virus where the bulk of the genome was derived from one strain, but included a fragment from a different strain.  The ability of these reassortant strains to kill Vero cells was then monitored in real-time by the xCELLigence system.  As seen in the below figure, parental strain 8 kills the target cells much more efficiently than does parental strain 6.  Interestingly, adding a fragment of the strain 8 genome to the strain 6 genome does not improve its cytopathogenicity; the killing kinetics of this chimera are slower.  In contrast, adding a fragment of strain 6 to the genome of strain 8 yields a virus with superior cell killing capabilities.

The ability to track these different viral phenotypes with precision enabled the authors to predict how bluetongue virus likely behaves in vivo.  Of special importance is the notion that a live attenuated strain of bluetongue virus being used to vaccinate animals could potentially rearrange with other strains in vivo, and thereby move beyond the attenuated phenotype to achieve viremias high enough to result in bona fide disease.

 

New Techniques in Viral CPE Assessment using Real Time Cell Analysis
Watch the webinar to learn how xCELLigence Real Time Cell Analysis technology can be used for vaccine development.
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Viral Fitness Publications

 

Featured xCELLigence RTCA Systems for Viral CPE Assays

Dual PurposeSingle PlateMulti PlateHigh Throughput
RTCA DPRTCA SPRTCA MPRTCA HT
3×16 wells1×96 wells6×96 wellsUp to 4×384 wells