Virus Watch: Counting Viruses

Summary

In this episode of Virus Watch, Vincent Rock explains how virologists measure the number of viruses in a sample, focusing on the plaque assay method. He contrasts it with other techniques like electron microscopy, PCR, and immunological assays, highlighting that only infectivity assays like the plaque assay can measure the number of infectious virus particles. The process involves infecting cultured cells, creating plaques with an agar overlay, and counting the plaques to determine the virus titer. While labor-intensive, the plaque assay remains a gold standard in virology for its accuracy in measuring infectious viruses.

Takeaways

• 😀 The Zika virus example introduces the challenge of measuring virus quantities in a sample.
• 😀 There are two general approaches to virus quantification: measuring virus particles or infectious virus particles.
• 😀 Electron microscopy is a method for measuring virus particles, but it is expensive and time-consuming.
• 😀 PCR (Polymerase Chain Reaction) allows rapid measurement of viral genomes but doesn't determine the number of infectious viruses.
• 😀 The plaque assay is a key method for counting infectious virus particles, particularly useful for viruses like poliovirus.
• 😀 In a plaque assay, viruses infect cultured cells, and the spread of infection forms visible plaques in the agar-covered medium.
• 😀 Agar overlays prevent virus particles from spreading too far, ensuring plaques remain localized and countable.
• 😀 Plaques are clear areas formed when virus infection kills cells, which can be counted to calculate virus titer in plaque-forming units (pfu).
• 😀 The plaque assay is more labor-intensive than PCR but provides accurate data about the infectivity of a virus sample.
• 😀 In experiments, careful dilution of the virus and precise plating are essential for obtaining countable plaques, with an ideal range of 10 to 100 plaques per plate.
• 😀 Despite being more labor-intensive, the plaque assay remains the preferred method for virologists to measure infectious virus particles over PCR.

Q & A

What is the main challenge in measuring the number of viruses in a sample?

The main challenge is determining how many infectious viruses are present, as methods like electron microscopy, PCR, and immunological assays measure total virus particles, which include both infectious and non-infectious particles.

What is the significance of the plaque assay in virology?

The plaque assay is crucial because it measures only the number of infectious virus particles in a sample, distinguishing them from non-infectious particles, unlike other methods such as PCR that measure total virus particles.

How does a plaque assay work?

A plaque assay works by infecting cultured cells with virus dilutions, covering them with a semi-solid agar medium, and observing the formation of plaques, which are clear zones where cells have been killed by the virus.

What role does agar play in a plaque assay?

Agar restricts the spread of viruses to neighboring cells, ensuring that each plaque originates from a single infected cell, allowing for accurate counting of infectious virus particles.

Why is PCR not sufficient for measuring infectious virus particles?

PCR only measures the number of viral genomes present in a sample, not whether those genomes are associated with infectious virus particles. It cannot distinguish between infectious and non-infectious viruses.

What is the meaning of 'titer' in the context of virus measurement?

Titer refers to the concentration of infectious virus particles in a sample, typically expressed as plaque-forming units (pfu) per milliliter, based on the number of plaques observed in a plaque assay.

What are some advantages and disadvantages of using a plaque assay?

Advantages of a plaque assay include its ability to measure infectious virus particles directly, while disadvantages include its labor-intensive nature and the time required for incubation and plaque formation.

Why is the ratio of infectious to non-infectious virus particles important?

The ratio is important because it affects the effectiveness of viral treatments or vaccines. A sample with a higher ratio of infectious viruses is more potent, while a higher number of non-infectious particles may reduce the sample's efficacy.

How do virologists ensure that the cells used in a plaque assay are susceptible and permissive to the virus?

Virologists select cell lines that have receptors for the virus and are capable of supporting the complete replication cycle, ensuring that the virus can infect and reproduce within the cells.

How do the incubation conditions affect the results of a plaque assay?

Incubation conditions such as temperature (37°C for virus adsorption and 37°C incubator for cell culture) and the presence of 5% CO2 are critical for maintaining cell health and supporting proper viral replication and plaque formation.