Sub-Topic 7: Virus Quantification Methods

Plaque assay principles, serial dilution and titer calculation.

7.1 Terminologies and principles

Key terminologies:

1. Plaque Assay: Quantitative assay in which each visible plaque corresponds to infection by a single infectious virus particle.
2. Plaque Forming Unit (PFU): Standard unit of infectious virus measured in plaque assays.
3. TCID₅₀ (Tissue Culture Infectious Dose 50): Virus dilution that infects 50% of cultures; statistical measure of infectivity.
4. Hemagglutination (HA) Units: Measure of virus particles capable of agglutinating red blood cells.
5. Genome Copies: Total viral nucleic acid quantified by qPCR, independent of infectivity.
6. Focus-Forming Units (FFU): Infectious units detected by immunostaining of infected cells.
7. ELISA: Immunological assay detecting viral proteins in a sample.

Principles:

1. Infectivity-Based Quantification: Plaque, TCID₅₀, and FFU assays measure functional, infectious virus capable of replicating in host cells.
2. Particle-Based Quantification: HA, ELISA, and qPCR detect viral components (protein or nucleic acid) independent of infectivity.
3. Serial Dilution: Essential to bring virus concentration into a measurable range and ensure accurate quantification.
4. Endpoint or Linear Range: Reliable counts or measurements occur within defined ranges—too dense or too dilute measurements reduce accuracy.
5. Assay Selection: Choice of method depends on virus type, infectivity requirement, detection sensitivity, and available equipment.

Worked Examples

7.2 Plaque Assay (PFU/mL)

A virus sample is serially diluted 10⁻⁴, 10⁻⁵, and 10⁻⁶. 0.1 mL of the 10⁻⁵ dilution is plated on a cell monolayer. After incubation, 75 plaques are counted. Calculate the viral titer in PFU/mL of the original stock.

Solution:

1. Formula:
   PFU/mL = (Number of plaques × dilution factor) ÷ volume plated (mL)
2. Plug in values:
   PFU/mL = (75 × 10⁵) ÷ 0.1
   PFU/mL = (7.5 × 10⁶) ÷ 0.1
   PFU/mL = 7.5 × 10⁷ PFU/mL

Answer: 7.5 × 10⁷ PFU/mL

7.3 TCID₅₀
In a 96-well plate assay, a virus is serially diluted 10-fold across 8 wells per dilution. Cytopathic effect (CPE) is observed in 8, 7, 5, 3, 1, 0, 0, 0 wells, respectively. Using the Reed–Muench method, calculate the TCID₅₀ per mL. (For simplicity, assume final TCID₅₀ falls at 10⁻⁴ dilution.)

Solution (simplified):

1. TCID₅₀ = reciprocal of the dilution giving 50% of wells infected.
2. 50% of 8 wells = 4 wells showing CPE.
3. From observations, 10⁻⁴ dilution is closest to infecting 4 wells.

Answer: TCID₅₀ ≈ 10⁴ per 0.1 mL
Convert to per mL: 10⁴ ÷ 0.1 mL = 1 × 10⁵ TCID₅₀/mL

7.4 Hemagglutination Assay (HA)
A virus suspension shows hemagglutination up to a 1:128 dilution. Calculate the HA titer (HA units/mL) if 50 µL of each dilution was tested.

Solution:

1. HA titer = reciprocal of the highest dilution showing complete agglutination ÷ volume tested in mL.
2. Volume tested = 50 µL = 0.05 mL
3. Reciprocal of dilution = 128
4. HA units/mL = 128 ÷ 0.05 = 2560 HA units/mL

Answer: 2.56 × 10³ HA units/mL

7.5 Hemagglutination Inhibition (HI) Assay

A researcher wants to measure the antibody titer against influenza virus using an HI assay. The virus stock has a hemagglutination titer of 1:64 HA units. Serial two-fold dilutions of a serum sample are prepared (1:10, 1:20, 1:40, 1:80, 1:160, 1:320). Equal volumes of virus (4 HA units) are added to each serum dilution, followed by red blood cells. After incubation, complete inhibition of hemagglutination is observed up to the 1:80 serum dilution.

a) Determine the HI titer of the serum.
b) Explain how the HI titer is interpreted in terms of antibody concentration.

Solution

Given:

• Virus stock: 1:64 HA units
• Serum dilutions tested: 1:10, 1:20, 1:40, 1:80, 1:160, 1:320
• Equal volume of virus (4 HA units) added to each dilution
• Complete inhibition observed up to 1:80 dilution

a) Identify the HI titer of the serum
The HI titer is defined as the highest dilution of serum that completely inhibits hemagglutination.

From the experiment:

• Complete inhibition occurs up to 1:80 dilution
• Next dilution (1:160) shows hemagglutination

Answer: HI titer = 1:80

b) Interpret the HI titer

• The HI titer reflects the antibody concentration in the serum.
• A higher HI titer (e.g., 1:160) indicates a higher antibody level capable of neutralizing more virus.
• In this example, 1:80 titer means the serum can inhibit hemagglutination when diluted 80-fold, showing a moderate level of specific antibodies against the virus.

7.6 qPCR / RT-qPCR
A qPCR assay detects 2 × 10⁶ viral genome copies in a 5 µL sample of RNA extracted from 100 µL of viral supernatant. Calculate the viral genome copies per mL of supernatant.

Solution:

1. Copies per µL of RNA = 2 × 10⁶ ÷ 5 µL = 4 × 10⁵ copies/µL
2. Total copies in 100 µL supernatant = 4 × 10⁵ × 100 = 4 × 10⁷ copies per 100 µL
3. Convert to per mL: 4 × 10⁷ × (1000 ÷ 100) = 4 × 10⁸ copies/mL

Answer: 4 × 10⁸ genome copies/mL

7.7 ELISA (Viral Protein Concentration)
An ELISA standard curve shows 1.0 absorbance unit corresponds to 10⁶ viral particles. A virus sample gives an absorbance of 0.45. If 100 µL of the sample was tested, calculate the viral particles per mL.

Solution:

1. Viral particles in 100 µL = 0.45 × 10⁶ = 4.5 × 10⁵ particles
2. Convert to per mL: 4.5 × 10⁵ ÷ 0.1 mL = 4.5 × 10⁶ particles/mL

Answer: 4.5 × 10⁶ viral particles/mL

Practice Questions

1. Why count 20–100 plaques?
2. Define PFU.

Answers

1. Accuracy range.
2. Infectious virus unit.