Sub-Topic 5: Viral Infection Principles

MOI-based infection calculations and adsorption principles.

5.1 Terminologies and principles

Key terminologies:

• Multiplicity of Infection (MOI): Ratio of infectious virus particles (PFU) to target cells.
• Plaque Forming Unit (PFU): Measure of infectious virus capable of forming plaques.
• Adsorption: Initial attachment and entry of virus into cells.
• Cytopathic Effect (CPE): Visible changes in cells due to viral infection.
• Virus Stock Titer: Concentration of infectious virus in stock preparation.

Principles:

1. MOI Determines Infection Synchrony: High MOI (>5) ensures all cells infected simultaneously for one-step growth curves.
2. Accurate Volume Pipetting: Critical for reproducible infections; adjust dilutions for practical volumes.
3. Adsorption Efficiency: Affected by temperature, mixing, and incubation time.

5.2 Multiplicity of Infection (MOI)

Core Formula:

PFU required=MOI×Number of cells

MOI = PFU/Number of cells

Worked example

A virology laboratory plans to infect a culture containing 2.0 × 10⁶ cells at a desired MOI of 0.5. The available virus stock has a titer of 1.0 × 10⁸ PFU/mL.

a) Calculate the total PFU required to achieve the desired MOI.
b) Calculate the volume (in mL and µL) of virus stock needed for the infection.

Step-by-step solution

Given

Number of cells = 2.0 × 10⁶
MOI = 0.5

Virus stock titer = 1.0 × 10⁸ PFU/mL

a) Calculate the PFU required

PFU required = MOI × Number of cells

PFU required = 0.5 × 2.0 × 10⁶
= 1.0 × 10⁶ PFU

b) Use the virus stock titer to calculate volume required

Virus stock titer = 1.0 × 10⁸ PFU/mL

Volume required = PFU required ÷ Stock titer

= (1.0 × 10⁶ PFU) ÷ (1.0 × 10⁸ PFU/mL)
= 0.01 mL

Convert volume to microlitres

Volume = 0.01 mL = 0.01 x 1,000 = 10 µL

5.3 Practical dilution adjustment

Intermediate dilutions improve pipetting accuracy.

Worked example

A virology laboratory plans to infect a cell culture but wants to avoid pipetting very small volumes that are prone to error. You are given the following information:
Virus stock concentration = 2 × 10⁶ PFU/mL
Number of target cells = 2 × 10⁵ cells
Desired MOI = 0.5

a) Calculate the number of PFU required for the infection.
b) Calculate the volume of virus stock needed to achieve this MOI.
c) Explain why this volume is impractical

d) describe a suitable dilution strategy to overcome the problem.

Step-by-step solution

a) Calculate the PFU required
PFU required = MOI × number of cells

PFU required = 0.5 × 2 × 10⁵
PFU required = 1 × 10⁵ PFU

b) Calculate the volume of virus stock required
Volume required = PFU required ÷ stock concentration

Volume = (1 × 10⁵ PFU) ÷ (2 × 10⁶ PFU/mL)
Volume = 0.05 mL

Convert to microlitres:
0.05 mL = 50 µL (too small)

c) Identify the practical problem
A volume of 50 µL is relatively small and increases the risk of pipetting error, especially in routine infection assays.

d) Practical dilution adjustment
To improve accuracy, the virus stock can be diluted before infection. For example, prepare a 1:10 dilution of the virus stock:

New virus concentration = 2 × 10⁵ PFU/mL

Recalculate volume needed:
Volume = (1 × 10⁵ PFU) ÷ (2 × 10⁵ PFU/mL)
Volume = 0.5 mL (500 µL) (easier to pipette)

General Rule: If calculated volume < 10% of final volume or < 10 µL, make appropriate dilution.

Infection Procedure:

1. Prepare cells at 70-90% confluence
2. Remove medium, wash with PBS if serum present
3. Add virus inoculum in minimal volume (just covering monolayer)
4. Adsorb for 1-2 hours at 37°C with occasional rocking
5. Remove inoculum, add fresh complete medium
6. Monitor for CPE at appropriate intervals

Practice Questions

1. Define MOI.
2. Effect of high MOI?

Answers

1. Virus-to-cell ratio.
2. Synchronous infection.