Lecture 20: Cultivation and Quantification of Viruses

Cultivation of Viruses

Different systems and methods are used to grow and propagate viruses for various reasons.
Applications include quantifying viruses and determining the number of viral particles in a sample.

Reasons to Culture Viruses

Isolation and Identification:
- To isolate and identify viruses from clinical samples to determine the cause of a disease.
- Isolation doesn't necessarily confirm causation.
- Example: Poliovirus vaccine (live attenuated) replicates in the oropharyngeal mucosa and adjacent tract, shedding viruses without causing disease.
Study of Viral Structure and Features:
- To study the structure of viruses, including the capsid, and their replication strategies.
Vaccine Production:
- To prepare vaccines, especially inactivated or live attenuated vaccines.
- Genetic engineering allows vaccine production without growing viruses directly, but a gene is still needed, typically sourced from a virus.
- Synthetic biology allows synthesizing a gene from scratch.
- Poliovirus was the first virus with a chemically synthesized RNA genome used to produce new viruses.

Virus Culture Systems

Two main categories:
- In vivo culture systems
- In vitro culture systems

In vivo Culture Systems

Uses host organisms:
- Animals (mice, monkeys, rabbits, hamsters)
- Plants
- Bacteria
- Chicken eggs
Animals:
- Animals are inoculated with a virus and observed for symptoms or death.
- Knockout animals can be used to introduce specific receptors that allow infection by a virus that wouldn't normally infect them.
- Infected animals can be sacrificed, and tissues/organs where the virus replicates are harvested for virus isolation, purification, or vaccine production.
Chicken Embryos:
- Chicken embryos have multiple compartments that support the growth of many viruses.
- Preferred for influenza vaccine production due to excellent yield.
- Compartments: Chorion allantoin fluid, amniotic fluid, yolk sac.
- Viruses are aspirated and purified from compartments for use as vaccines.
- Symptoms observed: Pox or lesions on membranes, changes in fluid, or death of the embryo.

In vitro Culture Systems

Use cell lines or cell cultures, which are cells isolated and prepared from organs and tissues of hosts.
Types of Cell Lines:
- Primary Cell Culture
- Continuous Cell Lines

Primary Cell Culture

Cells freshly prepared from tissues or organs (animals or humans).
Examples: Monkey kidney cells, human embryonic kidney cells, chicken embryo cells.
Preparation involves mincing tissues and using enzymes to break cell-to-cell bonds, yielding isolated cells that are added to flasks or petri dishes with broth media and incubated to allow them to grow.
Cells grow until they touch each other, stopping replication due to contact inhibition, which prevents over-proliferation of cells, preventing clumps.
A monolayer of cells is preferred for viral infection.
Primary cells have a short lifespan (2-3 days) and must be maintained or sub-cultured (passaged).
Sub-culturing involves:
- Detaching cells from the flask/petri dish.
  - Cells stick and attach to the surface of the flask or petri dish.
- Transferring them to another flask/petri dish.
- Replacing the growth media.
Cells will grow again, forming a monolayer.
Primary cells have a limited number of divisions, so they cannot be passaged indefinitely.

Continuous Cell Lines

Cells obtained from naturally occurring cancers.
Cancer cells can divide indefinitely, but still need to be sub-cultured to prevent clumping.
Cells are detached, split into sections, and each section is sub-cultured.
Theoretically, they can be sub-cultured indefinitely.
Cannot be used to produce vaccines due to abnormal chromosome numbers and irregularities.
Can be used to study viral replication and diagnose viruses.
Examples:
- HeLa cells: Obtained from a woman (Henrietta Lacks) who had cervical cancer in 1951. The cells were used without her permission. They have been used extensively in research and are still alive and can be passaged.

Cytopathic Effect (CPE)

Changes in the appearance or features of cells due to viral replication.
Observable distinct changes in shape, appearance, and morphological features in infected cells.
Use a control of uninfected, healthy cells of the same cell line to compare.
Examples:
- Vero cells (monkey kidney cells) infected with poliovirus:
  - Normal cells are attached to the surface, elongated with no gaps.
  - Infected cells become round, detach from the surface, and gaps appear.
  - Cell movement in the culture media fluid indicates dead, detached cells.
  - Rounding and detachment are typical of poliovirus.
Syncytia (Syncytium):
- Multinucleated giant cells formed by the fusion of multiple cells.
- Normal cells have stained cytoplasm and nucleus.
- Measles virus can cause cells to fuse together.
- Specific to enveloped viruses, which enter cells by fusion of the plasma membrane.
- Viruses can bind to neighboring cells before completely exiting, leading to fusion.
- Measles, mumps, herpes viruses, and HIV can cause syncytia.
Inclusion Bodies:
- Negri bodies: Found by Adelechi Negri in neurons infected with rabies.
  - Strange compartments within the cytoplasm (pink).
  - Sites of viral replication, specifically for RNA viruses that replicate in the cytoplasm.
- Nuclear inclusion bodies:
  - Found within the nucleus, indicating DNA virus replication.
  - Adenovirus replicates in the nucleus.
Specific Examples:
- Poliovirus causes complete lysis and destruction of cells; people infected shed a large amount of viruses.
- Shrinking of the nucleus with DNA viruses.
- Vacuoles in the cytoplasm with papillomavirus.
- Apoptosis: Immune system recognizes infected cells as foreign and kills them.

Quantification of Viruses

Used to determine the number of viruses.
Methods:
- Electron Microscope
- ELISA (Enzyme-Linked Immunosorbent Assay)
- Immunofluorescence
- Hemagglutination Assay
- Plaque Assay

Electron Microscope

Magnification power of 100,000x.
Used for the first identification of viruses as viral particles.
Difficult sample preparation and virus identification
Rarely used for diagnosis, but used for the structure of the capsule and the arrangement of the capsule.

ELISA

Based on the binding between antigens and antibodies.
One component is labeled with an enzyme.
The antigen can be the whole virus or part of the virus, such as the capsid.
Rapid tests for COVID are based on ELISA (lateral flow assay).
In COVID-infected nasal samples, the virus or capsid proteins act as antigens and bind to specific antibodies.

Immunofluorescence

Same principle as ELISA, but antibodies or antigens are labeled with an immunofluorescent agent (e.g., green).
Detection of respiratory syncytial virus (RSV) in lung tissues using labeled antibodies against viral proteins.
Green fluorescence indicates the location of the virus or viral proteins.

PCR

Detects viral nucleic acid (DNA or RNA).
If the virus is RNA, reverse transcriptase PCR (RT-PCR) is used
Methods like ELISA, immunofluorescence, and PCR do not differentiate between infectious and non-infectious viruses.
They are not considered infectivity assays.

Hemagglutination Assay

Determines the number of viruses based on their ability to agglutinate red blood cells (RBCs).
Some viruses (e.g., influenza, mumps, measles) have surface viral proteins (attachment proteins) that bind to and cross-link RBCs.
The viral attachment protein of influenza virus is called hemagglutinin.
The test involves incubating a suspension of the virus with RBCs.
* If viruses are present, they will bind to RBCs and form a red layer due to agglutination.
If viruses are absent, RBCs will precipitate, forming a button in the center of the tube.
The test is semi-quantitative.
Serial dilutions of the virus are prepared and incubated with a fixed amount of RBCs.
- Higher concentrations of virus lead to agglutination, while increased dilution leads to less agglutination.
The titer is the highest dilution that causes complete agglutination.
Example: A hemagglutination test on three patient samples with increasing dilutions.
- The goal is to determine the dilution that gives complete agglutination.
- Which patient has more viruses is determined by how diluted a sample can be while still causing agglutination of the red blood cells.
- Hemagglutination does not take into account the infectious viral particles.

Plaque Assay

Used to determine the number of infectious viral particles (infectivity assay).
Fred Rector observed that some bacteriophages could kill and lyse bacterial cells, forming zones of lysis (plaques).
Plaques are groups of dead cells that have been infected with viruses.
Renato del Becco designed a plaque assay based on these observations in 1952.
Procedure:
- A monolayer of susceptible cells is prepared in a petri dish or multi-well plate.
- A virus suspension is added, followed by a semi-solid agar overlay.
- The plate is incubated.
Viruses will infect cells and replicate.
- Each viral particle infects one cell, replicates, and produces many viruses.
- Viral replication leads to cell lysis (CPE).
- Viruses from one cell spread to neighboring cells, infecting them.
Small holes or zones of lysis (plaques) become visible.
- The formation of new plaques are restricted by the semi-solid agar, which is essential to make sure that the viruses cannot travel too far.
The number of plaques is proportional to the number of viral particles.
- One plaque is considered representative of one plaque-forming unit (PFU).

Formula and Calculation

First you need to dilute a sample.
Black Assay (Number of PFU / mL) = (Number of plaques) x (dilution factor) / (volume of inoculum (mL))
- Dilution factor:
  - Serial Dilution
  - ${10^{-7}}$
- Volume of inoculum:
  - Volume (mL) of diluted virus added to cells.
  - 0. 25 mL