Virus Culture Technology

I. Overview

Viruses are obligate intracellular parasites. Due to their lack of autonomous replication enzyme systems and inability to independently perform metabolic processes, viruses must rely on host cells or cellular components to synthesize nucleic acids and proteins. Therefore, viruses can only replicate within susceptible host cells and cannot grow in cell-free culture media. Consequently, the cultivation of animal viruses must be conducted in cells, fertilized eggs, or experimental animals.

II. Techniques for Propagating Viruses in Cells

Cell culture remains a simple and effective method for virus cultivation and is widely used. Common cell lines include BHK-21, Vero, Marc 145, ST, MDCK, BT, CRFK, F81, Hela, IBRS-2, MDBK, MA104, and PK-15, as well as primary or secondary monolayer cells prepared from chicken embryos or animal tissues.

(I) Nutritional Requirements for Cell Culture

During replication, viruses require living cells to supply energy and materials for biosynthesis. Cell culture media primarily consist of inorganic ions, carbohydrates, amino acids, vitamins, proteins, and antibiotics.

1. Inorganic Ions
   Sodium chloride in the culture medium maintains normal osmotic pressure. Weak bicarbonate buffers are commonly used to adjust the pH, typically maintained at 7.2–7.4 for optimal cell growth. In open containers, CO₂ must be introduced or cultures incubated in a CO₂-enriched incubator to prevent pH elevation due to CO₂ loss. Alternatively, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) can serve as a buffer. Essential ions such as sodium, potassium, calcium, magnesium, iron, carbonate, and phosphate support osmotic balance, enzyme activity, metabolism, and cell adhesion.

2. Carbohydrates
   Glucose is the primary energy source. Complex media may include additional sugars or compounds like lactate, pyruvate, or acetate as alternatives.

3. Amino Acids
   Mammalian cell cultures require L-isomer amino acids. The 13 essential amino acids are arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, valine, cysteine, and glutamic acid. Media such as Eagle’s MEM, RPMI 1640, and M199 may include additional amino acids for enhanced growth.

4. Vitamins
   Vitamins act as coenzymes or cofactors critical for metabolism. B vitamins (e.g., biotin, folate, niacin) are commonly used. Serum-free media may include lipid-soluble vitamins and antioxidants like glutathione.

5. Proteins
   Serum (e.g., fetal bovine serum) provides essential proteins, growth factors, and adhesion-promoting components. Batches must be heat-inactivated (56°C for 30 minutes) and tested for cytotoxicity.

6. Antibiotics
   Antibiotics (e.g., penicillin, streptomycin, amphotericin B) prevent bacterial or fungal contamination but do not contribute to cell nutrition.

For a simplified summary, refer to Table 1.

Table 1: Cell Culture Nutritional Requirements

(II) Basic Conditions for Cell Culture

Cell growth depends on inoculation density, medium composition, pH, gas conditions, temperature, sterility, and vessel preparation.

1. Inoculation Density
   Optimal densities: chicken fibroblasts (1 million/mL), rodent kidney cells (500,000/mL), passaged cell lines (100,000–300,000/mL). Monolayers typically form in 1–3 days.

2. Culture Medium
   Synthetic media (e.g., Eagle’s MEM, RPMI 1640) have replaced natural media.

3. pH and Gas Conditions
   Optimal pH: 7.0–7.4. Buffering relies on bicarbonate/CO₂ systems. Acidic conditions favor cell adhesion.

4. Temperature
   Incubate at the host species’ body temperature. Temperatures >2–3°C above optimal can kill cells within 24 hours.

5. Sterility
   Strict aseptic techniques are critical. Contamination sources include media, equipment, tissues, personnel, and air.

6. Vessel Preparation
   Glassware: cleaned with detergent, rinsed, and autoclaved. Plasticware: treated with NaOH/HCl, rinsed, UV-sterilized. Rubber stoppers: boiled in NaOH/HCl, autoclaved.

For a simplified summary, refer to Table 2.

Table 2: Basic Conditions for Cell Culture

(III) Requirements for Passaged Cell Lines

Established cell lines (seed stock system) are preferred over primary mammalian cells for virus propagation. Cell lines derived from tumors or normal tissues must undergo rigorous quality control.

1. Cell Bank System

   • Master Cell Bank (MCB): Uniform cells tested for contaminants (bacteria, mycoplasma, viruses).
   • Working Cell Bank (WCB): Expanded from MCB, limited to defined passage numbers.
   • Production cells must not exceed validated passage limits.

2. Cell Bank Management
   Records include origin, karyotype, tumorigenicity, and storage conditions. Non-production cells must be segregated.

3. Cell Line Testing
   Tests for MCB/WCB:

   • Microscopy, sterility, mycoplasma, adventitious viruses.
   • Karyotyping (≥50 mitotic cells): Chromosomal markers must match MCB.
   • Tumorigenicity: Inject 10⁷ cells into nude mice; monitor for nodules/tumors for 12 weeks.

4. Subculture Protocol
   Confluent monolayers are washed, trypsinized (0.25% trypsin/EDTA), resuspended in growth medium, and split 1:3–1:4.

For a simplified summary, refer to Table 3.

Table 3: Requirements for Passaged Cell Lines

(IV) Cell Preservation, Recovery, and Transport

1. Cryopreservation
   Cells (1–2 million/mL) in freezing medium (20% serum, 5–10% DMSO) are stored in liquid nitrogen.

2. Thawing
   Rapidly thaw cells in 37–40°C water, dilute to 500,000/mL, and incubate.

3. Transport
   Monolayers: fill vessels to prevent detachment. Suspensions: 1 million/mL at 15–25°C.

(V) Cell Culture Methods

1. Static Culture
   Cells are grown in sealed flasks. After monolayer formation, virus is added and incubated until 75% CPE is observed.

2. Roller Bottle Culture
   Cells are grown in rotating bottles (5–10 rpm). Used for large-scale production (e.g., foot-and-mouth disease virus).

(VI) Observing Virus Growth in Cells

• Optimal temperature: 35–37°C (some at 33°C).
• CPE Scoring:
  +: ≤25%, ++: ~50%, +++: ~75%, ++++: ~100%.
• CPE Types: Rounding, necrosis, syncytia, inclusion bodies (cytoplasmic/nuclear, acidophilic/basophilic).
• Detection: pH shifts, hemadsorption, hemagglutination, immunofluorescence, ELISA.

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