1. Purpose
Viral inactivation refers to physical or chemical treatments that render viruses non-infectious. It is commonly used to prepare viral antigens and to detoxify virus-contaminated cells or tissues. Typical approaches include:
- Formaldehyde (paraformaldehyde) inactivation: crosslinks proteins to disrupt viral structures.
- β-Propiolactone (BPL) inactivation: alkylates and damages nucleic acids, inhibiting replication.
- Ultraviolet (UV) inactivation: induces nucleic-acid lesions that block viral proliferation.
2. Scope
- Applicable to chemical inactivation of a wide range of DNA/RNA, enveloped/non-enveloped viruses.
- Applicable to virus-containing matrices such as cell-culture supernatants, tissue homogenates, serum/protein solutions, and vaccine bulks.
- Applicable to preparation of inactivated antigens (e.g., vaccine or diagnostic antigens) and to deactivation of materials contaminated by viruses.
- Suitable where preservation of protein epitopes is important (subject to project-specific validation).
- Suitable for process flows that can accommodate BPL hydrolysis/residual-removal steps.
3. Biosafety and Responsibilities
- Biosafety level:H5/H7 highly pathogenic avian influenza viruses and H2N2 subtype: perform in BSL-3 laboratories.Other influenza viruses: perform in BSL-2 laboratories.
- Personal protective equipment (PPE): compliant lab gown, disposable cap/shoe covers, double latex/nitrile gloves, N95 or equivalent respiratory protection, and safety goggles/face shield.
- Chemical safety (BPL): BPL is a strong mutagen and a suspected carcinogen; it is volatile and highly reactive. All preparation and additions must be performed in a fume hood with an ice bath. Avoid skin/mucosal contact and inhalation. Prepare a spill kit (absorbent materials, alkaline neutralizer, sealable waste containers).
4. Materials and Equipment
4.1 Reagents and Consumables
- 9–10-day-old SPF chicken embryos
- β-Propiolactone (BPL): prepare fresh 2% (w/v) solution (2 g BPL in 100 mL double-distilled water pre-chilled on ice). Keep on ice and work in a fume hood throughout.
- 0.5 M disodium hydrogen phosphate (Na₂HPO₄)
- 7% NaHCO₃ solution (for pH adjustment)
- Sterile PBS (pH 7.2–7.4)
- 75% ethanol
- T75 cell-culture flasks (for subsequent safety testing/culture)
- Red blood cells and HA plates (for hemagglutination assay)
- Sterile centrifuge tubes, pipettes/sterile tips, labels, etc.
4.2 Instruments
Candling lamp, egg opener, balance, ultracentrifuge/bench centrifuge, 37 °C water bath (with 15-min mixing reminders), 4 °C refrigerator and ice-bath setup, biological safety cabinet, fume hood, pH meter.
5. Procedure (BPL Inactivation)
General: Perform all open manipulations in a biological safety cabinet. Prepare and add BPL only in a fume hood with an ice bath.
（1）Virus preparation: Obtain clarified viral fluid. Record batch number, titer, volume, and source embryo information.
（2）Pre-inactivation setup: Pre-chill double-distilled water and ice bath. In a fume hood, prepare 2% BPL (2 g/100 mL ice-cold ddH₂O), mix, and keep on ice. Prepare 0.5 M Na₂HPO₄ and 7% NaHCO₃. Set the 37 °C water bath and set a 15-min mixing reminder.
（3）Dosing and addition: Transfer 38 parts of virus fluid to a labeled sterile container. Add 1 part 0.5 M Na₂HPO₄ and mix gently. In the fume hood on ice, add 1 part of 2% BPL (final BPL concentration 0.05%). Mix immediately and thoroughly to ensure contact with viral particles. Record all volumes and addition times.
（4）Inactivation reaction (choose one; do not combine):
- Option A: Incubate at 37 °C for 2 h, gently invert several times every 15 min.
- Option B: Incubate overnight (≥12 h) at 4 °C without agitation.
Note:Keep containers closed to prevent volatilization and exposure.
（5）pH adjustment: After reaction, in the biosafety cabinet slowly titrate with 7% NaHCO₃ to adjust the mixture to pH 7.3–7.4 (verify with a calibrated pH meter). Mix gently.
（6）Verification of inactivation: Blind-pass in chicken embryos for three generations; proceed only when hemagglutination (HA) testing is negative at every passage, at which point declare “inactivation complete” and allow progression to subsequent steps/release.