Viruses and cells
The vaccine strain Bartha K-61, also known as PRV (K), wild-type virulent strain PRV (B), and PK-15 cells were obtained from the College of Veterinary Medicine, South China Agricultural University. The purified wild-type virulent strain PRV (B) and the attenuated vaccine strain PRV (K) were propagated in PK-15 cells. The reproductive titer of the mutant (PRV (B)) can exceed 8, while that of the classical strain (PRV (K)) can only reach approximately 6.
Effect of temperature on PRV activity
A total of 50 µL of virus solution was aliquoted into PCR tubes. A total of 23 temperature gradients were set using a PCR machine. The processing temperature range was 40–72°C. The heat treatment time was between 0 s and 1200 s. After heat treatment, each sample was inoculated into a 96-well plate with a monolayer of PK-15. The cytopathic condition was observed and recorded 42 h after virus inoculation. 100% inactivation was consideration of the time value. All test samples were set up in triplicate.
Effect of pH on PRV activity
The virus was diluted 20 times into different pH solutions without buffer. Small volume of virus didn’t change the pH of the solution, and the pH was compared before and after treatment, which didn't change very much. The pH levels for exposure in this study were pH = 3, pH = 4, pH = 5, pH = 6, pH = 9, pH = 10, and pH = 11, and the treatment times were 0, 10, 20, and 30 min. The experiment was conducted in a room with a temperature in the range of 25-30°C. The reagents used to adjust and neutralize the pH of the virus solution were 1% sodium hydroxide (NaOH), 5% NaOH, and 3.6-3.8% hydrochloric acid (HCl). After the different pH treatments, the pH value of the sample was adjusted to 7-8 with the corresponding neutralizing agent after reaching the preset treatment time.
Effect of UV on PRV activity
The PRV-containing cell culture solution was aliquoted into 33-mm-diameter plates at 200 µL per plate, the cover of the plate was opened, and it was irradiated with different UV intensities. The UV treatment was conducted in a biological safety hood with an ambient temperature of approximately 26°C without ventilation. In an environment with an average C-band UV (UVC) intensity of 135 or 270 µW/cm2, the treatment times were 0, 1, 3, 5, 7, 9, 12, and 15 min. In an environment with µWnon-C-band ultraviolet light, the treatment times were 0, 5, 15, 35, 60, 90, 120, 150, and 180 min.
Effect of humidity on PRV activity
The PRV cell culture solution with known titers and containing double antibiotics was plated evenly in 33-mm-diameter plates, with 50 µL per plate. Three holes with diameters of 2 mm were punched on the lid to keep airflow with the outside. The plates were then quickly air-dried or naturally air-dried in a biological safety cabinet with a temperature of 26℃. After air-drying, three small holes with a diameter of 2 mm were punched on the lid to keep airflow with the outside. After reaching the preset treatment time, the resulting dried residue was rehydrated. Each sample was added 150 µL of DMEM cell culture medium containing 1% penicillin/streptomycin antibiotics, which was then washed and mixed. The sample was inoculated into a 96-well plate along with pk-15 cells. The cells were observed for the presence or absence of cytopathic effects after 24-48 h.. The control group was a sealed plate containing a known titer of PRV cell culture medium to prevent the virus solution from evaporating and drying, and the control plate was placed in a dry indoor environment.
Effect of freeze-thawing on PRV activity
In the PRV freeze-thaw experiment, the freezing temperature was -80°C, and two thawing temperatures were set: 26°C in air and a 37°C water bath. A total of 100 μL of diluted pseudorabies virus was placed in each tube in a -80°C freezer. The tubes were taken out quickly and thawed either in a 26°C air environment or in a 37°C water bath. The freeze-thaw cycles were repeated in this manner, and samples with a freeze-thaw number of 1, 5, 10, 15, 20, 25, 30, 35, and 40 were taken for titer measurement.
Effects of storage conditions on PRV activity
PRV solution was aliquoted into 1.5 mL EP tubes at 200 µL per tube, and then stored at 26℃, 4℃, -20℃, -80℃, respectively. Samples were removed according to the experimental time points for virus titer determination, and the titer curves were drawn.
Effects of sunlight exposure on PRV activity
A PRV stock solution of known titer was aliquoted into PCR tubes with 50 µL per tube, and the tubes were exposed to outdoor sunlight in an open and windless outside space. The control group was blocked from the sunlight with black cardboard while keeping the air flow unobstructed. Samples were taken at preset time points and were stored in the refrigerator at 4°C for virus titer measurement. At the same time, the temperature of the experimental table, air temperature, ultraviolet intensity, and light intensity were recorded. Data logger illuminance meter (TESTES Inc, Taiwan, China, ) was used to measure the illumination intensity.
Effect of chemical disinfectants on PRV activity
The following chemical disinfectants were selected in this study. Chlorine-containing disinfectant: sodium dichloroisocyanurate (SDIC), brand 84 Disinfectant. Ionic surfactants: sodium dodecyl sulfonate (SDS), powder laundry detergent, and benzalkonium bromide. Strong oxidant: potassium permanganate (KMnO4). Tissue fixatives: formaldehyde (HCHO) and ethanol (CH3CH2OH). Acid-base reagents: NaOH, HCl (HCl content: 36% to 38%), and acetic acid (CH3COOH).
Various chemical drugs were formulated into a 2.5% solution according to mass fraction or volume fraction, then a series of 2-fold dilutions were performed with distilled water. The different concentrations were obtained by dilution, and then mixed with PRV solution with a known titer at a 1:1 ratio. The mixture was allowed to stand for 30 min, which is the national standard for the evaluation of the effectiveness of disinfectants, and then it was inoculated into a 96-well cell plate with a monolayer of PK-15 with 100 µL per well, with three replicates per gradient. The plate was then incubated at 37°C in a 5%-CO2 incubator for 1.5 h. The supernatant was then discarded, and the plate was washed three times in phosphate-buffered saline (PBS). Next, 100 µL of fresh Dulbecco’s Modified Eagle’s Medium was added, followed by incubation for 36 h in a 37°C incubator with 5% CO2. Whether cytotoxicity occurred in cells was observed and recorded.