Background

Pseudorabies (PR), also known as Aujeszky’s disease or “mad itch”, is caused by pseudorabies virus (PRV), which is an enveloped DNA virus. The main susceptible animals are swine, cows, sheep, rabbits, mice, cats, and other mammals, while pigeons and chickens can resist PRV infection [1,2,3,4]. Not only are there reports of PRV isolated from people presumed to be infected with PRV but there is also direct evidence that PRV can cross hosts to infect humans [Effect of chemical disinfectants on PRV activity

The following chemical disinfectants were used 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: 36–38%), and acetic acid (CH3COOH).

Various chemical drugs were formulated into a 2.5% solution according to mass fraction or volume fraction, and then, a series of 2-fold dilutions were performed with distilled water. Different concentrations were obtained by dilution, and then, the dilutions were mixed with PRV solution with a known titer at a 1:1 ratio. The mixtures were allowed to stand for 30 min, which is the national standard for evaluation of the effectiveness of a disinfectant, and then inoculated at 100 µL per well into a 96-well cell plate containing a monolayer of PK-15 cells, 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 discarded, and the plate was washed three times in phosphate-buffered saline (PBS). Next, 100 µL of fresh DMEM 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.

Results

Effect of temperature on PRV activity

PRV had a certain resistance to temperature, especially in a neutral liquid environment. When PRV was in the temperature range of 48–55 °C, the virus titer decreased as temperature rose, accompanied by a significant decrease in infectivity. When the temperature reached above 70 °C, PRV was inactivated within a few seconds. PRV (B) and PRV (K) had different temperature tolerances in the range of 48–60℃, with PRV (K) having a slightly stronger tolerance (Fig. 1).

Fig. 1
figure 1

Heat resistance curve. Note: Diluted virus solution was aliquoted into 8-strip PCR tubes at 50 µL per tube. The heat treatment temperature and period were set using a PCR machine. The processing treatment temperature range was 40 °C to 80 °C, with a total of 23 temperature gradients. The heat treatment time was between 0 s and 1200s, with a total of 23 gradients. All test samples were analyzed in triplicate for each temperature and time point. After the heat treatment, 50 µL of DMEM was added to each tube. After mixing, the contents of each tube were inoculated into a 96-well plate with a monolayer of PK-15 cells. The cytopathic conditions were observed and recorded 42 h after virus inoculation. The time point was set as the time of 100% inactivation of the virus at the given temperature without any cytopathic effect (CPE)

Full size image

Effect of pH on PRV activity

A neutral liquid environment or a liquid environment with a pH of 6 to 9 had little effect on the activity of PRV, and the PRV toxicity was maintained for a long time. When PRV was in a liquid environment with a pH value of 4–6 or 9–11, the virus titer decreased slowly. When the pH value of the liquid water environment was lower than 4 or higher than 11, the PRV was rapidly denatured and inactivated, losing the ability to infect cells. The details can be seen in Fig. 2.

Fig. 2
figure 2

Effect of pH on PRV stability. a: PRV (K). B: PRV (b). 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. Every level in the experiment was repeated three times, and the mean values are presented in the figure

Full size image

Effect of UV on PRV activity

UVC effectively inactivated PRV, while UVA or UVB did not inactivate PRV. The ability of UVC to inactivate PRV depended on the intensity and time of irradiation. The greater the intensity was, the faster the inactivation occurred, and the longer the irradiation time was, the better the inactivation effect was. PRV was completely inactivated in 7 min at an intensity of 270 µW/cm2. The PRV (B) strain was more resistant to UV than the PRV (K) strain (Fig. 3).

Fig. 3
figure 3

C-band UV irradiation time–titer curve. A: The average UVC intensity was 135 µW/cm2. B: The average UVC intensity was 270 µW/cm2. C: Non-C-band UV irradiation. 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 non-C-band ultraviolet light, the treatment times were 0, 5, 15, 35, 60, 90, 120, 150, and 180 min. The viral titer in each group was determined by a TCID50 assay

Full size image

Effect of humidity on PRV activity

The activity of PRV was greatly affected by environmental humidity. A dry environment was not conducive to virus survival, and the virus activity decreased rapidly. When the virus was in a humid environment, the virus remained active for a longer time. The PRV (B) solution with a volume of 50 µL and TCID50 = 106-107/mL was dried quickly at room temperature (26℃) in a room with natural ventilation, and the infectivity was only maintained for 24 h. The infectivity of PRV (K) under the above conditions was maintained for 36 h, slightly longer than PRV (B). Both strains survived for six days after being air-dried. In a sealed and humid environment, the PRV infection ability was maintained for more than 14 days (Fig. 4).

Fig. 4
figure 4

PRV survival time under different humidity conditions. Plates were quickly air-dried with a fan set to the maximum setting or allowed to air-dry naturally in a biological safety cabinet at a temperature of 26 °C. A sealed plate containing a known titer of PRV cell culture medium was set as a control. The viral titer in each group was determined by a TCID50 assay. The experiment was repeated three times

Full size image

Effect of freeze-thawing on PRV activity

Repeated freeze-thawing had a certain effect on the activity of PRV, but the effect was not significant. Thawing temperatures of 20 °C and 37 °C did not significantly affect PRV activity. Therefore, PRV can tolerate repeated freeze-thaw cycles at certain temperatures (Fig. 5).

Fig. 5
figure 5

Freeze-thaw cycle–titer curve of PRV. Note: 20℃ and 37℃ represent the thawing temperatures. The freezing temperature was − 80 °C, and two thawing temperatures were set: 20 °C in air and 37 °C in a 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

Full size image

Effects of different storage conditions on PRV activity

Under short-term storage, four different storage conditions had no significant effect on the PRV titer. With long-term storage, different storage conditions had a significant effect on PRV activity. When the virus solution was stored at room temperature (26 °C), PRV (K) maintained infection ability for approximately 20 days, and PRV (B) maintained infection ability for approximately 35 days. When stored at -20 °C and − 80 °C, PRV (K) and PRV (B) viruses maintained high titers for more than 60 days. Based on analysis of the rate of titer decline, the ultralow temperature environment of -80 °C is the ideal temperature for storing PRV, which is similar to the biological characteristics of most other viruses (Fig. 6).

Fig. 6
figure 6

Storage time vs. virus titer curves. a: PRV (B). b: PRV (K). PRV solution was aliquoted into 1.5 mL EP tubes at 200 µL per tube and then stored at 26℃, 4℃, -20℃, or -80℃. Samples were removed according to the experimental time points for virus titer determination, and the titer curves were drawn. The experiment was repeated three times

Full size image

Effects of sunlight exposure on PRV activity

Under direct sunlight exposure with a lowest temperature of 38 °C, a highest temperature of 39 °C, an average temperature of 38.16 °C, a lowest experimental table temperature of 38 °C, a highest experimental table temperature of 45 °C, and an average experimental table temperature of 42 °C, PRV (B) and PRV (K) lost the ability to infect cells after approximately 10 min. Under the same temperature conditions, the virus samples blocked by cardboard survived for more than 30 min. Therefore, at the same temperature, sunlight exposure will accelerate virus inactivation, and sunlight can kill PRV (Table 1).

Table 1 Effect of sunlight exposure on PRV activity
Full size table

Effect of chemical disinfectants on PRV activity

The common disinfection chemicals had good PRV disinfection ability. Among them, ionic surfactants and strong oxidants had the best disinfection effect on PRV because they had good disinfection effects at lower concentrations. Powder laundry detergent and potassium permanganate had good disinfection effects and high safety ranges (Table 2).

Table 2 Effect of chemical disinfectants on PRV infectivity
Full size table

Discussion

At present, there are two main ways to prevent and control PR. One is to immunize swine herds with vaccines to reduce herd susceptibility; and the other is to increase the biosafety level of farms, eliminate pathogens, and cut off the spread of pathogens. Biosafety prevention and control have the advantages of low comprehensive cost and significant prevention and control effects. Such practices will be the future of prevention and control of PR and other diseases. The use of biosafety prevention and control of PRV requires an in-depth understanding of the biological characteristics of the pathogen. In addition, PRV (B) and PRV (K) are quite different in pathogenicity. We have reason to speculate that there are also differences in their biological characteristics. Therefore, this study compares the biological characteristics of different strains at the same time, laying a theoretical foundation for scientific and rational formulation of prevention and control measures.

To study the effect of temperature on the activity of PRV, many previous experiments used a water bath for heat treatment, but this method has many problems. During a pilot study of heat treatment, we found that only the temperature near the thermal sensor of the water bath was close to the chosen experimental temperature. Heat treatment of virus samples in a water bath can also easily lead to sample contamination. In the case of a large number of samples, it is difficult to accurately control the heat treatment time. When there are many samples, it places high operation standards on the experimenters, and the repeatability of the experimental data is low. To get around the above problems of heat treatment in water baths, we used a PCR machine to heat-treat virus samples. Using the PCR machine, slight differences in heat tolerance between different strains can be distinguished.

The sensitive temperature range of PRV is 50–70℃, and its activity decreases rapidly with increasing temperature. Heat resistance data have a very important guiding role in production processes. For example, when heating the feed, we can choose the optimal processing temperature and duration according to the actual production process to completely inactivate the virus and ensure achievement of multiple goals, such as no loss of nutritional content, minimum energy consumption, and reduced burden on production techniques. In addition, in terms of swine farm biosafety control, different heat treatment methods are used according to the needs of the farm to achieve thorough disinfection and save cost.

The pH value is affected by the concentration of the pH-regulating buffers and the buffering substances in the virus cell culture medium. We analyzed the rate of viral titer decline to determine the strength of the effects of different pH values on PRV activity. For example, the PRV titer decreased faster at pH = 4 than at pH = 6, and thus, the effect of pH = 4 on PRV activity is more significant than that of pH = 6. PRV is sensitive to acidic and alkaline environments. When cultivating and storing PRV, we must pay attention to control of pH. For example, when expanding PRV in the laboratory, we should choose the optimal harvest time based on the pH of the culture solution to ensure that cell metabolism does not lead to the formation of an acidic culture medium and lower the vitality of PRV. In actual production, the acid/base tolerance of PRV also plays an important role. For example, PRV is sensitive to acidic environments. Therefore, when disinfecting the environment, in addition to using conventional acid disinfectants, we can also choose acid-producing bacteria to regulate the pH of swine feed and drinking water, thereby controlling the growth and residual presence of harmful microorganisms. When studying the effect of environmental humidity on PRV activity, due to experimental error, some virus samples were contaminated with acid-producing bacteria, and thus, the virus samples became acidic. We found that virus samples contaminated with acid-producing bacteria showed a faster drop in titer than uncontaminated samples, which confirmed our hypothesis. Some highly acid-producing bacteria dropped the pH to 3.6 after fermentation at 37 °C for 24 h, the total acid production reached 35.3 g/L, and the pH dropped to below 3.6 after 48 h of fermentation. Theoretically, these acids can be used to disinfect the environment, but there are still many problems, such as which acid-producing bacteria should be selected, what the concentration of acid-producing bacteria should be, how to use them, and how to ensure efficient survival of acid-producing bacteria in the environment.

The effect of UV on PRV activity varies greatly depending on UV wavelength. UVC has the best inactivation effect on PRV but has the disadvantages of weak penetration and short killing distance. Due to the lack of instruments for measuring the intensity of UVA and UVB in the laboratory, when studying the effects of UVA and UVB on the activity of PRV, the intensity cannot be quantified, and only the power of the light source and the irradiation distance can be compared. In production, attention must be paid to UV wavelengths and intensity, the cleanliness of the air, and whether the items for disinfection are shielded or overlap with each other when UV disinfection is applied. UVC also exists in natural light. In sunny, cloudless weather, the UVC intensity is generally 20–40 µW/cm2. We can make full use of this natural UV light to disinfect the environment. The idea of sunlight exposure for antivirus treatment is based on this principle.

Humidity significantly affects PRV activity. For other pathogens, environmental humidity also affects pathogen spread, survival, and infection. For example, most viruses can lose the ability to infect cells when exposed to a dry environment for 36 h but can survive for more than 2 weeks in a liquid environment. Whether the viral surface antigen structure is damaged due to lack of water or whether the liquid water blocks the oxygen in the air and prevents some of the virus structures from being oxidized is not known, and the exact mechanism needs to be further investigated. During production, kee** the air humidity in the swine farm pens within a suitable range and kee** the ground, pens, and internal passages dry will help cut off the PRV transmission path and eliminate PRV from the environment.

Some disinfection agents have a single chemical structure, such as potassium permanganate, and some contain multiple chemical components, such as compound disinfectants and powder laundry detergent. Most disinfection agents have a certain effect on PRV, including ionic surfactants, strong oxidants, chlorine-containing disinfectants, and organic acids and bases. However, these disinfectants can cause harm to humans and animals and pollute the environment. When using disinfectant in actual production, it is necessary to coordinate the use of different disinfectants to achieve synergy, efficiency, economy, and environmental protection.

The influence of physical and chemical factors on PRV activity in vitro is often not determined by a single factor. In actual production, all factors must be fully considered in order to develop comprehensive, economical, and efficient biosafety measures to achieve scientific disinfection and prevent and control epidemic diseases.

Conclusions

In conclusion, the activity of PRV in vitro is affected by factors such as environmental temperature, pH, UV, humidity, freeze-thawing, storage conditions and sunlight. The higher the environmental temperature, the faster the PRV inactivation, and the lower the temperature, the more conducive to the maintenance of virus activity. The greater the environmental humidity, the longer the survival time of PRV in the environment, and the drier the environment, the faster the virus inactivation. PRV has certain resistance to weak acid or weak alkaline environment, and is rapidly inactivated in strong acid or strong alkaline environment. The influence of UV on the activity of PRV is mainly related to the frequency, intensity and irradiation time of UV. The higher the frequency, the stronger the intensity, and the longer the irradiation time, the better the inactivation effect. The effect of freezing-thawing on the infectivity of PRV is not significant, and the virus can still maintain a high titer after repeated freezing-thawing. Storage environment and temperature have an impact on the infectivity of PRV. The lower the storage temperature, the slower the decrease of PRV titer. Conventional disinfectants have a good disinfection effect on PRV, and the disinfection effect is related to the concentration of the disinfectant. The higher the concentration of the disinfectant, the better the disinfection effect. Sunlight exposure can inactivate PRV, which is mainly affected by factors such as temperature, sunlight intensity, ultraviolet intensity and environmental humidity. There are differences in the biological characteristics of PRV in the natural environment. The infectivity of PRV in vitro is affected by genetic genes and environmental physical and chemical factors.