Inactivation rate of African swine fever virus by a formaldehyde-based product

Abstract

African swine fever (ASF) is an important transboundary animal disease with a high mortality rate. The high African swine fever virus (ASFV) titers in the excretion of infected wild boar possibly contaminate the feed ingredient. Once contaminated, it could support persistent residual titer of the ASFV. The chemical inactivation of imported feed ingredients is a precautionary risk management measure to restrict the import risk of ASFV through international trade. The log ASFV titers were linearly reduced as a function of the inactivation time after exposure to 0.03%, 0.05%, 0.1%, and 0.2% formaldehyde-based product (FBP). A four-log reduction of ASFV titer was achieved after exposure to 0.2% FBP and 0.03%–0.1% FBP for 30-min and 60-min inactivation times, respectively. The decimal reduction time or D value is defined as the time required to inactivate the virus titer by 1 log. The ASFV inactivation rate from the independent experiment of FBP concentration was converted to a D value. The observed mean D_0.2%, D_0.1%, D_0.05%, and D_0.03% of FBP were 13.4, 44.9, 45.0, and 45.3 min per log reduction of ASFV, respectively. The interpretation of D_0.2% of FBP is that the ASFV titer is inactivated by 1 log after being exposed to 0.2% FBP for every 13.4 min. A more effective chemical has a lower D value because of a shorter inactivation time required to achieve the same 1-log reduction. In addition, the hypothetical inactivation time by any chemical additive is scenario-specific and is calculated by the product of D value (at a certain concentration) and log titers of residual ASFV. This study introduces the concept and application of the D value to compare the virucidal activity of chemicals and to determine the hypothetical inactivation time of chemicals depending on the chemical concentration including the virus titer in the feed.

The virucidal activity of 0.03%–0.2% formaldehyde-based product against African swine fever virus.

Introduction

African swine fever (ASF) is an important transboundary animal disease (TAD) of domestic pigs worldwide (OIE, 2021a). The highly virulent African swine fever virus (ASFV) causes severe clinical symptoms followed by sudden death in susceptible pigs. The routes of transmission of ASFV are either direct contact with infected animals or indirect contact with mechanical vectors and the contaminated environment (Olesen et al., 2020; OIE, 2021a). The high and wide ASFV titers in blood, nasal fluid and rectal fluid are in the range of 6–8.7 log haemadsorption (HAD)₅₀/mL, 1–4 log HAD₅₀/mL, and 1–2 log HAD₅₀/mL, respectively (Guinat et al., 2014; Mazur-Panasiuk et al., 2019). The excretion of the wild boar infected with ASFV possibly contaminates the field for plant-based feed ingredients and may have led to the ASF epidemic in Latvia in 2014 (Olsevskis et al., 2016). Once contaminated with ASFV derived from the excretion of infected wild boar, the plant-based feed commodity potentially serves as a TAD of ASF (Olsevskis et al., 2016). Since residual ASFV is highly contagious and persistent, border control of contaminated commodities to minimize the likelihood of ASFV importation incidentally leads to international trade restrictions (Beltran-Alcrudo et al., 2017; Niederwerder et al., 2021).

ASFV titer is significantly reduced to less than 2 log TCID₅₀/mL after exposure to 0.07% caprylic acid (C₈), 0.09% capric acid (C₁₀), 0.10% lauric acid (C₁₂), and 0.14% glycerol monolaurate (GM) in a suspension test at 37 °C for 60 min (P < 0.01). ASFV is resistant to a 0.25%–2.0% mixture of medium-chain fatty acids consisting of caprylic, capric, and lauric acids while ASFV is significantly inactivated no more than 1.0 log TCID₅₀/mL after being exposed to 2.0% GM in commercial swine feed at room temperature for at least 30 min (P < 0.01) (Jackman et al., 2020). An aqueous formaldehyde-based additive at 0.03% and 0.3% inactivates ASFV titer 0.8 and 3.5 logs TCID₅₀/mL at room temperature in 30-min inactivation time, respectively (Niederwerder et al., 2021). The effectiveness of this antiviral feed additive is concentration-dependent. Note that 10 times more concentrated additive (0.3% vs. 0.03% formaldehyde) does not intuitively correspond to 10 times more reduction (equivalent to 1 log reduction additional) as 0.3% formaldehyde inactivates 2.7 log additionally to 0.8 log titer by 0.03% formaldehyde.

The comparative effectiveness of chemical disinfectants inactivating ASFV in the environment or feed was previously reported for a few disinfection methods in a specified condition tested (De Lorenzi et al., 2020; Niederwerder, 2021; OIE, 2021a). The residual titer of ASFV in the commodity is sometimes unpredictable in the context of import risk analysis (OIE, 2021b). Therefore, the inactivation time and/or concentration of chemical disinfectant should be dynamic, as opposed to a one-size-fits-all procedure of disinfection, to completely inactivate the diverse residual titer of ASFV in the imported commodities.

The inactivation rate of the virus follows first-order kinetics (Sullivan et al., 1971; Kamolsiripichaiporn et al., 2007). The D value (decimal reduction time: DRT) is the time required to inactivate the virus titers by 1 log at a specified condition and also represents the inactivation rate (Cowan et al., 2015; Kalmar et al., 2018). By the definition of the D value, the (total) hypothetical inactivation time of a chemical to completely inactivate the residual virus titer is the product of the D value of the chemical and log virus titer. Next, the z value is the chemical concentration required to change the D value by 1 log (Grasteau et al., 2015). The z value is used to calculate unknown D values (Kamolsiripichaiporn et al., 2007). A more effective chemical has a lower D value because of a shorter inactivation time required to achieve the same 1-log reduction. In other words, to inactivate the (same) virus titer, the contact (inactivation) time of the chemical is supposed to be shortened by applying a more concentrated chemical. Therefore, the D and z values of the chemicals were used to adjust the appropriate concentration and/or inactivation time of the chemicals corresponding to the different residual titers of ASFV, thus completely inactivating ASFV titer in the feed ingredient. The D and z values of chemicals, particularly feed additives inactivating ASFV are rarely reported. Therefore, the objectives of this study were to evaluate the effectiveness of various concentrations of a liquid formaldehyde-based product (FBP) inactivating ASFV in terms of the D and z values by a suspension test at room temperature without interfering substances and to introduce the concept of applying D and z values to completely inactivate varied residual virus titer.

Materials and Methods

Ethics statement and consent to participate

Animal experiments regarding blood collection for the primary swine macrophages were performed under animal biosafety level 2 at the National Institute of Animal Health (NIAH), Bangkok, Thailand. All procedures were carried out in compliance with the Animal for Scientific Purpose Act 2015 (B.C. 2558). The ARRIVE guidelines 2.0 were followed for the care and use of laboratory animals. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at NIAH (Approval number EA-009/64(R)).

Cell and virus

Primary porcine alveolar macrophages (PAM) were collected from the lungs of healthy pigs (8–10 wks old). Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco^TM: Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Gibco^TM: Thermo Fisher Scientific, Waltham, MA, USA) and 1% antibiotic–antimycotic (Gibco^TM: Thermo Fisher Scientific, Waltham, MA, USA) were used for PAM cells. The ASFV strain VNUA/HY-ASF1/Vietnam/2019 was propagated in PAM cells and used for the experiment (Le et al., 2019). Virus titers expressed as the amount of virus causing hemadsorption in 50% of infected cultures (HAD₅₀/mL) were calculated according to the method of Reed–Muench (Reed and Muench, 1938).

Chemical inactivating ASFV

The ASFV virucidal activity of FBP (PINK Solution, PINK^TM: PVTM, Pathumthani, Thailand) was evaluated. The active ingredient of this FBP is formaldehyde stabilized with organic acid and surfactant. The FBP solutions were prepared in sterile water and mixed with an equal volume of ASFV suspension (10⁸ HAD₅₀/mL). The negative control (0% FBP) was performed in the same manner but FBP was replaced by sterile water. After the intervals of inactivation time, the virus suspension was prepared in 10-fold serial dilutions in RPMI medium and each dilution was then inoculated in three wells of PAM cells prepared in 96-well plates for virus titration. Each inactivation experiment was duplicated. The titer (log HAD₅₀/mL) of the recovered virus was calculated by the method of Reed–Muench (Reed and Muench, 1938).

Inactivation curve

The viral inactivation rate is assumed to follow first-order kinetics (Cowan et al., 2015; Kalmar et al., 2018). A linear inactivation curve is fitted to the observed viral reductions in the experiments as a logarithmic function of inactivation time at a constant concentration of FBP. The negative reciprocal of the slope of the inactivation curve is the D value as shown in the following equation:

$$\log \left(\frac{N_t}{N_0}\right)=\frac{1}{D}t,$$ (1)

where N_t and N₀ are the ASFV titers at inactivation times at t and zero, respectively. The D value is the time required to reduce the virus titer by 1 log at a certain condition particularly the concentration of the chemical. The D value of a chemical is specific to a certain condition of a suspension test such as concentration, pH, and temperature. The D value is used to compare the virucidal activity across concentrations and over chemical additives. The virucidal activity (inactivation rate) is negatively correlated with the D value. Since the D value is concentration specific, in this study, the subscript of the D value is the concentration of FBP (D_C), for example, D_0.2% is the D value of FBP concentration at 0.2%.

Decimal reduction time curve

Upon performing the inactivation experiments for FBP with multiple concentrations, the DRT curve is then derived from fitting multiple D values on a semi-logarithmic scale across concentrations of FBP. The linear DRT curve is fitted to the rate of change of D value as a logarithmic function of concentrations of chemical additive (Grasteau et al., 2015). Analogous to the D value, the z value is the negative reciprocal of the slope of the DRT curve. Therefore, the z value is the chemical concentration required to change the D value by 1 log. The unknown D value of non-experimental concentrations of chemical additive can be calculated by the z value together with a known D value using the following equation:

$$log\left(\frac{D_2}{D_1}\right)=\frac{1}{z}\left(C_1-C_2\right),$$ (2)

where D₁ and D₂ are the D values of ASFV at chemical concentrations C₁ and C₂, respectively. According to equation (2), for a certain z value the D value decreases as the chemical concentration increases.

Statistical analyses

Regression analysis was used to determine the statistical significance of both the inactivation curve and the DRT curve by an F test at a 5% level of significance. The goodness-of-fit (gof) of both the inactivation curve and the DRT curve was determined by the correlation coefficient (r²) and the root-mean-square error (RMSE) (Kamolsiripichaiporn et al., 2007). The concentration effect of FBP was determined by the statistical difference of ASFV inactivation rates (D values) across four concentrations by one-way analysis of variance (ANOVA). The Tukey’s multiple comparison test was followed to determine the pair-wise differences of FBP. IBM^® SPSS^® Statistics version 22 (SPSS Inc., Chicago, IL, USA—licensed to Chulalongkorn University) software was used to perform statistical analyses.

Results

ASFV inactivation by a chemical additive

The mean titer and the detection limit of ASFV in this study were 8.13 log and 2.5 log HAD₅₀/mL, respectively. This range of ASFV titer is wide enough to monitor the gradual change of ASFV titer and to examine the virucidal activity of the chemical additive. The ASFV titers were reduced as a function of inactivation time after being exposed to 0.03%, 0.05%, 0.1%, and 0.2% FBP as shown in Figure 1. A mean 5.63 log HAD₅₀/mL ASFV titer was inactivated after being exposed to 0.2 and 0.05%–0.1% FBP for 60 min and 180 min, respectively while the ASFV titer exposed to 0.03% FBP did not decrease below the detection limit even at 180-min inactivation time. In the context of an effective disinfectant, 0.2% FBP and 0.03%–0.1% FBP inactivates more than 4-log ASFV titer with 30-min and 60-min inactivation time, respectively. The more concentrated FBP with a smaller D value inactivated the same virus titer within a shorter contact (inactivation) time, that is, the same contact time as the more concentrated FBP with a smaller D value could inactivate more virus titer.

Figure 1.

Best-fit inactivation curves of ASFV exposed to (A) 0.2% FBP, (B) 0.1% FBP, (C) 0.05% FBP, (D) 0.03% FBP (P value < 0.05), and (E) 0% FBP in the suspension test at 25 °C. The error bars indicate the standard deviation of the mean ASFV titers.

D value representing the ASFV inactivation rate

The overall rate of ASFV inactivation was demonstrated by fitting an inactivation curve (linear regression) to the observed log reduction of ASFV titer (N_t) as a function of an FBP inactivation time (t) at a specified FBP concentration. The inactivation rate of ASFV by FBP was determined by the slope of this inactivation curve, which is the regression coefficient of the inactivation time variable (x-axis). The best-fit slope of the inactivation curve was always negative since the ASFV titers (y-axis) supposedly decrease along the inactivation time of FBP as shown in Figure 1. The mean and 95% confidence interval (CI) of D values, inactivation curves, and gof of FBP across 4 concentrations are shown in Table 1. The range of mean D_0.2%, D_0.1%, D_0.05%, and D_0.03% of FBP was between 13.4 min and 45.3 min. As expected, the mean D value of more concentrated FBP is lower than that of less concentrated FBP. The inactivation curves across three concentrations of FBP are highly significant (P < 0.015), indicating that ASFV is sensitive to 0.03%–0.2% FBP at room temperature. The inactivation curves of a lower concentration of FBP had a better fit to the log reduction of ASFV titer than that of a higher concentration because of the better gof (higher r², lower RMSE, and lower P value) of a more diluted FBP (Table 1).

Table 1.

D value, inactivation curve and gof of liquid FBP inactivating ASFV at 25°C.

% FBP	D value (min)a		Inactivation curveb	gof		P value
	Mean	95% CI		r 2	RMSE
0.20	13.4A	7.8–46.1	N t = −0.0747 t + 6.37	0.66	1.46	0.0137
0.10	44.9B	28.3–108.7	N t = −0.0223 t + 5.80	0.59	1.32	0.0035
0.05	45.0B	30.4–86.7	N t = −0.0222 t + 6.24	0.68	1.08	0.0009
0.03	45.3B	31.4–81.4	N t = −0.0221 t + 6.54	0.72	0.99	0.0005

^aIn the same column, mean D values with different letters imply that there are statistically significant differences (P < 0.01) among the different concentrations (letters A and B).

^bASFV titer N_t (log) at inactivation time t (min).

The one-way ANOVA indicated the significant difference among D_0.2%, D_0.1%, D_0.05%, and D_0.03% of FBP (P < 0.01). Tukey’s multiple comparisons of D values across FBP concentrations inactivating ASFV were performed and are shown in Table 1. The D_0.2% of FBP was significantly lowest (P < 0.05). However, D_0.1%, D_0.05%, and D_0.03% of FBP were not significantly different (P > 0.05) because the FBP concentrations tested were 2-fold serially diluted, and then the percent intervals at the more diluted FBP from the starting concentration of 0.2% FBP were getting smaller.

Z value calculated from multiple D values

The DRT curve was determined by plotting the logarithmic D values of FBP (y-axis) from Table 1 against four corresponding concentrations of FBP (x-axis) as shown in Figure 2.

Figure 2.

Log–linear DRT curve fitted to the mean log D values of FBP inactivating ASFV. The errors bar indicate the standard deviation of the mean log D values.

The mean and 95% CI of z values, DRT curve equation, and gof of FBP are shown in Table 2. Even though from Table 1D_0.1%, D_0.05%, and D_0.03% of FBP were not statistically different due to the narrow difference of FBP concentrations, the regression analysis of the log–linear DRT curve was statistically significant (P = 0.001). This indicates that the ASFV virucidal effect of FBP was concentration dependent.

Table 2.

Z value, decimal reduction curve and gof of liquid FBP inactivating ASFV at 25 °C.

z value (%)		DRT curvea	gof		P value
Mean	95% CI		r 2	RMSE
0.31	0.22–0.53	log DC = −3.21 C + 1.83	0.85	0.10	0.001

^aLog D_C (min) for the disinfectant concentration C (%).

Adjusted D values from the DRT curve

Even though the ASFV virucidal activity of FBP in terms of D value was independently observed across four concentrations with a certain variation, the log–linear regression of the DRT curve was fitted to eliminate the experimental bias or error across multiple log D values. The DRT curve equation was used to calculate the adjusted D values across four concentrations as shown in Table 3. Adjusted D_0.2%, D_0.05%, and D_0.03% of FBP increased while adjusted D_0.1% of FBP decreased.

Table 3.

Adjusted and mean D values (min) of liquid FBP inactivating ASFV at 25 °C

% FBP	Adjusted D value by DRT curve	Mean D value by observed virus reduction
0.20	15.4	13.4
0.10	32.3	44.9
0.05	46.7	45.0
0.03	54.2	45.3

Discussion

Since residual ASF is highly contagious and persistent in the contaminated commodity, border control of potentially contaminated commodity is needed to minimize the likelihood of TAD of ASF, thus also restricting international trade (Beltran-Alcrudo et al., 2017; Niederwerder et al., 2021). Chemical inactivation of an imported products is the recommended precautionary risk management measure to reduce the risk of ASFV introduction (Niederwerder, 2021). In this study, the virucidal activity of the formaldehyde-based additive (PINK Solution, PINK^TM) was investigated and its potential as an anti-ASFV chemical additive was assessed.

The ASFV virucidal activity of FBP was demonstrated by an at least 5-log reduction of ASFV titer after being exposed to 0.2 and 0.05%–0.1% FBP for 60 min and 180 min, respectively (Figure 1). The more concentrated FBP is more effective at inactivating ASFV than the less concentrated FBP since more ASFV titer was inactivated within the same inactivation time. This resulted in the concentration-dependent or dose-dependent reduction of FBP against ASFV and was in line with a previous study investigating the effectiveness of a liquid formaldehyde-based feed additive (Sal CURB^®: Kemin Industries, Inc., USA) against ASFV in a cell culture model at 25°C to mitigate the risk of ASF in feed (Niederwerder et al., 2021). Note that the virucidal activities of 0.2% FBP with 60-min contact time achieving a 4-log virus reduction were compatible with the requirement of an effective disinfectant by European standard EN 14675: 2015.

The D value is used to compare the virucidal activity across concentrations of the chemical. From Table 3, the mean D_0.1% of FBP inactivating ASFV was 44.9 min. This suggests that ASFV was inactivated by 1 log after being exposed to 0.1% FBP for 44.9-min inactivation time, while mean D_0.2% of FBP required only 13.4-min inactivation time to reduce ASFV by 1 log. This indicates that the ASFV virucidal activity of 0.2% FBP is more effective than that of 0.1% FBP. Therefore, for a certain chemical, a lower D value of a higher chemical concentration usually has a higher virucidal activity than a higher D value of a lower chemical concentration. From Table 3, the mean D_0.2% of FBP was the lowest and this was followed by higher D_0.1%, D_0.05%, and D_0.03%, respectively. This study demonstrated that 0.2% FBP had the highest ASFV virucidal activity and this was followed by 0.1%, 0.05%, and 0.03% FBP, respectively.

An additional advantage of using the D value to compare virucidal activity is that the D value is not based solely on an observed virus titer reduction by a single inactivation time. However, the D value of the chemical is determined by virus titer reductions based on a series of inactivation times at a constant concentration, thus eliminating the bias of experimental design or human error as shown in Figure 1. This approach does not apply to the experimental design examining only a virus titer reduction per inactivation time per concentration of a chemical.

The D value was used not only to compare the virucidal activity (across concentrations and over chemicals) but also to determine the hypothetical inactivation time of the chemical required to eliminate the highest virus titer possible in the feed ingredient. The reported highest ASFV (Lithuania LT14/1490 or Georgia 2007/1) titer possible in the blood from an infected swine at 6 to 11 days post inoculation was 8.7 log HAD₅₀/mL (Guinat et al., 2014; Gallardo et al., 2017). Such excretion of swine might contaminate the feed ingredient field for plant-based feed and this led to the ASF epidemic in Latvia in 2014 (Olsevskis et al., 2016). Therefore, the target of chemical treatment was to eliminate the highest titer of ASFV at 8.7 log HAD₅₀/mL. As an illustration, if we were to use 0.1% FBP and the mean D_0.1% of FBP is 44.9 min/log (Table 3). The hypothetical inactivation time of the chemical required to eliminate the virus titer in the commodity is the product of mean D_0.1% of FBA (44.9 min/log) and 8.7 log titer which is approximately 391 min (44.9 min/log × 8.7 log) or 6.5 h.

Conclusion

The ASFV titers were reduced as a function of inactivation time after being exposed to 0.03%, 0.05%, 0.1%, and 0.2% FBP(PINK Solution, PINK^TM). A four-log reduction of ASFV titer was achieved after being exposed to 0.2% FBP and 0.03%–0.1% FBP for 30-min and 60-min inactivation times, respectively. The D value represents the inactivation rate of ASFV by FBP and it was used to compare the virucidal activity across concentrations and chemicals. The scenario-specific examples illustrate the application of the D value to determine the hypothetical inactivation time of chemicals depending on the chemical concentration.

Glossary

Abbreviations

ANOVA

one-way analysis of variance

ASF

African swine fever

ASFV

African swine fever virus

CI

confidence interval

DRT

decimal reduction time

FBP

formaldehyde-based product

gof

goodness-of-fit

HAD

hemadsorption

OIE

World Organisation for Animal Health

PAM

primary porcine alveolar macrophage

RMSE

root mean square error

RPMI

Roswell Park Memorial Institute

TAD

transboundary animal disease

TCID

tissue culture infective dose

Conflict of Interest Statement

The authors declare no real or perceived conflicts of interest.