The inactivation effects of physicochemical factors on African swine fever virus
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摘要:目的
了解我国非洲猪瘟病毒(African swine fever virus，ASFV)的生物学特性及理化抗性，提高猪场生物安全水平以防控非洲猪瘟。
方法通过红细胞吸附试验和qPCR试验验证不同理化因素(包括静置、温度、UVC照射、室内外干燥、阳光暴晒和消毒剂)对ASFV的灭活效果。
结果UVC照射30 min即可灭活病毒，照射时间越长，ASFV核酸降解越严重；室内干燥2.5 d、室外干燥1.5 d、阳光暴晒30 min均可灭活ASFV，但不能降解ASFV核酸；常见消毒剂对ASFV的杀灭效果良好，各消毒剂按照推荐稀释浓度室温作用15或30 min，除碘酸混合溶液外均能使ASFV完全失活；温度升高(4、25和37 ℃)会增强消毒剂的消毒作用；有机物FBS的存在会削弱消毒剂的作用，且随FBS体积分数增加(0、10%和30%)消毒剂的消毒效果会降低。
结论本文系统研究了常见的理化因素对ASFV灭活效果的影响，有助于全面了解ASFV生物学特性，对非洲猪瘟的防控具有重要指导意义。
Abstract:ObjectiveThe purpose of this research was to understand the biological characteristics and physicochemical resistance of African swine fever virus (ASFV) in China, so as to improve the biosafety level of pig farms for the prevention and control of African swine fever.
MethodHemadsorption test and qPCR test were used to verify the inactivation effects of different physicochemical factors (including static treatment, temperature, UVC irradiation, indoor and outdoor drying, sun exposure and disinfectant) on ASFV.
ResultUVC irradiation for 30 min could inactivate the virus, the longer the irradiation time, the more serious degradation of ASFV nucleic acid. Indoor drying for 2.5 d, outdoor drying for 1.5 d or sun exposure for 30 min could inactivate ASFV, but these three physical factors had no effect on ASFV nucleic acid degradation. ASFV was susceptible to common disinfectants tested in study, except iodate mixed solution, all disinfectants could completely inactivate ASFV when treated at room temperature at the recommended diluted concentration for 15 or 30 min. An increase in temperature (4, 25 and 37 ℃) enhanced the inactivation effect of disinfectants. The presence of organic compound FBS could weaken the effect of disinfectants, and an increase in the volume fraction of FBS (0, 10% and 30%) could reduce the inactivation effect.
ConclusionThis paper systematically studies the effects of common physicochemical factors on the inactivation of ASFV, which is helpful to fully understand the biological characteristics of ASFV, and has important guiding significance for clinical prevention and control of African swine fever.
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图 1 温度对5种消毒剂杀灭ASFV的影响
白色圆圈代表红细胞吸附试验结果为阴性，红色圆圈代表红细胞吸附试验结果为阳性
Figure 1. Influence of temperature on the ASFV inactivation effect of five kinds of disinfectants
White circle represents the negative result of the hemadsorption test, and red circle represents the positive result of the hemadsorption test
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图 2 不同体积分数FBS对5种消毒剂杀灭ASFV的影响
白色圆圈代表红细胞吸附试验结果为阴性，红色圆圈代表红细胞吸附试验结果为阳性
Figure 2. Influence of FBS with different volume fractions on the ASFV inactivation effect of five kinds of disinfectants
White circle represents the negative result of the hemadsorption test, and red circle represents the positive result of the hemadsorption test
表 1 ASFV P72基因和猪β-actin基因的qPCR引物与探针
Table 1 Primer and probe for qPCR of ASFV P72 gene and porcine β-actin gene
引物/探针 Primer/probe 序列 Sequence 位置/bp Location ASFV F: 5′-CCC AGG RGA TAA AAT GAC TG-3′ 893~912 R: 5′-CAC TRG TTC CCT CCA CCG ATA-3′ 940~960 5′-/56-FAM/ GGC CAG GA /36-TAMSp/-3′ 930~937 β-actin F: 5′-GGA TGC AGA AGG AGA TCA CG-3′ 1 022~1 041 R: 5′-ATC TGC TGG AAG GTC GAC AG-3′ 1 132~1 151 5′-/56-FAM/ GGC CAG GA/36-TAMSp/-3′ 1 121~1 128 
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表 2 ASFV在不同条件下静置的存活天数
Table 2 Survival days of ASFV standing under different conditions
d 样品 Sample 4 ℃ 25 ℃ 37 ℃ 猪静脉血 Swine venous blood 14 猪动脉血 Swine arterial blood 30 纯水 Pure water 36 22 17 生理盐水 Normal saline >75 >60 40
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表 3 UVC照射对含FBS细胞培养液和细胞培养液中ASFV感染力的影响
Table 3 Effect of UVC irradiation on ASFV infection in FBS-containing cell supernatant and cell supernatant
培养液 Supernatant t/min 红细胞吸附1)Hemadsorption Ct 含FBS细胞培养液 FBS-containing cell supernatant 0 + 26.65±0.37 30 − 28.03±0.95 60 − 29.32±0.72 90 − 31.34±0.89 120 − 32.44±1.11 150 − 32.83±0.82 180 − 32.77±0.91 210 − 31.61±1.88 细胞培养液 Cell supernatant 0 + 24.65±0.29 3 + 22.12±1.59 6 + 23.58±0.61 9 + 24.01±0.82 15 + 24.27±0.41 20 + 25.02±0.65 30 − 25.89±0.52 40 − 27.54±1.28 50 − 28.15±0.77 60 − 30.16±0.39 1)“+”：红细胞吸附，“−”：无红细胞吸附 1) “+”: Hemadsorption, “−”: No hemadsorption
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表 4 室外和室内干燥环境对ASFV感染力的影响
Table 4 Effects of outdoor and indoor drying environment on ASFV infection
环境 Environment t/d 红细胞吸附1)Hemadsorption lg(病毒滴度)/ (HAD50·mL−1) lg(Virus titer) Ct 室外 Outdoor 0 + 6.67±0.07 24.75±0.24 0.5 + 3.38±0.33 24.86±0.65 1.0 + 3.17±0.44 25.33±0.36 1.5 + 1.59±0.26 25.48±0.71 2.0 − 0 24.21±0.33 2.5 − 0 24.18±0.72 3.0 − 0 24.12±1.21 4.0 − 0 24.74±1.47 4.5 − 0 25.01±0.57 5.0 − 0 25.67±0.78 室内 Indoor 0 + 6.50±0.13 24.58±0.44 0.5 + 3.67±0.56 24.62±0.58 1.0 + 1.75±0.13 24.55±1.36 2.0 + 0 25.60±0.87 2.5 + 0 25.87±1.72 3.0 − 0 25.02±0.65 3.5 − 0 25.92±0.21 4.0 − 0 27.54±1.28 4.5 − 0 28.15±0.77 5.0 − 0 30.16±0.39 1)“+”：红细胞吸附，“−”：无红细胞吸附 1) “+”: Hemadsorption, “−”: No hemadsorption
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表 5 阳光暴晒对细胞培养液中ASFV感染力的影响
Table 5 Effects of sun exposure on ASFV infection in cell supernatant
t/min 红细胞吸附1)Hemadsorption lg(病毒滴度)/ (HAD50·mL−1) lg(Virus titer) Ct 0 + 6.63±0.33 24.98±0.56 5 + 6.55±0.38 24.35±1.09 10 + 4.34±0.47 24.39±0.76 15 + 2.71±0.29 25.10±0.35 20 + 2.84±0.82 24.06±1.55 30 − 1.67±0.19 24.53±0.93 35 − 0 24.27±0.95 50 − 0 24.98±0.79 60 − 0 24.87±2.12 1)“+”：红细胞吸附，“−”：无红细胞吸附 1) “+”: Hemadsorption, “−”: No hemadsorption
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表 6 消毒剂对ASFV红细胞吸附能力的影响
Table 6 Effects of disinfectants on ASFV hemadsorption capacity
名称 Name 类别 Classification 有效成分 Active ingredient 稀释倍数 Dilution ratio 红细胞吸附1)Hemadsorption 15 min 30 min 甲醛 Formaldehyde 醛类 Aldehydes 甲醛 Formaldehyde 1∶100 − − 1∶200 − − 1∶500 − − 柠檬酸 Citric acid 酸类 Acids 柠檬酸 Citric acid 1∶50 − − 1∶100 − − 1∶200 + + 烧碱 Caustic soda 碱类 Alkalines 氢氧化钠 Sodium hydroxide 1∶50 − − 1∶100 − − 1∶200 − − 卫可 Virkon 氧化剂 Oxidizers 过硫酸氢钾 Potassium hydrogen persulfate 1∶200 − − 1∶400 − − 1∶800 + + 84消毒液 84 disinfectant 氯制剂 Chlorine preparations 有效氯 Available chlorine 1∶100 − − 1∶200 − − 1∶400 − − 漂白粉 Bleaching powder 氯制剂 Chlorine preparations 次氯酸钙 Calcium hypochlorite 1∶250 − − 1∶500 − − 1∶1 000 − − 二氯异氰尿酸钠 Dichlorocyanuric acid 氯制剂 Chlorine preparations 有效氯 Available chlorine 1∶250 − − 1∶500 − − 1∶1 000 − − 百毒杀 Povidone iodine solution 季铵盐类 Quaternary ammonium salts 二癸基二甲基溴化铵 Didecyl dimethyl ammonium bromide 1∶300 − − 1∶600 + ± 1∶1 200 + + 戊二醛 Glutaraldehyde 醛类 Aldehydes 戊二醛 Glutaraldehyde 1∶1 000 − − 1∶2 000 − − 1∶5 000 + − 安灭杀 Anabasin 混合制剂 Mixed preparations 戊二醛、季铵盐 Glutaraldehyde, quaternary ammonium salt 1∶100 − − 1∶200 − − 1∶400 − − 复合酚 Compound phenol 混合制剂 Mixed preparations 酚、醋酸 Phenol, Ethylic acid 1∶100 − − 1∶200 − − 1∶400 − − 戊二醛癸甲溴铵溶液 Glutaral and deciquam solution 混合制剂 Mixed preparations 戊二醛、二癸基二甲基溴化铵 Glutaraldehyde, didecyl dimethyl ammonium bromide 1∶500 − − 1∶1 000 − − 1∶1 200 − − 复方戊二醛 Compound glutaraldehyde 混合制剂 Mixed preparations 戊二醛、苯扎氯铵 Glutaraldehyde, benzalkonium chloride 1∶150 − − 1∶300 − − 1∶600 − − 碘酸混合溶液 Iodine and acid mixed solution 混合制剂 Mixed preparations 碘、磷酸 Iodine, phosphoric acid 1∶100 + + 1∶200 + + 1∶400 + + 1)“+”：红细胞吸附，“−”：无红细胞吸附 1) “+”: Hemadsorption, “−”: No hemadsorption
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