Enhanced inactivation of avian influenza virus at −20°C by disinfectants supplemented with calcium chloride or other antifreeze agents

Abstract

Avian influenza outbreaks have occurred during winter months, and effective disinfection of poultry premises at freezing temperatures is needed. The commercial disinfectants Virkon and Accel, supplemented with an antifreeze agent [propylene glycol (PG), methanol (MeOH), or calcium chloride (CaCl₂)], were evaluated for their effectiveness in killing avian influenza virus (AIV) at −20°C or 21°C. An AIV suspension was applied to stainless steel disks, air-dried, and covered with a disinfectant or antifreeze agent for 5 to 30 min. Virkon (2%) and Accel (6.25%) with 30% PG, 20% MeOH, or 20% CaCl₂ inactivated 6 log₁₀ AIV within 5 min at −20°C and 21°C. At these temperatures PG and MeOH alone did not kill AIV, but the 20% CaCl₂ solution alone inactivated 5 log₁₀ AIV within 10 min. The results suggested that CaCl₂ is potentially useful to enhance the effectiveness of disinfection of poultry facilities after outbreaks of AIV infection in warm and cold seasons.

Avian influenza viruses (AIVs) continue to be a threat to the Canadian poultry industry. Since 2004, there have been at least 6 outbreaks of AIV infection in Canada that have caused substantial economic loss (1–3). Several of these outbreaks have occurred in regions where winter temperatures as low as −20°C are common. Canada’s eradication policy to control these outbreaks requires killing of all birds on infected premises, safe disposal of the carcasses and wastes, and then cleaning and decontamination of buildings, vehicles, and equipment.

Avian influenza viruses are enveloped and under warm conditions are readily inactivated by most chemical disinfectants, including those in laundry detergents (4–6). However, at temperatures below the freezing point, disinfectants may not be effective in killing viruses (7). For that reason, the use of disinfectants in high concentrations was evaluated for the capacity to kill virus within 5 min before freezing occurred at −10°C (8). However, such a strategy may be of limited value, as freezing may be almost instantaneous when disinfectants are applied to cold metal surfaces. Furthermore, prolonged contact may be required to kill viruses in organic matter (9).

Davison et al (10) were the first to dilute disinfectants with propylene glycol (PG) or methanol (MeOH) to prevent freezing and then to demonstrate the effectiveness of these preparations in killing AIV at −15°C. The efficacy of this approach to cold-weather disinfection was supported by the studies of Guan et al (9), which showed that Virkon (2%), domestic bleach (5.25%), and surface decontamination foam, supplemented with PG, could be effective in killing Newcastle disease and infectious bursal disease viruses at −10°C and −25°C. The objective of the present study was to extend those findings by evaluating the effectiveness of Virkon and Accel, supplemented with PG, MeOH, or calcium chloride (CaCl₂) as antifreeze agents, at inactivating a representative AIV strain at −20°C. Virkon is widely used as a virucidal disinfectant in various environments (8,9), and Accel is an environmentally sustainable disinfectant because hydrogen peroxide (HP) breaks down into water and oxygen (according to the manufacturer).

Virkon solution (2%, w/v) containing 0.43% (w/v) potassium monopersulfate (MPS) as an active ingredient was prepared from Virkon tablets (Antec International, Sudbury, England). Accel solution (5% or 6.25%, v/v) containing 0.35% or 0.44% (w/w) HP as an active ingredient was prepared from Accel Prevention Concentrate (Virox Technologies, Oakville, Ontario). Standard hard water used in the study was prepared to have a standard hardness of 400 parts per million of calcium carbonate (11). The antifreeze agents, PG (1,2-propanediol; Sigma-Aldrich, Oakville, Ontario), MeOH (Caledon, Georgetown, Ontario), and anhydrous CaCl₂ (Sigma-Aldrich), were added during preparation of the disinfectant solutions to final concentrations of 10% to 40% (v/v) for PG and MeOH and 15% to 30% (w/v) for CaCl₂. The solutions were used immediately after preparation. Aliquots (1 mL) of the disinfectant solutions containing 1 of the antifreeze agents were dispensed into 2-mL Nalgene cryogenic vials (Thermo Fisher Scientific, Ottawa, Ontario), and the vials were placed in a freezer with the temperature set at −20°C. After incubation for 1 h the solutions were observed to determine if they remained liquid or were frozen. The lowest concentrations at which the disinfectant solutions remained liquid (30% PG, 20% MeOH, and 20% CaCl₂) were selected for further testing.

An H6N2 strain of AIV (A/Turkey/Mass/3740/65) was used for this study. Virus stock was prepared with 10-day-old embryonated chicken eggs (ECEs) from a specific-pathogen-free flock of White Leghorn chickens maintained by the Canadian Food Inspection Agency and testing negative for AIV. The amount of virus was determined by limiting dilutions in ECEs (12) and expressed as the 50% embryo infectious dose (EID₅₀).

The 2nd-tier quantitative carrier test (13) was used to evaluate the virucidal activity of the disinfectant solutions. Disks (1 cm in diameter; 0.75 mm thick) of brushed stainless steel (AISI no. 430; Muzeen & Blythe, Winnipeg, Manitoba) were washed 3 times with distilled water and sterilized at 121°C for 25 min before use. Contact time course experiments containing time points of 10, 20, and 30 min and single time point experiments using a contact time of 5 min were carried out. For each time point, triplicate sample disks and control disks were prepared: 10 μL of the virus inoculum (containing approximately 5 to 6 log₁₀ EID₅₀ of AIV according to virus recovered from the control disks) was applied to the surface of each disk, the disk was air-dried in a biosafety cabinet for 1 h and then, with the inoculum side up, was placed in a 30-mL polypropylene straightside vial, and the vials were placed into wells of custom-made metal blocks preconditioned to maintain the test temperatures. For the sample disks, disinfectant solution (50 μL) preconditioned to the test temperatures was added to cover the dried inoculum. For the control disks, phosphate-buffered saline (PBS, pH 7.0) with or without antifreeze agent (50 μL) was added. The vials containing the disks were incubated at −20°C or 21°C, simulating cold and warm conditions for specific periods of up to 30 min. At the end of each contact time a neutralizer solution (950 μL) was immediately added to each vial (including vials with control disks) to stop the activity of the disinfectant. The neutralizer was a mixture of 9 volumes of Difco D/E neutralizing broth (Thermo Fisher Scientific) and 1 volume of antibiotic–antimycotic (Invitrogen Canada, Burlington, Ontario). The suspension from each vial was then serially diluted 10-fold and tested for AIV infectivity in ECEs (14). Before testing of the disinfectant solutions, preliminary experiments were conducted to test the effect of the antifreeze agents and the neutralizer on survival of the virus and the ECEs and the effect of the neutralizer on the activity of the disinfectants.

The data presented are the mean amounts of virus recovered from triplicate sets of test or control disks in duplicate experiments. One-way analysis of variance (15) was used to determine significant differences in the amount of virus recovered from disks incubated at the same temperature for the same contact time. The critical level for significance was set at P < 0.05.

As reported in Table I, the addition of 30% PG, 20% MeOH, or 20% CaCl₂ to the Virkon and Accel solutions was sufficient to prevent the solutions from freezing for at least 1 h at −20°C. The antifreeze agents had no adverse effect on the ECEs after neutralization. The virus was resistant to treatment with 30% PG or 20% MeOH alone for up to 30 min (Tables II and III); thus, 30% PG or 20% MeOH did not kill AIV. In contrast, when control disks were treated with 20% CaCl₂ in PBS for 5 or 10 min at −20°C, a reduction of 2.0 and 4.7 log₁₀ EID₅₀ of AIV (compared with control disks with PBS only) was observed (Tables II and III). The killing effect may have resulted from the interactions between calcium cations and viral proteins. Such interactions could cause protein aggregation, precipitation, and denaturation, as demonstrated in cheese and tofu production (16). It was reported 70 years ago that CaCl₂ at concentrations of 0.05 N and 0.5 N (equivalent to 0.55% and 5.5%, w/v) produced a slight to moderate reduction of influenza virus within hours (17). In the present study, a much higher concentration of CaCl₂ (20%, w/v) inactivated approximately 5 log₁₀ EID₅₀ of influenza virus within minutes (Table II). This capacity of a 20% CaCl₂ solution to denature viral proteins is consistent with the report that such a solution caused coagulative necrosis of live animal tissues (18). It is noteworthy that CaCl₂ is readily available and widely applied to roads in Canada in winter months to melt ice. However, it is highly corrosive to metal and must be washed off to prevent damage.

Table I.

State of disinfectant solutions with addition of antifreeze agent after 1 h at −20°C

Antifreeze agent, concentration	Disinfectant solution; statea
	Virkon (2%)	Accel (5%)	Accel (6.25%)
Propylene glycol (PG)
10%	S	S	S
20%	L	G	G
30%	L	L	L
40%	L	L	L
Methanol (MeOH)
10%	S	S	G
20%	L	L	L
30%	L	L	L
40%	L	L	L
Calcium chloride (CaCl2)
15%	S	G	G
20%	L	L	L
25%	L	L	L
30%	L	L	L

^aS — solid; L — liquid; G — gel.

Table II.

Effect of disinfectant solutions on avian influenza virus (AIV) at various temperatures and contact times

Solution and antifreeze agent	Amount of virus (log10 EID50) after treatmenta
	−20°C			21°C
	10 min	20 min	30 min	10 min	20 min	30 min
Virkon (2%)
PG (30%)	0	0	0	0	0	0
MeOH (20%)	0	0	0	0	0	0
CaCl2 (20%)	0	0	0	0	0	0
Accel (5%)
PG (30%)	0	0	0	0	0	0
MeOH (20%)	< 0.9	0	0	0	< 0.9	0
CaCl2 (20%)	0	NT	NT	0	NT	NT
PBS
PG (30%)	4.8	4.8	4.5	4.7	4.3	4.7
MeOH (20%)	4.7	4.7	4.6	4.5	4.6	4.7
CaCl2 (20%)	< 0.9	0	0	0	0	0
None	4.7	4.7	4.7	4.7	4.7	4.7

^aMean from triplicate samples in duplicate experiments with a standard deviation ≤ 0.5. 0 — no virus was isolated; < 0.9 — the amount of virus isolated was below the titration limit of 0.9 log₁₀ EID₅₀. The numbers in boldface are significantly different (P < 0.05) from those after treatment with the other agents at the same temperature and contact time.

EID₅₀ — 50% embryo infectious dose; NT — not tested; PBS — phosphate-buffered saline.

Table III.

Effect of disinfectant solutions on AIV with 5 min of contact

Solution and antifreeze agent	Amount of virus (log10 EID50) after treatmenta
	−20°C	21°C
Virkon (2%)
PG (30%)	0	< 0.9
MeOH (20%)	0	< 0.9
CaCl2 (20%)	0	0
Accel (5%)
PG (30%)	2.0	0
MeOH (20%)	< 0.9	< 0.9
CaCl2 (20%)	0	0
Accel (6.25%)
PG (30%)	0	< 0.9
MeOH (20%)	0	< 0.9
CaCl2 (20%)	0	0
PBS
PG (30%)	5.9	5.7
MeOH (20%)	5.9	5.9
CaCl2 (20%)	4.0	< 0.9
None	6.0	6.0

^aMean from triplicate samples in duplicate experiments with a standard deviation ≤ 0.5. 0 — no virus was isolated; < 0.9 — the amount of virus isolated was below the titration limit of 0.9 log₁₀ EID₅₀ (50% embryo infectious dose). The numbers in boldface are significantly different (P < 0.05) from those after treatment with the other agents at the same temperature and contact time.

PBS — phosphate-buffered saline.

In other studies a contact time of 5 to 10 min was sufficient for disinfectants to kill AIV and Newcastle disease virus at about 20°C (4,5,9). However, as the temperature drops, chemical reactions slow and the contact time required for effective disinfection increases (9). Increasing the concentration of a disinfectant may speed up the disinfection process (8), but at temperatures as low as −20°C rapid freezing, along with the organic load on surfaces and the resistance of pathogens, may render the disinfection process ineffective (9,19). In the present study Virkon solution supplemented with 30% PG, 20% MeOH, or 20% CaCl₂ produced complete inactivation of 6.0 log₁₀ EID₅₀ of H6N2 AIV within 5 min at −20°C (Table III). These results are in agreement with those of previous studies in which Virkon and other oxidizing disinfectants supplemented with antifreeze agents remained effective in killing AIV and Newcastle disease virus at subfreezing temperatures (9,10). In the present study, Accel solution (5%) reduced the AIV by approximately 6 log₁₀ EID₅₀ within 5 min with the addition of 20% MeOH or 20% CaCl₂ but by only 4 log₁₀ with the addition of 30% PG (Table III). When the Accel concentration was increased from 5% to 6.25% a 6-log₁₀ reduction within 5 min was achieved with the addition of any of the 3 antifreeze agents (Table III).

Virkon and Accel are oxidizing disinfectants containing MPS and HP, respectively, as active ingredients, and their effectiveness relies on their oxidizing strength. Organic compounds including organic antifreezes (e.g., PG and MeOH) would be expected to react with these disinfectants, consume their oxidizing strength, and reduce their effectiveness (20). According to the manufacturers, HP has a lower standard electrode potential or oxidizing strength than MPS; thus, PG might have a greater impact on HP than on MPS. Nevertheless, increasing the concentration of disinfectants could help restore their effectiveness in the presence of organic antifreeze or other organic material. In comparison, CaCl₂ is an inert compound with its own disinfection capacity and may prove to be superior as an antifreeze agent for maintaining and even enhancing the effectiveness of oxidizing disinfectants. This could explain the complete inactivation of 6 log₁₀ of AIV when we used disinfectants supplemented with CaCl₂, whereas with PG or MeOH as the supplement, there was some residual viral infectivity (Tables II and III). In the present study the disinfectant solutions were used immediately after preparation. It is not known what effect delaying their application would have on their effectiveness and on the nature of the products and the kinetics of chemical reactions derived from mixing disinfectants with the antifreeze agents.

In summary, this study showed that preparations of Virkon and Accel, supplemented with PG, MeOH, or CaCl₂ as antifreeze agents, could be effectively applied at temperatures as low as −20°C for disinfecting premises after an outbreak of AIV infection. Because the 20% CaCl₂ solution alone inactivated 5 log₁₀ EID₅₀ of AIV within 10 min at −20°C, this product may prove particularly useful in cleaning and disinfection operations aimed at killing AIV and possibly other enveloped RNA viruses at freezing temperatures.