Evaluation of the Viricidal Efficacy of Four Chemical Disinfectants against Emerged H7N9 Avian Influenza Virus in China

H7N9 viruses have been extensively detected among poultry and in the environment in live-poultry markets (LPMs) across China (Yu et al., 2014; Wu et al., 2015). Inevitably, the widespread presence of H7N9 low pathogenic avian influenza virus (LPAIV) strains accelerated virus mutation, resulting in an emergence of novel high pathogenic avian influenza (HPAI) H7N9 variants in 2017, which caused poultry farms outbreaks in eight provinces (Qi et al., 2017; MOA, 2017). These seemingly normal poultry were transport between poultry farms and LPMs, serving as ‘mixing vessels’ and provided an important ecosystem for the circulation and evolution of avian influenza virus (AIV) (Su et al., 2014; Su et al., 2017). Therefore, controlling the wide circulation of H7N9 LPAIV in domestic poultry farms and LPMs is essential for preventing virus spread and human infection.
       
Strict biosecurity measures are increasingly being recognized as an effective way to stop infection and transmission of pathogenic microorganisms (Ssematimba et al., 2013; Saravanan et al., 2020). Most of poultry farms and LPMs had poor biosecurity conditions, especially overlooking the construction of disinfection techniques. Disinfectants are widely used in antiviral, antibacterial, and animal husbandry production (Jónás et al., 2018; Fan et al., 2015; Montazeri et al., 2017). The choice of effective disinfectants for AIV is often hard. Because the disinfection efficacy depends upon the strain of the virus whereas the mutation of AIV is so fast that many producers have not had time to evaluate the disinfection effects on novel emerged AIV, such as H7N9 subtype (Zou et al., 2013). Therefore, it is of crucial importance to identify disinfection agents or commonly available inactivation methods that might be used for the protection of poultry farms and LPMs against the novel H7N9 subtype AIV in China.
       
Hence, in the present study, the efficacy of four common disinfectants used in poultry farms and LPMs against the novel H7N9 subtype AIV was evaluated. This study will guide the selection of disinfectants for the inactivation of H7N9 AIV.

Viruses and cells
 
A/chicken/Jiangsu/WJ-14/2015 (H7N9) was obtained from the Key Laboratory for Animal Infectious Diseases, Ministry of Agriculture, Yangzhou University, Jiangsu, China (Sun et al., 2017). Strain was propagated in 10-day-old specific-pathogen-free (SPF) embryonic chicken eggs. The virus titers, determined as the 50% tissue culture infectious dose (TCID₅₀)/0.1 mL, were evaluated according to a previous method (Reed and Muench, 1938). All the experiments were conducted in a biosafety level 3 facility for this study. Madin-Darby canine kidney (MDCK) cells were purchased from ATCC.
 
Disinfectants and neutralizing agents
 
Four common disinfectants were chosen and evaluated from large-scale poultry farms and LPMs in China, including deciquam solution, glutaral and benzalkonium bromide solution, sodium dichloroisocyanurate powder and peroxyacetic acid solution. The neutralizing agents against deciquam solution or glutaral and benzalkonium bromide solution were 0.3% Tween 80+0.03% Soft phospholipid +0.1% Glycine in PBS or 0.4% Tween 80+0.05% Glycine in PBS, respectively, and the neutralizing agent against sodium dichloroisocyanurate powder and peroxyacetic acid solution was 0.2 mol/L Sodium thiosulfate in PBS (details in Table 1). Each disinfectant conformed to the quality standard of China’s Ministry of Agriculture and acquired production licenses. Each disinfectant was prepared with hard water (hardness: 342 mg/L) (MOH, 2002).
 

 
Homologous and phylogenetic analysis
 
HA gene sequences of our isolate strain and representative H7N9 subtype AIV during 2013-2019 in China available from the GISAID and GenBank database were downloaded and analyzed for homology using NCBI BLAST analysis. Furthermore, sequences were performed multiple sequence alignment by MEGA (Version 6) and then maximum likelihood phylogenetic trees were inferred with 1000 bootstraps.
 
Evaluation of neutralizing agents and neutralizing products on the growth of MDCK cells and the viral titers
 
In order to eliminate toxicity of disinfectants for MDCK cells, according to Disinfection Technical Specification (the PRC Hygiene Ministry, 2002 edition) (MOH, 2002), several neutralizing agents that do not influence the cell growth and the viral titers were chosen. For cell growth, experiment was divided into six groups: 1. Disinfectants; 2. Neutralizing agents; 3. Neutralizing products (disinfectants + neutralizing agents); 4. The control of standard hard water; 5. The control of phosphate buffered saline (PBS); 6. The control of DMEM. After incubation for 3-4 h, the growth of MDCK cells was observed by optical microscopy.
       
For the detection of virus titers, experiment was also divided into six groups: a. Disinfectants + virus suspension; b. (Disinfectants + virus suspension) + neutralizing agents; c. Neutralizing products (disinfectants + neutralizing agents) + virus suspension; d. (Hard water + virus suspension) + neutralizing agents; e. (Hard water + virus suspension) + PBS; f. Hard water + PBS as mock group. The criteria for successful determination: 1. Group-a has no or less viruses; 2. Virus titer of group-b was more than or similar with that of group 1, but significantly less than group c, d, and e; 3. Virus titer of group c and d were similar with that of group e; 4. The cell growth of group f is normal.
 
Disinfectant inactivation assay
 
According to Disinfection Technical Specification (the PRC Hygiene Ministry, 2002 edition) (MOH, 2002), disinfectants were diluted to twice of the maximum concentration (Fig 2) by using standard hard water, absorbing solution 0.5 mL in a thimble tube, and placed in a water bath at 20^oC±1^oC for 5 min, then 0.5 mL virus suspension was added and blended. After 10, 30, or 60 min, 0.1 mL was immediately taken out and 0.9 mL neutralizer was added for 10 min incubation. The subsequent virus TCID₅₀ titer was performed. TCID₅₀ (log10/0.1 mL) of positive virus group is 2.5. The experiment was repeated three times. The average inactivation logarithmic value was calculated by the following formula: the average virus infection titers (TCID₅₀) from positive control group was N₀ and the average virus infection titers (TCID₅₀) from disinfectant groups was N_x. The average inactivation logarithmic value is equal to log N0-log Nx.

Homologous and phylogenetic analysis of H7N9 AIV
 
Surface protein of influenza virus is mainly interaction site with majority of disinfectants (Suarez et al., 2003). Hemagglutinin (HA), as an important surface protein of influenza virus, is most frequently variable, resulting in the failure of vaccines and disinfectants. Therefore, it was essential to perform homologous and phylogenetic analysis of the HA gene from our isolate strain of H7N9 AIV or representative strains during 2013-2019. Our results displayed that A/chicken/Jiangsu/WJ-14/2015 (H7N9) to current major epidemic clade (Fig 1). Hence, it was chosen to evaluate the efficacy of four common disinfectants used in poultry farms and LPMs.
 

 
Effects of neutralizing agents, disinfectants and neutralizing products on the growth of MDCK cells
 
The growth of MDCK cells is extremely sensitive to different disinfectants, therefore, the neutralizing efficacy of suitable neutralizing agents is necessary for evaluation. As shown in Fig 2, four disinfectants caused varying degrees of influence on the growth of MDCK cells, resulting in cells becoming shrunken and rounded, arranged loosely, gradually losing adhesion and at last majority of the cells floated in the nutrient medium. In contrast, cell growth in the groups with neutralizing products was similar to the control groups, suggesting that each neutralizing agent can well neutralize its corresponding disinfectant with twice the test concentration.
 

 
The effect of neutralizing agents and neutralizing products on the viral titers
 
Whether neutralizing agents and neutralizing products influence the viral titers is key point to our cellular evaluation model. As expected, the results of group a, b and f conformed to our judgment standards. Importantly, the viral titers of group d were same as that of group e, suggesting that neutralizing agents did not influence viral titers. Additionally, the viral titers of group c were same as that of group e, suggesting that neutralizing products did not influence viral titers (Table 2).
 

 
Inactivation efficacy of deciquam solution against H7N9 AIV
 
The inactivation efficacy of deciquam solution with different dilution and different time intervals against the epidemic H7N9 AIV strain is shown in Table 3. The average inactivation logarithmic value was 2.5 when virus treated under the concentrations of 0.0075% deciquam solution for 10 min or 0.005% for 30 min, suggesting that H7N9 AIV was completely inactivated.
 

 
Inactivation efficacy of glutaral and benzalkonium bromide solution against H7N9 AIV
 
The inactivation efficacy of glutaral and benzalkonium bromide solution against H7N9 AIV was also evaluated (Table 4). The results showed that the average inactivation logarithmic value was 2.5 when virus treated under the concentrations of 0.05% for 10 min or 0.033% for 30 min or 0.025% for 60 min, suggesting that H7N9 AIV was completely inactivated.
 

 
Inactivation efficacy of sodium dichloroisocyanurate powder against H7N9 AIV
 
Next, the inactivation efficacy of sodium dichloroisocyanurate powder solution against H7N9 AIV was determined. As shown in Table 5, the average inactivation logarithmic value was 2.5 when virus exposed to the concentration of 0.24 g/L for 30 min, suggesting that H7N9 AIV was completely inactivated.
 

 
Inactivation efficacy of peroxyacetic acid solution against H7N9 AIV
 
Our data indicated that the average inactivation logarithmic value for peroxyacetic acid solution was 2.5 when virus exposed to the concentrations of 0.05% for 10 min, suggesting that H7N9 AIV was completely inactivated (Table 6).
 

       
The long-lasting circulation of the H7N9 virus in LPMs has often preceded the emergence of zoonotic influenza outbreaks (Peiris et al., 2016). Therefore, it is important to clean up H7N9 virus in LPMs in China. Most of common disinfectants that widely used in poultry farms and LPMs were not scientific evaluated. In our study, four common disinfectants, including deciquam solution, glutaral and benzalkonium bromide solution, sodium dichloroisocyanurate powder and peroxyacetic acid solution, were tested in inactivating the H7N9 AIV that emerged in China.
       
Deciquam, a cationic surfactant, has the characteristic of quick, efficient and broad anti-microbial properties. In addition, it also has stable performance and the ability of readily degradable. Therefore, deciquam is widely used as disinfectant and applied to veterinary field (Bin and Qun, 2008; Cursons et al., 1980). However, the efficacy of deciquam in inactivating AIV is seldom evaluated. In our study, we found that the novel avian influenza H7N9 viruses could be completely inactivated by deciquam under the concentrations of 0.0075% for 10 min or 0.005% for 30 min.
       
Benzalkonium bromide, a quaternary ammonium compounds, is wildly used as disinfectant for sterilization of non-living objects or surfaces (He et al., 2017). The usage of this disinfectant is the vital way to inhibit the spread of pathogens. Although quaternary ammonia compounds are easy absorbed by viral proteins with subsequent inactivation (Grossgebauer, 1970), its effectiveness is diminished in the presence of organic matter (Quinn and Markey, 2001), such as faeces and feathers of poultry. Glutaraldehyde as a high level disinfectant is found to remain relatively unaffected in presence of high level organic materials (Gorman and Scott, 2001). Glutaraldehyde was also effective in completely inactivating H5N1 subtype AIV reference viruses (Wanaratana et al., 2010). Therefore, the combination of benzalkonium bromide and glutaraldehyde, even at low concentrations, would be a good disinfectant selection to kill enveloped and non-enveloped viruses within field conditions of poultry farms and LPMs (Figueroa et al., 2017). Our data indicated that glutaral and benzalkonium bromide solution under the concentrations of 0.05% for 10 min can completely kill H7N9 AIV.
       
Sodium dichloroisocyanurate is a chlorinated derivative of cyanuric acid. When dissolved in water it rapidly hydrolyses to form cyanuric acid and releases free available chlorine in the form of hypochlorous acid (Patel and Jones, 2007). A study found that sodium dichloroisocyanurate under manufacturers’ recommendations was fully effective against H9N2 AIVs (A/chicken/Korea/MS96/1996) (Kim et al., 2018). However, we found that the concentration needed was 0.24 g/L for 30 min in order to completely kill H7N9 AIV, which was higher than that of any manufacturers’ recommendations. Therefore, sodium dichloroisocyanurate was not very sensitive to current H7N9 AIV in China. Subtype differences of AIV or extremely fast mutation ability (Jiao and Xiufan, 2016) might influence the efficacy of disinfectant against different stains.
       
Peracetic acid is a widely used environmentally friendly antimicrobial agents of the environmental protection chemicals, whose registered applications have expanded to include in sanitation at food processing and agricultural premises, as well as the disinfection of medical supplies, and as a water purifier and disinfectant (Wang et al., 2015). Our result showed that peracetic acid diluted to 0.05% (less than manufacturers’ recommendations) can still inactivate H7N9 AIV, suggesting that it was very sensitive to our virus strain used. Coincidently, peraclean® (peroxyacetic acid and hydrogen peroxide) was found to be an effective disinfectant against two distinct Egyptian subclades of H5N1 avian influenza virus, including variant subclade 2.2.1.1 and classic subclade 2.2.1/C (Rohaim, 2015). It is therefore inferred that peracetic acid, as a strong oxidant, might be suitable for inactivating broad spectrum of influenza viruses.

The goal of the present study was to compare and determine the ability of four common disinfectants that used in poultry farms and LPMs to inactivate current H7N9 AIV in China. Our results suggested that glutaral and benzalkonium bromide solution as well as peracetic acid was very sensitive to H7N9 virus than other two disinfectants (deciquam solution and sodium dichloroisocyanurate powder).

This work was supported by the National Key Research and Development Program of China (2016YFD0501602), the National Natural Science Foundation of China (31602057), the Jiangsu Provincial Natural Science Fund for Excellent Young Scholars, the Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province (CX(19)3001), the Key R&D Project of Jiangsu Province (BE2018358), Six Talent Peaks Project in Jiangsu Province (NY-131) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education (PAPD).