New possibilities for egg white lysozyme: heat-denatured lysozyme partially inactivates select foot-and-mouth disease virus strains

Foot-and-mouth disease (FMD) is one of the most contagious diseases of cloven-hoofed animals. Disinfectants are used to inactivate FMD virus (FMDV) in Japan. Reports that heat-denatured lysozyme inactivates bacteria as well as viruses, such as norovirus and hepatitis A virus, led us to determine its effects on FMDV. We show here that heat-denatured lysozyme partially inhibited the infectivity of FMDV O/JPN/2010-1/14C but of FMDVs A/TAI/46-1/2015 and Asia1/Shamir (ISR/3/89). Further, heat-denatured lysozyme variably reduced RNA loads of FMDVs O/JPN/2010-1/14C, O/MOG/2/Ca/BU/2017, O/Taiwan/1997, Asia1/Shamir (ISR/3/89), Asia1/TUR/49/2011, SAT1/KEN/117/2009, SAT2/SAU/6/2000 and SAT3/ZIM/3/83 but could not those of O/JPN/2000, A/TAI/46-1/2015, A22/IRQ/24/64, A15/TAI/1/60 and C/PHI/7/84. These findings indicate that heat-denatured lysozyme may serve as a new disinfectant against FMDV.


Discussion
Lysozyme degrades the bacterial cell walls 3 and is mainly against gram-positive bacteria 3,12 . Heat-denaturation of lysozyme changes its conformation, and its antimicrobial spectrum includes gram-negative bacteria and others 5,13 . Although heat-denaturation inactivates the enzymatic activity of lysozyme, specific constituent amino acids may be affected, which confer antimicrobial properties 5 . Further, heat-denatured lysozyme inactivates MNV and hepatitis A virus [8][9][10] . Here we show that heat-denatured lysozyme inactivated FMDV. Specifically, heat-denatured lysozyme inhibited the infectivity of FMDV O/JPN/2010-1/14C, in a concentration-dependent manner, by as much as 10 3 TCID 50 units.
In Japan, 4% sodium carbonate solution and several commercial products are generally used as disinfectants to inactivate FMDV 11 . However, the effects of these disinfectants are reduced by contact with organic matter 14 , which limits their use in food production. When these disinfectants are used to sterilize food products, they must be washed or treated before consumption for safety purposes. In contrast, heat-denatured lysozyme is safer than these disinfectants because lysozyme is derived from egg white. To our knowledge, this is the first report to show that heat-denatured lysozyme can partially reduce the infectivity of FMDV.
Propidium monoazide (PMA) enters damaged cells and inhibits PCR assays by binding to nucleic acids 15 . Further, through the same mechanism, PMA is used to effectively measure virus enumeration 16 . Here we used real-time RT-PCR combined with PMA to measure the reduction of viral RNA loads of FMDV strains treated with heat-denatured lysozyme. We found that heat-denatured lysozyme reduced the viral RNA loads of FMDV O/Taiwan/1997 by as much as 2.7 logs. The viral RNA loads of several FMDVs were variably reduced as well. However, the results acquired using real-time RT-PCR combined with PMA did not completely correlate with findings of reduced viral infectivity. These results may be explained by findings that PMA cannot completely bind all nucleic acids 17 .
The mechanism of inactivation of FMDV by heat-denatured lysozyme is unknown. However, assays employing real-time RT-PCR combined with PMA show that heat-denatured lysozyme disrupts the capsid proteins of FMDV, which may contribute to the mechanism of the inhibitory effects of heat-denatured lysozyme. For example, the size of MNV incubated with heat-denatured lysozyme expanded compared with that of unincubated www.nature.com/scientificreports/ MNV or MNV incubated with native lysozyme 8 . Therefore, changes of the conformations of capsid proteins increase the size of the virion, which accounts for the mechanism of inactivation of MNV by heat-denatured lysozyme. In addition, antiviral activity of lysozyme appears to be independent from its bacteriolytic activity, and is related to its positive charge that induces instability in cellular membranes 6,18 . Egg white lysozyme stops the typical cell fusion induced by herpes simplex virus; however, addition of negative charges to this molecule drastically reduced its antiviral activity 18 . Furthermore, the hydrophobicity of heat-denatured lysozyme tends to increase as the pH increased and contributes to MNV inactivation by heat-denatured lysozyme 19 . Further investigations are required to determine whether FMDV is inactivated through a similar mechanism. The amino acid sequences of lysozyme and α-lactalbumin are approximately 70% identical 20 , which is reflected by their similar secondary structures. Although the structural properties of lysozyme and α-lactalbumin are similar (secondary and tertiary structures, constituent amino acid ratios, and specific amino acid regions), the latter does not inactivate MNV 8 . These results suggest that inactivation of MNV by heat-denatured lysozyme is caused by a specific domain within lysozyme, rather than its entire structure. When the amino acid sequence of lysozyme was divided into three regions according to its secondary structure, one polypeptide inhibited MNV significantly more than the other peptides. Further, there is no significant difference between the inhibitory activities of the native and heat-denatured peptides, indicating that the primary amino acid sequence is responsible for inactivation of MNV. In addition, heat denaturation may also change the conformation of lysozyme, which confers its inhibitory activity. In contrast, α-lactalbumin lacks a corresponding sequence, which likely accounts for its inability to inactivate MNV.
Here we show that heat-denatured lysozyme did not significantly reduce the infectivities of it did reduced the RNA loads of Asia1, SAT1, SAT2 and SAT3 FMDVs to varying extents. Therefore, heat-denatured lysozyme cannot be used alone as a disinfectant for FMDV; however, it may be used to supplement other substances to inactivate FMDV. Therefore, comparative analyses of nucleotide and amino acid sequences of FMDV strains may disclose key determinants of heat-denatured lysozyme that inactivate FMDV. Further investigations are therefore required to improve the ability of heat-denatured lysozyme to inactivate FMDV.

Methods
Cells and viruses. IB-RS-2 21 , BHK-21 22 and LFBK-αvβ6 cells 23   Inactivation of the infectivity of FMDV by the heat-denatured lysozyme. Egg white lysozyme was dissolved at concentration of 4% (w/v) in distilled water (pH 6.5 and 7.0) and denatured in a water bath for 30 min at 100 °C. The solution of 4% of heat-denatured lysozyme was further diluted serially twofold from 2 to 0.5% by distilled water. FMDVs O/JPN/2010-1/14C, A/TAI/46-1/2015 and Asia1/Shamir (ISR/3/89) were treated as follows: equal volumes of FMDV and heat-denatured lysozyme were mixed, placed at room temperature for 1 min and then immediately diluted tenfold with Dulbecco's Modified Eagle Medium/Nutrient Mixture F12 supplemented with 10% fetal bovine serum to stop inactivation by the heat-denatured lysozyme. Virus titers were determined using LFBK-αvβ6 cells as described previously 29,30 .
Reduction of viral RNA loads of FMDV by heat-denatured lysozyme. The FMDV strains listed above were treated as follows: equal volumes of FMDV and the heat-denatured lysozyme were mixed and placed at room temperature for 1 min and then immediately diluted tenfold with Dulbecco's Modified Eagle Medium/ Nutrient Mixture F12 supplemented with 10% fetal bovine serum. PMA (Biotium, Hayward, CA, USA) was added to the mixture to 50 μM. The mixture was incubated in the dark for 5 min at room temperature and then irradiated for 15 min using a 375 nm UV-light source. Viral RNAs were extracted from the mixture using a High Pure Viral RNA Kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. Real-time RT-PCR assays were conducted using TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) with primer-set 3D Forward (5′-ACT GGG TTT TAC AAA CCT GTGA-3′) and 3D Reverse (5′-GCG AGT CCT GCC ACGGA-3′), and 3D Probe (5′-TCC TTT GCA CGC CGT GGG AC-3′) as previously described 29,31 . Viral RNA loads were determined by comparison with a standard curve prepared from a positive amplification control containing a segment of the 3D gene of O/JPN/2010-1/14C. The control sequence was transcribed using the T7 promoter with an mMESSAGE mMACHINE T7 Ultra Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The resulting preparation was purified using extraction with a mixture of phenol:chloroform:isoamyl alcohol (25:24:1) and then chloroform and precipitated using isopropanol. The RNA preparations were subjected to RT-PCR in the absence of reverse transcriptase to confirm the complete removal of contaminating DNAs. The RNAs were then quantified using an Ultrospec 2100 Pro (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Serial ten-fold dilutions of the RNA preparation were used to generate a standard curve for real-time RT-PCR.