What type of viruses contain the enzyme lysozyme

This study is the first report on the norovirus-inactivating effects of a thermally denatured lysozyme. Transmission electron microscope analysis showed that virus particles exposed to thermally denatured lysozymes were expanded, compared to the virus before exposure.

The amino acid sequence of the lysozyme was divided into three sections and the peptides of each artificially synthesised, in order to determine the region responsible for the inactivating effect.

These results suggest that thermal denaturation of the lysozyme changes the protein structure, activating the region responsible for imparting an inactivating effect against the virus.

Human norovirus is a non-enveloped virus of the Caliciviridae family, which is known to cause acute gastroenteritis 1 , 2. The virus infects humans through the consumption of raw or undercooked contaminated, food, through contact with the stool or vomit of an infected person 1.

The virus is highly infectious, with as few as 10 to individual viruses causing the onset of symptoms 4. Oysters and other bivalves have been widely reported 5 , 6 as the principal carrier food of norovirus. However, the past few years have seen reports of food poisoning being caused due to the ingestion of unheated food products ready-to-eat RTE products such as salads 7.

This is believed to be due to the cultivation of fresh vegetables in virus-contaminated water 8 , or due to post-harvest handling with contaminants 5 , 7. In recent years, most cases of viral food poisoning reported in Japan were caused by norovirus 9.

In addition, norovirus-related food poisoning cases are the highest, in terms of the number of patients, among all food poisoning cases, including bacterial food poisoning cases 9. This virus reportedly displays high environmental resistance 10 , 11 , 12 , and can survive for a long time on the surfaces of unclean utensils with leftover food particles Therefore, preventing the spread of norovirus infection necessitates an inactivating agent that can reliably damage or destroy norovirus particles.

Ethanol, which is currently the most-commonly used inactivating agent at food manufacturing sites, has little to no effect on norovirus Sodium hypochlorite is believed to be the most effective agent for norovirus inactivation However, the influence of organic matter food ingredients reduces or eliminates its effect 11 and can only be applied to a limited number of fields This has necessitated the development of a highly effective inactivating agent to replace these.

A lysozyme is an enzyme that hydrolyses the peptidoglycan that make up the cell wall of gram-positive bacteria Lysozymes are found in human tears and breast milk, and are industrially extracted and purified from egg white They describe a broad range of applications, with examples of their use including food and pharmaceutical science In recent years, Ibrahim et al.

This is thought to be due to the change in steric structure of the enzyme caused by thermal denaturation, which results in the expression of antibacterial activity even against non-gram-positive bacteria, due to the nature of the peptide itself However, no previous reports have studied a potential interaction between norovirus and lysozyme. In addition, nothing is known about the norovirus-inactivating effects of thermally denatured lysozyme.

In this study, we discovered that thermally denatured lysozymes express an anti-norovirus effect. This study clarifies the relationship between lysozyme heating conditions and the inactivating effect against norovirus. We have also attempted to identify the mechanism of the inactivating effect of lysozyme, as well as the amino acid sequence responsible for this action. To our knowledge, this study is the first report on the inactivation of norovirus by a lysozyme.

This study used murine norovirus MNV-1 , as a surrogate for human norovirus. Murine macrophage cells RAW Once sufficient cytopathic effect was observed, the cells were frozen and thawed four times and the virus particles eluted from the cells. A plaque assay was used to measure the infectivity.

Following this, they were diluted ten-fold with D-MEM and the infectivity measured using a plaque assay. The plaque assay was performed according to the method detailed by Gonzalez-Hernandez et al. Approximately 10 5 cells of RAW Post the incubation time, the culture solutions were extracted from the plates, and diluted stepwise in order to produce samples. Finally, the stain solution was extracted, the plaque count measured, and the infectivity calculated. The untreated virus solution, virus solution exposed to lysozyme, and virus solution exposed to thermally denatured lysozyme were all analysed by a TEM.

Micrographs were obtained and the outer diameters of ten randomly selected virus particles from each test group were measured, in order to calculate the size of the virus particles from the magnification power of the microscope. Real-time PCR was used to measure the virus particle count, in order to confirm the killing of the virus by thermally-denatured lysozyme.

Following exposure, the virus and lysozyme mixtures were diluted ten-fold with D-MEM, in order to stop the inactivating effect of the lysozyme. The PCR was performed with 2. The threshold cycle number was used to quantify the number of MNV particles compared to the standard curve.

Stool specimen was collected from a patient who suffered from diarrhea caused by norovirus. The suspension was used in subsequent experiments. Following exposure, PMA treatment was performed as previously described. The genomic copy number for human norovirus was estimated by comparison to a standard curve for the GII-positive control DNA supplied with the kit.

Following this, a plaque assay was used to measure the infectivity. The amino acid sequence constituting the lysozyme was divided broadly into three regions according to the motif of secondary structure, and the expression of MNV-inactivating peptide s was investigated for each region. The amino acid sequence of egg white lysozyme accession no. All peptides were synthesised by Eurofins Genomics K. Tokyo, Japan. The artificially synthesised peptides were attenuated to express the same concentration by molar ratio, and then introduced to the virus.

Following this, the inactivation effect was measured using the same procedure as in the previous tests. We first studied the relationship between the thermal denaturation temperature for the lysozyme and the inactivating effect against MNV Fig.

As shown in Fig. Therefore, the inactivation effect against MNV was correlated to the concentration and the heating temperature of lysozyme solution used. In comparison to the MNV particles not exposed to the lysozyme solution, which expressed a particle size of A greater degree of expansion was observed However, viral particles exposed to non-denatured lysozyme solution were not significantly different Real-time PCR was used to measure the virus particle count Fig.

Heat-treated lysozyme solutions were mixed with human norovirus and the viral particles were quantified using real-time PCR Fig. Therefore, in order to investigate the amino acid sequence of lysozyme responsible for viral inactivation, we broadly divided the amino acid sequence of the lysozyme into three sections by the motif of secondary structure of lysozyme, and studied their respective MNV-inactivating abilities Fig.

Of the peptides obtained by artificial synthesis, the first set displayed a higher inactivating effect, compared to that observed in the remaining two regions 58—81 residues, 98— residues. This peptide region reduced the infectivity of MNV by 2. The inactivating effect of the remaining two regions was 0. Lysozyme is an enzyme that breaks down the cell walls of bacteria 15 , and has known antimicrobial properties, mainly against gram-positive bacteria 15 , In recent years, it has been reported that heat-treatment of lysozyme changes the steric structure of the protein, broadening its antimicrobial spectrum to include gram-negative bacteria and others 16 , It is speculated that, although heating inactivates lysozyme as an enzyme, some of its specific constituent amino acids are affected, leading to the development of antimicrobial properties In this study, we focused on this action and formulated the idea that the thermally denatured lysozyme could be used to inactivate norovirus.

Viral infectivity was successfully reduced by as much as 4. A correlation was noted between the lysozyme concentration and reduction in viral infectivity, leading to the conclusion that reduced MNV infectivity was due to the lysozyme action. Because norovirus has high alcohol resistance 13 , hypochlorous acid has been generally used to inactivate this virus.

However, the effect of hypochlorous acid is known to reduce due to contact with organic matter Therefore, its uses and applications in the field of food production are limited. When it is used to sterilise food products, it needs to be washed or treated prior to consumption, for safety purposes. Thermally denatured lysozyme on the other hand is safer than hypochlorous acid, because it is a protein derived from egg white.

To our knowledge, this is the first report to state that thermally denatured lysozyme causes reduction of the infectivity of norovirus. Therefore, we decided to study the mechanism behind this phenomenon in further detail. Some viral particles exposed to non-denatured lysozyme were also observed to have expanded, but these were fewer in proportion.

In addition, there was a significant difference between the two, when the mean particle size was taken into consideration. These observations suggest that heat-treated lysozyme effects some action on the capsid protein of the viral surface, causing expansion. Real-time PCR investigation also showed a significant difference between virus exposed to the lysozyme and unexposed virus.

We verified the destruction of the virus particles by real-time PCR, using propidium monoazide. PMA is a reagent that has been extensively used in determining bacterial cell death 21 ; it flows into damaged cells, binding to the nucleic acid and inhibiting PCR.

A perfect method has not been reported for the application of PMA to virus enumeration. However, Escudero-Abarca et al. There are limitations to the amount of nucleic acid that can be blocked by PMA 23 , and this test did not exactly match the effectiveness of the plaque assay. Answers of these questions are given at the end of this blog post. Last updated on June 19th, In this blog, I have posted few multiple-choice questions regarding Hepatitis virus properties, lab diagnosis, vaccine, etc.

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