Bio-based disinfectants and their applications

Abstract

The present invention relates to the field of disinfectants, and more specifically, to a bio-based disinfectant and its application. The bio-based disinfectant contains polylysine, lysozyme and / or lysostaphin, lauroyl arginine ethyl ester hydrochloride and glycerol; in the bio-based disinfectant, the concentration of polylysine is 0.01-5% by weight, the concentration of lysozyme and / or lysostaphin is 0.01-5% by weight, the concentration of lauroyl arginine ethyl ester hydrochloride is 0.01-5% by weight, and the concentration of glycerol is 0.01-10% by weight. The disinfectant provided by the present invention has the characteristics of high antibacterial rate, high safety, wide antibacterial spectrum, wide applicability and mild and lasting.

Description

Technical Field

[0001] The present invention relates to the field of disinfectants, and more particularly to a bio-based disinfectant and application thereof. Background Art

[0002] At present, the disinfection products used in daily life are generally chemical preparations. According to their main effective ingredients, they can be divided into chlorine-containing disinfectants, iodine-containing disinfectants, peroxide disinfectants, aldehyde disinfectants, alcohol disinfectants, phenol disinfectants, biguanide disinfectants, quaternary ammonium disinfectants, etc. Aldehyde, phenol, and biguanide disinfectants have high bactericidal effects, but their ingredients are toxic; chlorine-containing disinfectants and peroxide disinfectants are irritating to a certain extent, and there will be safety hazards if they are not used properly; alcohol disinfectants, iodine-containing disinfectants, and quaternary ammonium disinfectants are relatively safe, but the bactericidal spectrum is narrow. At the same time, these chemical disinfectants also have disadvantages such as irritating odor and strong corrosiveness.

[0003] As people's demands for health become increasingly urgent, new green biological disinfectants have begun to receive widespread attention. Compared with traditional chemical disinfectants, biological disinfectants have the advantages of strong bactericidal specificity, mild action conditions, not easy to produce drug-resistant strains, no irritation to objects, no irritation to skin and mucous membranes, no residual harmful substances, not easy to burn, and safe to use.

[0004] However, the bactericidal effect of current biomaterial-based disinfectants needs to be further improved. Summary of the invention

[0005] The object of the present invention is to provide a bio-based disinfectant with high bactericidal effect and application thereof.

[0006] In order to achieve the above-mentioned purpose, on the one hand, the present invention provides a bio-based disinfectant, which contains polylysine, lysozyme and / or lysostaphin, lauroyl arginine ethyl ester hydrochloride and glycerol; wherein, in the bio-based disinfectant, the concentration of polylysine is 0.01-5% by weight, the concentration of lysozyme and / or lysostaphin is 0.01-5% by weight, the concentration of lauroyl arginine ethyl ester hydrochloride is 0.01-5% by weight, and the concentration of glycerol is 0.01-10% by weight.

[0007] In a second aspect, the present invention provides the use of the above-mentioned bio-based disinfectant in in vitro bacteriostasis.

[0008] The disinfectant provided by the present invention has the following beneficial effects:

[0009] High antibacterial rate - the disinfectant of the present invention can kill Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans with a logarithmic value greater than 5 in 1 minute, that is, it kills more than 99.999% of the bacteria, and has a significant bactericidal effect;

[0010] High safety - using natural raw materials as the main ingredients, safe and non-toxic;

[0011] Wide antibacterial spectrum - can effectively kill various pollutants in daily life;

[0012] Wide applicability - can be used for disinfection of hands, skin, surface of objects, etc.;

[0013] Gentle and long-lasting - no need to wash after use, little damage to skin or objects, no irritating odor, and the active ingredients are not easy to volatilize and can continuously inhibit bacteria, with a shelf life of 2 years at room temperature.

[0014] Other features and advantages of the present invention will be described in detail in the following detailed description. DETAILED DESCRIPTION

[0015] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0016] In a first aspect, the present invention provides a bio-based disinfectant, which contains polylysine, lysozyme and / or lysostaphin, lauroyl arginine ethyl ester hydrochloride and glycerol; wherein, in the bio-based disinfectant, the concentration of polylysine is 0.01-5% by weight, the concentration of lysozyme and / or lysostaphin is 0.01-5% by weight, the concentration of lauroyl arginine ethyl ester hydrochloride is 0.01-5% by weight, and the concentration of glycerol is 0.01-10% by weight.

[0017] In the present invention, polylysine, lysozyme and / or lysostaphin, and lauroyl arginine ethyl ester hydrochloride are used in combination to achieve a good synergistic effect, thereby achieving a better disinfection effect with a lower dosage during disinfection.

[0018] In the present invention, polylysine is also referred to as ε-polylysine, and its molecular formula is C 180 H 362 N 60 O 31 , a light yellow powder, is commercially available.

[0019] In the bio-based disinfectant of the present invention, the concentration of polylysine is 0.01-5% by weight, for example, it can be 0.01% by weight, 0.02% by weight, 0.03% by weight, 0.04% by weight, 0.05% by weight, 0.06% by weight, 0.07% by weight, 0.08% by weight, 0.09% by weight, 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1% by weight, 2% by weight, 3% by weight, 4% by weight, or 5% by weight; preferably, it is 0.05-3% by weight, more preferably, it is 0.1-1% by weight, and even more preferably, it is 0.3-0.6% by weight.

[0020] Most preferably, in the bio-based disinfectant of the present invention, the concentration of polylysine is 0.45-0.55 weight %, for example, it can be 0.41 weight %, 0.42 weight %, 0.43 weight %, 0.44 weight %, 0.45 weight %, 0.46 weight %, 0.47 weight %, 0.48 weight %, 0.49 weight %, 0.5 weight %, 0.51 weight %, 0.52 weight %, 0.53 weight %, 0.54 weight %, 0.55 weight %, 0.56 weight %, 0.57 weight %, 0.58 weight %, 0.59 weight %, or 0.6 weight %.

[0021] In the present invention, the term "lysozyme" refers to an enzyme that can hydrolyze mucopolysaccharides in bacteria, also known as muramidase and N-acetyl muramid polysaccharide hydrolase, which can be obtained commercially.

[0022] In the present invention, the term "lysostaphin" refers to an enzyme that can hydrolyze the glycine peptide bond in the peptidoglycan of bacterial cell walls to kill the bacteria, and can be commercially available.

[0023] According to the present invention, in the bio-based disinfectant of the present invention, the concentration of lysozyme and / or lysostaphin is 0.01-5% by weight, for example, it can be 0.01% by weight, 0.02% by weight, 0.03% by weight, 0.04% by weight, 0.05% by weight, 0.06% by weight, 0.07% by weight, 0.08% by weight, 0.09% by weight, 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1% by weight, 2% by weight, 3% by weight, 4% by weight, 5% by weight; preferably, it is 0.02-2% by weight, more preferably, it is 0.03-0.5% by weight, and further preferably, it is 0.04-0.15% by weight.

[0024] Most preferably, in the bio-based disinfectant of the present invention, the concentration of lysozyme and / or lysostaphin is 0.05-0.1 wt%, for example, 0.05 wt%, 0.055 wt%, 0.06 wt%, 0.065 wt%, 0.07 wt%, 0.075 wt%, 0.08 wt%, 0.085 wt%, 0.09 wt%, 0.095 wt%, 0.1 wt%.

[0025] In the present invention, when the bio-based disinfectant contains both lysozyme and lysostaphin, the above content refers to the total content of the two; when the bio-based disinfectant contains one of lysozyme and lysostaphin, the above content refers to the content of one of them.

[0026] According to the present invention, when the bio-based disinfectant contains both lysozyme and lysostaphin, the dosage ratio of the two can be selected within a wide range. However, in order to improve the bactericidal effect of the bio-based disinfectant, preferably, the mass ratio of lysozyme to lysostaphin is 1:1-3, more preferably 1:1.5-2.5, for example, it can be 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5.

[0027] In the present invention, the molecular formula of lauroyl arginine ethyl ester hydrochloride is C 20 H 41 ClN4O3 is commercially available.

[0028] According to the present invention, in the bio-based disinfectant of the present invention, the concentration of lauroyl arginine ethyl ester hydrochloride is 0.01-5% by weight, for example, it can be 0.01% by weight, 0.02% by weight, 0.03% by weight, 0.04% by weight, 0.05% by weight, 0.06% by weight, 0.07% by weight, 0.08% by weight, 0.09% by weight, 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1% by weight, 2% by weight, 3% by weight, 4% by weight, 5% by weight; preferably, it is 0.02-1.5% by weight, more preferably, it is 0.02-0.5% by weight, and further preferably, it is 0.02-0.2% by weight.

[0029] Most preferably, in the bio-based disinfectant of the present invention, the concentration of lauroyl arginine ethyl ester hydrochloride is 0.04-0.15 weight %, for example, it can be 0.04 weight %, 0.05 weight %, 0.06 weight %, 0.07 weight %, 0.08 weight %, 0.09 weight %, 0.1 weight %, 0.11 weight %, 0.12 weight %, 0.13 weight %, 0.14 weight %, 0.15 weight %.

[0030] According to the present invention, in the bio-based disinfectant of the present invention, the concentration of glycerol is 0.01-10 weight%, for example, it can be 0.01 weight%, 0.02 weight%, 0.03 weight%, 0.04 weight%, 0.05 weight%, 0.06 weight%, 0.07 weight%, 0.08 weight%, 0.09 weight%, 0.1 weight%, 0.2 weight%, 0.3 weight%, 0.4 weight%, 0.5 weight%, 0.6 weight%, 0.7 weight%, 0.8 weight%, 0.9 weight%, 1 weight%, 2 weight%, 3 weight%, 4 weight%, 5 weight%, 6 weight%, 7 weight%, 8 weight%, 9 weight%, 10 weight%; preferably, it is 0.1-2 weight%, more preferably, it is 0.05-1 weight%, and further preferably, it is 0.2-0.8 weight%.

[0031] Most preferably, in the bio-based disinfectant of the present invention, the concentration of glycerol is 0.4-0.6 weight %, for example, it can be 0.4 weight %, 0.42 weight %, 0.44 weight %, 0.46 weight %, 0.48 weight %, 0.5 weight %, 0.52 weight %, 0.54 weight %, 0.56 weight %, 0.58 weight %, or 0.6 weight %.

[0032] According to a preferred embodiment of the present invention, the bio-based disinfectant contains polylysine, lysozyme, lauroyl arginine ethyl ester hydrochloride and glycerol, and more preferably consists of polylysine, lysozyme, lauroyl arginine ethyl ester hydrochloride and glycerol; wherein, in the bio-based disinfectant, the concentration of polylysine is 0.45-0.55% by weight, the concentration of lysozyme is 0.05-0.1% by weight, the concentration of lauroyl arginine ethyl ester hydrochloride is 0.04-0.15% by weight, and the concentration of glycerol is 0.4-0.6% by weight. Under this preferred embodiment, the synergistic effect of polylysine, lysozyme and lauroyl arginine ethyl ester hydrochloride can be further improved.

[0033] According to the present invention, the bio-based disinfectant may further contain at least one of nisin, glycine, betaine and derivatives thereof.

[0034] In the present invention, Nisin, also known as nisin or transliterated as Nisin, is a polypeptide substance produced by lactococcus, composed of 34 amino acid residues, with a molecular weight of about 3500Da and a molecular formula of C 143 H 230 N 42 O 37 S7, commercially available.

[0035] Preferably, in the bio-based disinfectant, the concentration of nisin is 0.01-0.5 weight%, for example, it can be 0.01 weight%, 0.02 weight%, 0.03 weight%, 0.04 weight%, 0.05 weight%, 0.06 weight%, 0.07 weight%, 0.08 weight%, 0.09 weight%, 0.1 weight%, 0.2 weight%, 0.3 weight%, 0.4 weight%, 0.5 weight%, more preferably 0.02-0.2 weight%.

[0036] Preferably, in the bio-based disinfectant, the concentration of glycine is 0.01-0.5 weight%, for example, it can be 0.01 weight%, 0.02 weight%, 0.03 weight%, 0.04 weight%, 0.05 weight%, 0.06 weight%, 0.07 weight%, 0.08 weight%, 0.09 weight%, 0.1 weight%, 0.2 weight%, 0.3 weight%, 0.4 weight%, 0.5 weight%, more preferably 0.02-0.3 weight%.

[0037] In the present invention, the term "betaine" is an alkaloid with strong hygroscopic properties, so it is often treated with an anti-caking agent in the production process. Its molecular structure and application effect are not significantly different from those of natural betaine. It is a chemically synthesized natural equivalent with a molecular formula of C5H 11 NO2 is commercially available.

[0038] The methyl group in betaine can be replaced by other substituents to obtain many alkyl aromatic betaines or alkyl amide propyl betaines, etc.; the carbon bridge connecting the positive and negative charge centers can be extended to obtain propyl betaine, butyl betaine, etc.; the acetate group can also be replaced by other groups to obtain sulfoethyl, sulfopropyl betaine or sulfate (ethyl) betaine, phosphate (ethyl) betaine and other amphoteric surfactants.

[0039] According to a preferred embodiment of the present invention, the betaine derivative is an alkyl betaine with the molecular formula RN+(CH3)2CH2COO-, wherein the carbon number of R is 12-18. More preferably, the betaine derivative is dodecyl dimethyl betaine (BS-12), which can be obtained commercially.

[0040] Preferably, in the bio-based disinfectant, the concentration of betaine and / or its derivatives is 0.01-2% by weight, for example, it can be 0.01% by weight, 0.02% by weight, 0.03% by weight, 0.04% by weight, 0.05% by weight, 0.06% by weight, 0.07% by weight, 0.08% by weight, 0.09% by weight, 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1% by weight, 2% by weight, preferably 0.05-1% by weight, further preferably 0.1-0.15, for example, it can be 0.1% by weight, 0.11% by weight, 0.12% by weight, 0.13% by weight, 0.14% by weight, 0.15% by weight.

[0041] According to a preferred embodiment of the present invention, the bio-based disinfectant contains polylysine, lysostaphin, lauroyl arginine ethyl ester hydrochloride, glycerol and betaine BS12, or consists of polylysine, lysostaphin, lauroyl arginine ethyl ester hydrochloride, glycerol and betaine BS12.

[0042] According to a preferred embodiment of the present invention, the bio-based disinfectant further contains an organic acid, for example, at least one of acetic acid, lactic acid and citric acid. In the case of containing an organic acid, the disinfection effect can be further improved.

[0043] The concentration of acetic acid may be 0.001-0.05 wt %, preferably 0.005-0.01 wt %.

[0044] The concentration of lactic acid may be 0.001-0.8 wt %, preferably 0.1-0.5 wt %.

[0045] The concentration of citric acid may be 0.001-0.8 wt %, preferably 0.1-0.5 wt %.

[0046] According to the present invention, the pH of the bio-based disinfectant is preferably 2.5-7, for example, it can be 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, preferably 3-4.

[0047] According to the present invention, the bio-based disinfectant can be prepared according to conventional methods, for example, by directly mixing the components in water or a buffer solution according to predetermined amounts.

[0048] Wherein, the water can be pure water or deionized water.

[0049] Wherein, the buffer may be at least one of an acetic acid buffer, a lactic acid buffer and a citric acid buffer, and the concentration of each acid in the buffer is as described above.

[0050] According to a preferred embodiment of the present invention, the organic acid is prepared according to the concentration and adjusted to the corresponding pH (for example, using sodium hydroxide), and the corresponding components are mixed after the buffer solution is prepared.

[0051] In a second aspect, the present invention provides the use of the above-mentioned bio-based disinfectant in in vitro bacteriostasis.

[0052] According to the present invention, the bacteria can be bacteria and fungi, for example, can be Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and the like.

[0053] According to the present invention, the outside of the body can be hands, skin, the surface of an object, etc.

[0054] Example

[0055] The following examples will further illustrate the present invention, but are not intended to limit the present invention.

[0056] In the following examples and comparative examples:

[0057] Polylysine was purchased from Zhejiang Yinuo Biotechnology Co., Ltd., food grade polylysine;

[0058] Lysozyme was purchased from Zhejiang Yinuo Biotechnology Co., Ltd., food grade lysozyme;

[0059] Nisin was purchased from Zhejiang Yinuo Biotechnology Co., Ltd., food grade nisin;

[0060] Lysostaphin is a Lysostaphin protein (UniprotKB-P10547) from Staphylococcus simulans expressed in Escherichia coli. The expression method is conventional in the field of genetic engineering, such as constructing in a pET28a vector and transferring into a BL21 (DE3) expression strain, inducing expression, and then purifying the protein.

[0061] Examples and Comparative Examples

[0062] The bio-based disinfectant is prepared according to the components and dosages of each embodiment in Table 1. Specifically, at room temperature, the organic acid is prepared according to the concentration, and sodium hydroxide is used to adjust the pH to the corresponding pH. After the buffer solution is prepared, the corresponding components are mixed to obtain the disinfectant.

[0063] Table 1

[0064]

[0065]

[0066] Note: The units of each component in the table are weight %.

[0067] Example 7

[0068] This example is used to illustrate the disinfectant provided by the present invention.

[0069] The disinfectant was prepared according to the method of Example 1, except that deionized water was used to replace the buffer solution.

[0070] Comparative Example 4

[0071] This comparative example is used to illustrate the preparation of the reference disinfectant

[0072] The disinfectant was prepared according to the method of Example 1, except that lauroyl arginine ethyl ester hydrochloride was replaced by an equal amount of lysozyme.

[0073] Comparative Example 5

[0074] This comparative example is used to illustrate the preparation of the reference disinfectant

[0075] The disinfectant was prepared according to the method of Example 1, except that lysozyme was replaced by an equal amount of lauroyl arginine ethyl ester hydrochloride.

[0076] Test Example 1

[0077] 1) Sterilization test:

[0078] According to the suspension quantitative sterilization test operating procedures of the quantitative killing experiment in Part 2.1.1 of the "Technical Specifications for Disinfection" (Ministry of Health 2002 edition), quantitative killing experiments of bacteria and fungi and virus inactivation experiments were carried out. The results of the quantitative killing experiments are shown in column a of Table 2; Example 1 was selected for the virus inactivation experiment, and the results are shown in Table 3.

[0079] 2) Stability test:

[0080] The stability test of the disinfectant was carried out according to the stability evaluation method of the "Technical Specifications for Disinfection". The disinfectant was placed at 37°C for 90 days, and samples were taken for sterilization experiments. The results are shown in column b of Table 2.

[0081] Table 2

[0082]

[0083]

[0084] Note: Large intestine: Escherichia coli;

[0085] Staphylococcus aureus: Staphylococcus aureus

[0086] Pseudomonas aeruginosa: Pseudomonas aeruginosa;

[0087] Candida albicans: Candida albicans

[0088] a is the killing test directly after preparation, and b is the bactericidal effect of the stability evaluation test. Because the killing rate in some experimental conditions was 100% and the live bacteria count was 0, the accurate value could not be calculated, so the calculated killing logarithm value exceeding 6 was uniformly marked as >6.

[0089] 3) Virus elimination test

[0090] The disinfectant prepared in Example 1 of the present invention was brought into contact with the viruses listed in Table 3 at room temperature for 10 minutes. The disinfection results are shown in Table 3.

[0091] Table 3

[0092] Experimental virus Host cells Action time Average log inactivation value Average inactivation rate Influenza A virus H1N1 MDCK cell line 10min >4 99.99％ Influenza A virus H3N2 MDCK cell line 10min >4 99.99％ Enterovirus-71 VERO cell line 10min 1.22 94.12％ Murine norovirus GV Raw264.7 10min 0.89 88.18％

[0093] As can be seen from Table 2, the disinfectant provided by the present invention can achieve a killing logarithm value of more than 5 for Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans in 1 minute, that is, it kills more than 99.999% of the bacteria, and the bactericidal effect is significant, which complies with the "Technical Specifications for Disinfection". In addition, the combination of polylysine, lysozyme, and lauroyl arginine ethyl ester hydrochloride can achieve a good synergistic effect, so that a better disinfection effect can be obtained with a lower dosage in disinfection, and at the same time, the antiviral effect can be further improved in an acidic buffer.

[0094] According to the results shown in Table 3, taking Example 1 as an example, the disinfectant provided by the present invention also has an inactivation logarithm value of more than 4 for lipophilic viruses such as influenza virus, that is, more than 99.99% of the viruses are inactivated.

[0095] Therefore, the disinfectant of the present invention has a broad-spectrum, high-efficiency bactericidal effect, and the disinfection effect does not change significantly after being placed at 37°C for 90 days. According to the "Technical Specifications for Disinfection", it can be considered that the shelf life at room temperature reaches 2 years.

[0096] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0097] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0098] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A bio-based disinfectant, characterized in that: The bio-based disinfectant consists of polylysine, lysozyme and / or lysostaphin, lauroyl arginine ethyl ester hydrochloride, glycerol and buffer solution; wherein, in the bio-based disinfectant, the concentration of polylysine is 0.3-0.5% by weight, the concentration of lysozyme and / or lysostaphin is 0.02-0.1% by weight, the concentration of lauroyl arginine ethyl ester hydrochloride is 0.02-0.1% by weight, the concentration of glycerol is 0.4-0.6% by weight, and the remainder is buffer solution; the pH value of the bio-based disinfectant is 3-5.

2. The bio-based disinfectant according to claim 1, wherein: The bio-based disinfectant is additionally added with at least one of nisin, glycine, betaine and derivatives thereof.

3. The bio-based disinfectant according to claim 2, wherein: In the bio-based disinfectant, the concentration of nisin is 0.01-0.5% by weight; the concentration of glycine is 0.01-0.1% by weight; and the concentration of betaine and / or its derivatives is 0.01-0.1% by weight.

4. The bio-based disinfectant according to claim 3, wherein: In the bio-based disinfectant, the concentration of nisin is 0.02-0.2% by weight.

5. The bio-based disinfectant according to claim 2, wherein: The betaine derivative is an alkyl betaine.

6. The bio-based disinfectant according to claim 5, wherein: The betaine derivative is betaine BS12.

7. The bio-based disinfectant according to any one of claims 2 to 6, wherein: The bio-based disinfectant is additionally supplemented with an organic acid.

8. The bio-based disinfectant according to claim 7, wherein: The organic acid is selected from at least one of acetic acid, lactic acid and citric acid.

9. Use of the bio-based disinfectant according to any one of claims 1 to 8 in in vitro bacteriostasis or in vitro virus inhibition; in, The bacteria include bacteria and fungi; the bacteria are selected from at least one of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa; the fungi are Candida albicans; The virus is selected from at least one of influenza A virus H1N1, influenza A virus H3N2, enterovirus-71 and murine norovirus GV.

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