DISTINCTION OF INFECTIOUS VIRUS BASED ON MOLECULAR BIOMARKER AND NEUTRALIZATION OF VIRUS CAUSING FOOD POISONING

Abstract

Discloses are a method for detecting Norovirus using a Norovirus animal model, a method for screening an antivial agent against Norovirus, and a composition for neutralizing the infection with an enteric virus, containing concanavalin A as an active ingredient, so that the method for detecting Norovirus can allow the distinction between infectious Norovirus and non-infectious Norovirus, and the composition can neutralize a virus causing food poisoning.

Claims

1. A method for detecting Norovirus using a Norovirus animal model, the method comprising: (a) administering Norovirus to zebrafish (Danio rerio); and (b) detecting the expression level of a gene or protein selected from the group consisting of heat shock protein 90α (HSP90α), heat shock cognate 71 (HSC71), and transferrin receptor-1b (Tfr-1b), from the zebrafish in step (a).

2. The method of claim 1, wherein the Norovirus is infectious Norovirus or non-infectious Norovirus.

3. The method of claim 1, wherein step (b) is performed by a gene amplification reaction or an antigen-antibody reaction.

4. The method of claim 1, wherein step (b) is performed on one of days 2 to 6 after the infection in step (a).

5. A method for screening an antiviral agent against Norovirus using a Norovirus animal model, the method comprising: (a) administering Norovirus to zebrafish (Danio rerio); (b) administering an antiviral agent candidate against Norovirus to the zebrafish in step (a); and (c) detecting the expression level of a gene or protein selected from the group consisting of heat shock protein 90α (HSP90α), heat shock cognate 71 (HSC71), and transferrin receptor-1b (Tfr-1b), from the zebrafish in step (a), to determine efficacy of the antiviral agent candidate.

6. The method of claim 5, wherein the Norovirus is infectious Norovirus.

7. The method of claim 5, wherein step (c) is performed by a gene amplification reaction or an antigen-antibody reaction.

8. A composition for neutralizing the infection with an enteric virus, the composition containing concanavalin A (Con A) as an active ingredient.

9. The composition of claim 8, wherein the enteric virus is Norovirus, Hepatitis A virus (HAV), or Rotavirus.

10. The composition of claim 8, wherein the concanavalin A neutralizes Norovirus to 70-100%.

11. The composition of claim 8, wherein the concanavalin A inhibits the expression of transferrin receptor-1b (Tfr-1b) of zebrafish.

12. The composition of claim 8, wherein the concanavalin A has a K.sub.D value of 3.75×10.sup.−7 M with respect to Norovirus.

13. The composition of claim 8, wherein the concanavalin A neutralizes Hepatitis A virus to 80-100%.

14. The composition of claim 8, wherein the concanavalin A binds between viral protein 1 (VP1) domain and VP2 domain of Hepatitis A virus.

15. The composition of claim 8, wherein the concanavalin A has a K.sub.D value of 1.28×10.sup.−6 M with respect to Hepatitis A virus.

16. The composition of claim 8, wherein the concanavalin A neutralizes Rotavirus to 60-90%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The above and other objects, features, and advantages of the present invention will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0077] FIG. 1 shows results of confirming distinguishability between infectious Norovirus and non-infectious Norovirus through a gene amplification reaction;

[0078] FIG. 2 is a diagram showing a protein change using ingenuity pathway analysis (IPA) on the basis of proteins analyzed through proteomic analysis;

[0079] FIG. 3 shows results of confirming the expression change of heat shock protein 90α (HSP90α) and heat shock cognate 71 (HSC71) in zebrafish (Danio rerio) infected with infectious Norovirus, through western blot;

[0080] FIG. 4 shows results of confirming the possibility of HSP90α, HSC71, and Tfr-1 b as biomarkers by treatment with HSP090α inhibitor (17AAG) and HSC71 inhibitor (KNK437);

[0081] FIG. 5 shows results of confirming the expression changes of HSP90α and HSC71 in zebrafish infected with non-infectious Norovirus, through western blot;

[0082] FIG. 6 shows results of confirming the expression changes of HSP90α, HSC71 and Tfr-1b in zebrafish administered with infectious viruses and Con A;

[0083] FIG. 7 shows results of confirming binding affinity by treating the Hepatitis A virus (HAV)-immobilized amine reactive 2nd generation biosensor (ARG2) chip with Con A;

[0084] FIG. 8 shows results of confirming the suppression of Con A on HAV infection in Frhk-4 cells infected with HAV;

[0085] FIG. 9 shows the binding structure of Con A and HAV;

[0086] FIG. 10 shows results of confirming the binding between Con A and Norovirus through RT-PCR;

[0087] FIG. 11 shows results of confirming the binding affinity by treating Norovirus-immobilized ARG2 chip with Con A;

[0088] FIG. 12 shows results of confirming binding affinity of the antibody against Norovirus and Con A;

[0089] FIG. 13 shows results of comparing an epitope of the antibody against Norovirus and an epitope of Con A;

[0090] FIG. 14 shows results of confirming the binding of con A and Rotavirus; and

[0091] FIG. 15 shows results of Rotavirus neutralization by Con A.

DETAILED DESCRIPTION

[0092] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1: Confirmation of Norovirus Through PCR

[0093] Human infectious Norovirus (genotype GII-4) was heated at temperatures of 50 to 90° C. for 10 minutes, and then RNA was obtained. RNA isolation was conducted by using TRIzol (Invitrogen) according to the general RNA preparation protocol (Chomczynski P, Mackey K. Short technical report. Modification of the TRIzol reagent procedure for isolation of RNA from Polysaccharide- and proteoglycan-rich sources. Biotechniques 19(6): 942-945. 1995). 700 μl of Trizol was placed in 1.5 ml tubes, followed by light vortexing, and then 200 μl of chloroform was dispensed in each tube, followed by vortexing, and then each tube was allowed to stand for 5 minutes. After centrifugation at 12,000×g for 15 minutes at 4° C., the supernatant was transferred into new 1.5 ml tubes, and an equal volume of isopropanol was dispensed for each tube, followed by vortexing, and then each tube was allowed to stand for 10 minutes. After centrifugation at 12,000×g for 10 minutes at 4° C., the supernatant was removed, and 500 μl of 75% ethanol was dispensed, followed by light vortexing. After centrifugation at 7,500×g for 5 minutes at 4° C., the supernatant was removed, and RNA was obtained by using 10 μl of RNase-free water. To 10 μl of the obtained RNA, 1 μl of random primer, 4 μl of 5×M_MLV RTase buffer, 2 μl of 5×DTT, 2 μl of dNTP, 0.5 μl of RNase inhibitor, and 1 μl of M_MLV RTase were dispensed, followed by reaction at 65° C. for 10 minutes, at 37° C. for 1 hour, and at 72° C. for 5 minutes. RT-PCR was conducted by reaction of PCR premix (Bioneer), 16 μl of DNase-free water, and 2 μl of cDNA, which was synthesized by adding forward and reverse primers of 1 μl for each. PCR conditions were: 95° C. for 5 minutes, 40 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 45 seconds, and 72° C. for 5 minutes for last extension.

[0094] As can be confirmed in FIG. 1, the electrophoresis results obtained by conducting RT-PCR of Norovirus heated at different temperatures of 50-90° C. also confirmed gene products in the heated non-infectious Norovirus. Therefore, it was confirmed that the infectious Norovirus and non-infectious Norovirus could not be distinguished by gene amplification.

Example 2: Selection of Biomarker Candidate of Infectious Norovirus

[0095] Zebrafish (Danio rerio) were purchased from a local aquarium, and five zebrafish were placed in each 1 L water bath using a reverse osmosis (RO) system water for an acclimation period of time. 400 mg of Tricaine (ethyl 3-aminobenzoate methanesulfonate salt, Sigma) reagent used in the fish anesthesia, and 2.1 ml of 1 M Tris were dissolved in 97.9 ml of distilled water, followed by adjustment to pH 7, thereby preparing an anesthesia solution. 4.2 ml of the anesthesia solution was dissolved in 100 ml of RO system water, and the fish were anesthetized. The previously prepared sterile phosphate buffer saline (PBS) and Norovirus (genotype G II-4) diluted in sterile PBS of 20 μl for each (titer 1×10.sup.6 copy number) were intraperitoneally administered to the zebrafish. A lysis buffer (10 mM tris, 2 mM EDTA, 150 mM NaCl, 10% Triton X-100 10%, 10% NP40) was prepared, and a proteinase inhibitor and a phosphate were added to the lysis buffer at concentration ratios of 1:200 and 1:100, respectively. The mixture was dispensed into each 1.5 ml tube, and the zebrafish were rapidly frozen in liquefied nitrogen and then placed in the prepared 1.5 ml tubes. The zebrafish were cut into small tissues using sterile dissection scissors, and then homogenized using a homogenizer. These works were conducted in ice. The homogenized zebra tissues were subjected to vortexing once for every five minutes, followed by reaction in ice for 20 minutes. Thereafter, the centrifugation was conducted at 15,000 rpm for 13 minutes at 4° C., thereby obtaining supernatant. The zebrafish were sampled by date, and proteins were extracted. The protein change was confirmed by Coomassie Brilliant Blue. The protein change was confirmed using proteomic analysis in the zebrafish on day 3 on which there was the most significant difference.

[0096] As can be confirmed in FIG. 2 showing the protein change using the ingenuity pathway analysis (IPA) on the basis of the analyzed proteins, several important factors in the proteins were confirmed through protein analysis. Of these, three kinds of biomarkers, heat shock protein 90α (HSP90α), heat shock cognate 71 (HSC71), and transferrin receptor (Tfr) were confirmed to be overexpressed by infectious Norovirus.

Example 3: Confirmation on Biomarker Candidates of Infectious Norovirus

[0097] Biomarkers were specified according to the results of FIG. 2 showing the protein change analyzed by obtaining proteins of the zebrafish infected with human infectious Norovirus. 30 fig of each zebrafish protein was loaded on SDS-gel, and then the protein was transferred to the PVDF membrane (Immobilon-P, Millipore). Each membrane was blocked with TBST (1% Tween20 TBS) mixed with 5% skim milk for 1 hour. Each primary antibody of HSC71 (anti-HSC71 antibody, rabbit, Cell Signaling), HSP90α (anti-HSP90α antibody, rabbit, Anaspec), and Tfr-1b (anti-Tfr-1b antibody, rabbit, Anaspec) was dispensed at a concentration of 1:1000 in TBST mixed with 5% skim milk, followed by reaction overnight in a refrigerator. After washing three times with TBST for 10 minutes, secondary antibody (Polyclonal Goat, anti-rabbit immunoglobulins HRP, Dako) was dispensed at a concentration 1:2000 in TBST mixed with 5% skim milk, followed by reaction at room temperature for 2 hours. After washing five times with TBST for 5 minutes, the protein change was confirmed through a reaction with a western blot substrate (Luminata Crescendo, Millipore).

[0098] FIGS. 3a and 3b show the results of confirming the expression of biomarkers, HSP90α and HSC71, by date through western blot. On day 3 of the infection, the protein expression between Mock (sterile PBS inoculation group) and Norovirus infection group were greatly differentiated, and on day 4 of the infection, the expression level was significantly reduced in both of the Mock group and the Norovirus infection group, and thus the effect could not be confirmed. Therefore, it was investigated the possibility as biomarkers of HSP90α and HSC71 by using inhibitors of HSP90α and HSC71, which are considered to be biomarkers, in the zebrafish proteins on day 3 of the Norovirus infection. FIGS. 4a and 4b show the results of reducing the protein expression, which was increased due to the Norovirus infection, through HSP90α inhibitor (17AAG, Sigma) and HSC71 inhibitor (KNK437, Sigma), and thus confirmed the functions as biomarkers. In addition, when the Tfr-1 b (transferrin receptor 1 b and Anaspec) antibody was used, the proteins were expressed in the Norovirus infection group, and when using together with HSP90α inhibitor and HSC71 inhibitor, the protein expression was reduced, but the protein expression was still confirmed (FIGS. 4c and 4d).

Example 4: Confirmation on Norovirus Infection in Zebrafish

[0099] Norovirus was heated at different temperatures for 10 minutes, and thus zebrafish were infected with non-infectious state Norovirus. On day 3 of the infection, the proteins were extracted from the zebrafish, followed by western blot, thereby confirming the protein change. The expression levels of HSP90α and HSC71 were increased in the Norovirus heated at 50° C. and wild type (WT) Norovirus, but similar levels of proteins were expressed in the Norovirus heated at 70° C. and 90° C. and in the zebrafish inoculated with only sterile PBS. Therefore, HSP90α and HSC71 could be confirmed as biomarkers that can distinguish between human infectious and non-infectious Norovirus. In addition, the expression of Tfr-1 b occurred in only groups infected with Norovirus heated at 50° C. and WT Norovirus, and thus HSP90α and HSC71 could be confirmed as more accurate biomarkers.

Example 5: Norovirus Neutralization of Con A

[0100] The previously prepared sterile phosphate buffer saline (PBS) and Norovirus diluted in sterile PBS of 20 μl for each case were intraperitoneally administered to the zebrafish anesthetized using an anesthetic solution. In addition, 100 μg/ml Con A (concanavalin A, sigma) was allowed to react with human infectious Norovirus (genotype G II-4, titer 1×10.sup.6 copy number, reaction in a rotator at room temperature for 1 hour), and 20 μl of the resultant material was intraperitoneally administered to zebrafish. Zebrafish proteins were obtained 3 days after the infection, followed by western blot.

[0101] The expression levels of HSP90α and HSC71 were shown to still increase by about 2-fold in the group infected with only Norovirus (FIG. 6a). However, the proteins of the zebrafish infected with Norovirus plus Con A were expressed at similar levels compared with the group infected with only sterile PBS. In addition, as for Tfr-1b antibody, the zebrafish infected with Norovirus plus Con A showed a different tendency as compared with the treatment with inhibitors.

[0102] That is, the transferrin receptor was not expressed (FIG. 6b). This is thought to suppress the Norovirus infection per se.

Example 6: HAV and Con A Binding

[0103] The infection and culturing of Hepatitis A virus (HAV) occur in FRhk-4 cells (Rhesus monkey kidney, ATCC), and thus the FRhk-4 cells were utilized as a cell line capable of suppressing the infection mechanism. The FRhk-4 cells were cultured in a medium prepared by supplementing Dulbecco Modified Eagle Medium (DMEM, WELGENE) with 10% fetal bovine serum (FBS, WELGENE) and 1% penicillin streptomycin (Sigma). The FRhk-4 cells were dispensed in a 96-well plate at 8×10.sup.3 cells/well, and after 24 hours, when the the cells reached about 80-90% of confluence, the virus inoculation was carried out. HAV was added at 1×10.sup.5 unit/well, and an equal volume of HAV and 100 μg/ml Con A 100 were allowed to react each other, and the reaction material was dispensed to each well. RNA was obtained from the FRhk-4 cells treated with viruses by date, to investigate the copy number of HAV. RNA isolation was carried out by the method as in example 1.

[0104] The binding affinity according to the concentration of Con A was investigated after HAV was immobilized to the ARG2 chip, and it was verified that the higher the Con A concentration, the more the Con A bound to the HAV-immobilized chip (FIG. 7). In the HAV infection group, the binding affinity was not largely changed until day 5 of infection, but on day 6 and day 7 of the infection, the binding affinity was increased by a value of about 2 log and the copies were 3.3×10.sup.6 unit (FIGS. 8a and 8b). However, in the group infected with Con A plus HAV, the concentration of HAV was 2.7×10.sup.4 unit, which corresponded to a lower level than the inoculation concentration. In addition, from the images, the cytopathic effect (CPE) was observed in the group treated with HAV, but the CPE was not observed in the group treated with HAV plus Con A. It is thought that Con A obstructs the intercellular penetration of HAV to suppress replication of HAV.

[0105] Con A is bound between HAV VP1 (structure forming protein) domain and HAV VP2 domain, and the N-terminus of the HAV VP2 domain binds with two molecules of Con A to stabilize the structure binding (FIG. 9).

Example 7: Norovirus and Con A Binding

[0106] It was investigated through RT-PCR and BioLayer interferometry (BLI) assay whether human infectious Norovirus and Con A bind to each other. RT-PCR was carried out according to the concentration of Norovirus to obtain the Ct value for each concentration of Norovirus. Each concentration of Norovirus was mixed with 100 μl of Con A-bound sepharose 4B resin (25 mg Con A/ml; C7275, Sigma), followed by reaction for 10 minutes. After the reaction, the resin was subjected to centrifugation (2000 g, 3 minutes, 4° C.) to remove the supernatant, and then the resin was washed three times with the PBS solution, followed by RT-PCR.

[0107] For the BLI assay, a BLItz system (ForteBio Inc., CA) was employed, and an amine reactive biosensor (ARG2) chip and a protein A chip were used. After the Norovirus was immobilized to the ARG2 chip, the binding was investigated at Con A concentrations of 0-10 μM. The test was carried out as follows: initial reference value, distilled water, 30 seconds: 20 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 10 mM sulfo-N-hydroxysuccinimide (s-NHS), 240 seconds; loading: Norovirus dissolved in NaOAc (pH 4), 360 seconds; blocking: 1 M ethanolamine (pH 8.5), 240 seconds; reference value: 10 mM PBS (pH 7.4), 60 seconds; association: Con A dissolved in PBS, 180 seconds; dissociation: 10 mM PBS (pH 7.4) 240 seconds. Sensorgrams are shown in FIGS. 10 and 11 using data analysis software 7.1.0.36 for the reference value (60 seconds), association (180 seconds), and dissociation (240 seconds).

[0108] A competitive reaction test was carried out using the protein A chip in order to investigate whether the binding portions of the antibody against Norovirus and Con A with respect to Norovirus are the same as or different from each other. After the antibody was immobilized to the protein A chip, respective sensorgrams were investigated for samples of Con A, Norovirus, and Norovirus mixed with Con A (0.1 uM and 5 uM). The test on the protein A chip was carried out as follows: initial reference value: PBS, 30 seconds; loading: antibody dissolved in PBS, 120 seconds; reference value: 10 mM PBS (pH 7.4), 60 seconds; association: sample (FIG. 12) dissolved in PBS, 120 seconds; disassociation: 10 mM PBS (pH 7.4) 300 seconds. Sensorgrams are shown in FIG. 12 using data analysis software 7.1.0.36 for the baseline (60 seconds), association (120 seconds), and dissociation (300 seconds).

[0109] FIG. 10 shows the results of confirming the binding of Con A and Norovirus through RT-PCR. When Ct values for 1×10.sup.3 to 1×10.sup.8 copies/rat of Norovirus were compared with Ct values for Norovirus collected from Con A-bound sepharose 4B resin, the Ct value was reduced in a Norovirus concentration-dependent manner, and when Ct values of respective concentrations of Norovirus were compared with Ct values of Norovirus collected from Con A-bound sepharose 4B resin, the collection rate was confirmed to be 94.1% on average.

[0110] FIG. 11 shows the binding affinity according to the concentration of Con A after Norovirus was immobilized to the ARG2 chip, and it was verified that the higher the Con A concentration, the more the Norovirus bound to the Norovirus-immobilized chip. After the antibody and Con A were immobilized to ARG2 chips, respectively, the binding of Norovirus was investigated through the sensorgrams (FIG. 12). When sensorgrams of the antibody and Con A were compared with each other for the same concentration of Norovirus, it was verified that Con A bound to the Norovirus stronger than the antibody by 3-fold.

[0111] FIG. 13 shows the results through a competition test whether the binding portion between the antibody and Norovirus is the same as or different from the binding portion between Con A and Norovirus. After the Norovirus antibody was immobilized to the protein A chip, sensorgrams for Con A, Norovirus, and Norovirus plus Con A (0.1 μM and 5 μM) were compared with each other. As a result, it was verified that the Norovirus bound to the antibody-immobilized chip by 0.23 nm in the Norovirus sample as a positive control, and Con A did not bind to the antibody-immobilized chip in the Con A sample as a negative chip. Through these results, the sensorgrams for the binding with antibody in the Norovirus concentrations used in the test were obtained, and it could be verified that there was no binding between Con A and the antibody. Next, it was verified that the Norovirus bound to the antibody-immobilized chip by 0.2 nm in the respective samples of Norovirus and con A (0.1 μM and 5 μM), which were almost similar to sensorgrams in the positive control. In addition, the fact that the Norovirus was bound to the antibody-immobilized chip regardless of the binding of Con A and the Norovirus seems that the binding sites of Con A and the antibody with respect to the Norovirus were different from each other.

Example 8: Rotavirus and Con A Binding

[0112] In order to investigate whether Rotavirus among the poisoning viruses binds to Con A, a viral detection probe was manufactured by linking biotinylated Con A to streptavidin connected to magnetic beads as a magnetic body material. For the biotinylation of Con A, first, a labeling reaction was carried out by transferring 100 μl of Con A (1 mg/ml) into a reaction tube (component C). 1/10 of 1 M sodium bicarbonate was added, and mixed by pipetting, and then 1 μl of biotin-XX SSE was added and mixed, followed by reaction at room temperature for 15 minutes. Next, for the separation of Con A, the gel resin was allowed to fill an upper chamber, and 800 μl of resin was allowed fill a column, followed by centrifugation at 16,000 G for 15 seconds. The filling of resin was carried out using a centrifuge. After the resin was washed with PBS solution, the biotin-bound con A reaction material was placed in the spin column filled with the resin, followed by centrifugation at 16,000 G for 1 minute, thereby obtaining a reaction material. The thus reacted biotinylated Con A was linked to streptavidin-bound magnetic beads to manufacture a viral detection probe, and the manufacturing procedure thereof are as follows. Rotavirus was placed in the tube with the manufactured viral detection probe, followed by reaction at room temperature for 10 minutes. After the immunological reaction, the antigen-lectin binding portion was attached to magnets of the Con A-bound magnetic beads. The Con A-bound magnetic beads were washed three times with 200 μl of PBS to remove non-specifically bound impurities, and then floated in 100 μl of PBS, and the suspension was transferred to a new 1.5 ml tube to remove the supernatant. In order to separate streptavidin-bound antigen-Con A conjugate in the new tube, the conjugate was eluted with an eluent (50 mM glycine, pH 2.8), and neutralized to pH 7.5 with 100 mM Tris. In addition, RT-PCR was carried out for the pure separation of Rotavirus, and the virus was identified.

[0113] In order to investigate whether Rotavirus is detectable by immunoprecipitation using magnetic beads, RT-PCR of the eluted product was carried out to detect viruses. As Rotavirus primers used in the polymerase chain reaction (PCR) for the use of viral identification, RoV_VP4_F (5′-ATT TCG GAC CAT TTA TAA CC-3′) and RoV_VP4_R (5′-TGG CTT CGC CAT TTT ATA GAC A-3′) were used. The size of the PCR products amplified through the primers is 877 bp.

[0114] The RoV PCR conditions for viral identification were as follows. RoV PCR conditions: 35 cycles of denaturation at 94° C. for 30 seconds, annealing at 55° C. for 30 seconds, and elongation at 72° C. for 30 seconds, and the final elongation at 72° C. for 7 minutes. After RT-PCR, the PCR products were subjected to electrophoresis to monitor bands.

[0115] FIG. 14 shows the results of electrophoresis after RT-PCR in order to investigate the binding of Con A and Rotavirus using the Con A-bound magnetic beads. Column 1 shows RT-PCR results of the solution obtained by performing elution on the magnetic beads, which were collected after the reaction with Rotavirus, using an eluent, and column 2 shows RT-PCR results of the Rotavirus stock solution. Column 3 shows a negative control for confirming the success or not of PCR. As a result, the band corresponding to Rotavirus amplification product of 877 bp was confirmed at the same position as in column 2 as a positive control. These results confirmed that Con A was bound to Rotavirus.

Example 9: Rotavirus and Con A Binding

[0116] The infection and culturing of Rotavirus occur in MA-104 cells (Green monkey kidney, ATCC), and thus the MA-104 cells were utilized as a cell line capable of suppressing the infection mechanism. The MA-104 cells were cultured in a medium prepared by supplementing Dulbecco Modified Eagle Medium (DMEM, WELGENE) with 10% fetal bovine serum (FBS, WELGENE) and 1% penicillin streptomycin (Sigma). The MA-104 cells were dispensed in a 96-well plate at 1×10.sup.4 cells/well, and after 24 hours, when the the cells reached about 80-90% of confluence, the virus inoculation was carried out. Rotavirus was added at 1×10.sup.5 unit/well, and an equal volume of Rotavirus and 100 μg/ml Con A 100 were allowed to react each other at room temperature for 1 hour, and the reaction material was dispensed to each well. RNA was obtained from the MA-104 cells treated with viruses by date, to investigate the copy number of Rotavirus. RNA isolation was carried out by the method as in example 1.

[0117] In the Rotavirus infection group, the binding affinity was not largely changed until day 1 of infection, but on day 3 and day 5 of the infection, the binding affinity was increased by a value of about 1.5 log and the copies were 2.7×10.sup.5 unit. However, in the group infected with Con A plus Rotavirus, the concentration of Rotavirus was 6.4×10.sup.4 unit, which was reduced by a value of 1 log compared with the Rotavirus infection group. These results showed similar tendencies compared with the test using HAV plus Con A, indicating that the Con A neutralized Rotavirus to suppress the Rotavirus infection (FIG. 15).

[0118] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.