TWI531651B - Novel enterovirus 71 and application thereof - Google Patents

Description

Enterovirus 71 novel virus strain and application thereof

The present invention relates to a novel enterovirus 71 strain, an immunological composition containing the virus, a test kit and use.

Enterovirus is Enterovirus in the family Picaraviridae. Enterovirus is a group of positive single-stranded RNA (ssRNA) viruses of only 20-30 nm in size. It has an icosahedral structure and does not have an envelope and only contains a capsid. Enterovirus has a total of type 67 serotypes, including a total of type 23 serotypes of Coxsackievirus group A, a total of type 6 serotypes of Coxsackievirus group B, and Echovirus. Type 31 serotype, poliovirus (Poliovirus) type 3, and enterovirus type 68 to 71.

Enterovirus 71 was first isolated in California in 1969, and has since been reported in various parts of the world, including Australia, Japan, Hong Kong, Malaysia, Sweden, Bulgaria, Hungary, and France. Taiwan has experienced many different serotypes of enterovirus pandemics since 1994. Among them, 405 cases of severe cases and 78 deaths caused by enterovirus 71 in 1998 were the most serious. In the same year, about 1.4 million children were estimated to have hand and foot. Mouth and pharyngitis. From 1998 to 2012, at least 250 children have died of enteroviruses in Taiwan, and the cause is still mainly EV71.

The virological genotype of Enterovirus 71 can be divided into four types: A, B, C, and D. Among them, type B can be further divided into B1 to B5, and type C can be divided into subtypes such as C1 to C5. In recent years, enterovirus 71 is mainly prevalent in Southeast Asia, the Western Pacific and China. Enterovirus is highly contagious, mainly through the gastrointestinal tract (fecal mouth, contaminated water or food) or the respiratory tract (droplets, coughing or sneezing), and can also be infected by contact with the patient's secretions or blisters on the skin. .

Because enterovirus is suitable for survival and spread in a wet and hot environment, Taiwan is located in the subtropical zone. There are cases of infection throughout the year, so enterovirus infection is already one of the endemic diseases in Taiwan. According to the monitoring data of Taiwan's past years, young children are high risk groups with serious complications and death, and the severe mortality rate is between 3.8% and 25.7%. The type of enterovirus infection complicated with severe disease is mainly enterovirus 71 type, and the esaqi virus is secondary; the main common symptoms of common enterovirus infection are hand, foot and mouth disease or rash angina. The enterovirus epidemic began to rise from about the end of March every year. After reaching the peak in late May to mid-June, it slowly decreased, and then there was another wave of epidemics after the start of school in September. According to the age group analysis, the majority of the children were under 5 years old, accounting for 90% of all severe cases; in terms of deaths, the number of young children under 5 years old was the most.

Therefore, the development of an effective enterovirus vaccine is urgent for the prevention of enterovirus and it is an important matter.

The present invention provides, in the first part, a novel Enterovirus 71 (EV71) virus strain having VP1, VP2 of the amino acid sequence shown in SEQ ID NOs: 6, 8, 10, respectively. VP3 structural protein; after identification, the virus was confirmed to be a novel enterovirus 71 (EV71) strain, and was named enterovirus 71 (EV71) EV71-1501-3-M3 strain. The virus strain was deposited in the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Research Institute. The registration date was June 26, 103, and the registration number was BCRC970065.

In the second part of the invention, an immunological composition comprising the novel enterovirus 71 (EV71) strain is provided. In certain embodiments, the immunological composition further comprises a pharmaceutically acceptable carrier. In certain embodiments, the immunological composition further comprises at least one pathogenic antigen, including but not limited to: Coxsackie virus, Echovirus, Poliovirus, intestine Enterovirus, Hepatitis B virus, Mycobacterium bovis, Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis, influenza bloodthirsty Haemophilus influenza, varicella-zoster virus, Measles virus, Mumps virus, Rubella virus, Japanese Encephalitis virus, round Virus (Rotavirus) and Influenza virus (Influenza virus).

In the third aspect of the present invention, an antibody against EV71 (EV71) is produced by the enterovirus 71 (EV71) strain of the present invention. In certain embodiments, the antibody comprises, but is not limited to, a monoclonal antibody, a plurality of antibodies, and a recombinant antibody.

The present invention provides, in a fourth aspect, a polynucleotide comprising an amino acid sequence encoding a structural protein of the enterovirus 71 (EV71) strain of the present invention, the amino acid sequence of the structural protein comprising at least One of the following: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10. In certain embodiments, the sequence comprises at least one of the following: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.

In the fifth part of the present invention, a detection kit for enterovirus 71 (EV71) is provided, which comprises a detection unit, which comprises but is not limited to: a virus of the enterovirus 71 (EV71) strain of the invention. An antigen, an antibody prepared by the enterovirus 71 (EV71) strain of the present invention, a protein having an amino acid sequence as shown in SEQ ID NOs: 2, 4, 6, 8, 10, and a coding having A polynucleotide of a protein of the amino acid sequence as shown in SEQ ID NOs: 2, 4, 6, 8, 10 . In certain embodiments, the antibody comprises at least one of the following: a monoclonal antibody, a polyclonal antibody, and a recombinant antibody. In certain embodiments, the DNA sequence of the polynucleotide comprises at least one of the following: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.

In part, the present invention provides a novel enterovirus 71 (EV71) virus strain having VP1, VP2, VP3 structural proteins having amino acid sequences as shown in SEQ ID NOs: 6, 8, 10; After the identification, the virus was confirmed to be a novel enterovirus 71 (EV71) virus strain, and was named enterovirus 71 (EV71) EV71-1501-3-M3 virus strain. The virus strain was deposited in the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Research Institute. The registration date was June 26, 103, and the registration number was BCRC970065.

In part, the invention provides an immunological composition comprising the novel enterovirus 71 (EV71) strain. In some embodiments, the immunological composition is a vaccine that is inactivated by the above-described enterovirus 71 (EV71) virus strain, or is weakened by a virus strain, and is then pharmaceutically acceptable. Vehicle) is prepared into a dosage form suitable for use in the vaccine of the present invention. However, the dosage form of the vaccine includes, but is not limited to, a dead (non-activated) vaccine, a live (attenuated) vaccine, a sub-unit vaccine, a DNA vaccine, or the novel enterovirus 71 (EV71) strain or A vaccine prepared by a nucleotide sequence or an amino acid sequence.

The inactivated treatment of the present invention includes, but is not limited to, deactivator treatment, heat treatment, and the like, which are not activated by the present invention. The deactivating agent includes, but is not limited to, formaldehyde, paraformaldehyde, beta-propionolactone (BPL), binary ethyleneimine (BEI), or the present invention. Deactivator and the like.

Wherein the pharmaceutically acceptable carrier comprises one or more agents selected from the group consisting of: solvents, emulsifiers, suspending agents, decomposers, binding agents. , excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, interfacial activity Surfactant, adjuvant, and other carriers similar or suitable for use in the present invention.

Wherein the adjuvant includes, but is not limited to, an oil adjuvant (eg, mineral oil, vegetable oil, animal oil, Freund's complete adjuvant, Freund's incomplete adjuvant, etc.), water adjuvant (eg, aluminum hydroxide), double Phase oil adjuvant (eg, water-in-oil-in-water dosage form, w/o/w), bio-type adjuvant (eg, CpG oligonucleotide, bacterial toxoid). Wherein the dual phase oily adjuvant comprises a surfactant and an oil phase material; the surfactant comprises one or more of the following selected groups: sorbitol fatty acid ester; sorbitol fatty acid Ester and ethylene oxide or propylene oxide concentrate; mannitol fatty acid ester; mannitol fatty acid ester and ethylene oxide or propylene oxide concentrate; mannitol fat Acid esters with the following selected hydrophilic groups: carboxylic acid, amine, amide, alcohol, polyester, ether, oxygen Oxide conjugate; anhydrous mannitol fatty acid ester; anhydrous mannitol fatty acid ester with the following selected hydrophilic groups: carboxylic acid, amine, decylamine, alcohol, polyester polyol, ether a conjugate of an oxy group; a saccharose fatty acid ester; a sucrose fatty acid ester with an ethylene oxide or propylene oxide concentrate; a glycerin fatty acid ester; a glycerin fatty acid ester with ethylene oxide or propylene oxide Concentrate; fatty acid and ethylene oxide or propylene oxide concentrate; fatty alcohol and epoxy Dioxane or propylene oxide concentrate; and glycerophospholipids (glycerophospholipid). The oil phase material includes one or more of the following selected groups: mineral oil, vegetable oil, and animal oil.

In some embodiments, the immunological composition of the present invention may further comprise one or more pathogenic antigens, including but not limited to: Coxsackie virus, Echovirus, Pediatrics Poliovirus, Enterovirus, Hepatitis B virus, Mycobacterium bovis, Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis ), Haemophilus influenza, varicella-zoster virus, Measles virus, Mumps virus, Rubella virus, Japanese encephalitis vaccine Japanese Encephalitis virus), Rotavirus and Influenza virus.

In part, the present invention provides an antibody against EV71 (EV71) which is produced by the enterovirus 71 (EV71) strain of the present invention. In certain embodiments, the antibody comprises, but is not limited to, at least one of the following: a monoclonal antibody, a polyclonal antibody, and a recombinant antibody.

The anti-intestinal virus type 71 (EV71) antibody provided by the present invention can be produced by a technique known in the art of the present invention. In some specific embodiments, the antibody is a plurality of antibodies, and the preparation method thereof comprises, but is not limited to: The antigenic protein of the deactivated enterovirus 71 strain of the present invention or a specific antigen fragment thereof (eg, VP0, VP1, VP2, VP3, VP4) is mixed with a suitable adjuvant (eg, Freund's complete adjuvant) And animals (such as but not limited to: rats, rabbits, poultry (eggs), pigs, goats, cattle, aquatic animals) for the first immunization, after appropriate intervals (eg, 2 to 3 weeks), as needed The second immunization or even the third immunization or more immunization is administered with the non-activated virus solution and a suitable adjuvant (eg, Freund's incomplete adjuvant) to increase the antibody titer. After appropriate time interval (eg, 2 to 3 weeks), the serum of the immunized animal is collected, and an antibody against the enterovirus 71 type is prepared. In certain embodiments, the multi-strain antibody can be combined with a developer or fluorescent light as desired.

In some embodiments, the antibody is a monoclonal antibody, and the preparation method thereof comprises, but is not limited to, a concentrated enterovirus strain of the present invention, or a specific antigen fragment thereof (eg, VP0, VP1, VP2, VP3, The antigenic protein of VP4) is administered to an animal (e.g., a mouse), and an appropriate adjuvant (e.g., Freund's complete adjuvant) may be added as needed to perform the first immunization. After an appropriate time interval (eg, 2 to 3 weeks), a second immunization is administered as needed with an inactivated virus (or antigenic protein) and a suitable adjuvant (eg, Freund's incomplete adjuvant). After appropriate time intervals (eg, 2 to 3 weeks), serum from immunized animals (eg, mice) is collected to assess mice suitable for collecting spleen cells. The spleen cells and the myeloma cells (e.g., the FO cell strain, the NS cell strain) are collected from the suitable mouse for cell fusion with PEG (Polyethylene Glycol, such as PEG 1500). After screening for a secreted fusion tumor from a fused cell and monoculture, a fusion cell line suitable for producing a monoclonal antibody against the enterovirus 71 strain of the present invention can be obtained. The antibody obtained by the above preparation can be used for an immunoassay reagent, a therapeutic agent, and the like.

In a part, the present invention provides a polynucleotide comprising an amino acid sequence encoding a structural protein of the enterovirus 71 (EV71) strain of the present invention, the amino acid sequence of the structural protein comprising at least the following One: SEQ ID NO: 2. SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10. In certain embodiments, the sequence comprises at least one of the following: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.

In one aspect, the present invention provides a detection kit for enterovirus type 71 (EV71), comprising a detection unit comprising, but not limited to: a viral antigen of an enterovirus 71 (EV71) strain of the invention An antibody prepared by the enterovirus 71 (EV71) strain of the present invention, a protein having an amino acid sequence as shown in SEQ ID NOs: 2, 4, 6, 8, 10, and an encoding having SEQ ID NOs: A polynucleotide of a protein of the amino acid sequence shown in 2, 4, 6, 8, 10. In certain embodiments, the antibody comprises at least one of the following: a monoclonal antibody, a polyclonal antibody, and a recombinant antibody. In certain embodiments, the DNA sequence of the polynucleotide comprises at least one of the following: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.

The form of the test kit includes, but is not limited to, an enzyme-linked immunosorbent assay (ELISA) kit, a microchip test kit (Microchip kit), and an immunofluorescence assay (IFA) assay. Kits, polymerase chain reaction (PCR) test kits, or other test kits made by the enterovirus 71 (EV71) strain of the invention.

In some embodiments, the test kit comprises at least one antigenic disk containing the enterovirus 71 (EV71) strain of the invention, which can be used to test whether the sample contains antibodies against EV71 (EV71). The preparation method of the test kit includes, but is not limited to, adding a suspension of cells sensitive to enterovirus (such as, but not limited to, Vero cells, RD cells) to a micro-flat plate, and inoculating an appropriate amount of the enterovirus of the present invention. Type 71 (EV71) virus strain was incubated at 37 ° C, 5% CO2 for a suitable period of time, washed with PBS, fixed at room temperature with acetone at the appropriate concentration, washed with PBS, and placed in inverted 37 Dry in a °C incubator and store at -20 °C for later use. The antigen disk can be used to detect whether the sample contains an antibody against EV71 (EV71).

In certain embodiments, the test kit is an antigenic disk containing a recombinant VP0, VP1, VP2, VP3 or VP4 structural protein of the Enterovirus 71 (EV71) strain of the invention. The recombinant VP0, VP1, VP2, VP3, and VP4 structural proteins each have an amino acid sequence as shown in SEQ ID NOs: 4, 6, 8, 10, 12, and serve as an antigen on an antigenic disk. Preparation of recombinant VP0, VP1, VP2, VP3 or VP4 structural proteins can be obtained by methods known in the art, including but not limited to: synthesis by a protein synthesizer, molecular cloning, with The nucleotide sequence of the structural protein (such as, but not limited to, SEQ ID NOs: 3, 5, 7, 9, 11) is inserted into the expression vector and propagated into the expression host to express purification of the protein. Then, the obtained protein was adjusted to an appropriate concentration, coated on a micro-flat plate, and coated at 37 ° C for a suitable time, then coated at 4 ° C overnight; after washing with PBS, BSA blocking solution was added. The microplate flat plate was blocked, placed at 37 ° C for a suitable period of time, washed with PBS, and stored at 4 ° C as an enterovirus 71 (EV71) test kit.

The novel enterovirus 71 (EV71) virus strain of the present invention should also include its subcultured progeny, or mutant strain, but still have the characteristics, genomic, and genomic characteristics of the virus strain of the present invention. Or the same pathogenicity.

The "DNA (nucleic acid)", "polynucleotide", "amino acid", "peptide", "polypeptide" described in the present invention may be naturally occurring, isolated, recombinant, or artificially synthesized. .

All of the technical and scientific terms described in this specification, unless otherwise defined, are intended to be understood by those of ordinary skill in the art.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

Embodiment 1 Enterovirus 71 type (EV71) Separation and identification

1. Separation and identification of clinical samples of enterovirus strains

The patient's specimen was taken with a throat swab and inoculated into human embryonic fibroblast, LLC-MK2, HEp-2, and rhabdomyosarcoma (RD) cells, and more than 50% of the cells showed cytopathic effects. In the case of a Cytopathic effect (CPE), the cells are scraped off to obtain a sample virus solution. Indirect fluorescent antibody staining was performed on the test virus solution with an enterovirus-specific antibody (Chemicon International Inc., Temecula, CA, USA) to confirm the presence of enterovirus in the sample. On the other hand, the sample virus virus is sequenced into the enterovirus P1 structural protein gene, which is obtained by reverse transcription polymerase chain reaction (RT-PCR) and semi-nested reverse-polymerization (Semi-nested RT-PCR). The PCR product was purified and sequenced by ABI 3730XL auto-sequencer to isolate the virus, and the obtained enterovirus was numbered EV71-1501-3, and the P1 structural protein amino acid sequence was as shown in SEQ ID NO: 13.

2. Re-amplification and identification of enterovirus strains

The generation of the enterovirus EV71-1501-3 selected from the above samples was defined as P ₀ generation. Then, Vero cells (African green monkey kidney cells, ATCC CCL81) to ^{5.0 x 10 5 cells / 2ml /} well were seeded in 6-well plates at 37 ^o C, 5% CO ₂ incubator 16- after 20 hours, the medium was removed and the phosphate buffer solution (phosphate buffered saline, PBS) cells were washed, followed by addition of 100 μl of the specimen virus EV71-1501-3 virus solution (P ₀ generation) and 400 μl MEM medium, after infection at 37 ^o C, 5% CO ₂ incubator for 2 hours, the sample was added to each cell containing 2 ml MEM medium with 2% FBS, and at 37 ^o C, 5% CO ₂ incubator 2- 4 days.

The above-mentioned Vero cells infected with enterovirus 71 type EV71-1501-3 (P ₀ generation) were observed under a microscope. When more than 50% of the cells showed a Cytopathic effect (CPE), the cells were collected and thawed (freeze) -thaw) method to destroy the cells, centrifuge to collect the supernatant containing enterovirus, repeat the above-mentioned substeps 8 times, and then determine the viral power price by Reed and Muench method, and select the virus with the highest price (2x10 ^8.6 TCID ₅₀ /ml The number is EV71-1501-3-M3 (P ₈ generation) and continues to be analyzed.

Further, it was identified by Western blotting whether the enterovirus strain EV71-1501-3-M3 proliferated by Vero cells was an enterovirus strain. Western blot analysis was performed by electrophoresis of 10 μl of the purified sample with a 5 – 12% gradient of sodium n-dodecane-polyacrylamide colloid at a voltage of 180 mV for 40 minutes. Using a wet transfer tank, the protein sample was transferred from the colloid to the nitrocellulose membrane, and a 5% skim milk powder solution was added to the fiber membrane. After 4 °C shaking overnight, the anti-intestinal virus type 71 was diluted 1:1000. Antibodies were added to the fiber membrane and washed for 1 hour at room temperature and washed three times with 0.1% Tween 80/PBS to remove unbound antibody. A 1:5000 dilution of HRP-conjugated anti-mouse-IgG antibody was added and incubated at 37 ° C for 1.5 hours at room temperature and washed three times with 0.1% Tween 80/PBS to remove unbound antibody. An appropriate amount of ECL coloring agent is poured onto the fiber membrane for development, fixing, and tableting.

Western blot analysis results are shown in Figs. 1A and 1B, the enterovirus strain EV71-1501-3-M3 of the present invention and the enterovirus strain E49-1149 of the positive control group (which is an EV71 C4 gene subtype, by the Ministry of Health and Welfare of Taiwan) The disease control agency provides) anti-Enterovirus 71 VP1 monoclonal antibody (Abonva, Taiwan) (Figure 1A), anti-Enterovirus 71 VP0 monoclonal antibody (Millpore, Massachusetts, USA) (Figure 1B), anti-Enterovirus 71 VP2 monoclonal antibody (Millpore Inc., Massachusetts, USA) (Fig. 1B) identified and confirmed that the virus strain isolated by the present invention was an enterovirus strain.

3. Gene sequence analysis of enterovirus strains

The RNA of the enterovirus 71 strain EV71-1501-3-M3 (P ₈ generation) obtained above is extracted, and the viral gene is subjected to nucleic acid after reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis revealed that the DNA sequence of the P1 structural protein is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

Comparison of the P1 structural protein amino acid sequence of the above-mentioned EV71-1501-3 (P ₀ generation) isolated from the specimen and its sub-enteric EV71-1501-3-M3 (P ₈ generation) The alignment results are shown in Figure 2. There are 4 amino acid residues in the amino acid sequence of the P1 structural protein of EV71-1501-3 (P ₀ generation) and EV71-1501-3-M3 (P ₈ generation). The difference is 114, 269, 467, 684 amino acid residues, respectively. The results of the comparison showed that after passage, the enterovirus EV71-1501-3 was mutated, and the P1 structural protein shared the above four amino acid residues, which was different from the enterovirus EV71-1501-3 strain. EV71-1501-3-M3 virus strain.

Next, the DNA sequence and the amino acid sequence of the above EV71-1501-3-M3 strain were compared with the National Center for Biotechnology Information (NCBI) gene database (GenBank), and the results are shown in Table 1 and Table 2, respectively. The gene sequences of the enterovirus 71 strain EV71-1501-3-M3 of the present invention are similar to the enterovirus 71 strain (EV71), and the similarity can be shown. Up to 99%, but the GenBank database did not find the presence of the Enterovirus 71 strain with the same genetic sequence as the Enterovirus 71 strain EV71-1501-3-M3 (100% similarity). Therefore, from the results of the comparison, the EV71-1501-3-M3 strain of the EV71-1501-3-M3 obtained in the present invention belongs to the C4 gene subtype and is a novel virus strain.

Table 1 Comparison of DNA sequences of P1 structural proteins of Enterovirus 71 strain EV71-1501-3-M3 in NCBI [Table 1]         <TABLE style="BORDER-BOTTOM: medium none; BORDER-LEFT: medium none; BORDER-COLLAPSE: collapse; BORDER-TOP: medium none; BORDER-RIGHT: medium none; mso-border-alt: double windowtext 1.5pt; Mso-yfti-tbllook: 1184; mso-padding-alt: 0cm 5.4pt 0cm 5.4pt; mso-border-insideh: 1.5pt double windowtext; mso-border-insidev: 1.5pt double windowtext"_0004"MsoTableGrid" border=" 1" cellSpacing="0" cellPadding="0"><TBODY><tr><td> Accession No. </td><td> Description </td><td> Max score < /td><td> Total score </td><td> Max ident. </td></tr><tr><td> KF444676.1 </td><td> Enterovirus A71 strain C4-NHRIEV95493-V060270365 -TW </td><td> 4700 </td><td> 4700 </td><td> 99% </td></tr><tr><td> JX678877.1 </td><td > Human enterovirus 71 strain FY17.08-6/AH/CHN/2008 </td><td> 4590 </td><td> 4590 </td><td> 99% </td></tr>< Tr><td> JX678876.1 </td><td> Human enterovirus 71 strain FY17.08-5/AH/CHN/2008 </td><td> 4573 </td><td> 4573 </td> <td> 98% </td></tr><tr><td> FJ606448.1 </td><td> Human enterovirus 71 strain BJ08-Z011-4 </td><td> 4567 </td><td> 4567 </td><td> 98% </td></tr><tr><td> EU703813.1 < /td><td> Human enterovirus 71 strain EV71/Fuyang.Anhui.PRC/17.08/2 </td><td> 4567 </td><td> 4567 </td><td> 98% </td> </tr></TBODY></TABLE>

Table 2 Results of the amino acid sequence of the P1 structural protein of Enterovirus 71 strain EV71-1501-3-M3 in NCBI [Table 2]         <TABLE style="BORDER-BOTTOM: medium none; BORDER-LEFT: medium none; BORDER-COLLAPSE: collapse; BORDER-TOP: medium none; BORDER-RIGHT: medium none; mso-border-alt: double windowtext 1.5pt; Mso-yfti-tbllook: 1184; mso-padding-alt: 0cm 5.4pt 0cm 5.4pt; mso-border-insideh: 1.5pt double windowtext; mso-border-insidev: 1.5pt double windowtext"_0005"MsoTableGrid" border=" 1" cellSpacing="0" cellPadding="0"><TBODY><tr><td> Accession No. </td><td> Description </td><td> Max score < /td><td> Total score </td><td> Max ident. </td></tr><tr><td> AFQ91919.1 </td><td> polyprotein [Enterovirus A71] </td ><td> 1803 </td><td> 1803 </td><td> 99% </td></tr><tr><td> AHA38102.1 </td><td> polyprotein [Enterovirus A71 ] </td><td> 1802 </td><td> 1802 </td><td> 99% </td></tr><tr><td> AGH30718.1 </td><td> Polyprotein [Enterovirus A71] </td><td> 1801 </td><td> 1801 </td><td> 99% </td></tr><tr><td> AHX57762.1 </td ><td> putative genome polyprotein [Enterovirus A71] </td><td> 1801 </td><td> 1801 </td ><td> 99% </td></tr><tr><td> AHX57721.1 </td><td> genome polyprotein [Enterovirus A71] </td><td> 1800 </td><td > 1800 </td><td> 99% </td></tr></TBODY></TABLE>

The result of the analysis shows that the DNA sequence of the P1 structural protein of the enterovirus 71 strain EV71-1501-3-M3 of the present invention is as shown in SEQ ID NO: 1, and the amino acid sequence thereof is shown in SEQ ID NO: 2; The P1 structural protein can be further hydrolyzed into VP0, VP1, VP3 structural proteins, and the VP0 structural protein can be further hydrolyzed into VP2, VP4 structural proteins. The DNA sequences of the VP0, VP1, VP2, VP3, and VP4 structural proteins of the enterovirus 71 strain EV71-1501-3-M3 of the present invention are shown in SEQ ID NOs: 3, 5, 7, 9, 11 respectively. The amino acid sequences are shown in SEQ ID NOs: 4, 6, 8, 10, 12, respectively.

The above results confirmed that the enterovirus 71 strain EV71-1501-3-M3 provided by the present invention is a novel enterovirus 71 strain, which was deposited on June 26, 103, in the Republic of China. Bioresource Conservation and Research Center, Institute of Food Industry Development, the registration number is BCRC970065.

Embodiment 2 Enterovirus 71 type (EV71) Do not Preparation of activated vaccine

Monolayers of Vero cells infected with enterovirus strain EV71-1501-3-M3 of the present invention were cultured in MEM medium containing 2% FBS in a 5% CO ₂ incubator at 37 ° C to more than 50% of cells. In the case of cytopathic effect (CPE), cells were harvested and disrupted by freezing and thawing, and the supernatant containing enterovirus was collected by centrifugation, and the viral power was determined by Reed and Muench method.

Formaldehyde was added to the above enteric virus harvesting solution to a final concentration of 0.01%, and deactivated by shaking at 37 ° C on a shaking table. During the reaction, 50% Tissue Culture Infection Dose (TCID ₅₀ ) was periodically sampled to observe changes in viral power until no virus survived.

Next, take 100~120 ml of deactivated virus solution, centrifuge at 27000 rpm for 2-4 hours, remove the supernatant, and resuspend the virus pellet in 1-3 ml PBS. The suspension was added to a liquid surface layer containing 20 mL of a 15% sucrose buffer centrifuge tube, centrifuged at 23,000 rpm for 16 hours, and the supernatant was removed, and the pellet was dispersed in 20 ml of PBS, and then centrifuged at 27,000 rpm. After 4 hours, the supernatant was removed, and the pellet was dispersed in 1 ml of PBS to obtain a virus-free purification solution.

The resulting inactivated virus purification solution can be used directly as an inactivated vaccine, or can be formulated as an inactivated vaccine containing an adjuvant with an AlPO ₄ adjuvant.

Embodiment 3 Enterovirus 71 type (EV71) Do not Efficacy test of activated vaccine 1

Mouse immunization program

50 healthy female ICR strain mice of 6 to 8 weeks old were used as experimental animals, and the mice were purchased from the Animal Center of Taiwan University (Taipei, Taiwan) and tested at the Animal Center of Taiwan University. The serum of all mice showed a negative enterovirus antibody status before the test. The 50 mice were randomly divided into 10 groups, 5 rats in each group, and the enterovirus vaccine was administered according to the following groups, and immunized by intraperitoneal injection. The injection dose was 100 ml/time/time, respectively, on the 0th day of the experiment. On the 14th and 28th day, the first immunization, the second immunization, and the third immunization were performed, and blood was collected on the 0th day, the 7th day, the 27th day, and the 42nd day of the test, and each group 5 The blood samples of only the mice were combined into one, and the serum samples were separated. The test groups were as follows: Group 1: 1 μg enterovirus strain EV71-1501-3-M3 viral protein; Group 2: 1 μg enterovirus strain EV71-1501-3-M3 viral protein + 30 μg AlPO ₄ adjuvant; Group 3: 5 μg enterovirus strain EV71-1501-3-M3 viral protein; Group 4: 5 μg enterovirus strain EV71-1501-3-M3 viral protein + 30 μg AlPO ₄ adjuvant; Group 5: 1 Gg enterovirus strain E49-1149 viral protein; group 6: 1 μg enterovirus strain E49-1149 viral protein + 30 μg AlPO ₄ adjuvant; group 7: 5 μg enterovirus strain E49-1149 viral protein; group 8 : 5 μg enterovirus strain E49-1149 viral protein + 30 μg AlPO ₄ adjuvant; Group 9: PBS negative control group; Group 10: PBS + 30 μg AlPO ₄ adjuvant negative control group.

2. Neutralizing antibody test:

The RD cell line (rhinodomoma cell line, Rhabdomyosarcoma, ATCC CCL-136) was seeded at a density of 5 x 10 ⁴ cells/well in a 96-well cell culture dish and cultured at 37 ° C in a 5% CO ₂ incubator. -20 hours. On the other hand, each of the collected serum samples was placed in a 56 ° C water bath at a constant temperature for 30 minutes, and then the serum samples were subjected to 2-fold serial dilution on a 96-well cell culture plate using MEM medium containing 2% FBS. An equal volume of 100 TCID ₅₀ enterovirus (C4 subtype enterovirus strain EV71-1501-3-M3, or C4 subtype enterovirus strain E49-1149, or B4 subtype enterovirus strain 2847) was added to each sample. Serum samples were used as a serum control group, and MEM medium containing 2% FBS was used as a cell control group. Then, each sample was placed in a 37 ° C, 5% CO ₂ incubator for neutralization for 2 hours, and after washing with PBS, the neutralized serum sample was transferred to a 96-well cell culture plate containing RD cells. Medium (100 μl/well), cultured in a 37 ° C, 5% CO ₂ incubator for 5 days, after crystal violet staining, the results were visually interpreted, and the virus was inhibited at the highest dilution concentration. The magnification is the neutralizing antibody titer.

The results of the neutralizing antibody test are shown in Figs. 3A, 3B, and 3C; a mouse serum sample containing 1 mg of the enterovirus strain EV71-1501-3-M3 virus protein of the present invention containing AlPO ₄ adjuvant was diluted 1024 times. It still has the ability to neutralize the C4 subtype enterovirus strains EV71-1501-3-M3, the C4 subtype enterovirus strain E49-1149, and the B4 subtype enterovirus strain 2847, so that the cells are protected from enterovirus infection. The mouse serum sample containing 5 mg of the enterovirus strain EV71-1501-3-M3 virus protein containing the AlPO ₄ adjuvant was also neutralized with the C4 subtype enterovirus strain EV71- after 512 times dilution. The ability of 1501-3-M3, C4 subtype enterovirus strain E49-1149 and B4 subtype enterovirus strain 2847. In addition to the neutralizing antibody against the enterovirus strain EV71-1501-3-M3, the enterovirus strain EV71-1501-3-M3 of the present invention can also induce the C4 subtype enterovirus strain E49-1149. And the cross-protective ability of B4 subtype enterovirus strain 2847. The enterovirus strain EV71-1501-3-M3 of the present invention clearly had a higher neutralizing antibody titer than the neutralizing antibody titer of the control enterovirus strain E49-1149.

Embodiment 4 Enterovirus 71 type (EV71) Do not Efficacy test of activated vaccine 2

Rabbit immune program

Four female New Zealand female rabbits, 6 to 8 weeks old, were used as experimental animals. The experimental rabbits were purchased from the Animal Center of Taiwan University (Taipei, Taiwan) and tested at the Animal Center of Taiwan University. The sera of all the test rabbits showed a state in which the enterovirus antibody was negative before the test. The 4 test rabbits were randomly divided into 2 groups (test group and negative control group), 2 in each group. The experimental group was intraperitoneally injected with 100 μg enterovirus strain EV71-1501-3 containing 30 μg of AlPO ₄ adjuvant. -M3 viral protein, while the negative control group was intraperitoneally injected with 30 μg of AlPO ₄ adjuvant in PBS; the dose of each test rabbit was 600 ml / time / time, respectively, on the 0th day, 14th On the 28th day, the first immunization, the second immunization, and the third immunization were performed, and blood samples were taken on the 0th day, the 14th day, the 28th day, and the 42nd day of the test to obtain a serum sample.

2. Neutralizing antibody test

The Vero cell strain was seeded at a density of 2.5 x 10 ⁴ cells/well in a 96-well cell culture dish and cultured at 37 ° C in a 5% CO ₂ incubator for 16-20 hours. On the other hand, each of the collected serum samples was placed in a 56 ° C water bath at a constant temperature for 30 minutes, and then the serum samples were subjected to 2-fold serial dilution on a 96-well cell culture plate using MEM medium containing 2% FBS. An equal volume of 100 TCID ₅₀ enterovirus (C4 subtype enterovirus strain EV71-1501-3-M3, or C4 subtype enterovirus strain A, B, C, D, or B4 subtype enterovirus strain 2847 was added to each sample. Serum samples were used as a serum control group, and MEM medium containing 2% FBS was used as a cell control group. Then, each sample was placed in a 37 ° C, 5% CO ₂ incubator for neutralization for 2 hours, and after washing with PBS, the neutralized serum sample was transferred to a 96-well cell culture plate containing Vero cells. Medium (100 μl/well), cultured at 37 ° C, 5% CO ₂ incubator for 7 days, the results were visually interpreted, and the serum was at the highest dilution level, and the virus-infected cells were still inhibited. potency.

The results of the neutralizing antibody test are shown in FIGS. 4A and 4B; the rabbit serum sample containing 100 mg of the enterovirus strain EV71-1501-3-M3 virus protein of the present invention containing AlPO ₄ adjuvant is still diluted after 512 times. The ability to neutralize the C4 subtype enterovirus strain EV71-1501-3-M3 protects cells from enterovirus infection. The above two rabbit serum samples still have the ability to neutralize the C4 subtype enterovirus strain B when diluted 512 times; the above two rabbit serum samples still have the neutralizing C4 subtype when diluted 1024 times and 512 times, respectively. The ability of enterovirus strain C; the above two rabbit serum samples still have the ability to neutralize the C4 subtype enterovirus strain D when diluted 2048 times and 512 times respectively; the above two rabbit serum samples were diluted 1024 times and 256 respectively. At times, it still has the ability to neutralize the subtype B enterovirus strain. From this, it can be seen that the enterovirus strain EV71-1501-3-M3 of the present invention can induce the enterovirus of the strain C4 subtype, in addition to the neutralizing antibody against the enterovirus strain EV71-1501-3-M3. And the cross-protection ability of the subtype B enterovirus strain.

The detailed description of the preferred embodiments of the present invention is intended to be limited to the scope of the invention, and is not intended to limit the scope of the invention. The patent scope of this case.

Figure 1 shows the results of confirmation of enterovirus samples by Western blotting. Fig. 1A shows that the enterovirus strain EV71-1501-3-M3 (lane 1) of the present invention and the enterovirus strain E49-1149 of the positive control group were identified by anti-Enterovirus 71 VP1 monoclonal antibody (Abonva Co., Ltd., Taiwan) (2nd) As a result of the tract, the arrow points to the VP1 protein. Figure 1B shows the recognition of the enterovirus strain EV71-1501-3 of the present invention with an anti-Enterovirus 71 VP0 monoclonal antibody (Millpore Inc., Massachusetts, USA), an anti-Enterovirus 71 VP2 monoclonal antibody (Millpore Inc., Massachusetts, USA). M3 (lane 1) and the result of the positive control enterovirus strain E49-1149 (lane 2).

Figure 2 shows the alignment of the P1 structural protein amino acid sequence of enterovirus EV71-1501-3 (P ₀ generation) and EV71-1501-3-M3 (P ₈ generation). Query is enterovirus EV71-1501 -3 (P ₀ generation) P1 structural protein amino acid sequence, Subject is the P1 structural protein amino acid sequence of enterovirus EV71-1501-3-M3 (P ₈ generation), the box is labeled as a different amine Base acid residue.

Figure 3 shows the results of neutralization antibody titer analysis of blood samples collected on the 42nd day after the test (after the third immunization) of mice after immunization with different enterovirus 71 strains. Fig. 3A is a serum sample of mice immunized with the enterovirus strain EV71-1501-3-M3 inactivated vaccine of the present invention, respectively, against C4 subtype enterovirus strain EV71-1501-3-M3, C4 subtype enterovirus strain E49- The results of the neutralization reaction were carried out in 1149 and B4 subtype enterovirus strain 2847. Fig. 3B is a serum sample of mice immunized with the control vaccine strain E49-1149 inactivated vaccine, respectively, against C4 subtype enterovirus strain EV71-1501-3-M3, C4 subtype enterovirus strain E49-1149 and B4 sub- The enterovirus strain 2847 was the result of a neutralization reaction. Fig. 3C is a serum sample of mice injected with PBS solution, and neutralized C4 subtype enterovirus strains EV71-1501-3-M3, C4 subtype enterovirus strain E49-1149 and B4 subtype enterovirus strain 2847, respectively. The result.

Figure 4 is a blood sample collected from the 42nd day after the rabbit (after the third immunization), and the C4 subtype enterovirus strain, respectively, after the rabbit was immunized with the enterovirus strain EV71-1501-3-M3 inactivated by the vaccine of the present invention. EV71-1501-3-M3, C4 subtype enterovirus strains A, B, C, D, and B4 subtype enterovirus strain 2847 were subjected to neutralization antibody titer analysis. Figure 4A is a graph showing the results of neutralization antibody titer analysis of serum samples of the first test rabbit. Figure 4B is a graph showing the results of neutralization antibody titer analysis of serum samples of the second test rabbit.

TW Republic of China Food Industry Development Institute Biological Resources Conservation and Research Center 2014/06/ 26 BCRC970065

<110> Kea Biotechnology Co., Ltd. <120> Enterovirus 71 novel virus strain and its application <130> P14-0037 <160> 12 <170> PatentIn version 3. 5     <210>   1   <211>   2586   <212>   DNA   <213>   Enterovirus 71 (Enterovirus   71)     <400>   1   Atgggttcgc   Aagtgtctac   Tcagcgctcc   Ggttctcacg   Aaaattcaaa   Ttcagccact   60   Gagggttcta   Ccataaacta   Caccaccatt   Aattactaca   Aagactccta   Cgctgccaca   120   Gcaggcaaac   Agagtctcaa   Gcaggatcca   Gacaagtttg   Caaatcctgt   Taaagacatc   180   Ttcactgaaa   Tggcagcgcc   Actgaagtcc   Ccatccgctg   Aggcatgtgg   Atacagtgat   240   Cgagtggcgc   Aattaactat   Tggcaactcc   Accatcacca   Cgcaagaagc   Ggcaaacatc   300   Atagtcggtt   Atggtgagtg   Gccttcctac   Tgctcagatt   Ctgacgctac   Agcagtggat   360   Aaaccaacgc   Gcccagatgt   Ttcagtgaac   Aggttttaca   Cattggacac   Taaattgtgg   420   Gagaaatcgt   Ccaagggatg   Gtactggaag   Ttcccggatg   Tgttaactga   Aactggggtt   480   Tttgggcaaa   Atgcacaatt   Ccactacctc   Taccgatcag   Ggttctgcat   Ccacgtgcag   540   Tgcaacgcca   Gtaaattcca   Ccaaggagca   Ctcctagtcg   Ctgtcctacc   Agaggtgtc   600   Attgggacag   Tggcaggcgg   Tacagggacg   Gaagataccc   Acccccctta   Caagcagacc   660   Caacccggcg   Cggatggttt   Cgagttgcaa   Cacccgtacg   Tgcttgatgc   Tggcatccca   720   Atatcgcagt   Taacagtgtg   Cccacaccag   Tggattaatt   Tgaggaccaa   Caattgtgct   780   Acaataatag   Tgccatacat   Taacacactg   Cctttgtatt   Ctgccttgaa   Ccattgcaac   840   Tttggcctgt   Tagttgtgcc   Tattagccca   Ctagactacg   Accaaggagc   Gacgccagta   900   Atccctataa   Ctatcacatt   Ggccccaatg   Tgttctgaat   Tcgcaggtct   Taggcaggca   960   Gtcacgcaag   Ggttccccac   Cgagctaaaa   Cctggcacaa   Atcaattttt   Aaccaccgat   1020   Gatggcgttt   Cagcacctat   Tctaccaaac   Ttccacccca   Ccccgtgtat   Ccacatacct   1080   Ggtgaagtta   Ggaacttgct   Agaggtatgc   Caggtggaga   Ccattctgga   Ggttaacaat   1140   Gtgcccacga   Atgccactag   Cttaatggag   Agactgcgct   Tcccggtctc   Agcacaagca   1200   Gggaaaggtg   Aactgtgtgc   Ggtatttaga   Gccgatcctg   Ggcgaaatgg   Accatggcaa   1260   Tccaccttac   Tgggtcagtt   Gtgcgggtac   Taaccccaat   Ggtcaggatc   Attggaagtt   1320   Accttcatgt   Ttactggatc   Cttcatggct   Accggcaaga   Tgctcatagc   Ctatacaccg   1380   Ccagggggtc   Ctctgcccag   Ggaccgggcg   Actgccatgt   Tgggcacgca   Tgtcatctgg   1440   Gattttgggc   Tgcaatcgtc   Tgttaccctt   Gtaataccat   Ggatcagtaa   Tactcattat   1500   Agagcacatg   Cccgagatgg   Agtgtttgac   Tattacacca   Cagggttagt   Cagtatatgg   1560   Taccagacaa   Attacgtggt   Tccaatcggt   Gcgcccaaca   Cagcctatat   Aatagcacta   1620   Gcggcagccc   Aaaagaactt   Cactatgaaa   Ttgtgcaagg   Atgctagtga   Tatcctgcag   1680   Acgggcacca   Tccagggaga   Tagggtggca   Gatgtaattg   Aaagttccat   Aggagatagc   1740   Gtgagcagag   Ccctcactca   Cgctctacca   Gcacccacag   Gccaaaacac   Acaggtgagc   1800   Agtcatcgac   Tggatacagg   Caaggttcca   Gcactccaag   Ctgctgaaat   Tggagcatca   1860   Tcaaatgcta   Gtgacgagag   Catgattgaa   Acacgctgtg   Ttcttaactc   Gcacagtaca   1920   Gctgagacca   Ctcttgatag   Tttcttcagt   Agggcgggat   Tagttggaga   Galatatctc   1980   Cctcttgagg   Gcacaactaa   Cccaaatggt   Tatgccaact   Gggacataga   Tataacaggt   2040   Tacgcgcaac   Tgcgtagaaa   Ggtagagcta   Ttcacctaca   Tgcgttttga   Tgcagagttc   2100   Acttttgttg   Cgtgcacacc   Cactggggga   Gttgtcccac   Aattgctcca   Atatatgttt   2160   Gtgccacctg   Gagcccctaa   Gccagattct   Agggaatccc   Ttgcatggca   Aaccgctacc   2220   Aacccctcag   Tttttgtcaa   Gctgtcagac   Cctccggcgc   Aggtttcagt   Gccattcatg   2280   Tcacctgcga   Gtgcttatca   Atggttttat   Gagggatatc   Ccacattcgg   Agaacacaaa   2340   Caggagaaag   Atcttgaata   Cggggcatgt   Ctataataaca   Tgatgggcac   Attctcagtg   2400   Cggactgtgg   Ggacctccaa   Gtccaagtac   Cctttagtgg   Ttaggattta   Catgagaatg   2460   Aagcacgtca   Gggcgtggat   Acctcgcccg   Atgcgcaacc   Agaactacct   Attcaaagcc   2520   Aacccaaatt   Atgctggcaa   Ctccattaag   Ccaactggtg   Ccagtcgcac   Agcgatcacc   2580   Actctt   2586       <210>   2   <211>   862   <212>   PRT   <213>   Enterovirus 71 (Enterovirus   71)     <400>   2   Met   Gly   Ser   Gln   Val   Ser   Thr   Gln   Arg   Ser   Gly   Ser   His   Glu   Asn   Ser   1   5   10   15   Asn   Ser   Ala   Thr   Glu   Gly   Ser   Thr   Ile   Asn   Tyr   Thr   Thr   Ile   Asn   Tyr     20   25   30   Tyr   Lys   Asp   Ser   Tyr   Ala   Ala   Thr   Ala   Gly   Lys   Gln   Ser   Leu   Lys   Gln     35   40   45   Asp   Pro   Asp   Lys   Phe   Ala   Asn   Pro   Val   Lys   Asp   Ile   Phe   Thr   Glu   Met     50   55   60   Ala   Ala   Pro   Leu   Lys   Ser   Pro   Ser   Ala   Glu   Ala   Cys   Gly   Tyr   Ser   Asp   65   70   75   80   Arg   Val   Ala   Gln   Leu   Thr   Ile   Gly   Asn   Ser   Thr   Ile   Thr   Thr   Gln   Glu     85   90   95   Ala   Ala   Asn   Ile   Ile   Val   Gly   Tyr   Gly   Glu   Trp   Pro   Ser   Tyr   Cys   Ser     100   105   110   Asp   Ser   Asp   Ala   Thr   Ala   Val   Asp   Lys   Pro   Thr   Arg   Pro   Asp   Val   Ser     115   120   125   Val   Asn   Arg   Phe   Tyr   Thr   Leu   Asp   Thr   Lys   Leu   Trp   Glu   Lys   Ser   Ser     130   135   140   Lys   Gly   Trp   Tyr   Trp   Lys   Phe   Pro   Asp   Val   Leu   Thr   Glu   Thr   Gly   Val   145   150   155   160   Phe   Gly   Gln   Asn   Ala   Gln   Phe   His   Tyr   Leu   Tyr   Arg   Ser   Gly   Phe   Cys     165   170   175   Ile   His   Val   Gln   Cys   Asn   Ala   Ser   Lys   Phe   His   Gln   Gly   Ala   Leu   Leu     180   185   190   Val   Ala   Val   Leu   Pro   Glu   Tyr   Val   Ile   Gly   Thr   Val   Ala   Gly   Gly   Thr     195   200   205   Gly   Thr   Glu   Asp   Thr   His   Pro   Pro   Tyr   Lys   Gln   Thr   Gln   Pro   Gly   Ala     210   215   220   Asp   Gly   Phe   Glu   Leu   Gln   His   Pro   Tyr   Val   Leu   Asp   Ala   Gly   Ile   Pro   225   230   235   240   Ile   Ser   Gln   Leu   Thr   Val   Cys   Pro   His   Gln   Trp   Ile   Asn   Leu   Arg   Thr     245   250   255   Asn   Asn   Cys   Ala   Thr   Ile   Ile   Val   Pro   Tyr   Ile   Asn   Thr   Leu   Pro   Phe     260   265   270   Asp   Ser   Ala   Leu   Asn   His   Cys   Asn   Phe   Gly   Leu   Leu   Val   Val   Pro   Ile     275   280   285   Ser   Pro   Leu   Asp   Tyr   Asp   Gln   Gly   Ala   Thr   Pro   Val   Ile   Pro   Ile   Thr     290   295   300   Ile   Thr   Leu   Ala   Pro   Met   Cys   Ser   Glu   Phe   Ala   Gly   Leu   Arg   Gln   Ala   305   310   315   320   Val   Thr   Gln   Gly   Phe   Pro   Thr   Glu   Leu   Lys   Pro   Gly   Thr   Asn   Gln   Phe     325   330   335   Leu   Thr   Thr   Asp   Asp   Gly   Val   Ser   Ala   Pro   Ile   Leu   Pro   Asn   Phe   His     340   345   350     Pro   Thr   Pro   Cys   Ile   His   Ile   Pro   Gly   Glu   Val   Arg   Asn   Leu   Leu   Glu     355   360   365   Leu   Cys   Gln   Val   Glu   Thr   Ile   Leu   Glu   Val   Asn   Asn   Val   Pro   Thr   Asn     370   375   380     Ala   Thr   Ser   Leu   Met   Glu   Arg   Leu   Arg   Phe   Pro   Val   Ser   Ala   Gln   Ala   385   390   395   400   Gly   Lys   Gly   Glu   Leu   Cys   Ala   Val   Phe   Arg   Ala   Asp   Pro   Gly   Arg   Asn     405   410   415   Gly   Pro   Trp   Gln   Ser   Thr   Leu   Leu   Gly   Gln   Leu   Cys   Gly   Tyr   Tyr   Thr     420   425   430   Gln   Trp   Ser   Gly   Ser   Leu   Glu   Val   Thr   Phe   Met   Phe   Thr   Gly   Ser   Phe     435   440   445   Met   Ala   Thr   Gly   Lys   Met   Leu   Ile   Ala   Tyr   Thr   Pro   Pro   Gly   Gly   Pro     450   455   460   Leu   Pro   Arg   Asp   Arg   Ala   Thr   Ala   Met   Leu   Gly   Thr   His   Val   Ile   Trp   465   470   475   480   Asp   Phe   Gly   Leu   Gln   Ser   Ser   Val   Thr   Leu   Val   Ile   Pro   Trp   Ile   Ser     485   490   495   Asn   Thr   His   Tyr   Arg   Ala   His   Ala   Arg   Asp   Gly   Val   Phe   Asp   Tyr   Tyr     500   505   510   Thr   Thr   Gly   Leu   Val   Ser   Ile   Trp   Tyr   Gln   Thr   Asn   Tyr   Val   Val   Pro     515   520   525   Ile   Gly   Ala   Pro   Asn   Thr   Ala   Tyr   Ile   Ile   Ala   Leu   Ala   Ala   Ala   Gln     530   535   540   Lys   Asn   Phe   Thr   Met   Lys   Leu   Cys   Lys   Asp   Ala   Ser   Asp   Ile   Leu   Gln   545   550   555   560   Thr   Gly   Thr   Ile   Gln   Gly   Asp   Arg   Val   Ala   Asp   Val   Ile   Glu   Ser   Ser     565   570   575   Ile   Gly   Asp   Ser   Val   Ser   Arg   Ala   Leu   Thr   His   Ala   Leu   Pro   Ala   Pro     580   585   590   Thr   Gly   Gln   Asn   Thr   Gln   Val   Ser   Ser   His   Arg   Leu   Asp   Thr   Gly   Lys     595   600   605   Val   Pro   Ala   Leu   Gln   Ala   Ala   Glu   Ile   Gly   Ala   Ser   Ser   Asn   Ala   Ser     610   615   620   Asp   Glu   Ser   Met   Ile   Glu   Thr   Arg   Cys   Val   Leu   Asn   Ser   His   Ser   Thr   625   630   635   640   Ala   Glu   Thr   Thr   Leu   Asp   Ser   Phe   Phe   Ser   Arg   Ala   Gly   Leu   Val   Gly     645   650   655   Glu   Ile   Asp   Leu   Pro   Leu   Glu   Gly   Thr   Thr   Asn   Pro   Asn   Gly   Tyr   Ala     660   665   670   Asn   Trp   Asp   Ile   Asp   Ile   Thr   Gly   Tyr   Ala   Gln   Leu   Arg   Arg   Lys   Val     675   680   685   Glu   Leu   Phe   Thr   Tyr   Met   Arg   Phe   Asp   Ala   Glu   Phe   Thr   Phe   Val   Ala     690   695   700   Cys   Thr   Pro   Thr   Gly   Gly   Val   Val   Pro   Gln   Leu   Leu   Gln   Tyr   Met   Phe   705   710   715   720   Val   Pro   Pro   Gly   Ala   Pro   Lys   Pro   Asp   Ser   Arg   Glu   Ser   Leu   Ala   Trp     725   730   735   Gln   Thr   Ala   Thr   Asn   Pro   Ser   Val   Phe   Val   Lys   Leu   Ser   Asp   Pro   Pro     740   745   750   Ala   Gln   Val   Ser   Val   Pro   Phe   Met   Ser   Pro   Ala   Ser   Ala   Tyr   Gln   Trp     755   760   765   Phe   Tyr   Asp   Gly   Tyr   Pro   Thr   Phe   Gly   Glu   His   Lys   Gln   Glu   Lys   Asp     770   775   780     Leu   Glu   Tyr   Gly   Ala   Cys   Pro   Asn   Asn   Met   Met   Gly   Thr   Phe   Ser   Val   785   790   795   800   Arg   Thr   Val   Gly   Thr   Ser   Lys   Ser   Lys   Tyr   Pro   Leu   Val   Val   Arg   Ile     805   810   815   Tyr   Met   Arg   Met   Lys   His   Val   Arg   Ala   Trp   Ile   Pro   Arg   Pro   Met   Arg     820   825   830   Asn   Gln   Asn   Tyr   Leu   Phe   Lys   Ala   Asn   Pro   Asn   Tyr   Ala   Gly   Asn   Ser     835   840   845     Ile   Lys   Pro   Thr   Gly   Ala   Ser   Arg   Thr   Ala   Ile   Thr   Thr   Leu     850   855   860       <210>   3   <211>   969   <212>   DNA   <213>   Enterovirus 71 (Enterovirus   71)     <400>   3   Atgggttcgc   Aagtgtctac   Tcagcgctcc   Ggttctcacg   Aaaattcaaa   Ttcagccact   60   Gagggttcta   Ccataaacta   Caccaccatt   Aattactaca   Aagactccta   Cgctgccaca   120   Gcaggcaaac   Agagtctcaa   Gcaggatcca   Gacaagtttg   Caaatcctgt   Taaagacatc   180   Ttcactgaaa   Tggcagcgcc   Actgaagtcc   Ccatccgctg   Aggcatgtgg   Atacagtgat   240   Cgagtggcgc   Aattaactat   Tggcaactcc   Accatcacca   Cgcaagaagc   Ggcaaacatc   300   Atagtcggtt   Atggtgagtg   Gccttcctac   Tgctcagatt   Ctgacgctac   Agcagtggat   360   Aaaccaacgc   Gcccagatgt   Ttcagtgaac   Aggttttaca   Cattggacac   Taaattgtgg   420   Gagaaatcgt   Ccaagggatg   Gtactggaag   Ttcccggatg   Tgttaactga   Aactggggtt   480   Tttgggcaaa   Atgcacaatt   Ccactacctc   Taccgatcag   Ggttctgcat   Ccacgtgcag   540   Tgcaacgcca   Gtaaattcca   Ccaaggagca   Ctcctagtcg   Ctgtcctacc   Agaggtgtc   600   Attgggacag   Tggcaggcgg   Tacagggacg   Gaagataccc   Acccccctta   Caagcagacc   660   Caacccggcg   Cggatggttt   Cgagttgcaa   Cacccgtacg   Tgcttgatgc   Tggcatccca   720   Atatcgcagt   Taacagtgtg   Cccacaccag   Tggattaatt   Tgaggaccaa   Caattgtgct   780   Acaataatag   Tgccatacat   Taacacactg   Cctttgtatt   Ctgccttgaa   Ccattgcaac   840   Tttggcctgt   Tagttgtgcc   Tattagccca   Ctagactacg   Accaaggagc   Gacgccagta   900   Atccctataa   Ctatcacatt   Ggccccaatg   Tgttctgaat   Tcgcaggtct   Taggcaggca   960   Gtcacgcaa   969       <210>   4   <211>   323   <212>   PRT   <213>   Enterovirus 71 (Enterovirus   71)     <400>   4   Met   Gly   Ser   Gln   Val   Ser   Thr   Gln   Arg   Ser   Gly   Ser   His   Glu   Asn   Ser   1   5   10   15   Asn   Ser   Ala   Thr   Glu   Gly   Ser   Thr   Ile   Asn   Tyr   Thr   Thr   Ile   Asn   Tyr     20   25   30   Tyr   Lys   Asp   Ser   Tyr   Ala   Ala   Thr   Ala   Gly   Lys   Gln   Ser   Leu   Lys   Gln     35   40   45   Asp   Pro   Asp   Lys   Phe   Ala   Asn   Pro   Val   Lys   Asp   Ile   Phe   Thr   Glu   Met     50   55   60   Ala   Ala   Pro   Leu   Lys   Ser   Pro   Ser   Ala   Glu   Ala   Cys   Gly   Tyr   Ser   Asp   65   70   75   80   Arg   Val   Ala   Gln   Leu   Thr   Ile   Gly   Asn   Ser   Thr   Ile   Thr   Thr   Gln   Glu     85   90   95   Ala   Ala   Asn   Ile   Ile   Val   Gly   Tyr   Gly   Glu   Trp   Pro   Ser   Tyr   Cys   Ser     100   105   110   Asp   Ser   Asp   Ala   Thr   Ala   Val   Asp   Lys   Pro   Thr   Arg   Pro   Asp   Val   Ser     115   120   125   Val   Asn   Arg   Phe   Tyr   Thr   Leu   Asp   Thr   Lys   Leu   Trp   Glu   Lys   Ser   Ser     130   135   140   Lys   Gly   Trp   Tyr   Trp   Lys   Phe   Pro   Asp   Val   Leu   Thr   Glu   Thr   Gly   Val   145   150   155   160   Phe   Gly   Gln   Asn   Ala   Gln   Phe   His   Tyr   Leu   Tyr   Arg   Ser   Gly   Phe   Cys     165   170   175   Ile   His   Val   Gln   Cys   Asn   Ala   Ser   Lys   Phe   His   Gln   Gly   Ala   Leu   Leu     180   185   190   Val   Ala   Val   Leu   Pro   Glu   Tyr   Val   Ile   Gly   Thr   Val   Ala   Gly   Gly   Thr     195   200   205   Gly   Thr   Glu   Asp   Thr   His   Pro   Pro   Tyr   Lys   Gln   Thr   Gln   Pro   Gly   Ala     210   215   220   Asp   Gly   Phe   Glu   Leu   Gln   His   Pro   Tyr   Val   Leu   Asp   Ala   Gly   Ile   Pro   225   230   235   240   Ile   Ser   Gln   Leu   Thr   Val   Cys   Pro   His   Gln   Trp   Ile   Asn   Leu   Arg   Thr     245   250   255   Asn   Asn   Cys   Ala   Thr   Ile   Ile   Val   Pro   Tyr   Ile   Asn   Thr   Leu   Pro   Phe     260   265   270   Asp   Ser   Ala   Leu   Asn   His   Cys   Asn   Phe   Gly   Leu   Leu   Val   Val   Pro   Ile     275   280   285   Ser   Pro   Leu   Asp   Tyr   Asp   Gln   Gly   Ala   Thr   Pro   Val   Ile   Pro   Ile   Thr     290   295   300   Ile   Thr   Leu   Ala   Pro   Met   Cys   Ser   Glu   Phe   Ala   Gly   Leu   Arg   Gln   Ala   305   310   315   320   Val   Thr   Gln       <210>   5   <211>   891   <212>   DNA   <213>   Enterovirus 71 (Enterovirus   71)     <400>   5   Ggagataggg   Tggcagatgt   Aattgaaagt   Tccataggag   Atagcgtgag   Cagagccctc   60   Actcacgctc   Taccagcacc   Cacaggccaa   Aacacacagg   Tgagcagtca   Tcgactggat   120   Acaggcaagg   Ttccagcact   Ccaagctgct   Gaaattggag   Catcatcaaa   Tgctagtgac   180   Gagagcatga   Ttgaaacacg   Ctgtgttctt   Aactcgcaca   Gtacagctga   Gaccactctt   240   Gatagtttct   Tcagtagggc   Gggattagtt   Ggagagatag   Atctccctct   Tgagggcaca   300   Actaacccaa   Atggttatgc   Caactgggac   Atagatataa   Caggttacgc   Gcaactgcgt   360   Gaagaaggtag   Agctattcac   Ctacatgcgt   Tttgatgcag   Agttcacttt   Tgttgcgtgc   420   Acacccactg   Ggggagttgt   Cccacaattg   Ctccaatata   Tgtttgtgcc   Acctggagcc   480   Cctaagccag   Attctaggga   Atcccttgca   Tggcaaaccg   Ctaccaaccc   Ctcagttttt   540   Gtcaagctgt   Cagaccctcc   Ggcgcaggtt   Tcagtgccat   Tcatgtcacc   Tgcgagtgct   600   Tatcaatggt   Tttatgacgg   Atatcccaca   Ttcggagaac   Acaaacagga   Gaaagatctt   660   Gaatacgggg   Catgtcctaa   Taacatgatg   Ggcacattct   Cagtgcggac   Tgtggggacc   720   Tccaagtcca   Agtaccctt   Aggtgttagg   Atttacatga   Gaatgaagca   Cgtcagggcg   780   Tggatacctc   Gcccgatgcg   Caaccagaac   Tacctattca   Aagccaaccc   Aaattatgct   840   Ggcaactcca   Ttaagccaac   Tggtgccagt   Cgcacagcga   Tcaccactct   t   891       <210>   6   <211>   297   <212>   PRT   <213>   Enterovirus 71 (Enterovirus   71)     <400>   6   Gly   Asp   Arg   Val   Ala   Asp   Val   Ile   Glu   Ser   Ser   Ile   Gly   Asp   Ser   Val   1   5   10   15   Ser   Arg   Ala   Leu   Thr   His   Ala   Leu   Pro   Ala   Pro   Thr   Gly   Gln   Asn   Thr     20   25   30   Gln   Val   Ser   Ser   His   Arg   Leu   Asp   Thr   Gly   Lys   Val   Pro   Ala   Leu   Gln     35   40   45   Ala   Ala   Glu   Ile   Gly   Ala   Ser   Ser   Asn   Ala   Ser   Asp   Glu   Ser   Met   Ile     50   55   60   Glu   Thr   Arg   Cys   Val   Leu   Asn   Ser   His   Ser   Thr   Ala   Glu   Thr   Thr   Leu   65   70   75   80   Asp   Ser   Phe   Phe   Ser   Arg   Ala   Gly   Leu   Val   Gly   Glu   Ile   Asp   Leu   Pro     85   90   95   Leu   Glu   Gly   Thr   Thr   Asn   Pro   Asn   Gly   Tyr   Ala   Asn   Trp   Asp   Ile   Asp     100   105   110   Ile   Thr   Gly   Tyr   Ala   Gln   Leu   Arg   Arg   Lys   Val   Glu   Leu   Phe   Thr   Tyr     115   120   125   Met   Arg   Phe   Asp   Ala   Glu   Phe   Thr   Phe   Val   Ala   Cys   Thr   Pro   Thr   Gly     130   135   140   Gly   Val   Val   Pro   Gln   Leu   Leu   Gln   Tyr   Met   Phe   Val   Pro   Pro   Gly   Ala   145   150   155   160   Pro   Lys   Pro   Asp   Ser   Arg   Glu   Ser   Leu   Ala   Trp   Gln   Thr   Ala   Thr   Asn     165   170   175   Pro   Ser   Val   Phe   Val   Lys   Leu   Ser   Asp   Pro   Pro   Ala   Gln   Val   Ser   Val     180   185   190   Pro   Phe   Met   Ser   Pro   Ala   Ser   Ala   Tyr   Gln   Trp   Phe   Tyr   Asp   Gly   Tyr     195   200   205   Pro   Thr   Phe   Gly   Glu   His   Lys   Gln   Glu   Lys   Asp   Leu   Glu   Tyr   Gly   Ala     210   215   220     Cys   Pro   Asn   Asn   Met   Met   Gly   Thr   Phe   Ser   Val   Arg   Thr   Val   Gly   Thr   225   230   235   240   Ser   Lys   Ser   Lys   Tyr   Pro   Leu   Val   Val   Arg   Ile   Tyr   Met   Arg   Met   Lys     245   250   255   His   Val   Arg   Ala   Trp   Ile   Pro   Arg   Pro   Met   Arg   Asn   Gln   Asn   Tyr   Leu     260   265   270   Phe   Lys   Ala   Asn   Pro   Asn   Tyr   Ala   Gly   Asn   Ser   Ile   Lys   Pro   Thr   Gly     275   280   285     Ala   Ser   Arg   Thr   Ala   Ile   Thr   Thr   Leu     290   295       <210>   7   <211>   762   <212>   DNA   <213>   Enterovirus 71 (Enterovirus   71)     <400>   7   Tccccatccg   Ctgaggcatg   Tggatacagt   Gatcgagtgg   Cgcaattaac   Tattggcaac   60   Tccaccatca   Ccacgcaaga   Agcggcaaac   Atcatagtcg   Gttatggtga   Gtggccttcc   120   Tactgctcag   Attctgacgc   Tacagcagtg   Gataaaccaa   Cgcgcccaga   Tgtttcagtg   180   Aacaggtttt   Acacattgga   Cactaaattg   Tgggagaaat   Cgtccaaggg   Atggtactgg   240   Aagttcccgg   Atgtgttaac   Tgaaactggg   Gtttttgggc   Aaaatgcaca   Attccactac   300   Ctctaccgat   Cagggttctg   Catccacgtg   Cagtgcaacg   Ccagtaaatt   Ccacacaagga   360   Gcactcctag   Tcgctgtcct   Accagagtat   Gtcattggga   Cagtggcagg   Cggtacaggg   420   Acggaagata   Cccacccccc   Ttacaagcag   Acccaacccg   Gcgccgatgg   Tttcgagttg   480   Caacacccgt   Acgtgcttga   Tgctggcatc   Ccaatatcgc   Agttaacagt   Gtgcccacac   540   Cagtggatta   Atttgaggac   Caacaattgt   Gctacaataa   Tagtgccata   Cattaacaca   600   Ctgccttttg   Attctgcctt   Gaaccattgc   Aactttggcc   Tgttagttgt   Gcctattagc   660   Ccactagact   Acgaccaagg   Agcgacgcca   Gtaatcccta   Taactatcac   Attggcccca   720   Atgtgttctg   Aattcgcagg   Tcttaggcag   Gcagtcacgc   Aa   762       <210>   8   <211>   254   <212>   PRT   <213>   Enterovirus 71 (Enterovirus   71)     <400>   8   Ser   Pro   Ser   Ala   Glu   Ala   Cys   Gly   Tyr   Ser   Asp   Arg   Val   Ala   Gln   Leu   1   5   10   15   Thr   Ile   Gly   Asn   Ser   Thr   Ile   Thr   Thr   Gln   Glu   Ala   Ala   Asn   Ile   Ile     20   25   30   Val   Gly   Tyr   Gly   Glu   Trp   Pro   Ser   Tyr   Cys   Ser   Asp   Ser   Asp   Ala   Thr     35   40   45   Ala   Val   Asp   Lys   Pro   Thr   Arg   Pro   Asp   Val   Ser   Val   Asn   Arg   Phe   Tyr     50   55   60   Thr   Leu   Asp   Thr   Lys   Leu   Trp   Glu   Lys   Ser   Ser   Lys   Gly   Trp   Tyr   Trp   65   70   75   80   Lys   Phe   Pro   Asp   Val   Leu   Thr   Glu   Thr   Gly   Val   Phe   Gly   Gln   Asn   Ala     85   90   95   Gln   Phe   His   Tyr   Leu   Tyr   Arg   Ser   Gly   Phe   Cys   Ile   His   Val   Gln   Cys     100   105   110   Asn   Ala   Ser   Lys   Phe   His   Gln   Gly   Ala   Leu   Leu   Val   Ala   Val   Leu   Pro     115   120   125     Glu   Tyr   Val   Ile   Gly   Thr   Val   Ala   Gly   Gly   Thr   Gly   Thr   Glu   Asp   Thr     130   135   140   His   Pro   Pro   Tyr   Lys   Gln   Thr   Gln   Pro   Gly   Ala   Asp   Gly   Phe   Glu   Leu   145   150   155   160   Gln   His   Pro   Tyr   Val   Leu   Asp   Ala   Gly   Ile   Pro   Ile   Ser   Gln   Leu   Thr     165   170   175   Val   Cys   Pro   His   Gln   Trp   Ile   Asn   Leu   Arg   Thr   Asn   Asn   Cys   Ala   Thr     180   185   190   Ile   Ile   Val   Pro   Tyr   Ile   Asn   Thr   Leu   Pro   Phe   Asp   Ser   Ala   Leu   Asn     195   200   205   His   Cys   Asn   Phe   Gly   Leu   Leu   Val   Val   Pro   Ile   Ser   Pro   Leu   Asp   Tyr     210   215   220     Asp   Gln   Gly   Ala   Thr   Pro   Val   Ile   Pro   Ile   Thr   Ile   Thr   Leu   Ala   Pro   225   230   235   240   Met   Cys   Ser   Glu   Phe   Ala   Gly   Leu   Arg   Gln   Ala   Val   Thr   Gln     245   250         <210>   9   <211>   726   <212>   DNA   <213>   Enterovirus 71 (Enterovirus   71)     <400>   9   Gggttcccca   Ccgagctaaa   Acctggcaca   Aatcaatttt   Taaccaccga   Tgatggcgtt   60   Tcagcaccta   Ttctaccaaa   Cttccacccc   Accccgtgta   Tccacatacc   Tggtgaagtt   120   Aggaacttgc   Tagagttatg   Ccaggtggag   Accattctgg   Aggttaacaa   Tgtgcccacg   180   Aatgccacta   Gcttaatgga   Gagactgcgc   Ttcccggtct   Cagcacaagc   Agggaaaggt   240   Gaactgtgtg   Cggtatttag   Agccgatcct   Gggcgaaatg   Gaccatggca   Atccacctta   300   Ctgggtcagt   Tgtgcgggta   Ctacacccaa   Tggtcaggat   Cattggaagt   Taccttcatg   360   Tttactggat   Ccttcatggc   Taccggcaag   Atgctcatag   Cctatacacc   Gccagggggt   420   Cctctgccca   Gggaccgggc   Gactgccatg   Ttgggcacgc   Atgtcatctg   Ggattttggg   480   Ctgcaatcgt   Ctgttaccct   Tgtaatacca   Tggatcagta   Atactcatta   Tagagcacat   540   Gcccgagatg   Gagtgtttga   Ctattacacc   Acagggttag   Tcagtatatg   Gtaccagaca   600   Aattacgtgg   Ttccaatcgg   Tgcgcccaac   Acagcctta   Taatagcact   Agcggcagcc   660   Caaaagaact   Tcactatgaa   Attgtgcaag   Gatgctagtg   Atatcctgca   Gacgggcacc   720   Atccag   726       <210>   10   <211>   242   <212>   PRT   <213>   Enterovirus 71 (Enterovirus   71)     <400>   10   Gly   Phe   Pro   Thr   Glu   Leu   Lys   Pro   Gly   Thr   Asn   Gln   Phe   Leu   Thr   Thr   1   5   10   15   Asp   Asp   Gly   Val   Ser   Ala   Pro   Ile   Leu   Pro   Asn   Phe   His   Pro   Thr   Pro     20   25   30   Cys   Ile   His   Ile   Pro   Gly   Glu   Val   Arg   Asn   Leu   Leu   Glu   Leu   Cys   Gln     35   40   45   Val   Glu   Thr   Ile   Leu   Glu   Val   Asn   Asn   Val   Pro   Thr   Asn   Ala   Thr   Ser     50   55   60   Leu   Met   Glu   Arg   Leu   Arg   Phe   Pro   Val   Ser   Ala   Gln   Ala   Gly   Lys   Gly   65   70   75   80   Glu   Leu   Cys   Ala   Val   Phe   Arg   Ala   Asp   Pro   Gly   Arg   Asn   Gly   Pro   Trp     85   90   95   Gln   Ser   Thr   Leu   Leu   Gly   Gln   Leu   Cys   Gly   Tyr   Tyr   Thr   Gln   Trp   Ser     100   105   110     Gly   Ser   Leu   Glu   Val   Thr   Phe   Met   Phe   Thr   Gly   Ser   Phe   Met   Ala   Thr     115   120   125   Gly   Lys   Met   Leu   Ile   Ala   Tyr   Thr   Pro   Pro   Gly   Gly   Pro   Leu   Pro   Arg     130   135   140     Asp   Arg   Ala   Thr   Ala   Met   Leu   Gly   Thr   His   Val   Ile   Trp   Asp   Phe   Gly   145   150   155   160   Leu   Gln   Ser   Ser   Val   Thr   Leu   Val   Ile   Pro   Trp   Ile   Ser   Asn   Thr   His     165   170   175   Tyr   Arg   Ala   His   Ala   Arg   Asp   Gly   Val   Phe   Asp   Tyr   Tyr   Thr   Thr   Gly     180   185   190   Leu   Val   Ser   Ile   Trp   Tyr   Gln   Thr   Asn   Tyr   Val   Val   Pro   Ile   Gly   Ala     195   200   205   Pro   Asn   Thr   Ala   Tyr   Ile   Ile   Ala   Leu   Ala   Ala   Ala   Gln   Lys   Asn   Phe     210   215   220   Thr   Met   Lys   Leu   Cys   Lys   Asp   Ala   Ser   Asp   Ile   Leu   Gln   Thr   Gly   Thr   225   230   235   240   Ile   Gln       <210>   11   <211>   207   <212>   DNA   <213>   Enterovirus 71 (Enterovirus   71)     <400>   11   Atgggttcgc   Aagtgtctac   Tcagcgctcc   Ggttctcacg   Aaaattcaaa   Ttcagccact   60   Gagggttcta   Ccataaacta   Caccaccatt   Aattactaca   Aagactccta   Cgctgccaca   120   Gcaggcaaac   Agagtctcaa   Gcaggatcca   Gacaagtttg   Caaatcctgt   Taaagacatc   180   Ttcactgaaa   Tggcagcgcc   Actgaag   207       <210>   12   <211>   69   <212>   PRT   <213>   Enterovirus 71 (Enterovirus   71)     <400>   12   Met   Gly   Ser   Gln   Val   Ser   Thr   Gln   Arg   Ser   Gly   Ser   His   Glu   Asn   Ser   1   5   10   15   Asn   Ser   Ala   Thr   Glu   Gly   Ser   Thr   Ile   Asn   Tyr   Thr   Thr   Ile   Asn   Tyr     20   25   30   Tyr   Lys   Asp   Ser   Tyr   Ala   Ala   Thr   Ala   Gly   Lys   Gln   Ser   Leu   Lys   Gln     35   40   45   Asp   Pro   Asp   Lys   Phe   Ala   Asn   Pro   Val   Lys   Asp   Ile   Phe   Thr   Glu   Met     50   55   60   Ala   Ala   Pro   Leu   Lys   65       <210>   13   <211>   862   <212>   PRT   <213>   Enterovirus 71 (Enterovirus   71)     <400>   13   Met   Gly   Ser   Gln   Val   Ser   Thr   Gln   Arg   Ser   Gly   Ser   His   Glu   Asn   Ser   1   5   10   15   Asn   Ser   Ala   Thr   Glu   Gly   Ser   Thr   Ile   Asn   Tyr   Thr   Thr   Ile   Asn   Tyr     20   25   30   Tyr   Lys   Asp   Ser   Tyr   Ala   Ala   Thr   Ala   Gly   Lys   Gln   Ser   Leu   Lys   Gln     35   40   45   Asp   Pro   Asp   Lys   Phe   Ala   Asn   Pro   Val   Lys   Asp   Ile   Phe   Thr   Glu   Met     50   55   60   Ala   Ala   Pro   Leu   Lys   Ser   Pro   Ser   Ala   Glu   Ala   Cys   Gly   Tyr   Ser   Asp   65   70   75   80   Arg   Val   Ala   Gln   Leu   Thr   Ile   Gly   Asn   Ser   Thr   Ile   Thr   Thr   Gln   Glu     85   90   95   Ala   Ala   Asn   Ile   Ile   Val   Gly   Tyr   Gly   Glu   Trp   Pro   Ser   Tyr   Cys   Ser     100   105   110   Asp   Phe   Asp   Ala   Thr   Ala   Val   Asp   Lys   Pro   Thr   Arg   Pro   Asp   Val   Ser     115   120   125   Val   Asn   Arg   Phe   Tyr   Thr   Leu   Asp   Thr   Lys   Leu   Trp   Glu   Lys   Ser   Ser     130   135   140   Lys   Gly   Trp   Tyr   Trp   Lys   Phe   Pro   Asp   Val   Leu   Thr   Glu   Thr   Gly   Val   145   150   155   160   Phe   Gly   Gln   Asn   Ala   Gln   Phe   His   Tyr   Leu   Tyr   Arg   Ser   Gly   Phe   Cys     165   170   175   Ile   His   Val   Gln   Cys   Asn   Ala   Ser   Lys   Phe   His   Gln   Gly   Ala   Leu   Leu     180   185   190     Val   Ala   Val   Leu   Pro   Glu   Tyr   Val   Ile   Gly   Thr   Val   Ala   Gly   Gly   Thr     195   200   205   Gly   Thr   Glu   Asp   Thr   His   Pro   Pro   Tyr   Lys   Gln   Thr   Gln   Pro   Gly   Ala     210   215   220     Asp   Gly   Phe   Glu   Leu   Gln   His   Pro   Tyr   Val   Leu   Asp   Ala   Gly   Ile   Pro   225   230   235   240   Ile   Ser   Gln   Leu   Thr   Val   Cys   Pro   His   Gln   Trp   Ile   Asn   Leu   Arg   Thr     245   250   255   Asn   Asn   Cys   Ala   Thr   Ile   Ile   Val   Pro   Tyr   Ile   Asn   Ala   Leu   Pro   Phe     260   265   270   Asp   Ser   Ala   Leu   Asn   His   Cys   Asn   Phe   Gly   Leu   Leu   Val   Val   Pro   Ile     275   280   285   Ser   Pro   Leu   Asp   Tyr   Asp   Gln   Gly   Ala   Thr   Pro   Val   Ile   Pro   Ile   Thr     290   295   300   Ile   Thr   Leu   Ala   Pro   Met   Cys   Ser   Glu   Phe   Ala   Gly   Leu   Arg   Gln   Ala   305   310   315   320   Val   Thr   Gln   Gly   Phe   Pro   Thr   Glu   Leu   Lys   Pro   Gly   Thr   Asn   Gln   Phe     325   330   335   Leu   Thr   Thr   Asp   Asp   Gly   Val   Ser   Ala   Pro   Ile   Leu   Pro   Asn   Phe   His     340   345   350   Pro   Thr   Pro   Cys   Ile   His   Ile   Pro   Gly   Glu   Val   Arg   Asn   Leu   Leu   Glu     355   360   365   Leu   Cys   Gln   Val   Glu   Thr   Ile   Leu   Glu   Val   Asn   Asn   Val   Pro   Thr   Asn     370   375   380   Ala   Thr   Ser   Leu   Met   Glu   Arg   Leu   Arg   Phe   Pro   Val   Ser   Ala   Gln   Ala   385   390   395   400   Gly   Lys   Gly   Glu   Leu   Cys   Ala   Val   Phe   Arg   Ala   Asp   Pro   Gly   Arg   Asn     405   410   415   Gly   Pro   Trp   Gln   Ser   Thr   Leu   Leu   Gly   Gln   Leu   Cys   Gly   Tyr   Tyr   Thr     420   425   430   Gln   Trp   Ser   Gly   Ser   Leu   Glu   Val   Thr   Phe   Met   Phe   Thr   Gly   Ser   Phe     435   440   445   Met   Ala   Thr   Gly   Lys   Met   Leu   Ile   Ala   Tyr   Thr   Pro   Pro   Gly   Gly   Pro     450   455   460   Leu   Pro   Lys   Asp   Arg   Ala   Thr   Ala   Met   Leu   Gly   Thr   His   Val   Ile   Trp   465   470   475   480   Asp   Phe   Gly   Leu   Gln   Ser   Ser   Val   Thr   Leu   Val   Ile   Pro   Trp   Ile   Ser     485   490   495   Asn   Thr   His   Tyr   Arg   Ala   His   Ala   Arg   Asp   Gly   Val   Phe   Asp   Tyr   Tyr     500   505   510   Thr   Thr   Gly   Leu   Val   Ser   Ile   Trp   Tyr   Gln   Thr   Asn   Tyr   Val   Val   Pro     515   520   525   Ile   Gly   Ala   Pro   Asn   Thr   Ala   Tyr   Ile   Ile   Ala   Leu   Ala   Ala   Ala   Gln     530   535   540   Lys   Asn   Phe   Thr   Met   Lys   Leu   Cys   Lys   Asp   Ala   Ser   Asp   Ile   Leu   Gln   545   550   555   560   Thr   Gly   Thr   Ile   Gln   Gly   Asp   Arg   Val   Ala   Asp   Val   Ile   Glu   Ser   Ser     565   570   575   Ile   Gly   Asp   Ser   Val   Ser   Arg   Ala   Leu   Thr   His   Ala   Leu   Pro   Ala   Pro     580   585   590   Thr   Gly   Gln   Asn   Thr   Gln   Val   Ser   Ser   His   Arg   Leu   Asp   Thr   Gly   Lys     595   600   605   Val   Pro   Ala   Leu   Gln   Ala   Ala   Glu   Ile   Gly   Ala   Ser   Ser   Asn   Ala   Ser     610   615   620   Asp   Glu   Ser   Met   Ile   Glu   Thr   Arg   Cys   Val   Leu   Asn   Ser   His   Ser   Thr   625   630   635   640   Ala   Glu   Thr   Thr   Leu   Asp   Ser   Phe   Phe   Ser   Arg   Ala   Gly   Leu   Val   Gly     645   650   655     Glu   Ile   Asp   Leu   Pro   Leu   Glu   Gly   Thr   Thr   Asn   Pro   Asn   Gly   Tyr   Ala     660   665   670   Asn   Trp   Asp   Ile   Asp   Ile   Thr   Gly   Tyr   Ala   Gln   Met   Arg   Arg   Lys   Val     675   680   685     Glu   Leu   Phe   Thr   Tyr   Met   Arg   Phe   Asp   Ala   Glu   Phe   Thr   Phe   Val   Ala     690   695   700   Cys   Thr   Pro   Thr   Gly   Gly   Val   Val   Pro   Gln   Leu   Leu   Gln   Tyr   Met   Phe   705   710   715   720   Val   Pro   Pro   Gly   Ala   Pro   Lys   Pro   Asp   Ser   Arg   Glu   Ser   Leu   Ala   Trp     725   730   735   Gln   Thr   Ala   Thr   Asn   Pro   Ser   Val   Phe   Val   Lys   Leu   Ser   Asp   Pro   Pro     740   745   750   Ala   Gln   Val   Ser   Val   Pro   Phe   Met   Ser   Pro   Ala   Ser   Ala   Tyr   Gln   Trp     755   760   765   Phe   Tyr   Asp   Gly   Tyr   Pro   Thr   Phe   Gly   Glu   His   Lys   Gln   Glu   Lys   Asp     770   775   780   Leu   Glu   Tyr   Gly   Ala   Cys   Pro   Asn   Asn   Met   Met   Gly   Thr   Phe   Ser   Val   785   790   795   800   Arg   Thr   Val   Gly   Thr   Ser   Lys   Ser   Lys   Tyr   Pro   Leu   Val   Val   Arg   Ile     805   810   815   Tyr   Met   Arg   Met   Lys   His   Val   Arg   Ala   Trp   Ile   Pro   Arg   Pro   Met   Arg     820   825   830   Asn   Gln   Asn   Tyr   Leu   Phe   Lys   Ala   Asn   Pro   Asn   Tyr   Ala   Gly   Asn   Ser     835   840   845   Ile   Lys   Pro   Thr   Gly   Ala   Ser   Arg   Thr   Ala   Ile   Thr   Thr   Leu     850   855   860

Claims (9)

An enterovirus 71 (Entrovirus 71, EV71) virus strain, which is deposited in the Bioresource Conservation and Research Center of the Food Industry Development Research Institute, and its registration number is BCRC970065. An enterovirus 71 (EV71) strain according to claim 1, which is a VP1 structural protein having an amino acid sequence as shown in SEQ ID NO: 6. The enterovirus 71 (EV71) virus strain according to claim 1, which is a VP2 structural protein having an amino acid sequence as shown in SEQ ID NO: 8. The enterovirus 71 (EV71) virus strain according to claim 1, which is a VP3 structural protein having an amino acid sequence as shown in SEQ ID NO: 10. An immunological composition comprising the enterovirus 71 (EV71) strain of the invention according to claim 1. The immunological composition of claim 5, wherein the immunological composition further comprises a pharmaceutically acceptable carrier. The immunological composition according to claim 5, further comprising at least one pathogenic antigen which is a Coxsackie virus, an Echovirus, a Poliovirus, an enterovirus (Enterovirus), Hepatitis B virus, Mycobacterium bovis, Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis, Haemophilus influenzae (Haemophilus influenza), varicella-zoster virus, Measles virus, Mumps virus, Rubella virus, Japanese encephalitis virus, rotavirus (Rotavirus) and Influenza virus. A polynucleotide comprising an amino acid sequence encoding a structural protein of the enterovirus 71 (EV71) strain of claim 1, wherein the amino acid sequence of the structural protein comprises at least the following One: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10. The polynucleotide of claim 8, wherein the sequence comprises at least one of the following: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.