TWI225095B - A strain of enterovirus 71, YN3-4a, is used to develop a vaccine - Google Patents

Abstract

The present invention relates to a virulent strain of enterovirus 71 (EV71), YN3-4a, for vaccine development which was selected from the wild type strain, neu. The YN3-4a strain has superior vaccine features, such as high viral yield, strong immunogenicity, broad-based antigenic coverage and passage stability. The YN3-4a strain was more suitable for the development of an inactivates viral vaccine than neu strain.

Description

Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1225095 A7 __B7 V. Description of the invention (丨) The present invention relates to an autoimmune enterovirus virus mother strain (like 0), which can be used in the preparation of 71 enterovirus vaccine. Mutant virus strains and their special screening methods. 1W3-For example, the mutant virus strain has higher virus production, stronger immune-inducing ability, broader antigen-shielding ability and progeny stability than the parent strain (wew). This feature is more suitable for producing enterovirus-type inactivated enterovirus vaccines than its parent strain. Enteroviruses are classified virologically as belonging to the enterovims in the family picomaviriade. It is a group of RNA viruses' only 20-30 nm in size, with a total of 67 types of blood sacrifice, including poliovirus—a total of types 1 to 3, and Coxsackievirus A—a total of 23 types, that is, types 1 to 24 but not including type 23 ( Type 23 is reclassified as Echovirus 9), Coxsackievirus B—type 6 in total, Echovirus—type 31 in total, ie types 1 to 34 'but excluding types 8, 10, 28 and newly discovered Enterovims—type 4 in total, ie 68 to 71 models. The size of the enterovirus is about 30 nm. The structure is an icosahedron. It is a single-stranded RNA with no envelope and only the outer v-capsid (capsid), about 7.4Kb, plus strain—that is, Without the need of reverse transcription enzymes, only RNA-dependent RNA Polymerase is required to directly produce its own RNA. According to epidemiological studies, the incidence of enterovirus 71 infection has increased significantly in the Asia-Pacific region in recent years. Patients infected with type 71 enterovirus often experience severe brainstem encephalitis and complex pulmonary edema and other lesions. This often results in a very high probability of death for patients. Therefore, the development of vaccines has become a type 71 enterovirus. One of the important jobs of infection. In general, vaccines developed against whole strains of viruses mainly include live attenuated vaccines and inactivated vaccines, and standard manufacturing techniques for these two vaccines have been established. This makes it possible to use 4 paper sizes in China in accordance with China National Standard (CNS) A4 (210 X 297 mm) 45¾ (Please read the precautions on the back before filling this page) -------- Order --------- line. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, 1225095 V. Invention Description (>) The vaccine used to control diseases will be more efficient. However, some attenuated vaccines themselves are potentially viruient revertant 'e.g.' yellow fever vaccines. Although long-term safety reports have been used, their toxicity reversion mutations are still heard. Virological studies and clinical observations have revealed that many characteristics of enterovirus 71 are highly similar to poliovirus. Therefore, the model established for the development of poliovirus vaccines in the past should be effective in preventing and treating enterovirus 71 infection. However, since oral polio vaccines also have the potential for virulence back mutations, coupled with the current high levels of similarity and variability of various types of enterovirus strains, this has led to the development of type 71 enterovirus vaccines. When inactivated virus vaccine should be a more suitable choice. On the other hand, although the use of a deactivated virus vaccine is relatively safe, the immune response it can induce is also relatively weak, resulting in the need to use higher virus antigen doses in actual application in order to activate the immune system to make it Achieving acceptable immunity. According to the foregoing considerations, the first step in developing a deactivated vaccine is to find a strain that can activate the immune system and cause a stronger immune response in the host at a lower dose (high immune induction, Extensive antigen shielding). In the process of in vitro culture preparation, such a virus strain also needs to have the ability to grow in host cell lines, stable genetic traits (progeny inheritance stability), and the ability to generate high viral titer (high virus production). Quantity), so that it can be used in the production of inactive vaccines. The object of the present invention is to provide a type 71 enterovirus strain with progeny passage stability, high immune-inducing ability, extensive antigen shielding properties and high virus production characteristics, and a special screening method thereof. It is used for the development of a highly immune-inducible enterovirus 71 inactivated vaccine. This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) (Please read the precautions on the back before filling this page) Order --------- line · 1225095 Α7 Β7 V. Invention Explanation (q) Schematic description (please read the precautions on the back before filling this page). Figure 1: 1W of Enterovirus 71 and "The Origin and Evolution of Subgeneration from Virus Strains. All strains of the W strain were given a number to indicate the selected plaque, and the next two digits were used to indicate the number of passages. Figure 2: Kinetics of the virus infecting Vero cells at different multiplicity of infection (MOI) 〇 The viral force in the culture medium ((·) extracellular force) and the resuspension of the cells in the original volume Viral valence in the culture medium ((0) represents intracellular valence) was measured separately. The virus yields at the infection rates of 0.1 (a and b), 1 (c and d), and 5 (e and f) are shown in the figure, respectively. Figure 3: Survival rates of neonatal ICR mice infected with the prion virus. ⑻ mice are injected intraperitoneally with a dose of 106 TCID5G ml · 1 (the highest viral power available) of the prion mother strain; (b) mice are injected intraperitoneally with 102 to 106 TCID50ml · 1) W3- Such as virus strains. Figure 4: The performance of their respective growth kinetics when the strains were infected with Vero cell lines (M0I = 1) and cultured in media containing blood maggots and blood maggots that were printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. DMEM medium containing 2% fetal bovine blood pupa (FBS); (b) DMEM medium containing B-27 blood pupa auxiliary. All (_); extracellular (·); intracellular (0), the viral power is expressed as TCIDsomr1. Figure 5: Comparing the growth curves of the 4th, 6th, 9th, and 12th generations of 1TO A virus strains infected with Vero cell lines (M0I = 1). The size of this paper is applicable to Chinese National Standard (CNS) A4 (210 X 297 mm). ^ ^ 1225095 Α7 Β7 V. Description of the invention u) Implementation of the county body Culture and preservation of fine maggot strains and virus strains (please read first (Notes on the back then fill out this page)

Vero cell line (ATCC CCL81, green monkey kidney cell) culture: The methods and conditions for subculture and preservation are based on the ATCC (American Type Culture Collection) standard culture preservation method for Vero cell lines. The culture medium is DMEM (Dulbecco's modified Eagle medium) containing 5% fetal bovine serum (FBS). ◦ Culture and storage of the virus strain: Firstly, the Vero cell line is first cultured in DMEM medium containing 5% fetal bovine serum. In order to achieve a cell density (confluence) of about 80%. The virus strain to be cultured is adjusted to have a multiplicity of infection (MOI) of between 0.1 and 0.5, and then with the previously cultured monolayer Vero cell line, in a DMDM medium containing 2% fetal bovine blood pupa , 5% C02 and 37 ° C. The Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs printed a microscope to observe that when more than 80% of Vero cell lines were cultured with cytopathic effect (CPE) of enterovirus infection, the infected Vero cell lines in the culture medium, After "freeze-thaw" treatment (freeze at -70 ° C for 5 minutes, then move to 37 ° C water bath to thaw for 7 minutes) twice to destroy the cell membrane of the cell line and contain the virus contained in it Strains are released. Centrifuge at 2,000xg for 6 minutes. The precipitated cell debris was removed, and the virus stored in the supernatant was collected and stored at -70 ° C or liquid nitrogen for long-term storage. The method for screening and preparing virus strains includes the following steps: 1. The histopathological specimens of patients infected with type 71 enterovirus are grouped into 7 groups of paper at room temperature in accordance with Chinese National Standard (CNS) A4 (210 X 297) (Centi) 4 Μ 1225095 Α7 Β7 V. Description of the invention (ί) Weaving is placed in a MEM medium (modified minimal essential medium) containing 2% fetal bovine blood cymbal to fully tissue the tissue with a homogenizer (please read the back first) Note: Please fill in this page again) After grinding, it becomes a sample suspension. 〇 2. MRC5 cell line (ATCC CCL171) was cultured in MEM medium containing 10% fetal bovine blood pupa. 3. Remove the original medium of the above MRC5 cell line, rinse it once with PBS phosphate-buffered saline (pH 7.2), and place it in a 6-well tissue culture plate (105 cells /? L), add 2 Ml of MEM medium containing 2% fetal bovine blood maggot and 0.5 ml of the suspension of the pathological specimen in step 1 were cultured at 37 ° C. 4. Observe with a microscope, when more than 50% of the cell lines are cytopathic, collect the cell lines in the wells and process them with "freeze-thaw" (at -70 ° C Freeze in the freezer for 5 minutes, then move to a 37 ° C water bath to thaw for 7 minutes.) Treat twice to break the cells. 5. Centrifuge at 4,500 rpm for 5 minutes. The supernatant liquid was taken to obtain type 71 enterovirus virus strain, which was used as the mother virus strain for further screening. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 6. Take 1 ml of the supernatant of the mother strain containing the virus in step 5 and add 1 ml to a single layer of Vero cell line (green monkey kidney cells, ATCC CCL81, co-cultured in DMEM medium (Dulbecco's modified Eagle medium) (5 ml / well) containing 2% fetal bovine blood maggot, 5% CO2 and 37 ° C. 7. Observe with a microscope, When more than 80% of the cell lines show a cytopathic phenomenon, collect the cell lines in the wells and treat them twice with the aforementioned "freeze-thaw" treatment process to rupture the cells. 8. At 2,000 rpm Centrifugation speed for 6 minutes. This paper size is in accordance with China National Standard (CNS) A4 (210 X 297 mm). Χ »i * · α 5 η 1225095 A7 B7 V. Description of the invention (b) (Please read first (Notes on the reverse side, please fill in this page again) 9. Take 1 ml of the supernatant of the virus strain contained in step δ with a micropipette and a single layer of Vero cell line cultured in a well culture dish. DMEM medium (5 ml / well), 5% C02 and 37 ° C Co-cultivation in the environment. 10. Repeat steps 7 to 9 to passage the parent culture of the virus. 11. After 2 to 4 subcultures on the Vero cell line, remove from the resulting plaque Viral mother strain, adjust its multiplicity of infection (MOI) between 0.001 ~ 0.0001, and work with Vero cell line in DMEM medium containing 2% fetal bovine blood 淸, 5% C02 and 37 ° C. 12. After the virus strain of the previous step was prolonged to 21 to 30 days, the gene phenotype (the appearance of the lysolytic plaque) from the plaque generated was different from that of the plaque. A variant virus strain of its mother virus strain. 13. The mutant virus strain was subjected to the aforementioned "freeze-thaw" process to rupture the cells. Centrifuge at 2,000 rpm for 6 minutes. 14. Take 1 ml of the supernatant solution containing the mutant virus strain from step 13 and add it to a single layer of Vero cell line cultured in a 6-well culture plate in a micropipette. /? L), 5% CO2 and 37 ° C. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 15. Observe with a microscope. When more than 80% of the cell lines show cytopathic phenomena, collect the cell lines in the wells ^ 16. Repeat steps 13 to 15 to make the mutant virus strain proliferation. 17. The selected mutant virus strains were tested for their virus titers by viral titer analysis to confirm that their viral titers were higher than their original virus strains. 18. The mutant virus strain after the expansion culture will be adjusted to its virus infection rate so that the paper size is in accordance with the Chinese National Standard (CNS) A4 (210 X 297 mm) 4¾¾ 1225095 A7 ___________ B7 V. Description of the Invention (Smart) MOM, Co-cultured with Vem cell line in 2% fetal bovine blood 胎 DMEM medium, 5% CO2 and 37 ° C culture environment. Within 48 hours, the first mutant virus strain isolated from the host cell was isolated from the culture medium. 19. Confirm that the mutated virus strain isolated in step 18 is superior to the virus mother strain isolated from the pathological specimen in terms of virus power, growth curve, antibody neutralization power, and virus toxicity analysis. 20. The mutant virus strain obtained in step 18 is subcultured in the same manner as described above for the culture of the virus strain. 2ι. The growth progeny of the virus obtained in step 20 and the mutant virus strain obtained in step 18 are measured and analyzed for growth kinetics, gene sequence, and antibody neutralization to confirm step 18 The mutant virus strains purified in this method still maintained high virus yield and had stable genetic traits in subsequent subcultures. Viral titration measurement The viral titer measurement in the present invention is measured by the conventional Reed and Muench method. This assay uses the lowest virus dilution concentration that can cause more than 50% of Vero cell lines to exhibit typical enterovirus cytopathic phenomena as its measurement of viral power, which is called TCID50 (50% tissue culture infection dose; 50% tissue culture infective dose). The determination steps are as follows: DMEM medium containing no fetal bovine blood pupa is used as a dilution solution, and the tested virus strain is serially diluted ten times. A single layer of Vero fine in a 96-well plate. This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (Please read the precautions on the back before filling this page) 0 --- — — — — Order --------- line. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Printed by 1225095 A7 B7 V. Invention Description (纩) The cell line (104 cells / well) was co-cultured in DMEM medium containing 2% fetal bovine blood maggot, 5% C02, and 37X: in a culture environment. After culturing, the Vem cell line was infected with the virus strain, and the result of typical enterovirus cytopathic phenomenon was observed under the microscope to monitor the growth of the virus strain. When the typical enterovirus cytopathic phenomenon produced by the Vero cell line in the dish no longer increases (about 4-7 days), at this time, more than 50% of the Vero cell lines in the well present the lowest virus when the typical enterovirus cytopathic phenomenon occurs. The dilution concentration is the determination of TaD50 ml · 1. Virus growth analysis. Monolayer Vero cell lines were cultured in 24-well tissue culture plates (105 cells / well) in DMEM medium containing 2% fetal bovine blood maggots, respectively, with virus strains with different virus infection rates at 37 ° C. Co-culture for 1 hour. The original medium in the plate was replaced with 500 μl of fresh DMEM medium containing 2% fetal bovine blood pupa, and then co-cultured at 5% CO 2 and 37 ° C. At each measurement time point in the growth curve, collect all contents (including cell lines and culture medium) in the wells and centrifuge at 2,000 g for 6 minutes. The virus stored in the supernatant solution was collected and stored at -7 ° C to facilitate the subsequent determination of viral titer, which is the extracellular viral titer. In addition, the above-mentioned centrifugal pellet contains a culture medium and a cell residue ' The same volume of medium as the original medium is resuspended in it. Then, through the aforementioned freeze-thaw, the treatment process is processed twice to break the cell membrane 11 ___ This paper size applies Chinese national standards (CNS) A4 specification (210 X 297 mm) tree. (Please read the notes on the back before filling this page) 0 Order --------- line · 1225095 A7 B7 V. Description of invention (A) Centrifuge at 2,000xg for 6 minutes. Remove the deposited cell debris, collect the virus stored in the supernatant, and store it at -70 ° C for subsequent determination of extracellular virus power, which is the cell. Viral titer. All viral titers are measured and analyzed in the same batch by the viral titer analysis method. The process of stalk, body neutralization, and [(neutralization) test is performed using conventional enterovirus antibodies. And test method, which The general test procedure is as follows: Virus protein is purified by the conventional sucrose gradient method. Enterovirus 71 strain was infected with Vero cell strain at an infection rate of M0I = 1, and contained 2% fetal bovine blood.共同 DMEM medium, 5% C02, and 37 ° C. Co-culture under a culture environment. Observe with a microscope, when more than 50% of Vero cell lines exhibit typical enterovirus cytopathic phenomena, collect cells and medium. Use "" ; Freeze-thaw (freeze-thaw) process (frozen in a freezer at _70 ° C for 5 minutes, and then moved to a 37 ° C water bath to thaw for 7 minutes) twice to rupture the cells. 4,500 Centrifuge at rpm for 5 minutes, and take the supernatant containing the virus. Centrifuge at 40,000 rpm for 4 hours. After removing the supernatant, use 4 ° C TES buffer (0.01M Tris (pH 7) .2) + 0,002Mm) TA + (X15M sodium chloride) rinse the precipitate. After that, suspend it in TES buffer solution. Under the room temperature, centrifuge at 12,000 rpm, 12 paper scales suitable for China National Standard (CNS) A4 (210 X 297 mm) 4¾¾ (please first Read the notes on the back and fill in this page again) 0 IIIII 1 Order! III I-* 3 ^. Printed by the Employees 'Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1225095 A7 B7 Printed by the Employees' Cooperatives of the Ministry of Economic Affairs Note (π) for 4 minutes. Take 2 ml of the supernatant obtained in the previous step and add it to a 65%, 35%, and 10% discontinuous sucrose gradient at 35,000 rpm at 4 ° C. Centrifuge at 4 rpm. Collect a virus with a gradient between 35% and 10%. Add a 10-fold volume of PBS phosphate buffered saline (PhosPhate-buffered saline, pH 7.2) to obtain a virus solution. The virus solution was centrifuged at 4,500 rpm for 30 minutes, and the supernatant was collected. The supernatant solution was passed through an amicomY-10 column to condense the virus solution to obtain a purified viral protein. The purified viral protein was deactivated by heating at 56 ° C for 30 minutes. BALB / c female mice aged 4 to 6 weeks were intraperitoneally (i.p.) 10 pg of viral protein purified from the previous step. After four weeks, the above steps were repeated one more time to make the female mice have an immune response. One week after the second intraperitoneal injection, blood was collected from the tail of the injected female to collect blood. The collected blood was centrifuged at a centrifugal speed of 2,000 g for 10 minutes, and the mash was collected to obtain anti-blood mash. The obtained anti-blood pupa was heated at 56 ° C for 30 minutes. Then, DMEM medium containing no fetal bovine pupa was used as the diluent, and it was diluted 2 times (1 to 212). The diluted sample was placed in a DMEM medium (containing 2% fetal bovine blood pupa) of the same volume containing 100 TCID5Q virus dose and cultured at 4 ° C overnight. 13 This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) 4 ^ 4 (Please read the notes on the back before filling this page) 0 Order --------- line. 1225095 A7 B7 V. Description of the invention (Λ) The above-mentioned virus / anti-blood mixture was added to a 96-well tissue culture plate (104 cells / well) containing a single-layer Vero cell line (containing 2% fetal bovine Fresh DMEM medium of Hemophilus sibiricum) and incubate at 37 ° C for 1 hour. Remove the culture medium from the plate. Then, 100 μl of fresh DMEM medium containing 2% fetal bovine blood pupa was injected into the wells of the culture plate, and co-cultured at 5% CO 2 and 37 ° C. Observed with a microscope, when the typical enterovirus cytopathic phenomenon produced by the Vero cell line in the dish is no longer increased (about 4-7 days), at this time, less than 50% of the Vero cell line in the well can produce the typical enterovirus The highest anti-blood dilution factor of cytopathic phenomenon is the anti-blood force of the virus strain. Virulence analysis The measurement of the virus strain LD5G (Lethal dose) was performed using the conventional Reed and Muench method. The general operation steps are as follows: The virus strains are injected into the abdominal cavity of ICR newborn mice at different virus dose concentrations (100 μl containing 102 to 106 TCID5G), respectively. Observe and record the occurrence of paralysis and death of the hind limbs of mice every time, and draw the curve of the animal lethality caused by each dilution to find out the endpoint virus dilution concentration that caused 50% of the animal death. Concentrations are sufficient to cause death in 50% of animals. Based on this, the LD5Q analysis number of the virus strain was determined and calculated. Gene sequencing. Virus strains were first cultured in Vero cell lines and 14% containing 2% fetal bovine blood maggots. This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 4¾ (Please read the notes on the back first (Fill in this page again)-! Order 1 --------- Line. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1225095 A7 ________ B7 V. Description of the Invention (Λ >) In DMEM medium, reuse it on the market A readily available RNA (RNA) extraction kit (QIAamp viral RNA mini kit, Qiagen Inc., Santa Clara, CA) extracts viral RNA from the culture medium. Using the viral RNA as a template, the reverse transcription-polymerase chain reaction (RT-PCR) technology commonly used in general laboratories is used to copy the full-length virus gene into several fragments, and then directly enter the enterovirus gene. Sequence analysis (using a fully automatic analyzer). The untranslated region (UTR) sequences at the 3'- and 5 'ends of the virus use the commercially available 5' Race and 3 'Race system (GIBCO BRL Products) to tag the nucleotide sequence. At the tail of the full-length virus cDNA, this sequence is marked for PCR amplification and automatic sequence analysis (ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit, Pcrkin_Elmer Inc., CA). Sequence analysis was performed when the target was prime annealing. Specific Examples Example 1 Isolation and production of iW virus line 1. A histopathological specimen of a patient infected with enterovirus 71, the tissue was suspended in a MEM medium containing 2% fetal bovine blood cymbal at room temperature and a tissue honing machine was used. The tissue was fully ground into a suspension of pathological specimens. 2. MRC5 cell line previously cultured in MEM medium containing 10% fetal bovine blood pupa, after removing the culture medium, rinse it once with PBS phosphate-buffered saline (pH 7.2), and then place it in 6 wells Tissue culture dish. Add 2 ml of MEM medium containing 2% fetal bovine blood maggot to the well, and 0.5 ml of the pathological specimen suspension in the above steps. '15 This paper size applies to the Chinese National Standard (CNS) A4 (210 X 297 mm) 4 % (Please read the notes on the back before filling out this page) 0 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1225095 A7 B7 V. Description of the invention (A) The culture was carried out at 37 ° C. (Please read the precautions on the back before filling this page) 3. Observe with a microscope, when more than 50% of the cell lines show cytopathic phenomenon, collect the cell lines in the wells and place them in a freezer at -7 ° C Freeze for 5 minutes, then move to a 37 ° C water bath for 7 minutes, and then freeze and thaw again under the same conditions to destroy the cell membrane and release the virus strain in the cells. 4. Centrifuge at 4,500 rpm for 5 minutes. The supernatant was taken to obtain type 71 enterovirus virus strain, which was used as the mother virus strain for further screening, and was named here as the 〃ew strain. 5. Take 1 ml of the above-mentioned supernatant from the virus mother strain with a micropipette and a single layer of Vero cell line (green monkey kidney cells, ATCC CCL81) cultured in a 6-well culture plate in 2% fetal bovine The blood pupa was co-cultured in DMEM medium (5 ml / well), 5% 0) 2 and 37 ° C. 6. Then observe with a microscope. When more than 80% of the cell lines show cytopathic symptoms, collect the cell lines in the wells and treat them twice in the "freeze_thaw" process in step 3. 7. Centrifuge at 2,000 rpm for 6 minutes. The supernatant liquid was taken, and the virus strain contained therein was referred to herein as the parent virus strain (the first generation in the Vero cell line). Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs8. Take 1 ml of the supernatant of the mother strain containing the virus in step 7 and add 1 ml to a single-layer Vero cell line cultured in a well culture dish. S order contains 2 Co-cultured in DMEM medium (5 ml / well) of 5% fetal bovine blood maggot, 5% CO2 and 37 ° C. 9. Repeat steps 6 to 8 for passage cultures of the parent virus strain. After subculture, the virus mother strain (second generation), virus mother strain (third generation), "_ virus mother strain (fourth generation), and virus mother strain are applicable to Chinese national standards ( CNS) A4 specification (210 X 297 mm) 4 ^ 7 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1225095 A7 ___B7_ V. Description of the invention (A) (fifth generation) and virus mother strain (sixth generation) . (Figure 1) 10. Viral titer analysis was used to determine the viral titer of each parent virus. 11. After 2 passages on the Vero cell line, the virus mother strain (third passage) taken from the generated plaque was adjusted to adjust the virus infection rate ΜΟΜΙ.ΟΟΙ, and the Vero cell line contained 2% fetus. Bovine serum and DMEM medium containing 0.3% agar gum were co-cultured in a culture environment of 5% CO 2 and 37 ° C. 12. After prolonging the culture time to 21 days, 3 plaques were obtained. The mutant virus strains isolated from these three plaques were later named lineages, respectively, KW and 1W5. 13. The} W strain mutant virus strain cultured in the Vero cell line was treated twice with the aforementioned "freeze-thaw" processing procedure. Centrifuge at 2,000 rpm for 6 minutes. 14. Take 1 ml of the supernatant liquid containing the virus strain from step 13 and add it to a single layer of Vero cell line cultured in a 6-well culture plate in a micropipette in DMEM medium (5 ml / Wells), 5% CO2 and 37 ° C. 15. Observe with a microscope, when more than 80% of the cell lines exhibit cytopathic phenomena, collect the cell lines in the wells. 16. Repeat steps 13 to 15 for the mutant virus strain to proliferate. The descendants obtained after repeating this step twice are the third generation), layer d (the third generation of the layer, the muscle 3 (the third generation of the soup). W3-3 is the next generation. Available. 17. When measuring the viral titer of the virus by the aforementioned viral titer method, I found that] strain W (please read the precautions on the back before filling this page) ------- order ------ --- Line · National Standard (CNS) A4 specification (210 X 297 mm) 1225095 A7 B7 V. Description of the variant virus strain (λ <), its maximum viral strength is different from its parent virus strain (Table- ") 〇18 · Take the KV34 mutant virus strain, adjust its virus infection rate MOI = 1, and Ver〇 cell strain in DMEM medium containing 2% fetal bovine serum, 5% CO2 and 371: Co-culture under environmental conditions. Within 24 hours, the earliest virus strain released from the host cell was isolated from the culture medium and named it YN3-4a. 19. The KV34 mutant virus strain was determined according to the aforementioned method Analyze virus titer, growth curve, gene sequence, antibody neutralizing titer, and virus toxicity, etc. 20 · IWKa variant virus strains, specific The subculture of the virus strain in the usual way is carried out. 21 · Take the 6th, 9th (ZA ^^ P) and 12th (1W3 ± 72), and the mutant virus strains. 'Analyze and determine its growth kinetics, gene sequence, and antibody neutralization to confirm that the screened and purified H-cardiac variant virus strains still maintain high virus yield and inheritance in subsequent subcultures. Stable characteristics, etc. 22. The YN3-4a mutant virus strain isolated in step 18 and the ^ / 5 virus mother strain obtained in step 9 were replaced on July 3, 1991 and July 16, 1991. Deposited at the Food Industry Development Research Institute, the registration numbers are CCRC970033 and CCRC970034. This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 4¾¾ (Please read the precautions on the back before filling in this page) ------- Order ----- I --- Line · Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1225095 A7 B7 V. Description of the invention (&) Table 1 Different virus strains grow on Vero cell lines Virus (MOM)-Viral strain intracellular Maximum extracellular viral force 'YN3-4a-2 1 (TCID5〇mr1) 2.5 x 109 1.0 χίο6 YN4-2 6.4 χ 107 1.0 χ ίο6 YN5-2 2.0 χ ΙΟ7 3.2 χ 1〇5 YN3-3 2.0 χ ΙΟ7 3.2 χ 10.5 Neu5 1.0 χ 10 06 3.2 χ 10 5 a Data are taken from the number at the time when virus production was highest in the growth curve. Example 2 Gene sequence analysis and comparison of virus strains The known mutant virus strains obtained in Example 1 and "from 5 virus mother strains, according to the gene sequencing method described in the specific embodiment, were used to carry out the full length virus genes of the virus strains Sequencing analysis. After sequencing analysis, it was found that the mutant virus strain had a full-length nucleotide sequence of 7414 bases and a translation amino acid sequence consisting of 2,193 amino acids. Its complete sequence is not shown in the table. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) Table 2 cDNA nucleotide sequence of 1W3-cardiac mutation virus strain and its translated amino acid sequence ITO-cardiac mutation virus CDNA nucleotide sequence

1 TTAAAACAGC CTGTGGGTTG CACCCACCCA CAGGGCCCAC TGGGCGTTAG 51 CACTCTGGTA CTGAGGTACC TTTGTGCGCC TGTTTTTACT CCCCTTCCCC 101 CGAAGTAACT TAGAAGCTGT AAATCAACGA TCAATAGCAG GTGTGGCACA 151 CCAGTCATAC CTCGATCAAG CACTTCTGTT TCCCCGGACT GAGTATCAAT 201 AGGCTGCTCG CGCGGCTGAA GGAGAAAACG TTCGTTACCC GACCAACTAC present paper applies the Chinese national standard scale (CNS) A4 size (210 X 297 mm) Economy Printed by the Consumer Cooperatives of the Ministry of Intellectual Property Bureau 1225095 A7 ____ Β7_ V. Description of Invention (A)

251 TTCGAGAAGC TTAGTACCAC CATGAACGAG GCAGGGTGTT TCGCTCAGCA 301 CAACCCCAGT GTAGATCAGG CTGATGAGTC ACTGCAACCC CCATGGGCGA 351 CCATGGCAGT GGCTGCGTTG GCGGCCTGCC CATGGAGAAA TCCATGGGAC 401 GCTCTAATTC TGACATGGTG TGAAGAGCCT ATTGAGCTAG CTGGTAGTCC 451 TCCGGCCCCT GAATGCGGCT AATCCCAACT GCGGAGCACA TGCTCACAAA 501 CCAGTGGGTG GTGTGTCGTA ACGGGCAACT CTGCAGCGGA ACCGACTACT 551 TTGGGTGTCC GTGTTTCCTT TTATTCCTAT ATTGGCTGCT TATGGTGACA 601 ACCAAAGAGT TGTTACCATA TAGCTATTGG ATTGGCCATC CGGTGTGCAA 651 CAGGGCAATT GTTTACCTAT TTATTGGTTT TGTACCATTA TCACTGAAGT 701 CTGTGATCAC TCTCAAATTC ATTTTGACCC TCAACATAAT CAGACATGGG 751 CTCACAGGTG TCCACACAAC GCTCCGGTTC GCACGAAAAC TCTAACTCAG 801 CTACCGAGGG TTCCACTATA AACTATACTA CCATTAATTA CTATAAAGAT 851 TCCTATGCCG CCACAGCAGG TAAGCAGAGC CTTAAGCAGG ACCCAGACAA 901 GTTTGCAAAT CCTGTCAAAG ACATCTTCAC TGAAACGGCA GCGCCATTAA 951 AATCTCCATC TGCTGAGGCG TGTGGTTACA GCGATCGGGT GGCACAATTA 1001 ACTATTGGC A ATTCTACC AT CACTACGC AA G AAGCAGC AA AC ATC ATAGT 1051 TGGCTATGGT GAGTGGCCTT CCTACTGTTC GGACTCTGAT GCTACTGCAG 1101 TGGACAAACC AACGCGCCCA GATGTTTCGG TGAATAGGTT TTACACATTG 1151 GACACAAAAC TGTGGGAGAA ATCATCCAAG GGGTGGTACT GGAAATTCCC 1201 GGATGTGTTA ACTGAAACCG GGGTCTTTGG TCAAAATGCA CAGTTCCACT 1251 ACCTCTATCG GTCAGGGTTC TGCATTCACG TGCAGTGCAA TGCCAGTAAG 1301 TTCCACCAAG GAGCACTCCT AGTCGCTGTC CTCCCAGAGT ATGTCATTGG 1351 GACAGTGGCA GGTGGCACAG GGACGGAGGA TAGCCACCCC CCTTATATGC 1401 AGACTCAACC CGGTGCTGAT GGCTTCGAAT TGCAACAGCC GTACGTGCTT 1451 G ATGCTGGCA TTCC AATATC ACAATTAACA GTGTGCCCAC ATCAGTGG AT 1501 TAATTTGAGG ACCAACAATT GTGCCACAAT AATAGTGCCG TACATAAACG 1551 CACTACCCTT TGATTCTGCC TTGAACCATT GTAACTTTGG TCTGCTGGTT 1601 GTGCCTATTA GCCCGTTAGA TTATGACCAA GGTGCGACGC CAGTGATCCC 1651 CATTACTATC ACCTTGGCCC CAATGTGTTC TGAATTTGCA GGCCTTAGAC 1701 AAGCAGTTAC GCAAGGGTTT CCTACTGAGC TGAAACCTGG CACAAACCAA 1751 TTTTTAACCA CTGACGATGG CGTCTCAGCA CCCATTCTGC CAAACTTTCA 1801 CCCCACCCCG TGTATCCATA TACCCGGTGA AGTTAGAAAC TTGCTAGAGC 1851 TATGCCAGGT GG AG ACC ATT TTAGAGGTCA ACAATGTACC TACGAATGCC 1901 ACTAGCTTAA TGGAGA GACT GCGCTTCCCG GTCTCAGCTC AAGCCGGGAA 1951 AGGTGAGCTA TGTGC AGTGT TCAGAGCTG A CCCTGG ACGA AGTGGGCCAT 2001 GGCAGTCCAC CTTGTTGGGC CAGTTGTGCG GGTACTACAC CCAATGGTCA 2051 GGATCACTGG AAGTCACCTT CATGTTCACC GGCTCCGG China CC standards Please note this page before filling in this page) Order --------- line. 1225095 Α7 Β7 V. Description of the invention)

2101 CAAGATGCTC ATAGCATACA CACCACCAGG AGGCCCCTTA CCCAAGGACC 2151 GGGCGACCGC CATGTTGGGC ACGCACGTCA TCTGGGACTT TGGGCTGCAA 2201 TTGTCTGTCA CCCTTGTAAT ACCATGGATC AGCAACACTC ATTACAGAGC 2251 GCACGCTCGA GATGGTGTGT TTGACTACTA CACTACAGGT TTGGTTAGCA 2301 TATGGTACCA GACGAATTAT GTGGTTCCAA TTGGAGCACC CAATACAGCC 2351 TATATAATAG CATTGGCGGC AGCCCAGAAG AACTTCACCA TGAAATTGTG 2401 TAAGGATGCT AGTGATATCC TACAGACAGG CACTATCCAA GGAGATAGGG 2451 TGGCAGATGT GATTGAGAGT TCTATAGGGG ACAGTGTGAG CAGAGCCCTC 2501 ACCCGAGCTC TACCGGCACC TACCGGCCAA GACACACAGG TAAGCAGCCA 2551 TCGATTAGAT ACTGGTAAAG TTCCAGCACT CCAAGCCGCT GAAATTGGAG 2601 CATCATCAAA TGCTAGTGAT GAGAGTATGA TTGAGACACG GTGTGTTCTT 2651 AATTCACATA GCACAGCTGA GACCACTCTT GATAGCTTCT TCAGCAG AGC 2701 AGGATTAGTT GGAGAGATAG ACCTCCCTCT TGAAGGCACA ACCAACCCGA 2751 ATGGGTACGC AAACTGGGAC ATAGACATAA CAGGTTACGC GCAAATGCGT 2801 AGAAAGGTGG AGCTGTTCAC CTACATGCGT TTTGACGCAG AGTTCACCTT 2851 TGTTGCATGC ACCCCTACCG GGGAAGTTGT CCCGCAATTG CTCCAATATA 2901 TGTTTGTACC ACCCGGAGCC CCCAAGCCAG A CTCCAGAGA ATCTCTCGCA 2951 TGGCAAACTG CCACTAATCC CTCAGTTTTT GTGAAGCTGT CAGACCCCCC 3001 AGCACAGGTT TCCGTCCCAT TCATGTCACC AGCGAGCGCC TATCAATGGT 3051 TTTATGACGG GTACCCCACA TTCGGTGAAC ACAAACAGGA GAAAGACCTT 3101 GAATACGGGG CATGCCCAAA CAACATGATG GGTACGTTCT CAGTGCGGAC 3151 TGTAGGCACC TCGAAGTCCA AGTACCCATT GGTGATCAGG ATTTACATGA 3201 GGATGAAGCA CGTCAGGGCG TGGATACCTC GCCCAATGCG TAACCAGAAC 3251 TATCTATTCA AAGCCACCCC AAATTATGCT GGTAATTCTA TTAAACCAAC 3301 TGGTGCCAGT CGCACAGCAA TCACCACTCT CGGGAAATTT GGACAGCAGT 3351 CCGGAGCTAT CTACGTGGGC AACTTTAGAG TGGTTAACCG CCATCTTGCT 3401 ACTCATAATG ACTGGGCAAA CCTTGTTTGG GAAGACAGCT CCCGCGACTT 3451 GCTCGTATCA TCTACCACTG CTCAAGGTTG TGACACGATT GCTCGCTGCA 3501 ATTGCCAGAC AGGAGTGTAT TATTGTAACT CAATGAGAAA ACACTATCCG 3551 GTCAGTTTCT CGAAACCCAG TTTGATCTTC GTGGAGGCCA GCGAGTATTA 3601 TCCAGCTAGA TACCAGTCAC ATCTCATGCT TGCAGTGGGT CATTCGGAAT 3651 CAGGGGATTG CGGTGGCATT CTTAGGTGCC AACATGGCGT CGTAGGGGTA 3701 GTTTCCACCG GGGGAAACGG CCTGGTGGGG TTCGCCGATG TGAGGGATCT 3751 TCTGTGGTTG GAT GATGAAG CCATGGAGCA GGGCGTGTCT GATTACATTA 3801 AAGGGCTTGG AGATGCTTTT GGCATGGGGT TTACAGACGC AGTGTCAAGA 3851 GAAGTTGAAG CACTGAAAAG CCACTTGATC GGCTCAGAGG GTGCCGTGGA 3901 GAAGATTCTA AAGAACTTAG GTTC TCA CAT standard CTCTCTCT Read the notes on the back before filling this page)-1 !! 1 Order ·! Line. Printed by the Employees 'Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Printed by the Employees' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Printed by 1225095 A7 __B7_ V. Description of the Invention ( \gas)

3951 TCAGGAGTGA TTATGACATG GTCACATTGA CGGCAACACT TGCCCTGATC 4001 GGGTGCCACG GGAGCCCTTG GGCCTGGGTT AAGTCGAAGA CAGCATCAAT 4051 TTTGGGCATA CCGATGGCTC AGAAGCAGAG TGCCTCTTGG TTAAAGAAGT 4101 TCAACGATGC GGCGAGTGCC GCGAAGGGGC TTGAGTGGAT CTCCAACAAA 4151 ATCAGTAAAT TCATCGATTG GCTCAAGGAG AAAATCATAC CGGCTGCTAA 4201 AGAGAAAGTC GAGTTTCTAA ACAATCTAAA GCAACTCCCC TTATTGGAGA 4251 ACCAAATTTC TAATCTCGAA CAGTCAGCAG CTTCGCAGGA GGACCTTGAG 4301 GCGATGTTTG GCAACGTGTC TTATCTGGCC CACTTCTGCC GCAAATTCCA 4351 ACCCCTCTAT GCCACGGAAG CAAAGAGGGT GTACGCCCTA GAAAAGAGAA 4401 TGAATAATTA CATGCAGTTC AAGAGCAAAC ACCGTATTGA ACCTGTATGC 4451 CTAATCATCA GAGGCTCGCC TGGTACTGGG AAGTCCTTGG CAACAGGGAT 4501 TATTGCTAGA GCCATAGCAG ACAAGTACCA CTCCAGTGTG TATTCCTTAC 4551 CTCCAGACCC AGACCACTTT GACGGATACA AACAACAGAT CGTCACTGGT 4601 ATGGACGACC TATGCCAAAA CCCAGACGGG AAAGACATGT CACTATTTTG 4651 TCAGATGGTC TCCACAGTGG ATTTTATACC GCCTATGGCA TCTCTGGAGG 4701 AGAAGGGAGT CTCATTCACC TCCAAGTTTG TGATTGCCTC CACTAACGCC 4751 AGTAACATCA TAGTGCCAAC AGTCTCGGAT TC AGATGCCA TCCGTCGCCG 4801 GTTCTTTATG GACTGCGACA TTGAGGTGAC CGATTCCTAT AAGACAGAGC 4851 TGGGCAGACT TGATGCAGGG AGAGCAGCCA GGCTGTGCTC TGAGAACAAC 4901 ACTGCAAACT TTAAACGGTG CAGTCCATTA GTCTGTGGGA AAGCAATCCA 4951 GCTTAGGG AT AGG AAGTCC A AGGTGAG ΑΤΑ CAGTGTGGAC ACGGTAGTG A 5001 GTGAACTTAT CAGGGAGTAT AGCGACAGAT CAGTTGTTGG GAACGCCATT 5051 GAAGCTCTTT TCCAAGGAAC CCCTAAATTT AGTCCAATAA GGATTAGCTT 5101 AGAGGAGAAG CCCGCACCTG ATGCTATTAG TGGCTTATTA GCTAGTGTTG 5151 ATAGTGAAGA GGTTCGCCAA AACTGTAGAG ATCAGGGATG GATTATCCCT 5201 GAAACTCCCA CCAATGTTGA GCGACACCTC AACAGAGCGG TGCTAGTCAT 5251 GCAATCCATC GCTACTGTGG TTGCAGTTGT CTCACTGGTG TATGTTATTT 5301 ATAAGTTGTT CGCCGGGTTT CAAGGAGCAT ATTCCGGCGC CCCCAAGCAA 5351 ACACTCAAGA TACCAGTGCT GCGCACGGCA ACTGTGCAGG GGCCGAGCTT 5401 GGACTTCGCT CTATCTCTAC TTAGGAGGAA CATTAGGCAG GTCCAAACCG 5451 ACCAGGGCCA CTTTACAATG TTAGGAGTGC GAGACCGCTT GGCTGTGCTC 5501 CCCAGACACT CCCAACCAGG AAAGACCATC TGGGTTGAAC ACAAATTAGT 5551 GAAGATCGTA GATGCTGTGG AGTTAGTAGA CGAACAAGGG GTTAACTTAG 5601 AGCTCAC ACT GGTAACGCTT GATACTAATG AAAAATTTAG AGACATCACA 5651 AGATTCATAC CAGAAACAAT TAGTCCTGCT AGTGATGCCA CTTTAGTTAT 5701 AAATACTGAA CATATGCCCA GTATGTTTGT GCCAGTTGG A GATGTGGTCC 5 (AGTATGGGTT TTTGAACCTT AGTGGTAGGCCCCC China Standard ACACT) Note to fill out this page again)-— — — — — — — Order ·! !! !! Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1225095 A7 ____B7_ V. Description of Invention (> °)

5801 TACAATTTCC CAACAAAAGC AGGACAGTGT GGTGGTGTTG TGACTGCCGT 5851 GGGTAAAGTG ATTGGGATCC ACATTGGTGG CAACGGTAGG CAAGGTTTCT 5901 GCGCTGCCCT GAAGAGGGGA TACTTTTGCA GTGAACAAGG TGAGATCCAA 5951 TGGATGAAGC CCAACAAAGA AACTGGCAGG TTGAACATCA ACGGACCTAC 6001 TCGCACTAAG CTTGAACCAA GTGTCTTTCA CGATGTGTTC GAAGGCACTA 6051 AAGAGCCAGC AGTGCTGACT AGTAAAGACC CAAGGCTGGA AGTTGATTTT 6101 GAACAGGCTC TTTTTTCAAA ATACGTGGGG AACACGCTTC ATGAACCCGA 6151 CGAGTTTGTC AAGGAGGCGG CCTTACATTA TGCCAACCAA CTCAAGCAGT 6201 TAGATATCAA GACCACCAAG ATGAGCATGG AGGATGCATG TTACGGCACA 6251 GAGAACCTGG AAGCTATAGA TCTTCACACA AGTGCAGGAT ATCCATACAG 6301 TGCACTAGGC ATCAAGAAAA AGGACATTTT GGACCCAACA ACTCGCGATG 6351 TCAGCAAGAT GAAATTCTAC ATGGACAAGT ATGGGTTGGA TCTACCGTAC 6401 TCTACTTATG TTAAAGATGA ACTTAGGGCC ATCGACAAGA TCAAGAAAGG 6451 GAAGTCTCGT CTCATAGAAG CGAGCAGTCT AAATGACTCA GTGTACTTGA 6501 GAATGACATT TGGGCACCTT TATGAAGCTT TCCACGCCAA TCCAGGTACA 6551 ATCACTGGTT CAGCTGTTGG GTGCAACCCA GATGTGTTTT GGAGCAAGTT 6601 ACCAATTCTA CTTCCAGGGT CGCTTTTCGC GT TTGACTAC TCGGGGTATG 6651 ACGCTAGTCT CAGCCCAGTG TGGTTCAGGG CGCTGGAGAT AGTCCTGCGG 6701 GAAATTGGAT ACTCCGAAGA CGCAGTGTCT CTCATAGAAG GAATCAATCA 6751 CACCCATCAT GTGTACCGCA ATAAAACTTA TTGTGTTCTT GGGGGAATGC 6801 CCTCAGGTTG CTCAGGCACC TCCATTTTCA ACTCGATGAT CAACAATATC 6851 ATTATTAGAA CACTCCTGAT TAAAACATTC AAAGGGATAG ATCTAGATGA 6901 ACTGAACATG GTGGCCTACG GGGATGATGT GTTGGCTAGT TACCCCTTCC 6951 CAATTGACTG TCTGGAGTTG GCAAGAACAG GCAAGGAGTA TGGTCTAACT 7001 ATGACCCCTG CCGACAAGTC ACCCTGCTTT AATGAGGTTA CATGGGAGAA 7051 TGCCACTTTC TTGAAGAGAG GATTCTTGCC TGATCATCAA TTCCCGTTTC 7101 TCATCCACCC TACGATGCCA ATGAGGGGGA TTCACGAATC CATTCGTTGG 7151 ACCAAAGATG CACGAAGTAC TCAAGATCAC GTGCGCTCCC TCTGCTTATT 7201 AGC ATGGCAC AACGGG AA AG AGGAGTATG A AAAATTTGTG AGTGCAATCA 7251 GATCAGTTCC AATTGGAAAA GCATTGGCTA TACCAAATTA TGAGAATCTG 7301 AGAAGAAATT GGCTCGAATT GTTTTAAATT TACAGTTTGT AACTGAACCC 7351 CACCAGTAAT CTGGTCGTGT TAATGACTGG TGGGGGTAAA TTTGTTATAA 7401 CCAGAATAGC AAAAA AAAAA AAA 1W3-Amino Acid Translation Listed as:

1 MGSQVSTQRS GSHENSNSAT EGSTINYTTINYYKDSYAAT AGKQSLKQDP 51 DKFANPVKDIFTETAAPLKS PSAEACGYSD RVAQLTIGNS TITTQEAANI 23 This paper size applies to the Chinese National Standard (CNS) A4 (210 X 297 mm) r »^ (Please read the note on the back — please fill in this page again) — — — ^ 0 — — — — — — — 1225095 A7 B7 V. Description of the invention () \)

101 IVGYGEWPSY CSDSDATAVD KPTRPDVSVN RFYTLDTKLW EKSSKGWYWK 151 HPDVLTETGV FGQNAQEHYL YRSGFCIHVQ CNASKFHQGA LLVAVLPEYV 201 IGTVAGGTGT EDSHPPYMQT QPGADGFELQ HPYVLDAGIPISQLTVCPHQ 251 WINLRTNNCA TIIVPYINAL PFDSALNHCN FGLLVVPISP LDYDQGATPV 301 IPITITLAPM CSEFAGLRQA VTQGFPTELK PGTNQFLTTD DGVSAPILPN 351 FHPTPCIHIP GEVRNLLELC QVETILEVNN VPTNATSLME RLREPVSAQA 401 GKGELCAVFR ADPGRSGPWQ STLLGQLCGY YTQWSGSLEV TFMFTGSFMA 451 TGKMLIAYTP PGGPLPKDRA TAMLGTHVIW DFGLQLSVTL VIPWISNTHY 501 RAHARDGVED YYTTGLVSIW YQTNYVVPIG APNTAYIIAL AAAQKNFTMK 551 LCKDASDILQ TGTIQGDRVA DVIESSIGDS VSRALTRALP APTGQDTQVS 601 SHRLDTGKVP ALQAAEIGAS SNASDESMIE TRCVLNSHST AETTLDSFFS 651 RAGLVGEIDL PLEGTTNPNG YANWDIDITG YAQMRRKVEL FTYMRFDAEF 701 TFVACTPTGE VVPQLLQYMF VPPGAPKPDS RESLAWQTAT NPSVFVKLSD 751 PPAQVSVPFM SPASAYQWFY DGYPTFGEHK QEKDLEYGAC PNNMMGTFSV 801 RTVGTSKSKY PLVIRIYMRM KHVRAWIPRP MRNQNYLFKA TPNYAGNSIK 851 PTGASRTAIT TLGKFGQQSG AIYVGNFRVV NRHLATHNDW ANLVWEDSSR 901 DLLVSSTTAQ GCDTIARCNC QTGVYYCNSM RKHYPVSFSK PSLIFVEAS E 951 YYPARYQSHL MLAVGHSESG DCGGILRCQH GVVGVVSTGG NGLVGFADVR 1001 DLLWLDDEAM EQGVSDYIKG LGDAFGMGFT DAVSREVEAL KSHLIGSEGA 1051 VEKILKNLVK LISALVIVIR SDYDMVTLTA TLALIGCHGS PWAWVKSKTA 1101 SILGIPMAQK QSASWLKKFN DAASAAKGLE WISNKISKFIDWLKEKIIPA 1151 AKEKVEFLNN LKQLPLLENQISNLEQSAAS QEDLEAMFGN VSYLAHFCRK 1201 FQPLYATEAK RVYALEKRMN NYMQFKSKHRIEPVCLIIRG SPGTGKSLAT 1251 GIIARAIADK YHSSVYSLPP DPDHFDGYKQ QIVTGMDDLC QNPDGKDMSL 1301 FCQMVSTVDFIPPMASLEEK GVSFTSKFVIASTNASNIIV PTVSDSDAIR 1351 RREFMDCDIE VTDSYKTELG RLDAGRAARL CSENNTANFK RCSPLVCGKA 1401 IQLRDRKSKV RYSVDTVVSE LIREYSDRSV VGNAIEALFQ GTPKFSPIRI 1451 SLEEKPAPDAISGLLASVDS EEVRQNCRDQ GWIIPETPTN VERHLNRAVL 1501 VMQSIATVVA VVSLVYVIYK LFAGFQGAYS GAPKQTLKIP VLRTATVQGP 1551 SLDFALSLLR RNIRQVQTDQ GHFTMLGVRD RLAVLPRHSQ PGKTIWVEHK 1601 LVKIVDAVEL VDEQGVNLEL TLVTLDTNEK FRDITRHPE TISPASDATL 1651 VINTEHMPSM FVPVGDVVQY GFLNLSGKPT HRTMMYNFPT KAGQCGGVVT 1701 AVGKVIGIHI GGNGRQGFCA ALKRGYFCSE QGEIQWMKPN KETGRLNING 1751 PTRTKLEPSV FHDVFEGTKE PAVLTSKDPR LEVDFEQA LF SKYVGNTLHE 1801 PDEFVKEAAL HYANQLKQLDIKTTKMSMED ACYGTENLEAIDLHTSAGYP 1851 YSALGIKKKDILDPTTRDVS KMKFYMDKYG LDLPYSTYVK DELRAIDKIK 1901 KGKSRLIEAS SLNDSVYLRM TFGHLYEAFH ANPG • Order --------- Line-Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1225095 A7B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the Invention (> >)

1951 KLPILLPGSL FAFDYSGYDA SLSPVWFRAL EIVLREIGYS EDAVSLIEGI 2001 NHTHHVYRNK TYCVLGGMPS GCSGTSIFNS MINNIIIRTL LIKTFKGIDL 2051 DELNMVAYGD DVLASYPFPIDCLELARTGK EYGLTMTPAD KSPCFNEVTW 2101 ENATFLKRGF LPDHQFPFLIHPTMPMRGIH ESIRWTKDAR STQDHVRSLC 2151 LLAWHNGKEE YEKFVSAIRS VPIGKALAIP NYENLRRNWL ELF when KV3- and complete viral gene sequence variations and viral strains were analyzed and compared when the mother plant, It was found that the nucleotide sequence of the two had an identity of 95.4%. And the biggest different region is located in 3C region, in which the nucleotide sequence of the two is 92% identical and the translated amino acid sequence is only 87% identical (Table 3), and in the VP1 region The nucleotide sequence (3332 ~ 3781) is only 87% identical, while the translated amino acid sequence (863 ~ 1012 has 96% identity). When the two untranslated regions (UTR) are analyzed In comparison, the nucleotide sequence in the 5 'untranslated region is 98% identical, while it is only 92% in the 3' untranslated region. Table 3 1TO 'and the nucleus of the viral gene from the 5 virus strain The identity of the nucleotide sequence and the translated amino acid sequence (%) 5, UTR VP4 VP2 VP3 VP1 2A 2B 2C 3A 3B 3C 3D 3,% identity between UTR and wew virus strains Nucleotide 98 99 99 98 86 99 98 94 95 97 92 97 92 Amino acid 100 99 97 96 100 97 93 94 95 87 97 — UTR: untranslated region In addition, the mutant amino acid sequence is compared with the amino acid sequence translated by other enterovirus 71 At the same time, it was found that there are 13 positions of amines in the amino acid sequence translated by KV3-like mutant virus strain. The acid is obviously different from other 71 type virus, its location and the corresponding amino acids are listed in Table 4. In addition, the Chinese National Standard (CNS) A4 specification (210 x 297 mm) is applicable to 25 paper sizes ( Please read the notes on the back before filling out this page) — — — — — — 11111111 · 1225095 A7 B7 V. Description of the invention (for) The 5 'untranslated region of the nucleotide sequence also has a sequence of 4 positions Obviously different from other enteroviruses of type 71 (Table 4). Table 4] Specific differences in amino acid sequences of WKa variant virus strains and other types of enteroviruses of type 71 Sequence position 1W3-such as variant virus strains 71 other enterovirus strains VP4 64 Methionine Methionine VP2 218 Methionine Methionine VP1 841 Methionine Aspartame 2A 969 Proline Serine Acid 2C 1124 serine polymorphic 2C 1434 alanine cinnamic acid 2C 1426 serine aspartic acid, isoleucine 3A 1442 cinnamic acid proline 3A 1446 serine spermine Acid 3A 1463 Acid aspartic acid, alanine 3B 1539 isoleucine lysine 3D 2114 histamine polytype 3D 2128 glycine glutamate 5 'untranslated region with different nucleotide sequence positions and their corresponding cDNAs Nucleotide region sequence position variation virus strains Other 71 enterovirus strains 5, UTR 47 Thymic chewing cytoplasmic osteoporosis 5, UTR 163 Cytosine thymine 5, UTR 476 Dense thymine 5, UTR 743 Guanine adenine 26 This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling this page) Order --------- line · 1225095 A7 ____ B7 V. Explanation of the invention (vv) Example 3 compares the growth kinetics of viruses (growth kinetics) The ΓΛΤ mutant virus strain obtained in Example 1 is used as the experimental group, and the "⑼5 virus mother strain is used as the control Group, were cultured on monolayer Vero cells in a 24-well tissue culture plate containing 2% fetal bovine blood cymbal at different viral infection multiples (M0, B0, B, B, and 5), and thereafter According to the virus growth analysis described in the specific embodiment Fixed manner, sampling and culturing. Overall, the κνϋ mutant virus strain showed higher virus production than the parent virus strain at all sampling time points in all three infection rates (Figure 2). On the other hand, the time for the growth curve to reach its peak under each growth condition of the virus strain is inversely proportional to its infection rate, and the time required for the mutant virus strain to be shorter than that of the 5 virus mother strain. For example, in the group with an infection rate of 5, ΓΛ① knows that the peak time of the mutant virus strain and the mud mu virus mother strain after Vero cells infection is 6 hours and 20 hours (2e and f). In addition, in the lower infection rate group (Μοχ).), For example, the intracellular and extracellular viral titers of the mutant virus strains peaked at 30 and 40 hours after infection, respectively (Figure 2a), and 5 viruses The mother plant showed very slow growth kinetics, and did not reach a peak peak in the test time (Figure 2b). As for the infection rate groups of 1 and 5, the mother virus strain 5 showed similar growth kinetics as the mutant virus strain, but its maximum virus yield was about 1,000 times lower than that of the H such as the mutant virus strain ( 108 is higher than 10n) (Figure 2d and q. Example 4 Toxicity test of virus strains The Γϋ mutant virus strain obtained in Example 1 was used as the experimental group, and the mother virus strain was used as the control group to implement the virus Toxicity analysis, its specific 27 paper sizes are applicable to Chinese National Standard (CNS) A4 specifications (210 X 297 meals) (Please read the precautions on the back before filling this page) ϋ — · 1 ϋ I n _1 Mmemm III 1 · — Ϋ Line I-Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1225095 A7 B7 V. Description of Invention (&) The implementation method is as follows. When the virus toxicity analysis is performed on the tested virus strain using ICR newborn mice, ^ Heavy doses were used in 21 newborn mice to test the δ formula to observe and record the results. The results obtained after testing found that when the injection of a mutant virus strain with a valence of 103 or 104 tciD50ml · 1 Can cause 100% lethality in ICR newborn mice The mice injected with 1W3-such as the mutant virus strain will first develop severe hind limb paralysis and die within 10 days. Finally, the analysis results determined that the KV3-such as the mutant virus strain has a half lethal dose (LD50) of 2 · 3 X 101 TCID5 () ml · 1, while the age / 5 virus mother strain is 106 TCID50 ml-1 (Figure 3a). This result shows that the virus toxicity of 1W3-like mutant virus strains is much more 5 virus mother strains are large. Example 5 Virus strains were grown on blood-free maggot-free medium. The mutant virus strain obtained in Example 1 was used to infect (M0I = 1) a single layer of Vero cell line at 37 ° C. After the adsorption reaction was performed for 1 hour, the medium containing fetal bovine blood maggots was replaced with a medium containing B-27 blood cymbal supplement as the experimental group, and the control group was replaced with a fresh medium containing 2% fetal bovine blood maggots. Then, the virus growth analysis method in the specific embodiment was used to culture separately. When analyzing the highest virus power and growth kinetics of viruses produced on the two culture media, it can be found that the two media systems have the same capabilities. Can provide 1TO ' Heterovirus strains are required for growth, and can make the 1W3-A mutant virus strains produce the same high virus potency and similar growth kinetics (Figure 4a and b), showing that the virus strains can be grown in blood-free medium . 28 This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) (Please read the notes on the back before filling out this page)! 1 order Printed by the cooperative 1225095 Printed by the cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 Printed by the cooperative V. Description of the invention d) Example 6 Antigenicity and antigenicity of the virus strain KV3-such as the mutant virus strain obtained in Example 1 The experimental group, and the mother virus strain was used as the control group, and the viral protein purification method (sucrose gradient centrifugation method) in the specific embodiment was used to purify and obtain the viral protein, and then the antibody neutralization test method was used. The viral protein (l (^ g)) was injected into the mother rats by intraperitoneal injection. After four weeks, the l (^ g virus protein was injected once by intraperitoneal injection. Blood was collected from the tails of the mice one week later. blood The centrifuged at 2,000g, centrifuged for 10 minutes, was taken on to Qing Qing blood antibody respectively, followed by reaction of the antibody and tested to obtain their respective Qing titer antisera. A total of 17 types of enterovirus 71 isolates isolated from Malaysia, central Taiwan and Taipei were used as antigens for the infection of type 71 enterovirus strains. Enterovirus strains were subjected to antibody neutralization reactions to test the effectiveness of neutralizing antibodies against blood crickets. All samples must be tested in triplicate. If the results obtained by the triplicate are more than twice the difference, then the analysis should be repeated. In the test, coxsackievirus A16 (coxsackievirus A16) was used as a reference for the cross-reactive serotype to distinguish between enteroviruses of type 71 enterovirus and other non-types of enterovirus. The results showed that when 1TO 'mutant virus strain and its ^ / 5 virus mother strain were injected into BALB / c mice with the same amount of viral protein to generate immunity, the mutant virus strain could continue to induce mice to generate more virus mother strains. It is a high amount of auto-neutralizing antibodies (force values are 2560 and 640, Table 5). In addition, it can also be seen from Table 5 that the anti-blood maggots produced against the mutant virus strains also confirmed that when compared with the anti-blood maggots produced against their virus mother strains, the neutralized reaction of the tested Π virus strains was performed. , Has a significantly higher neutralizing force 29 This paper size applies Chinese National Standard (CNS) A4 specifications (21 × 297 mm) (Please read the precautions on the back before filling this page) — — — — — — — «— — — — — — — I—. 1225095 A7 _B7_ 5. Explanation of the invention (^) value, and has better coverage of heterologous antigens against most virus strains (ρ < 0 · 0001). In addition, when the autoantibodies of both were adjusted to the same valence of 640, the antiserum produced by the mutant virus strain also had 7 out of the 15 test virus strains, showing a better resistance against the diarrhea. Antiviral maggots produced by 5 virus mother strains have about twice the potency of heterologous neutralizing antibodies (Table 5). These data show that, for example, the immune effect produced by mutant virus strains is qualitatively or quantitatively more effective than that of the mother virus strains, and can more effectively induce the production of neutralizing antibodies in mice, and is more effective against a wide range of type 71 enteroviruses. Capacity (p < 0.0001) 〇 (Please read the notes on the back before filling out this page) Printed by the Employees' Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs This paper is printed in accordance with China National Standard (CNS) A4 (210 X 297 mm) 1225095 Printed by A7 ____B7 in the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (&f; Table 5) Table 5 Injection of KV3-such as mutant virus strains and mutant virus mother strains to produce immune response in mice Neutralization valence of different wild virus strains of the virus _______ _Neutralization reaction 8___ I. Propionate H such as antiserum YN3-4a 2560 (640) a 40 Neu6 640 (160) 640 Der 1280 (320) 160 1743 (B) > 10240 (2560) 320 1997/1998 Malaysia isolate 14343 Throat 640 (160) 40 13898 Medulla 2560 (640) 160 13898 Throat swab 1280 (320) 160 13898 Spinal cord 320 (80) 160 13898 Stools 320 (8 0) 160 13091 Brain 320 (80) 80 14245 Throat swab 160 (40) 40 16985 Stools 320 (80) 40 1998 from Central Taiwan isolate 5277 Vesicle swab (OP) 320 (80) 160 5414 Throat swab 640 (160) 160 5415 Stools 640 (160) 160 2000 from Taipei isolate 2K 0652 320 (80) 40 2K1148 1280 (320) 80 Cox Α16 80 40 a Adjust autoantibody blood pressure value to 640 (CNS) A4 size (210 X 297 mm) (Please read the precautions on the back before filling this page)

1225095 A7 B7 V. Description of the invention ^ \) Example 7] W3-Subsequent stability of mutant strains (please read the precautions on the back before filling this page) To detect the mutations obtained from Example 1 ™ Viral strains, the stability of their genes and phenotypes when their derived families are grown on Vero cell lines, the mutant virus strains were selected for the 6th generation (W3- and -6) and the 9th generation of Vero cell lines. Generation and I2 generation, separately [] sequence their genes according to the aforementioned method, and then perform gene comparison. The mice were induced to produce antiserums according to the method described above, and the antiserums and their descendant virus strains were crossed for neutralizing antibody response analysis. It was found that all the tested virus strains exhibited very similar growth kinetics (Figure 5). In addition, when analyzing the VP1 region, 3C region, 5 ', and 3'UTR regions of the gene sequences of these tested virus strains, it was also found that in these aligned gene sequence regions, all tested secondary virus strains showed their It is very similar in the change of gene sequence. For example, the translated amino acid sequences of all tested secondary virus strains in the VP1 region and the 3C region are completely the same (Table 6), and the 5 'untranslated region of the nucleotide sequence shows 98% identity, but the 3' untranslated region The translation area is exactly the same. In addition, in terms of antigen stability, by neutralizing the test of different generations such as mutant virus strains, the anti-blood maggots induced by each virus strain were used to fight against the ITO-ie virus strains. The results showed that all The anti-blood maggots had similar neutralizing response efficacy to all tested virus strains (printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, printed by the seventh). 32 This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) JT i II.. Rii: > 1225095 A7 B7 V. Description of the invention Table 6 1TO 'variant virus strains and their subsequent descendants The homology (%) of the VP1, 3C, 5'UTR, and 3 'UTR regions of the nucleotide and translated amino acid sequences of the VP1 3C 5, UTR 3, UTR nucleotide amino group Acid nucleotide amino acid (nucleotide) (nucleotide) homology (%) YN3-4a-6 99 100 100 100 98 100 ΥΝ3-4α-9 99 100 100 100 98 100 ΥΝ3-4α-12 99 100 100 100 98 100 (Please read the precautions on the back before filling out this page) Table 7 IW3- And the generations of mutant virus strains are tested for interaction with the anti-blood maggots induced by these generations. _ Source of anti-blood test: Bing Bingxiu 3-43-4-4 ΥΝ3-4α-6 ΥΝ3-4α-9 ΥΝ3-4α-12 ΥΝ3-4α-4 640 320 320 320 ΥΝ3-4α_6 640 640 320 320 ΥΝ3-4α- 9 320 320 640 320 3-43-43-4-12 640 320 320 640 --I ---- Order -------- * 5 ^ Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 33 Paper sizes Applicable to China National Standard (CNS) A4 (210 X 297 mm)

Claims (1)

Central Ministry of Economic Affairs rub quasi Bureau body workers consumer cooperatives printing equipment 1225095 • A8 B8 C8 D8 seven patented range of 601 ACCAAAGAGT TGTTACCATATAGCTATTGG ATTGGCCATC CGGTGTGCAA 651 CAGGGCAATT GTTTACCTAT TTATrGGTTT TGTACCATTA TCACTGAAGT 701 CTGTGATCAC TCTCAAATTC ATTTTGACCC TCAACATAAT CAGACATGGG 751 CTCACAGGTG TCCACACAAC GCTCCGGTTC GCACGAAAAC TCTAACTCAG 801 CTACCGAGGG TTCCACTATA AACTATACTA CCATTAATTA CTATAAAGAT 851 TCCTATGCCG CCACAGCAGG TAAGCAGAGC CTTAAGCAGG ACCCAGACAA 901 GTTTGCAAAT CCTGTCAAAG ACATCTTCAC TGAAACGGCA GCGCCATTAA 951 AATCTCCATC TGCTGAGGCG TGTGGTTACA GCGATCGGGT GGCACAATTA 1001 ACTAITGGCA ATTCTACCAT CACTACGCAA GAAGCAGCAA ACATCATAGT 1051 TGGCTATGGT GAGTGGCCTT CCTACTGITC GGACTCTGAT GCTACTGCAG 1101 TGGACAAACC AACGCGCCCA GATGTTTCGG TGAATAGGTTITACACATTG 1151 GACACAAAAC TGTGGGAGAA ATCATCCAAG GGGTGGTACT GGAAATTCCC 1201 GGATGTGTTA ACTGAAACCG GGGTCTTTGG TCAAAATGCA CAGTTCCACT 1251 ACCTCTATCG GTCAGGGTTC TGGAITCACG TGCAGTGCAA TGCCAGTAAG 1301 TTCCACCAAG GAGCACTCCT AGTCGCTGTC CTCCCAGAGT ATGTCATTGG 1351 GACAGTGGCA GGTGGCACAG GGACGGAGGA TAGCCACCCC CCTTATATGC 1401 AGACTCAACC CGGTGCTGAT GGCTTCGAAT TGCAACACCC GTACGTGCIT 1451 GATGCTGGCA TTCCAATATC ACAATTAACA GTGTGCCCAC ATCAGTGGAT 1501 TAATTTGAGG ACCAACAATT GTGCCACAAT AATAGTGCCG TACATAAACG 1551 CACTACCCTT TGATTCTGCC TTGAACCATT GTAACTTTGG TCTGCTGGTT 1601 GTGCCTATTA GCCCGTTAGA TTATGACCAA GGTGCGACGC CAGTGATCCC 1651 CATTACTATC ACCTTGGCCC CAATGTGTTC TGAATTTGCA GGCCTTAGAC 1701 AAGCAGTTAC GCAAGGGTTT CCTACTGAGC TGAAACCTGG CACAAACCAA 1751 TmTAACCA CTGACGATGG CGTCTCAGCA CCCATTCTGC CAAACTITCA 1801 CCCCACCCCG TGTATCCATATACCCGGTGA AGTTAGAAAC TTGCTAGAGC 1851 TATGCCAGGT GGAGACCATT TTAGAGGTCA ACAATGTACC TACGAATGCC 1901 ACrAGCTTAATGGAGAGACT GCGCTTCCCG GTCTCAGCTC AAGCCGGGAA 1951 AGGTGAGCTATGTGCAGTGT TCAGAGCTGA CCCTGGACGA AGTGGGCCAT 2001 GGCAGTCCAC CTTGTTGGGC CAGTTGTGCG GGTACTACAC CCAATGGTCA 2051 GGATCACTGG AAGTCACCTT CATGTTCACC GGGTCCTTTA TGGCTACCGG 2101 CAAGATGCTC ATAGCATACA CACCACCAGG AGGCCCCTTA CCCAAGGACC 2151 GGGCGACCGC CATGTTGGGC ACGCACGTCA TCTGGGACTT TGGGCTGCAA 2201 TTGTCTGTCA CCCTTGTAAT ACCATGGATC AGCAACACTC ATTACAGAGC 2251 GCACGCTCGA GATGGTGTGT TTG ACT ACTA CACTACAGGT TTGGTTAGCA 2301 TATGGTACCA GACGAATTAT GTGGTTCCAATTGGAGCACC CAATACAGCC 2351 TATATAATAG CATTGGCGGC AGCCCAGAAG AACTTCACCA TGAAATTGTG 2401 TAAGGATGCT AGTGATATCC TACAGACAGG CACTATCCAA GGAGATAGGG 35 Ben paper scale Xiao with China National Standard (CNS) A4 size (21 OX297 public (%) (Please read "Notes on the back side before filling in this card") Ministry of Economic Affairs Bureau of Standards Λ workers consumer cooperatives print «. 1225095, A8 / B8 C8 D8 hole, patented range of 2451 TGGCAGATGT GATTGAGAGT TCTATAGGGG ACAGTGTGAG CAGAGCCCTC 2501 ACCCGAGCTC TACCGGCACC TACCGGCCAA GACACACAGG TAAGCAGCCA 2551 TCGATTAGAT ACTGGTAAAG TTCCAGCACT CCAAGCCGCT GAAATTGGAG 2601 CATCATCAAATGCTAGTGAT GAGAGTATGATTGAGACACG GTGTGTTCTT 2651 AATTCACATA GCACAGCTGA GACCACTCTT GATAGCTTCT TCAGCAGAGC 2701 AGGATTAGTT GGAGAGATAG ACCTCCCTCT TGAAGGCACA ACCAACCCGA 2751 ATGGGTACGC AAACTGGGAC ATAGACATAA CAGGTTACGC GCAAATGCGT 2801 AGAAAGGTGG AGCTGTTCAC CTACATGCGT TTTGACGCAG AGTTCACCTT 2851 TGTTGCATGC ACCCCTACCG GGGAAGTTGT CCCGCAATTG CTCCAATATA 2901 TGTTTGTACC ACCCGGAGCC CCCAAGCCAG ACTCCAGAGAATCTCTCGCA 2951 TGGCAAACTG CCACTAATCC CTCAGTTTTT GTGAAGCTGT CAGACCCCCC 3001 AGCACAGGTT TCCGTCCCAT TCATGTCACC AGCGAGCGCC TATCAATGGT 3051 TTTATGACGG GTACCCCACA TTCGGTGAAC ACAAACAGGA GAAAGACCTT 3101 GAATACGQGG CATGCCCAAA CAACATGATG GGTACGTTCT CAGTGCGGAC 3151 TGTAGGCACC TCGAAGTCCA AGTACCCATT GGTGATCAGG ATITAC ATGA 3201 GGATGAAGCA CGTCAGGGCG TGGATACCTC GCCCAATGCGTAACCAGAAC 3251 TATCTATTCA AAGCCACCCC AAATTATGCT GGTAATTCTATTAAACCAAC 3301 TGGTGCCAGT CGCACAGCAA TCACCACTCT CGGGAAATTT GGACAGCAGT 3351 CCGGAGCTAT CTACGTGGGC AACTTTAGAG TGGTTAACCG CCATCTTGCT 3401 ACTCATAATG ACTGGGCAAA CCTTGTTTGG GAAGACAGCT CCCGCGACTT 3451 GCTCGTATCATCTACCACTG CTCAAGGTTG TGACACGATT GCTCGCTGCA 3501 ATTGCCAGAC AGGAGTGTATTATTGTAACT CAATGAGAAA ACACTATCCG 3551 GTCAGTTTCT CGAAACCCAG TTTGATCTTC GTGGAGGCCA GCGAGTATTA 3601 TCCAGCTAGA TACCAGTCAC ATCTCATGCT TGCAGTGGGT CATTCGGAAT 3651 CAGGGGATTG CGGTGGCATT CTTAGGTGCC AACATGGCGT CGTAGGGGTA 3701 GTTTCCACCG GGGGAAACGG CCTGGTGGGG TTCGCCGATG TGAGGGATCT 3751 TCTGTGGTTG GATGATGAAG CCATGGAGCA GGGCGTGTCT GATTACATTA 3801 AAGGGCTTGG AGATGCTTTT GGCATGGGGT TTACAGACGC AGTGTCAAGA 3851 GAAGTTGAAG CACTGAAAAG CCACTTGATC GGCTCAGAGG GTGGCGTGGA 3901 GAAGATTCTA AAGAACTTAG TTAAACTCAT CTCTGCGCTC GTCATCGTCA 3951 TCAGGAGTGATTATGACATG GTCACATTGA CGGCAACACT TGCCCTGATC 4001 GGGTGCCACG GGAGCCCTTG GGCCTGGGTT AA GTCGAAGA CAGCATCAAT 4051 TTTGGGCATA CCGATGGCTC AGAAGCAGAG TGCCTCTTGG TTAAAGAAGT 4101 TCAACGATGC GGCGAGTGCC GCGAAGGGGC TTGAGTGGAT CTCCAACAAA 4151 ATCAGTAAATTCATCGATTG GCTCAAGGAG AAAATCATAC CGGCTGCTAA 4201 AGAGAAAGTC GAGTTTCTAA ACAATCTAAA GCAACTCCCC TTATTGGAGA 4251 ACCAAATTTC TAATCTCGAA CAGTCAGCAG CTTCGCAGGA GGACCTTGAG 36 Ben paper with Chinese national scale Xiao Qiu quasi (CNS) A4 size (21 OX297 (Mm) (Please read the notes on the back before filling out this page) Central Ministry of Economic Affairs rub quasi Bureau anus consumer cooperatives printing equipment 1225095 A8 " B8 C8 D8 six patented range of 4301 GCGATGTTTG GCAACGTGTC TTATCTGGCC CACTTCTGCC GCAAATTCCA 4351 ACCCCTCTAT GCCACGGAAG CAAAGAGGGT GTACGCCCTA GAAAAGAGAA 4401 TGAATAATTA CATGCAGTTC AAGAGCAAAC ACCGTATTGA ACCTGTATGC 4451 CTAATCATCA GAGGCTCGCC TGGTACTGGG AAGTCCTTGG CAACAGGGAT 4501 TATTGCTAGA GCCATAGCAG ACAAGTACCA CTCCAGTGTG TATTCCTTAC 4551 CTCCAGACCC AGACCACTTT GACGGATACA AACAACAGAT CGTCACTGGT 4601 ATGGACGACC TATGCCAAAA CCCAGACGGG AAAGACATGT CACTATTTTG 4651 TCAGATGGTC TCCACAGTGG AITITATACC GCCTATGGCA TCTCTGGAGG 4701 AGAAGGGAGT CTCATTCACC TCCAAGTTTG TGATTGCCTC CACTAACGCC 4751 AGTAACATCA TAGTGCCAAC AGTCTCGGAT TCAGATGCCA TCCGTCGCCG 4801 GTTCTTTATG GACTGCGACA TTGAGGTGAC CGATTCCTAT AAGACAGAGC 4851 TGGGCAGACT TGATGCAGGG AGAGCAGCCA GGCTGTGCTC TGAGAACAAC 4901 ACTGCAAACT TTAAACGGTG CAGTCCATTA GTCTGTGGGA AAGCAATCCA 4951 GCITAGGGAT AGGAAGTCCA AGGTGAGATA CAGTGTGGAC ACGGTAGTGA 5001 GTGAACTTAT CAGGGAGTAT AGCGACAGAT CAGTTGTTGG GA ACGCCATT 5051 GAAGCTCTTT TCCAAGGAAC CCCTAAATTT AGTCCAATAA GGATTAGCTT 5101 AGAGGAGAAG CCCGCACCTG ATGCTATTAG TGGCTTATTA GCTAGTGTTG 5151 ATAGTGAAGA GGTTCGCCAA AACTGTAGAG ATCAGGGATG GATTATCCCT 5201 GAAACTCCCA CCAATGTTGA GCGACACCTC AACAGAGCGG TGCTAGTCAT 5251 GCAATCCATC GCTACTGTGG TTGCAGTTGT CTCACTGGTG TATGTTATIT 5301 ATAAGTTGTT CGCCGGGTTT CAAGGAGCAT ATTCCGGCGC CCCCAAGCAA 5351 ACACTCAAGATACCAGTGCT GCGCACGGCA ACTGTGCAGG GGCCGAGCTT 5401 GGACTTCGCT CTATCTCTAC TTAGGAGGAA CATTAGGCAG GTCCAAACCG 5451 ACCAGGGCCA CTTTACAATG TTAGGAGTGC GAGACCGCTT GGCTGTGCTC 5501 CCCAGACACT CCCAACCAGG AAAGACCATC TGGGTTGAAC ACAAATTAGT 5551 GAAGATCGTA GATGCTGTGG AGTTAGTAGA CGAACAAGGG GTTAACTTAG 5601 AGCTCACACT GGTAACGCTT GATACTAATG AAAAATTTAG AGACATCACA 5651 AGATTCATAC CAGAAACAATTAGTCCTGCT AGTGATGCCA CnTAGTTAT 5701 AAATACTGAA CATATGCCCA GTATGTTTGT GCCAGTTGGA GATGTGGTCC 5751 AGTATGGGTTTTTGAACCTT AGTGGTAAGC CCACTCACAG GACTATGATG 5801 TACAATITCC CAACAAAAGC AGGACAGTGT GGTGGTGTTG TGACTGCCGT 5851 GGGTAAAGTG ATTGGGATCC ACATTGGT GG CAACGGTAGG CAAGGTTTCT 5901 GCGCTGCCCT GAAGAGGGGATACTTTTGCA GTGAACAAGG TGAGATCCAA 5951 TGGATGAAGC CCAACAAAGA AACTGGCAGG TTGAACATCA ACGGACCTAC 6001 TCGCACTAAG CTTGAACCAA GTGTCTTTCA CGATGTGTTC GAAGGCACTA 6051 AAGAGCCAGC AGTGCTGACT AGTAAAGACC CAAGGCTGGA AGTTGATTIT 6101 GAACAGGCTC ΊΤΤΤΊΤΟΑΑΑ ATACGTGGGG AACACGCTTC ATGAACCCGA 37 Ben applicable Chinese national scale paper rubbing quasi (CNS) A4 size (21 OX297 Mm) (Please read the notes on the back before filling in this page) Central 1,225,095 Ministry of Economic Affairs knead rate Bureau negative working consumer cooperatives print «. A8 B8 C8 D8 patented range of 6151 CGAGTTTGTCAAGGAGGCGG CCTTACATTATGCCAACCAACTCAAGCAGT 6201 TAGATATCAA GACCACCAAG ATGAGCATGG AGGATGCATG TTACGGCACA 6251 GAGAACCTGG AAGCTATAGA TCTTCACACA AGTGCAGGAT ATCCATACAG 6301 TGCACTAGGC ATCAAGAAAAAGGACATnT GGACCCAACA ACTCGCGATG 6351 TCAGCAAGAT GAAATTCTAC ATGGACAAGT ATGGGTTGGA TCTACCGTAC 6401 TCTACTTATG TTAAAGATGA ACTTAGGGCC ATCGACAAGA TCAAG AAAGG 6451 GAAGTCTCGT CTCATAGAAG CGAGCAGTCT AAATGACTCA GTGTACTTGA 6501 GAATGACATT TGGGCACCTT TATGAAGCIT TCCACGCCAA TCCAGGTACA 6551 ATCACTGGTT CAGCTGTTGG GTGCAACCCA GATGTGTTTT GGAGCAAGTT 6601 ACCAATTCTA CTTCCAGGGT CGCTTTTCGC GTTTGACTAC TCGGGGTATG 6651 ACGCTAGTCT CAGCCCAGTG TGGTTCAGGG CGCTGGAGAT AGTCCTGCGG 6701 GAAATTGGAT ACTCCGAAGA CGCAGTGTCT CTCATAGAAG GAATCAATCA 6751 CACCCATCAT GTGTACCGCA ATAAAACTTATTGTGTTCIT GGGGGAATGC 6801 CCTCAGGTTG CTCAGGCACC TCCATTTTCA ACTCGATGAT CAACAATATC 6851 ATTATTAGAA CACTCCTGAT TAAAACATTC AAAGGGATAG ATCTAGATGA 6901 AC TGAACATG GTGGCCTACG GGGATGATGT GTTGGCTAGTTACCCCTTCC 6951 CAATTGACTG TCTGGAGTTG GCAAGAACAG GCAAGGAGTA TGGTCTAACT 7001 ATGACCCCTG CCGACAAGTC ACCCTGCnT AATGAGGTTA CATGGGAGAA 7051 TGCCACTITC TTGAAGAGAG GATTCITGCC TGATCATCAATTCCCGTTTC 7101 TCATCCACCC TACGATGCCA ATGAGGGGGA TTCACGAATC CATTCGTTGG 7151 ACCAAAGATG CACGAAGTAC TCAAGATCAC GTGCGCTCCC TCTGCTTATT 7201 AGCATGGCAC AACGGGAAAG AGGAGTATGA AAAATTTGTG AGTGCAATCA 7251 GATCAGTTCC AATTGGAAAA GCATTGGCTATACCAAATTATGAGAATCTG 7301 AG AAGAAATT GGCTCGAATT GTTTTAAATr TACAGTTTGT AACTGAACCC translated amino acid sequence of 7351 CACCAGTAAT CTGGTCGTGT TAATGACTGG TGGGGGTAAA TTTGTTATAA 7401 CCAGAATAGC AAAAAAAAAA AAA 1 MGSQVSTQRS GSHENSNSAT EGSTINYTO NYYKDSYAAT AGKQSLKQDP 51 DKFANPVKDIFTETAAPLKS PSAEACGYSD RVAQLTIGNS THTQEAANI 101 IVGYGEWPSY CSDSDATAVD KPTRPDVSVN RFYTLDTKLW EKSSKGWYWK 151 FPDVLTETGV FGQNAQFHYL YRSGFCIHVQ CNASKFHQGA LLVAVLPEYV 201 IGTVAGGTGT EDSHPPYMQT QPGADGFELQ HPYVLDAGIPISQLTVCPHQ 251 WINLRTNNCA TIIVPYINAL PFDS ALNHCN FGLLVVPISP LDYDQGATPV 301 ipithlapm csefaglrqa vtqgfptelk pgtnqflttd dgvsapilpn 351 FHPTPCIHIP GEVRNLLELC QVETILEVNN VPTNATSLME RLRFPVSAQA 401 GKGELCAVFR ADPGRSGPWQ STILLGQLCGY YTQWSGSLEA 4 TFMFTG China National Standards TFMFTG ) 1,225,095, Shen gas f Ministry of Economic Affairs Qiu quasi Board Consumer Cooperatives India with negative working A8 B8 C8 D8 jGPLPKDRA tamlgthviw dfglqlsvtl vipwisnthy 501 RAHARDGVFD YYTTGLVSIW YQTNYWPIG APNTAYnAL AAAQKNFTMK 551 LCKDASDILQ TGTIQGDRVA DVIESSIGDS VSRALTRALP APTGQDTQVS 601 SHRLDTGKVP ALQAAEIGAS SNASDESMIE TRCVLNSHST AETTLDSFFS 651 RAGLVGEIDL PLEGTTNPNG YANWDIDITG YAQMRRKVEL FTYMRFDAEF 701 TFVACTPTGE WPQLLQYMF VPPGAPKPDS RESLAWQTAT NPSVFVKLSD 751 PPAQVSVPFM SPASAYQWFY DGYPTFGEHK QEKDLEYGAC PNNMMGTFSV 801 RTVGTSKSKY PLVIRIYMRM KHVRAWIPRP MRNQNYLFKA TPNYAGNSIK 851 PTGASRTAIT TLGKFGQQSG AIYVGNFRVV NRHLATHNDW ANLVWEDSSR 901 DLLVSSTTAQ GCDTIARCNC QTGVYYCNSM RKHYPVSFSK PSLIFVEASE 951 YYPARYQSHL MLAVGHSESG DCGGILRCQH GVVGWSTGG NGLVGFADVR 1001 DLLWLDDEAM EQGVSDYIKG LGDAFGMGFT DAVSREVEAL KSHLIGSEGA 1051 VEKILKNLVK LISALVIVIR SDYDMVTLTA TLALIGCHGS PWAWVKSKTA 1101 SE. GIPMAQK QSASWLKKFN DAASAAKGLE WISNKISKH DWLKEKIIPA .1151 AKEKVEFLNN LKQLPLLENQISNLEQSAAS QEDLEAMFGN VSYLAHFCRK 1201 FQPLYATEAK RVYALEKRMN NYMQFKSKHRIEPVC LIIRG SPGTGKSLAT 1251 GIIARAIADK YHSS VYSLPP DPDHFDGYKQ QIVTGMDDLC QNPDGKDMSL 1301 FCQMVSTVDFIPPMASLEEK GVSFTSKFVIASTNASNEV PTVSDSDAIR 1351 RRFPMDCDIE VTDS YKTELG RLDAGRAARL CSENNTANFK RCSPLVCGKA 1401 IQLRDRKSKV RYSVDTWSE LIREYSDRSV VGNAffiALFQ GTPKFSPIRI 1451 SLEEKPAPDAISGLLASVDS EEVRQNCRDQ GWIIPETPTN VERHLNRAVL 1501 VMQSIATWA WSLVYVIYK LFAGFQGAYS GAPKQTLKDP VLRTATVQGP 1551 SLDFALSLLR RNIRQVQTDQ GHFTMLGVRD RLAVLPRHSQ PGKTIWVEHK 1601 LVKIVDAVEL VDEQGVNLEL TLVTLDTNEK FRDITRFIPE BSPASDATL 1651 VINTEHMPSM FVPVGD WQY GFLNLSGKPT HRTMMYNHPT KAGQCGGWT 1701 AVGKVIGIHIGGNGRQGFCA ALKRGYFCSE QGEIQWMKPN KETGRLNING 1751 PTRTKLEPSV FHDVFEGTKE PAVLTSKDPR LEVDFEQALF SKYVGNTLHE 1801 PDEFVKEAAL HYANQLKQLDIKTTKMSMED ACYGTENLEAIDLHTSAGYP 1851 YSALGIKKKDILDPTTRDVS KMKFYMDKYG LDLPYSTYVK DELRAIDKIK 1901 KGKSRLIEAS SLNDSVYLRM TOHLYEAFH ANPGUTGSA VGCNPDVFWS 1951 KLPILLPGSL FAFDYSGYDA SLSPVWFRAL EIVLREIGYS EDAVSLIEGI 2001 NHTHHVYRNK TYCVLGGMPS GCSGTSIFNS MINNIIIRTL LIKTFKGIDL 2051 DELNMVAYGD DVLAS YPFPIDCLELART GK EYGLTMTPAD KSPCFNEVTW 2101 ENATFLKRGF LPDHQFPFLIHPTMPMRGIH ESIRWTKDAR STQDHVRSLC 2151 LLAWHNGKEE YEKFVS AIRS VPIGKALAIP NYENLRRNWL ELF or a nucleotide sequence with the same translated amino acid sequence. 39 This paper is a Chinese standard (CNS) Α4 size (210X297 mm) (Please read the precautions on the back before filling this page) 1225095 B8 C8 D8 6. Scope of patent application 2. The virus strain described in item 1 of the scope of patent application, the KVKfl variant virus strain can be cultured in host cells suitable for its growth. 3. The virus strain according to item 2 of the patent application scope, wherein the host cell is a Vero cell strain. 4. The virus strain according to item 1 of the scope of the patent application, wherein the mutant virus strain can be replicated and proliferated in a serum-free medium after infecting the host cells. 5. The virus strain according to item 1 of the scope of the patent application, wherein the type 71 enterovirus vaccine comprises an inactivated vaccine and an attenuated vaccine, and a genetically engineered vaccine containing this genetic characteristic. 6. The virus strain according to item 1 of the scope of the patent application, wherein the KV5-like mutant virus strain and its successor are in the VP1 region of the nucleotide sequence with 99% or more identity. Printed by the Central Labor Bureau of the Ministry of Economic Affairs and Consumer Cooperatives (please read the precautions on the back before filling out this page) 7. The virus strain described in item 1 or 6 of the patent application scope, where the ™ mutant virus strain is related to Subsequent generations have similar highest viral titers, growth curves, and antiserum produced by them with similar broad-spectrum antigen shielding. 8. The virus strain described in item 1 of the scope of patent application, in which the YATKfl variant of this paper sheet is used in accordance with China National Standards (CNS) A4 (4) (210X1297 mm) 1225095 At B8 C8 D8 6. Scope of patent application The VP1 region of the cDNA nucleotide sequence of the virus strain and its translated amino acid sequence are as follows: The cDNA nucleotide sequence (3332 ~ 3781) of the VP1 region is: 3332 CGGGAAATTT GGACAGCAGT 3351 CCGGAGCTAT CTACGTGGGC AACTTTAGAG TGGTTAACCG CCATCTTGCT 3401 ACTCATAATG ACTGGGCAAA CCTTGTCT the amino acid sequence (863- translation of CCCGCGACTT 3451 GCTCGTATCATCTACCACTG CTCAAGGTTG TGACACGATT GCTCGCTGCA 3501 ATTGCCAGAC AGGAGTGTATTATTGTAACT CAATGAGAAAACACTATCCG 3551 GTCAGTTTCT CGAAACCCAG TTTGATCTTC GTGGAGGCCA GCGAGTATTA 3601 TCCAGCTAGA TACCAGTCAC ATCTCATGCT TGCAGTGGGT CATTCGGAAT 3651 CAGGGGATTG CGGTGGCATT CTTAGGTGCC AACATGGCGT CGTAGGGGTA 3701 GTTTCCACCG GGGGAAACGG CCTGGTGGGG TTCGCCGATG TGAGGGATCT 3751 TCTGTGGTTG GATGATGAAG CCATGGAGCA G VP1 region 1012) ··: 863 TLGKFGQQSG AIYVGNFRW NRHLATHNDW ANLVWEDSSR 901 DLLVSSTTAQ GCDTI ARCNC QTGVYYCNSM RKHYPVSFSK PSLIFVEASE 951 YYPARYQSHL MLAVGHSESG DCGGILRCQH GVVGWSTGG NGLVGFADVR 1001 DLLWLDDEAMEQ or a cDNA nucleotide sequence with the same translated amino acid sequence. Printed by the Central Government Bureau of the Ministry of Economic Affairs of the Shoulder Cooperative Consumer Cooperative (please read the notes on the back before filling out this page) 9. The virus strain described in item 1 or 9 of the scope of patent application, of which the recommended variant virus strain The corresponding amino acids in the translated amino acid sequences at positions 64, 218, 841, 969, 1124, 1426, 1434, 1442, 1446, 1463, 1539, 2114, and 2128 are respectively Thr, Methionine (Met), Tetamine (Thr), Serine (Ser), Serine (Ser), Serine (Aer), Alanine (Thr) , Serine, Gly, Ile, His, and Gly, among these 13 amino acids, no more than Corresponding amino acids at more than 3 positions are mutated. 41 This paper size is in accordance with China National Standards (CNS) A4 (210X297 mm) 1225095 6. Scope of patent application 10. The virus strain described in item 10 of the scope of patent application, wherein the ™? -Known variant virus strain is located at the 5′-end untranslated region (UTR) 47, 163, 476 of the nucleotide sequence and The cDNA nucleotides at position 743 are thymine, cytosine, cytosine, and guanine 〇 (Please read the precautions on the back before filling this page) Order . Printed by the Central Government Standards Bureau of the Ministry of Economic Affairs; printed by the Industrial and Consumer Cooperatives. The paper size applies to the Chinese National Standard (CNS) A4 (210X297 mm).