Heat-stable enterovirus group A type 71 virus strain and screening method and application thereof

Abstract

The present invention provides a heat-stable enterovirus group A71 strain, screening methods, and applications thereof. The heat-stable enterovirus group A71 strain, deposited with CCTCC No. V202233, exhibits stable cell growth after heat treatment at 52°C and exhibits high yield and high immunogenicity. Compared to wild-type or heat-unstable strains, the heat-stable strain exhibits five nucleotide mutations in its coding region, including three missense mutations and two synonymous mutations. This strain can be used in the screening and modification of inactivated vaccine strains, as well as in vaccine production.

Description

Technical Field

[0001] The present invention relates to the technical field of virology, and in particular to a heat-stable enterovirus group A type 71 virus strain, a screening method and an application thereof. Background Art

[0002] Hand, foot, and mouth disease (HFMD) is an acute infectious disease caused by various enteroviruses, most commonly affecting children under five years of age. Its clinical symptoms primarily include herpes sores on the hands, feet, and oral mucosa, sometimes accompanied by fever. A minority develop serious complications such as myocarditis, aseptic encephalitis, heart failure, pneumonia, pulmonary edema, and flaccid paralysis, sometimes leading to death.

[0003] The causative agents of HFMD include various serotypes of enterovirus, the most common of which are enterovirus A71 (EV-A71), coxsackievirus A16 (CV-A16), coxsackievirus A6 (CV-A6), and coxsackievirus A10 (CV-A10).

[0004] EV-A71 belongs to the genus Enterovirus in the family Picornaviridae. Enterovirus particles are icosahedral and spherically symmetrical, with a diameter of approximately 27-30 nm. The genome consists of approximately 7400 nt of single-stranded positive-strand RNA. The EV-A71 viral structural proteins consist of a precursor protein, which is cleaved by proteases into VP4, VP2, VP3, and VP1. VP1-VP3 are located on the surface of the virion, while VP4 is located within the virion, linked to the viral RNA. Enterovirus particles exist with two different sedimentation coefficients: hollow and solid. EP, containing three structural proteins: VP0, VP2, and VP3, is non-infectious. FP, containing four structural proteins: VP1-VP4. VP0 matures into VP4 and VP2, and the virion contains viral RNA, making it an infectious mature virus particle.

[0005] Currently, the thermal stability of common EV-A71 virus strains is poor, and the immunogenicity and virus particle production need to be improved. Summary of the Invention

[0006] Based on this, it is necessary to provide a heat-stable enterovirus A group 71 virus strain and its screening method and application, which can still proliferate on cells after being treated at 52°C for 30 minutes, and the virus particle yield and humoral immunogenicity are significantly improved.

[0007] The present invention adopts the following technical solutions:

[0008] The present invention provides a heat-stable enterovirus A group 71 virus strain, the cell phenotype of which is the ability to stably grow on Vero cells after heat treatment at 52°C. The mutated amino acid sites are as follows: the 44th amino acid of the VP1 protein is alanine, the 247th amino acid is valine; the 45th amino acid of the 2A protein is alanine. The EV-A71 virus strain was deposited with the China Center for Type Culture Collection on May 24, 2022, with the deposit number CCTCC No. V202233. Its nucleotide sequence is shown in SEQ ID NO. 20, and its amino acid sequence is shown in SEQ ID NO. 21.

[0009] The mother virus strain of the heat-stable enterovirus group A type 71 virus strain was isolated from sample No. 0227 of hand, foot and mouth disease patient in Peixian County, Jiangsu Province, China in 2019. After heat treatment at 52°C for 30 minutes, it was no longer infectious and could not grow on Vero cells. The 44th amino acid of its VP1 protein was valine, the 247th amino acid of the VP1 protein was leucine (L), and the 45th amino acid of the 2A protein was threonine.

[0010] The nucleotide and amino acid sequences of the two strains are identical in length, and their genomic nucleotide and amino acid sequences share the following characteristics: the genome consists of the non-coding regions 5'-UTR and 3'-UTR and the coding regions for the structural proteins VP4, VP2, VP3, VP1, and non-structural proteins 2A, 2B, 2C, 3A, 3B, 3C, and 3D. The genomes are both 7403 nucleotides long, with the 5'-UTR and 3'-UTR non-coding regions located at nucleotide positions 1-742 and 7325-7403, respectively. The 3'-UTR is followed by a poly(A) tail of undetermined length, encoding a polyprotein of 2194 amino acid residues.

[0011] Compared with wild-type or thermolabile strains, the heat-stable enterovirus A71 strain had five nucleotide mutations in its coding region, including three missense mutations and two synonymous mutations. The three missense mutations and corresponding amino acid positions were: VP1-T2568C / V44A, VP1-T3176G / L247V, and 2A-A3461G / T45A; the two synonymous mutations were: 2A-T3715C and 3A-T5065C.

[0012] The present invention also provides a screening method for the above-mentioned heat-stable enterovirus group A type 71 virus strain, comprising the following steps: treating the virus mother strain EV-A71TS- / JS PX / 2019 sample at 52°C for 30 minutes and immediately storing it at 4°C; after the heat-treated virus mother strain is cooled, inoculating Vero cells, and culturing them in a 37°C incubator until the cells show pathological changes; repeatedly freezing and thawing, and continuously heat treating for multiple generations by repeating the above steps to harvest the virus liquid screened by heat treatment; inoculating the virus liquid screened by heat treatment into Vero cells, performing plaque purification, and obtaining a heat-stable EV-A71TS+ / JS PX / 2019 virus strain harvest liquid.

[0013] In some embodiments, the plaque purification step is as follows: the virus solution screened by heat treatment is inoculated into Vero cells and then placed at 52°C for 30 minutes, and then the virus is diluted 10-fold multiple times, preferably in a total of 6 dilution gradients; the virus solution is added to 6-well plates, adsorbed at 37°C for a period of time, and then discarded; 2% low-melting point agarose and 2×DMEM maintenance solution are mixed at a ratio of 1:1 and equal volumes are added to each well of the 6-well plate, and placed in an incubator after solidification; the results are observed after 2-3 days, and plaques are picked at the lowest dilution; three consecutive plaque purifications are performed to obtain a cloned strain; the cloned strain is blindly propagated on Vero cells for three consecutive generations at a low MOI to obtain a heat-stable strain virus harvest liquid.

[0014] The present invention also provides a method for purifying virus particles of a heat-stable enterovirus group A type 71 virus strain, comprising the following steps: inoculating Vero cells with a heat-stable enterovirus group A type 71 virus strain; harvesting the virus when more than 90% of the cells develop pathological changes, and sequentially performing ultrafiltration concentration, sucrose-bottomed centrifugation, sucrose density gradient centrifugation, CsCl density gradient centrifugation, and ultracentrifugation for desalting to harvest the virus particles, detecting the antigen content of the hollow particles and the solid particles, and inactivating the virus particles.

[0015] The heat-stable enterovirus group A type 71 virus particles obtained by the above purification method are used as candidate vaccines.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention, for the first time, uses Vero cells for isolation, passage, and adaptive culture through heat treatment, obtaining a heat-stable enterovirus group A71 strain. The cell phenotype shows stable growth on the cells after heat selection pressure treatment at 52°C, and exhibits high yield and high immunogenicity. The present invention also provides methods for isolating and identifying the heat-stable EV-A71 strain, full particles (FP) and empty particles (EP), and a method for determining humoral immunogenicity. These methods can be applied to the screening, modification, and production of inactivated vaccine strains.

[0018] Furthermore, the nucleotide and amino acid sites in the VP1 gene and other gene sequences associated with thermostability identified in this invention can also be used to construct high-yielding, thermostable strains of other enteroviruses besides EV-A71. By altering these structural protein gene sequence sites, thermostable, high-yielding, and highly immunogenic virus-like particle (VLP) vaccine candidates for EV-A71 and other enteroviruses can be constructed, facilitating the development of whole-virus inactivated vaccines and VLP vaccines with strong humoral immunogenicity, long-lasting efficacy, and consistent quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison of the growth status of EV-A71TS- / JS PX / 2019 virus strain and EV-A71TS+ / JS PX / 2019 virus strain on Vero cells.

[0020] Figure 2 This is a diagram showing the growth status of EV-A71TS- / JS PX / 2019 virus strain and EV-A71TS+ / JS PX / 2019 virus strain after treatment at 52°C.

[0021] Figure 3 This is a statistical chart showing the differences in virus titers between the EV-A71TS- / JS PX / 2019 virus strain and the EV-A71TS+ / JS PX / 2019 virus strain after heat treatment at different temperatures.

[0022] Figure 4 These are the purification result identification diagrams of EV-A71TS- / JS PX / 2019 virus strains and EV-A71TS+ / JS PX / 2019 virus strains; among them, (a) is the side view corresponding to EV-71 sucrose gradient centrifugation, and (b) corresponds to the SDS-PAGE identification diagram after depletion of CsCl.

[0023] Figure 5Comparison of humoral immunogenicity between EV-A71TS- / JS PX / 2019 virus strain and EV-A71TS+ / JS PX / 2019 virus strain, where ns represents not significant (P≥0.05); * represents 0.01≤P<0.05; ** represents P<0.01; *** represents P<0.001 and **** represents P<0.0001. DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0025] The following embodiments are only used to illustrate the present invention, but are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention.

[0026] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0027] Source of test materials:

[0028] Thermostable virus strain EV-A71 TS- / JS PX / 2019: isolated from sample No. 0227 of hand, foot and mouth disease patient in Peixian County, Jiangsu Province, China in 2019 (JS-PX). The cell phenotype is: it cannot proliferate normally on Vero cells after treatment at 52°C (TS-).

[0029] The thermostable EV-A71 strain is the EV-A71TS+ / JS PX / 2019 strain. Its cell phenotype demonstrates stable growth after 52°C heat selection pressure and exhibits high yield and immunogenicity. This thermostable EV-A71 strain was deposited with the China Center for Type Culture Collection on May 24, 2022, under the CCTCC No. V20223. The deposit address is Wuhan University, China.

[0030] EV-A71TS- / JS PX / 2019 and EV-A71TS+ / JS PX / 2019 were inoculated into Vero cells at the same inoculum volume, and the cytopathic effects of the two strains were observed. Figure 1 shown.

[0031] The present invention treats the EV-71TS- / JS PX / 2019 virus strain at 52°C for 30 minutes and immediately stores it at 4°C. The treated virus sample is then thawed and inoculated into Vero cells for passage. After 5 consecutive heat treatments, the harvested virus liquid is heat treated again at 52°C and plaque purified to obtain the clone EV-A71TS+ / JS PX / 2019. The heat-unstable strain is obtained by directly subjecting the virus liquid that has undergone the same number of passages on Vero cells and has not been heat treated to three rounds of plaque purification. The two strains were verified for their heat resistance after passage through Vero cells. After further large-scale cultivation and purification of the two strains, the antigen production and humoral immunogenicity of the two strains were compared, and the nucleotide and amino acid sequences of the two phenotypically different strains were compared and analyzed.

[0032] The specific test plan is as follows:

[0033] Example 1

[0034] This embodiment provides a method for screening thermostable virus strains, comprising the following steps:

[0035] (1) The EV-A71 TS- / JS PX / 2019 virus sample was placed at 52°C for 30 min and immediately stored at 4°C.

[0036] (2) After the heat-treated sample is thawed, inoculate Vero cells and culture in a 37°C incubator until the cells show pathological changes (see Figure 1 ) and stored at -20℃.

[0037] (3) The virus solution was harvested by repeated freezing and thawing three times, centrifuged at 6000×g for 5 min, the cell debris precipitate was removed, and the supernatant was collected.

[0038] (4) Repeat (1) to (3) for the collected virus solution, and heat-treat and passage it 5 times.

[0039] Example 2

[0040] This embodiment provides a method for plaque purification of a thermostable virus strain, comprising the following steps:

[0041] (1) The prepared Vero cells were digested and then mixed at an appropriate ratio (5×10 5 cells / mL) were inoculated into 6-well cell culture plates.

[0042] (2) When the cells grow into a dense monolayer, discard the culture medium and wash twice with 0.01M PBS (pH≈7.2).

[0043] (3) The virus solution harvested from the fifth generation heat treatment in Example 1 was again treated at 52°C for 30 min, and the virus was serially diluted 10-fold, with a total of 6 dilution gradients.

[0044] (4) Add the virus solution to a 6-well plate and adsorb at 37°C for 2 h before discarding the virus solution.

[0045] (5) Mix 2% low-melting-point agarose and 2× DMEM maintenance solution in a 1:1 ratio of equal volumes, add 2 mL to each well of a 6-well plate, and place in an incubator after solidification.

[0046] (6) Observe the results after 2 to 3 days and pick out plaques at the lowest dilution.

[0047] (7) Repeat plaque purification three times to obtain cloned strains.

[0048] (8) The clones obtained from three rounds of plaque purification were blind-passaged three times on Vero cells at a low MOI to obtain the harvest fluid of the thermostable virus strain (named EV-A71TS+ / JS PX / 2019).

[0049] Example 3

[0050] This embodiment provides a method for testing the viral titer of a thermostable virus strain, comprising the following steps:

[0051] (1) Vero cells grown to a dense monolayer were digested and plated at 1×10 5 The cells were plated at a density of 100 μL / mL into a 96-well plate.

[0052] (2) The virus solution harvested in Example 2 was diluted in DMEM maintenance medium in a series of 10-fold gradients, with a total of 8 dilution gradients.

[0053] (3) After the cells adhere to the wall of the 96-well plate, add the diluted virus solution, 8 replicates per gradient, 100 μL per well. Mix the virus solution and cells thoroughly and incubate in a 37°C incubator.

[0054] (4) After 7 days, the pathological changes in the 96-well plate were observed and the virus titer was calculated according to the Reed-Mench formula.

[0055] (5) Repeat the titration 3 to 6 times to determine that the viral titer of the heat-stable strain EV-A71TS+ / JS PX / 2019 on Vero cells is 7.5 lgCCID 50 / ml.

[0056] Example 4

[0057] This example specifically compares the heat treatment capabilities of EV-A71TS+ / JS PX / 2019 and EV-A71 TS- / JS PX / 2019, including the following steps:

[0058] (1) The EV-A71TS+ / JS PX / 2019 virus strain and the EV-A71TS- / JS PX / 2019 virus strain were treated at 37°C, 42°C, 45°C, 48°C, 50°C, 52°C, and 54°C for 30 min, respectively, and immediately stored at 4°C.

[0059] (2) The two treated strains were seeded at the same MOI into 6-well plates filled with Vero cells, cultured in a 37°C incubator, and the pathological conditions were observed.

[0060] (3) Titrate the treated virus solution according to the virus titer determination method in Example 3.

[0061] (6) Draw the heat resistance treatment curve, the results are shown in Figure 2 and Figure 3 .

[0062] Depend on Figure 2 It can be seen that 2 days after virus inoculation, the EV-A71TS+ / JS PX / 2019 strain began to show lesions, while no obvious lesions were observed in the EV-A71TS- / JS PX / 2019 strain 4 days after inoculation.

[0063] Depend on Figure 3 It can be seen that the EV-A71TS+ / JS PX / 2019 virus strain and the EV-A71TS- / JS PX / 2019 virus strain were treated at 37℃~54℃ for 30min and then immediately cooled at 4℃. 50 The titer of the virus strain was detected on Vero cells by the method. When the EV-A71TS- / JS PX / 2019 virus strain was treated at 48°C, most of the viruses were inactivated and lost their infectivity. However, the EV-A71TS+ / JSPX / 2019 virus strain was still able to maintain a high titer after treatment at 50°C, and its thermal stability was significantly better than that of the EV-A71TS- / JS PX / 2019 virus strain.

[0064] Example 5

[0065] This example performs whole genome sequence testing on EV-A71TS+ / JS PX / 2019 and EV-A71 TS- / JS PX / 2019, respectively, including the following steps:

[0066] (1) Viral RNA extraction:

[0067] Two types of EV-A71 viral RNA were extracted and purified using QIAGEN's viral RNA extraction and purification kit QIAamp Viral RNA Mini Kit (Cat. No.: 52906).

[0068] (2) Reverse transcription to synthesize cDNA:

[0069] The cDNA synthesis reaction system for reverse transcription is shown in Table 1 and Table 2:

[0070] Table 1 Reaction system for the first step of reverse transcription synthesis of cDNA

[0071] Reagents Volume (μL) RNase-Free H2O 3.0 Oligo dT Primer 1.0 dNTP Mixture 1.0 RNA 5.0 Total Volume 10.0

[0072] Table 2 Reaction system for the second step of reverse transcription synthesis of cDNA

[0073]

[0074]

[0075] Add the reagents listed in Table 1 to the PCR tube, mix well, and place in a PCR instrument. React at 65°C for 5 minutes, then quickly cool on ice.

[0076] The reagents listed in Table 2 were added to the above reaction solution, mixed well, and placed in a PCR instrument. The reaction conditions were 30°C for 15 min; 42°C for 60 min; and 70°C for 15 min.

[0077] (3) PCR amplification

[0078] The primers for EV-A71 full sequence determination are shown in Table 3:

[0079] Table 3 Primers for EV-A71 full genome sequencing

[0080]

[0081]

[0082] PCR amplification was performed using KOD-Plus-Neo (Cat. No. KOD-401) produced by Toyobo Company, with cycles of 30 to 35 at 98°C for 10 min, 98°C for 30 sec, 57°C for 3 min 30 sec, and 68°C (extension rate 30 sec / kb).

[0083] (4) Agarose gel electrophoresis

[0084] After the PCR reaction is completed, the product is subjected to 1% agarose gel electrophoresis to identify the PCR amplified bands. Once the PCR product is correctly identified, it is sent to Sangon Bioengineering Technology Service Co., Ltd. for sequencing, and the fragments are spliced ​​to obtain the full genome sequence.

[0085] Sequencing results showed that the full genome sequence of the EV-A71TS- / JS PX / 2019 virus strain is as follows:

[0086] 1 ttaaaacagcctgtgggttgcacccacccacagggcctactgggcgcaagcactctggta

[0087] 61 tctcggtacctttgtgcgcctgttttataacccccccccaacgaaacttagaagcagcaa

[0088] 121 atcacgatcaatagcaggcataacgctccagttgtgtctcgatcaagcacttctgtttcc

[0089] 181 ccggactgagtatcaatagactgctcgcgcggttgaaggagaaaacgttcgttatccggc

[0090] 241 tagctacttcggaaaacctagtagcaccatgaaagttgcggagagcttcgttcagcactc

[0091] 301 ccccagtgtagatcaggtcgatgagtcaccgcattccccatgggcgaccgtggcggtggc

[0092] 361 tgcgttggcggcctgcccatggggtaacccatggggcgctctaatacggacatggtgtga

[0093] 421 agagtctactgagctagctagtagtcctccggcccctgaatgcggctaatcccaactgcg

[0094] 481 gagcacacgcccacaaaccagcgggtagtgtgtcgtaatgggtaactctgcagcggaacc

[0095] 541 gactactttgggtgtccgtgtttccttttatctttatattggctgcttatggtgacaatt

[0096] 601 aaagaattgttaccatatagctattggattggccatccggtgtgcaacagagcgattgtc

[0097] 661 tacctatttattggttttgttccattaactttgaattctgtgactacccttaattatatc

[0098] 721 ttgacccttaacacagctaaacatgggctcgcaagtgtctacacagcgctccgggtctca

[0099] 781 cgagaactcaaactcagccactgagggttctactataaactacaccaccattaattacta

[0100] 841 caaagactcctatgctgccacagcaggcaaacagagcctcaaacaggatccagacaagtt

[0101] 901 tgcaaatcctgttaaagacatctttactgaaatggcagcgccattgaagtccccatccgc

[0102] 961 tgaagcatgtgggtacagtgatcgagtggcgcagttaactattggcaactccaccatcac

[0103] 1021 tacgcaagaagcggctaacatcatagtcggttatggcgagtggccttcctactgctcaga

[0104] 1081 ttctgacgctacagcagtggataaaccaacgcgcccggatgtttcagtgaacaggtttta

[0105] 1141 cacattggacactaagttgtgggagaaatcgtctaaaggatggtactggaagttcccaga

[0106] 1201 tgtgctgactgaaactggggtttttgggcaaaatgcacaattccattacctctaccggtc

[0107] 1261 agggttttgcatccacgtacagtgcaatgctagtaaattccatcaaggagcactcctagt

[0108] 1321 cgccgtcctaccagagtatgtcattgggacagtggcaggcggtacagggacggaagatac

[0109] 1381 ccaccccccttacaagcagactcaacccggtgctgatggcttcgaactgcaacacccata

[0110] 1441 tgtgcttgatgctggcatcccaatatcacagttaacagtgtgcccacatcagtggatcaa

[0111] 1501 cctgaggaccaacaattgtgctacaataatagtgccatacatcaacgcactgccttttga

[0112] 1561 ctctgctttgaatcattgcaactttggcctgttagttgtgcctattagcccactagacta

[0113] 1621 cgaccaaggagcgacgccagtaatccctataactattacgttggccccaatgtgctctga

[0114] 1681 gttcgcaggtcttaggcaagcagtcacgcaaggattccccaccgagctgaaacctggtac

[0115] 1741 aaatcagtttctaaccactgacgatggcgtttcagcacctattctaccaaactttcaccc

[0116] 1801 caccccgtgtatccacatacctggtgaggttaggaacttgctagagttatgccaggtgga

[0117] 1861 gaccattctggaggtcaacaatgtgcccacgaatgccactagcttaatggagagactccg

[0118] 1921 cttcccggtttcggcacaagcagggaagggtgagctgtgtgcggtatttagagctgatcc

[0119] 1981 tgggcgaaatgggccatggcaatccaccttactgggtcagttgtgcgggtactacaccca

[0120] 2041 atggtcaggatcattggaagtcacctttatgtttactggatctttcatggctaccggtaa

[0121] 2101 gatgctcatagcctatacaccgccaggaggtcctctgcccaaggaccgggcgaccgccat

[0122] 2161 gttgggcacgcacgtcatctgggattttgggctgcaatcatctgttaccttagtaatacc

[0123] 2221 atggatcagcaacactcattatagagcacatgcccgggatggagtgtttgactactacac

[0124] 2281 cacggggttagttagtatatggtatcagacgaattacgtggttccaatcggtgcgcccaa

[0125] 2341 cacagcctatataatagcactagcagcagctcaaaagaacttcacaatgaaattgtgtaa

[0126] 2401 ggatgctagtgatatcctgcagacgggcactattcagggagacagggtggcagatgtaat

[0127] 2461 agaaagctccataggagacagcgtgagcagagctctcactcacgctctaccagcacccac

[0128] 2521 cggccagaacacacaggtgagcagccatcgactggatacaggtaaggttccagcactcca

[0129] 2581 ggctgccgagattggagcatcatcaaatgctagtgatgagagcatgattgaaacacgctg

[0130] 2641 tgttcttaactcgcacagcacagctgagaccactcttgatagtttcttcagcagggcggg

[0131] 2701 tttagttggagaaatagatctccctcttgaaggtacaactaacccaaatggttatgccaa

[0132] 2761 ctgggatatagatataacaggttacgcgcaaatgcgtagaaaggtggagttattcaccta

[0133] 2821 catgcgctttgatgcagagttcacttttgttgcgtgtacacccaccgggcaagttgtacc

[0134] 2881 acaattgctccaatatatgtttgtgccacctggagcccctaagccagattcaagggaatc

[0135] 2941 ccttgcatggcaaaccgccactaacccctcagtttttgtcaagttgtcagaccctccagc

[0136] 3001 gcaggtttcagtaccatttatgtcacctgcgagtgcttaccaatggttttatgacggata

[0137] 3061 tcccacattcggagagcacaagcaagagaaagatcttgaatatggagcatgtcctaataa

[0138] 3121 catgatgggcacgttctcggtacggactgtggggacctccaagtctaagtatcctttagt

[0139] 3181 agttaggatctacatgagaatgaagcacgttagggcgtggatacctcgcccgatgcgtaa

[0140] 3241 ccagaactacctattcaaagccaacccaaattatgctggcaactctattaagccaactgg

[0141] 3301 tgccagtcgcacagcgatcaccactcttgggaaatttggacaacagtctggggctattta

[0142] 3361 tgtgggtaattttagagtggtcaatcgacatcttgccactcacaatgattgggcaaatct

[0143] 3421 tgtttgggaagacagctcccgcgatctgcttgtgtcatccaccactgcccaaggttgcga

[0144] 3481 cacgattgctcgttgcgattgccagacaggggtgtactactgtaactcgatgagaaaaca

[0145] 3541 ctacccagtcagtttttcaaaacccagcttgatctatgtagaggctagcgagtattaccc

[0146] 3601 agccaggtaccaatcacatctcatgctcgcacagggtcactcagaacctggtgattgcgg

[0147] 3661 tggtatccttagatgtcaacatggcgtcgttggcatagtgtctactggtggcaatggact

[0148] 3721 cgttggttttgcagatgttagagacctcttgtggttagatgaggaagctatggaacaggg

[0149] 3781 tgtgtctgactacatcaagggacttggagatgcttttggaacaggcttcactgacgcagt

[0150] 3841 ctcaagagaggttgaagctctcaagacctatcttataggatctgaaggagcagttgagaa

[0151] 3901 aatcttgaaaaatctcattaaattaatctctgcactggtgattgtgatcaggagtgatta

[0152] 3961 tgacatggttaccctcactgcaaccttggcgctgataggctgtcatggtagtccttgggc

[0153] 4021 ttggatcaaagctaaaacagcctctatcttaggtatccctatcgctcagaaacagagcgc

[0154] 4081 ttcctggctcaagaaattcaatgacatggccaatgccgccaaggggttagagtgggtttc

[0155] 4141 taacaaaatcagcaaatttattgattggcttaaggagaaaatagtaccagcagccaggga

[0156] 4201 gaaagttgaattcctgaataacttgaaacagttgccactactagagaatcagatctcaaa

[0157] 4261 cttggaacagtctgctgcctcacaggaggaccttgaagtcatgtttggaaatgtgtcgta

[0158] 4321 cctagctcacttctgtcgcaagtttcaaccgttatacgccacagaagctaaaagagtcta

[0159] 4381 tgccctggagaagagaatgaacaactatatgcagttcaagagcaaacaccgaattgaacc

[0160] 4441 tgtatgtcttattattaggggttcaccaggcacagggaagtctctagccactggtatcat

[0161] 4501 tgctcgagcaatcgctgataagtaccactccagcgtgtactcgctcccaccagacccgga

[0162] 4561 tcattttgacggttacaagcaacaggtggttacagtgatggacgatttgtgtcaaaaccc

[0163] 4621 cgatggtaaggatatgtctctgttctgtcaaatggtatccaccgtggatttcattccacc

[0164] 4681 aatggcttctctcgaggagaagggagtttccttcacctctaagtttgtcatcgcatccac

[0165] 4741 taatgccagtaatatcatagtgccaacagtgtctgattctgacgctattcgccgcaggtt

[0166] 4801 ttacatggactgtgacattgaagtgacagactcatacaagacagatctaggtagactgga

[0167] 4861 tgcggggcgagccgctaaactgtgttctgaaaacaacactgcaaatttcaaacggtgcag

[0168] 4921 cccattagtgtgtgggaaagctatccaacttagagacagaaagtctaaagtcagatacag

[0169] 4981 tgtggacacggtagtttcggaacttattagggaatacagcaataggtccgccattggcaa

[0170] 5041 cacaatcgaggctcttttccaaggtccacccaagttcaggccaattaggattagccttga

[0171] 5101 agaaaaaccagctccagacgctattagcgatctccttgctagcgtagatagtgaagaggt

[0172] 5161 gcgccagtactgcagagatcaaggttggatcattcctgaagctcccaccaatgtagagcg

[0173] 5221 acacctcaatagagcggtgcttgttatgcagtccattgccacagtggtggcggttgtttc

[0174] 5281 attggtatatgtcatctacaagctctttgcagggtttcagggtgcgtactctggagctcc

[0175] 5341 taagcaagtgcttaagaagcctgctcttcgcacagcaacagtgcagggcccgagccttga

[0176] 5401 ctttgctctctccctactgagaaggaacatcaggcaagtccaaacagaccaggggcattt

[0177] 5461 caccatgctgggtgttagggatcgcttagcagtcctcccacgccactcgcaacctggcaa

[0178] 5521 agctatttggatcgagcacaaacttgtaaacgtccttgatgcagttgaactggtggatga

[0179] 5581 gcaaggagtcaacctggaattaaccctcatcactcttgacaccaacgaaaagtttaggga

[0180] 5641 tattaccaaattcatcccagaaaatattagcactgctagtgatgccaccctagtgatcaa

[0181] 5701 cacggagcacatgccatcaatgtttgtcccggtaggtgacgttgtgcagtatggcttttt

[0182] 5761 gaatctcagtggtaagcccacccatcgcactatgatgtacaactttcctactaaagcagg

[0183] 5821 gcagtgtggaggagtggtgacatctgttgggaagattgttggtattcacattggtggtaa

[0184] 5881 tggcagacaaggtttttgcgcaggcctcaaaaggagttattttgccagtgaacaaggaga

[0185] 5941 gatccaatgggttaagcccaataaagaaactggaagactcaacatcaatggaccaacccg

[0186] 6001 caccaagttagaacctagtgtgttccatgacatcttcgagggaaacaaagaaccagctgt

[0187] 6061 cttgcacagtaaggacccccgacttgaggtggattttgaacaggccctgttctctaagta

[0188] 6121 tgtgggaaacacactacatgagcctgatgagtacatcaaagaggcagctcttcattatgc

[0189] 6181 gaaccagttaaagcaactagaaattaatacctctcaaatgagcatggaggaggcctgcta

[0190] 6241 cggcactgaaaatcttgaggctattgatcttcacactagtgcaggttacccttatagtgc

[0191] 6301 tctggggataaagaaaagagacatcttagaccctaccactagggacgtgagtagaatgaa

[0192] 6361 gttttacatggacaagtatggtcttgatcttccttactccacttatgtcaaggatgagct

[0193] 6421 gcgctcgattgacaagattaagaaagggaagtcccgtctgatcgaagccagtagtctgaa

[0194] 6481 tgattcagtgtaccttagaatggctttcggacatttgtatgaggcctttcacgcaaatcc

[0195] 6541 tgggacgataactggatcagcagtggggtgcaatcctgacacattctggagtaagctgcc

[0196] 6601 aattttgctccctggttcactctttgcctttgactactcaggttatgatgccagtcttag

[0197] 6661 ccctgtctggttcagagcattagaattggttcttagggagatagggtatagtgaggaggc

[0198] 6721 aatttcactcattgaaggaatcaaccacacacaccatgtgtatcgtaacaagacctactg

[0199] 6781 tgtgcttggtgggatgccctcaggctgttcgggaacatccattttcaattcaatgatcaa

[0200] 6841 caacattattatcagagcgctgctcataaaaacatttaagggcattgacttggatgaact

[0201] 6901 caacatggttgcttatggagacgatgtgctcgctagttaccccttcccaatcgattgttt

[0202] 6961 ggaactagcaaagactggtaaggagtatggtctgaccatgactcctgctgataaatctcc

[0203] 7021 ttgttttaatgaggtcaattggggtaatgcgaccttcctcaaaaggggcttcttgcccga

[0204] 7081 cgaacagtttccatttttgattcaccctactatgccaatgagtgagatccatgagtccat

[0205] 7141 tcgatggactaaggacgcacgaaacactcaagatcatgtgcggtccttgtgcctcctggc

[0206] 7201 atggcataatggtaagcaagaatatgagaggtttgtgagcacaattaggtctgtcccagt

[0207] 7261 agggagagctttggctatcccaaattatgaaaatcttagacgcaattggctcgaattatt

[0208] 7321 ttagaggttatacacctcaaccccaccagaaatctggtcgtgaatatgactggtgggggt

[0209] 7381 aaatttgttataaccagaatagc(SEQ ID NO.18)。

[0210] The amino acid sequence of the polyprotein is as follows:

[0211] 1 MGSQVSTQRSGSHENSNSTATEGSTINYTTINYYKDSYAATAGKQSLKQDPDKFANPVKDI

[0212] 61 FTEMAAPLKSPSAEACGYSDRAVQLTIGNSTITTEAANIIVGYGEWPSYCSDSDATAVD

[0213] 121 KPTRPDVSVNRFYTLDTKLWEEKSSKGWYWKFPDVLTE TG VFGQNAQFHYLYRSGFCIHVQ

[0214] 181 CNASKFHQGALLVAVLPEYVIGTVAGGTGTEDTHPPYKQTQPGADGFELQHPYVLDAQIP

[0215] 241 ISQLTVCPHQWINLRTNNCATIIVPYINALPFDSALNHCNFGLLVVPISPLDYDQGATPV <(

[0216] 301 IPITITLAPMCSEFAGLRQAVTQG FPTELKP GTNQF LTTDDGVSAPILPNFHPTPCHIP

[0217] 361 GEVRNLLELCQVETILEVNNVPTNATSLMERLRFPVSAQAGKGELCAVFRADPGRNGPWQ

[0218] 421 STLLGQLCGYYTQWSGSLEVTFMFTGSFMATGKM LIAYTPPGGPLPKDRATAMLGT HVIW It should be noted that there seems to be a small error in the tag in line 26 where it should probably be

[0216] instead of <(

[0216] . This has been translated as best as possible with the given text.

[0219] 481 dfglqssvtlvipwisnthyrahardgvfdyyttglvsiwyqtnyvvpigapntayiial

[0220] 541 aaaqknftmklckdasdilqtgtiqgdrvadviessigdsvsralthalpaptgqntqvs

[0221] 601 shrldtgkvpalqaaeigassnasdesmietrcvlnshstaettldsffsraglvgeidl

[0222] 661 plegttnpngyanwdiditgyaqmrrkvelftymrfdaeftfvactptgqvvpqllqymf

[0223] 721 vppgapkpdsreslawqtatnpsvfvklsdppaqvsvpfmspasayqwfydgyptfgehk

[0224] 781 qekdleygacpnnmmgtfsvrtvgtskskyplvvriymrmkhvrawiprpmrnqnylfka

[0225] 841 npnyagnsikptgasrtaittlgkfgqqsgaiyvgnfrvvnrhlathndwanlvwedssr

[0226] 901 dllvssttaqgcdtiarcdcqtgvyycnsmrkhypvsfskpsliyveaseyyparyqshl

[0227] 961 mlaqghsepgdcggilrcqhgvvgivstggnglvgfadvrdllwldeeameqgvsdyikg

[0228] 1021 lgdafgtgftdavsrevealktyligsegavekilknliklisalvivirsdydmvtlta

[0229] 1081 tlaligchgspwawikaktasilgipiaqkqsaswlkkfndmanaakglewvsnkiskfi

[0230] 1141 dwlkekivpaarekveflnnlkqlpllenqisnleqsaasqedlevmfgnvsylahfcrk

[0231] 1201 fqplyateakrvyalekrmnnymqfkskhriepvcliirgspgtgkslatgiiaraiadk

[0232] 1261 yhssvyslppdpdhfdgykqqvvtvmddlcqnpdgkdmslfcqmvstvdfippmasleek

[0233] 1321 gvsftskfviastnasniivptvsdsdairrrfymdcdievtdsyktdlgrldagraakl

[0234] 1381 csenntanfkrcsplvcgkaiqlrdrkskvrysvdtvvselireysnrsaigntiealfq

[0235] 1441 gppkfrpirisleekpapdaisdllasvdseevrqycrdqgwiipeaptnverhlnravl

[0236] 1501 vmqsiatvvavvslvyviyklfagfqgaysgapkqvlkkpalrtatvqgpsldfalsllr

[0237] 1561 rnirqvqtdqghftmlgvrdrlavlprhsqpgkaiwiehklvnvldavelvdeqgvnlel

[0238] 1621 tlitldtnekfrditkfipenistasdatlvintehmpsmfvpvgdvvqygflnlsgkpt

[0239] 1681 hrtmmynfptkagqcggvvtsvgkivgihiggngrqgfcaglkrsyfaseqgeiqwvkpn

[0240] 1741 ketgrlngingptrtklepsvfhdifegnkepavlhskdprlevdfeqalfskyvgntlhe

[0241] 1801 pdeyikeaalhyanqlkqleintsqmsmeeacygtenleaidlhtsagypysalgikkrd

[0242] 1861 ildpttrdvsrmkfymdkygldlpystyvkdelrsidkikkgksrlieasslndsvylrm

[0243] 1921 afghlyeafhanpgtitgsavgcnpdtfwsklpillpgslfafdysgydaslspvwfral

[0244] 1981 elvlreigyseeaislieginhthhvyrnktycvlggmpsgcsgtsifnsminniiiral

[0245] 2041 liktfkgidldelnmvaygddvlasypfpidclelaktgkeygltmtpadkspcfnevnw

[0246] 2101 gnatflkrgflpdeqfpflihptmpmseihesirwtkdarntqdhvrslcllawhngkqe

[0247] 2161 yerfvstirsvpvgralaipnyenlrrnwl elf*(SEQ ID NO.19).

[0248] The complete genome sequence of the EV-A71 TS+ / JS PX / 2019 virus strain is as follows:

[0249] 1 ttaaaacagcctgtgggttgcacccacccacagggcctactgggcgcaagcactctggta

[0250] 61 tctcggtacctttgtgcgcctgttttataacccccccccaacgaaacttagaagcagcaa

[0251] 121 atcacgatcaatagcaggcataacgctccagttgtgtctcgatcaagcacttctgtttcc

[0252] 181 ccggactgagtatcaatagactgctcgcgcggttgaaggagaaaacgttcgttatccggc

[0253] 241 tagctacttcggaaaacctagtagcaccatgaaagttgcggagagcttcgttcagcactc

[0254] 301 ccccagtgtagatcaggtcgatgagtcaccgcattccccatgggcgaccgtggcggtggc

[0255] 361 tgcgttggcggcctgcccatggggtaacccatggggcgctctaatacggacatggtgtga

[0256] 421 agagtctactgagctagctagtagtcctccggcccctgaatgcggctaatcccaactgcg

[0257] 481 gagcacacgcccacaaaccagcgggtagtgtgtcgtaatgggtaactctgcagcggaacc

[0258] 541 gactactttgggtgtccgtgtttccttttatctttatattggctgcttatggtgacaatt

[0259] 601 aaagaattgttaccatatagctattggattggccatccggtgtgcaacagagcgattgtc

[0260] 661 tacctatttattggttttgttccattaactttgaattctgtgactacccttaattatatc

[0261] 721 ttgacccttaacacagctaaacatgggctcgcaagtgtctacacagcgctccgggtctca

[0262] 781 cgagaactcaaactcagccactgagggttctactataaactacaccaccattaattacta

[0263] 841 caaagactcctatgctgccacagcaggcaaacagagcctcaaacaggatccagacaagtt

[0264] 901 tgcaaatcctgttaaagacatctttactgaaatggcagcgccattgaagtccccatccgc

[0265] 961 tgaagcatgtgggtacagtgatcgagtggcgcagttaactattggcaactccaccatcac

[0266] 1021 tacgcaagaagcggctaacatcatagtcggttatggcgagtggccttcctactgctcaga

[0267] 1081 ttctgacgctacagcagtggataaaccaacgcgcccggatgtttcagtgaacaggtttta

[0268] 1141 cacattggacactaagttgtgggagaaatcgtctaaaggatggtactggaagttcccaga

[0269] 1201 tgtgctgactgaaactggggtttttgggcaaaatgcacaattccattacctctaccggtc

[0270] 1261 agggttttgcatccacgtacagtgcaatgctagtaaattccatcaaggagcactcctagt

[0271] 1321 cgccgtcctaccagagtatgtcattgggacagtggcaggcggtacagggacggaagatac

[0272] 1381 ccaccccccttacaagcagactcaacccggtgctgatggcttcgaactgcaacacccata

[0273] 1441 tgtgcttgatgctggcatcccaatatcacagttaacagtgtgcccacatcagtggatcaa

[0274] 1501 cctgaggaccaacaattgtgctacaataatagtgccatacatcaacgcactgccttttga

[0275] 1561 ctctgctttgaatcattgcaactttggcctgttagttgtgcctattagcccactagacta

[0276] 1621 cgaccaaggagcgacgccagtaatccctataactattacgttggccccaatgtgctctga

[0277] 1681 gttcgcaggtcttaggcaagcagtcacgcaaggattccccaccgagctgaaacctggtac

[0278] 1741 aaatcagtttctaaccactgacgatggcgtttcagcacctattctaccaaactttcaccc

[0279] 1801 caccccgtgtatccacatacctggtgaggttaggaacttgctagagttatgccaggtgga

[0280] 1861 gaccattctggaggtcaacaatgtgcccacgaatgccactagcttaatggagagactccg

[0281] 1921 cttcccggtttcggcacaagcagggaagggtgagctgtgtgcggtatttagagctgatcc

[0282] 1981 tgggcgaaatgggccatggcaatccaccttactgggtcagttgtgcgggtactacaccca

[0283] 2041 atggtcaggatcattggaagtcacctttatgtttactggatctttcatggctaccggtaa

[0284] 2101 gatgctcatagcctatacaccgccaggaggtcctctgcccaaggaccgggcgaccgccat

[0285] 2161 gttgggcacgcacgtcatctgggattttgggctgcaatcatctgttaccttagtaatacc

[0286] 2221 atggatcagcaacactcattatagagcacatgcccgggatggagtgtttgactactacac

[0287] 2281 cacggggttagttagtatatggtatcagacgaattacgtggttccaatcggtgcgcccaa

[0288] 2341 cacagcctatataatagcactagcagcagctcaaaagaacttcacaatgaaattgtgtaa

[0289] 2401 ggatgctagtgatatcctgcagacgggcactattcagggagacagggtggcagatgtaat

[0290] 2461 agaaagctccataggagacagcgtgagcagagctctcactcacgctctaccagcacccac

[0291] 2521 cggccagaacacacaggtgagcagccatcgactggatacaggtaaggctccagcactcca

[0292] 2581 ggctgccgagattggagcatcatcaaatgctagtgatgagagcatgattgaaacacgctg

[0293] 2641 tgttcttaactcgcacagcacagctgagaccactcttgatagtttcttcagcagggcggg

[0294] 2701 tttagttggagaaatagatctccctcttgaaggtacaactaacccaaatggttatgccaa

[0295] 2761 ctgggatatagatataacaggttacgcgcaaatgcgtagaaaggtggagttattcaccta

[0296] 2821 catgcgctttgatgcagagttcacttttgttgcgtgtacacccaccgggcaagttgtacc

[0297] 2881 acaattgctccaatatatgtttgtgccacctggagcccctaagccagattcaagggaatc

[0298] 2941 ccttgcatggcaaaccgccactaacccctcagtttttgtcaagttgtcagaccctccagc

[0299] 3001 gcaggtttcagtaccatttatgtcacctgcgagtgcttaccaatggttttatgacggata

[0300] 3061 tcccacattcggagagcacaagcaagagaaagatcttgaatatggagcatgtcctaataa

[0301] 3121 catgatgggcacgttctcggtacggactgtggggacctccaagtctaagtatcctgtagt

[0302] 3181 agttaggatctacatgagaatgaagcacgttagggcgtggatacctcgcccgatgcgtaa

[0303] 3241 ccagaactacctattcaaagccaacccaaattatgctggcaactctattaagccaactgg

[0304] 3301 tgccagtcgcacagcgatcaccactcttgggaaatttggacaacagtctggggctattta

[0305] 3361 tgtgggtaattttagagtggtcaatcgacatcttgccactcacaatgattgggcaaatct

[0306] 3421 tgtttgggaagacagctcccgcgatctgcttgtgtcatccgccactgcccaaggttgcga

[0307] 3481 cacgattgctcgttgcgattgccagacaggggtgtactactgtaactcgatgagaaaaca

[0308] 3541 ctacccagtcagtttttcaaaacccagcttgatctatgtagaggctagcgagtattaccc

[0309] 3601 agccaggtaccaatcacatctcatgctcgcacagggtcactcagaacctggtgattgcgg

[0310] 3661 tggtatccttagatgtcaacatggcgtcgttggcatagtgtctactggtggcaacggact

[0311] 3721 cgttggttttgcagatgttagagacctcttgtggttagatgaggaagctatggaacaggg

[0312] 3781 tgtgtctgactacatcaagggacttggagatgcttttggaacaggcttcactgacgcagt

[0313] 3841 ctcaagagaggttgaagctctcaagacctatcttataggatctgaaggagcagttgagaa

[0314] 3901 aatcttgaaaaatctcattaaattaatctctgcactggtgattgtgatcaggagtgatta

[0315] 3961 tgacatggttaccctcactgcaaccttggcgctgataggctgtcatggtagtccttgggc

[0316] 4021 ttggatcaaagctaaaacagcctctatcttaggtatccctatcgctcagaaacagagcgc

[0317] 4081 ttcctggctcaagaaattcaatgacatggccaatgccgccaaggggttagagtgggtttc

[0318] 4141 taacaaaatcagcaaatttattgattggcttaaggagaaaatagtaccagcagccaggga

[0319] 4201 gaaagttgaattcctgaataacttgaaacagttgccactactagagaatcagatctcaaa

[0320] 4261 cttggaacagtctgctgcctcacaggaggaccttgaagtcatgtttggaaatgtgtcgta

[0321] 4321 cctagctcacttctgtcgcaagtttcaaccgttatacgccacagaagctaaaagagtcta

[0322] 4381 tgccctggagaagagaatgaacaactatatgcagttcaagagcaaacaccgaattgaacc

[0323] 4441 tgtatgtcttattattaggggttcaccaggcacagggaagtctctagccactggtatcat

[0324] 4501 tgctcgagcaatcgctgataagtaccactccagcgtgtactcgctcccaccagacccgga

[0325] 4561 tcattttgacggttacaagcaacaggtggttacagtgatggacgatttgtgtcaaaaccc

[0326] 4621 cgatggtaaggatatgtctctgttctgtcaaatggtatccaccgtggatttcattccacc

[0327] 4681 aatggcttctctcgaggagaagggagtttccttcacctctaagtttgtcatcgcatccac

[0328] 4741 taatgccagtaatatcatagtgccaacagtgtctgattctgacgctattcgccgcaggtt

[0329] 4801 ttacatggactgtgacattgaagtgacagactcatacaagacagatctaggtagactgga

[0330] 4861 tgcggggcgagccgctaaactgtgttctgaaaacaacactgcaaatttcaaacggtgcag

[0331] 4921 cccattagtgtgtgggaaagctatccaacttagagacagaaagtctaaagtcagatacag

[0332] 4981 tgtggacacggtagtttcggaacttattagggaatacagcaataggtccgccattggcaa

[0333] 5041 cacaatcgaggctcttttccaaggcccacccaagttcaggccaattaggattagccttga

[0334] 5101 agaaaaaccagctccagacgctattagcgatctccttgctagcgtagatagtgaagaggt

[0335] 5161 gcgccagtactgcagagatcaaggttggatcattcctgaagctcccaccaatgtagagcg

[0336] 5221 acacctcaatagagcggtgcttgttatgcagtccattgccacagtggtggcggttgtttc

[0337] 5281 attggtatatgtcatctacaagctctttgcagggtttcagggtgcgtactctggagctcc

[0338] 5341 taagcaagtgcttaagaagcctgctcttcgcacagcaacagtgcagggcccgagccttga

[0339] 5401 ctttgctctctccctactgagaaggaacatcaggcaagtccaaacagaccaggggcattt

[0340] 5461 caccatgctgggtgttagggatcgcttagcagtcctcccacgccactcgcaacctggcaa

[0341] 5521 agctatttggatcgagcacaaacttgtaaacgtccttgatgcagttgaactggtggatga

[0342] 5581 gcaaggagtcaacctggaattaaccctcatcactcttgacaccaacgaaaagtttaggga

[0343] 5641 tattaccaaattcatcccagaaaatattagcactgctagtgatgccaccctagtgatcaa

[0344] 5701 cacggagcacatgccatcaatgtttgtcccggtaggtgacgttgtgcagtatggcttttt

[0345] 5761 gaatctcagtggtaagcccacccatcgcactatgatgtacaactttcctactaaagcagg

[0346] 5821 gcagtgtggaggagtggtgacatctgttgggaagattgttggtattcacattggtggtaa

[0347] 5881 tggcagacaaggtttttgcgcaggcctcaaaaggagttattttgccagtgaacaaggaga

[0348] 5941 gatccaatgggttaagcccaataaagaaactggaagactcaacatcaatggaccaacccg

[0349] 6001 caccaagttagaacctagtgtgttccatgacatcttcgagggaaacaaagaaccagctgt

[0350] 6061 cttgcacagtaaggacccccgacttgaggtggattttgaacaggccctgttctctaagta

[0351] 6121 tgtgggaaacacactacatgagcctgatgagtacatcaaagaggcagctcttcattatgc

[0352] 6181 gaaccagttaaagcaactagaaattaatacctctcaaatgagcatggaggaggcctgcta

[0353] 6241 cggcactgaaaatcttgaggctattgatcttcacactagtgcaggttacccttatagtgc

[0354] 6301 tctggggataaagaaaagagacatcttagaccctaccactagggacgtgagtagaatgaa

[0355] 6361 gttttacatggacaagtatggtcttgatcttccttactccacttatgtcaaggatgagct

[0356] 6421 gcgctcgattgacaagattaagaaagggaagtcccgtctgatcgaagccagtagtctgaa

[0357] 6481 tgattcagtgtaccttagaatggctttcggacatttgtatgaggcctttcacgcaaatcc

[0358] 6541 tgggacgataactggatcagcagtggggtgcaatcctgacacattctggagtaagctgcc

[0359] 6601 aattttgctccctggttcactctttgcctttgactactcaggttatgatgccagtcttag

[0360] 6661 ccctgtctggttcagagcattagaattggttcttagggagatagggtatagtgaggaggc

[0361] 6721 aatttcactcattgaaggaatcaaccacacacaccatgtgtatcgtaacaagacctactg

[0362] 6781 tgtgcttggtgggatgccctcaggctgttcgggaacatccattttcaattcaatgatcaa

[0363] 6841 caacattattatcagagcgctgctcataaaaacatttaagggcattgacttggatgaact

[0364] 6901 caacatggttgcttatggagacgatgtgctcgctagttaccccttcccaatcgattgttt

[0365] 6961 ggaactagcaaagactggtaaggagtatggtctgaccatgactcctgctgataaatctcc

[0366] 7021 ttgttttaatgaggtcaattggggtaatgcgaccttcctcaaaaggggcttcttgcccga

[0367] 7081 cgaacagtttccatttttgattcaccctactatgccaatgagtgagatccatgagtccat

[0368] 7141 tcgatggactaaggacgcacgaaacactcaagatcatgtgcggtccttgtgcctcctggc

[0369] 7201 atggcataatggtaagcaagaatatgagaggtttgtgagcacaattaggtctgtcccagt

[0370] 7261 agggagagctttggctatcccaaattatgaaaatcttagacgcaattggctcgaattatt

[0371] 7321 ttagaggttatacacctcaaccccaccagaaatctggtcgtgaatatgactggtgggggt

[0372] 7381 aaatttgttataaccagaatagc(SEQ ID NO.20)。

[0373] The amino acid sequence of the polyprotein is as follows:

[0374] 1 mgsqvstqrsgshensnsategstinyttinyykdsyaatagkqslkqdpdkfanpvkdi

[0375] 61 ftemaaplkspsaeacgysdrvaqltignstittqeaaniivgygewpsycsdsdatavd

[0376] 121 kptrpdvsvnrfytldtklweksskgwywkfpdvltetgvfgqnaqfhylyrsgfcihvq

[0377] 181 cnaskfhqgallvavlpeyvigtvaggtgtedthppykqtqpgadgfelqhpyvldagip

[0378] 241 isqltvcphqwinlrtnncatiivpyinalpfdsalnhcnfgllvvpispldydqgatpv

[0379] 301 ipititlapmcsefaglrqavtqgfptelkpgtnqflttddgvsapilpnfhptpcihip

[0380] 361 gevrnllelcqvetilevnnvptnatslmerlrfpvsaqagkgelcavfradpgrngpwq

[0381] 421 stllgqlcgyytqwsgslevtfmftgsfmatgkmliaytppggplpkdratamlgthviw

[0382] 481 dfglqssvtlvipwisnthyrahardgvfdyyttglvsiwyqtnyvvpigapntayiial

[0383] 541 aaaqknftmklckdasdilqtgtiqgdrvadviessigdsvsralthalpaptgqntqvs

[0384] 601 shrldtgkapalqaaeigassnasdesmietrcvlnshstaettldsffsraglvgeidl

[0385] 661 plegttnpngyanwdiditgyaqmrrkvelftymrfdaeftfvactptgqvvpqllqymf

[0386] 721 vppgapkpdsreslawqtatnpsvfvklsdppaqvsvpfmspasayqwfydgyptfgehk

[0387] 781 qekdleygacpnnmmgtfsvrtvgtskskypvvvriymrmkhvrawiprpmrnqnylfka

[0388] 841 npnyagnsikptgasrtaittlgkfgqqsgaiyvgnfrvvnrhlathndwanlvwedssr

[0389] 901 dllvssataqgcdtiarcdcqtgvyycnsmrkhypvsfskpsliyveaseyyparyqshl

[0390] 961 mlaqghsepgdcggilrcqhgvvgivstggnglvgfadvrdllwldeeameqgvsdyikg

[0391] 1021 lgdafgtgftdavsrevealktyligsegavekilknliklisalvivirsdydmvtlta

[0392] 1081 tlaligchgspwawikaktasilgipiaqkqsaswlkkfndmanaakglewvsnkiskfi

[0393] 1141 dwlkekivpaarekveflnnlkqlpllenqisnleqsaasqedlevmfgnvsylahfcrk

[0394] 1201 fqplyateakrvyalekrmnnymqfkskhriepvcliirgspgtgkslatgiiaraiadk

[0395] 1261 yhssvyslppdpdhfdgykqqvvtvmddlcqnpdgkdmslfcqmvstvdfippmasleek

[0396] 1321 gvsftskfviastnasniivptvsdsdairrrfymdcdievtdsyktdlgrldagraakl

[0397] 1381 csenntanfkrcsplvcgkaiqlrdrkskvrysvdtvvselireysnrsaigntiealfq

[0398] 1441 gppkfrpirisleekpapdaisdllasvdseevrqycrdqgwiipeaptnverhlnravl

[0399] 1501 vmqsiatvvavvslvyviyklfagfqgaysgapkqvlkkpalrtatvqgpsldfalsllr

[0400] 1561 rnirqvqtdqghftmlgvrdrlavlprhsqpgkaiwiehklvnvldavelvdeqgvnlel

[0401] 1621 tlitldtnekfrditkfipenistasdatlvintehmpsmfvpvgdvvqygflnlsgkpt

[0402] 1681 hrtmmynfptkagqcggvvtsvgkivgihiggngrqgfcaglkrsyfaseqgeiqwvkpn

[0403] 1741 ketgrlngingptrtklepsvfhdifegnkepavlhskdprlevdfeqalfskyvgntlhe

[0404] 1801 pdeyikeaalhyanqlkqleintsqmsmeeacygtenleaidlhtsagypysalgikkrd

[0405] 1861 ildpttrdvsrmkfymdkygldlpystyvkdelrsidkikkgksrlieasslndsvylrm

[0406] 1921 afghlyeafhanpgtitgsavgcnpdtfwsklpillpgslfafdysgydaslspvwfral

[0407] 1981 elvlreigyseeaislieginhthhvyrnktycvlggmpsgcsgtsifnsminniiiral

[0408] 2041 liktfkgidldelnmvaygddvlasypfpidclelaktgkeygltmtpadkspcfnevnw

[0409] 2101gnatflkrgflpdeqfpflihptmpmseihesirwtkdarntqdhvrslcllawhngkqe

[0410] 2161 yerfvstirsvpvgralaipnyenlrrnwl elf* (SEQ ID NO. 21).

[0411] The genome of the EV-A71 virus strain is 7403 nucleotides in length. The 5'-UTR and 3'-UTR of the non-coding regions are located at nucleotide positions 1-742 and 7325-7403, respectively. The 3'-UTR is followed by a poly(A) tail of undetermined length. The encoded polyprotein is 2194 amino acid residues in length. The amino acid sequences and positions of the structural and non-structural proteins of the virus are as follows:

[0412] The VP4 protein is the amino acid sequence from position 1 to 69;

[0413] The VP2 protein is the amino acid sequence from position 70 to 323;

[0414] The VP3 protein is the amino acid sequence from position 324 to 565;

[0415] The VP1 protein is the amino acid sequence from position 566 to 862;

[0416] The 2A protein is the amino acid sequence from position 863 to 1012;

[0417] The 2B protein is the amino acid sequence from positions 1013 to 1111;

[0418] The 2C protein is the amino acid sequence from positions 1112 to 1440;

[0419] The 3A protein is the amino acid sequence from position 1441 to 1526;

[0420] The 3B protein is the amino acid sequence from position 1527 to 1548;

[0421] The 3C protein is the amino acid sequence from position 1549 to 1731;

[0422] The 3D protein is the amino acid sequence from position 1732 to 2193.

[0423] Example 6

[0424] The complete genome sequences of EV-A71TS+ / JS PX / 2019 and EV-A71TS- / JS PX / 2019 were aligned using SnapGene software:

[0425] The 44th amino acid of the VP1 protein of the EV-A71TS- / JS PX / 2019 strain is valine (V); the 247th amino acid of the VP1 protein is leucine (L); and the 45th amino acid of the 2A protein is threonine (T). The 44th amino acid of the VP1 protein of the EV-A71TS+ / JS PX / 2019 strain is alanine (A); the 247th amino acid of the VP1 protein is valine (V); and the 45th amino acid of the 2A protein is alanine (A).

[0426] The nucleotide variations that cause the above amino acid changes and the variations of nonsense nucleotides in the genome are shown in Table 4 below:

[0427] Table 4 Key sites affecting the proliferation of strains in Vero cells after treatment at 52°C

[0428]

[0429] Example 7

[0430] This embodiment provides a method for purifying viral particles and preparing antigens, comprising the following steps:

[0431] (1) When the Vero cells in the ten-layer cell factory grew to a dense monolayer state, two EV-A71 strains (EV-A71TS+ / JS PX / 2019 virus strain and EV-A71TS- / JS PX / 2019 virus strain) were inoculated into the cell factory at the same MOI and cultured in a 37°C incubator.

[0432] (2) After more than 90% of the cells have become diseased, freeze and thaw three times repeatedly, harvest the supernatant of the virus solution by centrifugation at 4°C, 10,000×g for 30 minutes, and then concentrate it 10 times through ultrafiltration with a 100kDa ultrafiltration membrane. The virus concentrate is centrifuged at 4°C, 28,000rpm, and 20% sucrose as a bottom for 3 hours. The precipitate is resuspended in 0.01M PBS and then centrifuged at 4°C, 40,000rpm, and 15% to 55% sucrose density gradient (interval 10%) for 3 hours. The bands appearing in the 25% to 45% sucrose gradient are extracted. The extracted bands are mixed with cesium chloride to prepare a density of 1.29g / cm 3The CsCl solution was centrifuged at 48000 rpm and CsCl isodensity gradient at 4°C for 20 h. The bands were extracted and centrifuged to remove salts and harvest the virus particles.

[0433] The stratification of sucrose density gradient centrifugation is as follows Figure 4 As shown in (a): 4 opalescent bands appeared in the centrifuge tube.

[0434] After centrifugation to remove CsCl, the results of SDS-PAGE identification are as follows Figure 4 As shown in (b), EV-A71 EP and FP virus particles were obtained after purification. Under the same purification conditions, the yield of EV-A71 TS+ / JS PX / 2019 virus particles was significantly higher than that of EV-A71 TS- / JS PX / 2019 virus particles.

[0435] (3) The protein concentration of the purified virus particles was determined by the BCA method, and the total protein content of the harvested virus was calculated. The results of the EP and FP antigen content of the two strains are shown in Table 5 below:

[0436] Table 5 Comparison of antigen production of two virus strains

[0437]

[0438]

[0439] The yield of a single ten-layer cell factory for both EP and FP virus particles of the EV-A71TS+ / JS PX / 2019 strain was 0.9 mg; while the amount of antigen harvested by a single ten-layer cell factory for the EV-A71TS- / JS PX / 2019 strain was only 0.3 mg, which is about 1 / 3 of the yield of EV-A71TS+ / JS PX / 2019.

[0440] (4) 4% formaldehyde was added to the EP and FP particle suspensions of the two strains respectively for inactivation, inactivated at 37°C for 48 h, and then centrifuged using a 10 KDa ultrafiltration concentrator to remove the formaldehyde.

[0441] Example 8

[0442] This embodiment provides a method for animal immunization, comprising the following steps:

[0443] (1) The concentration of the formaldehyde-inactivated virus particles prepared in Example 7 was determined by the BCA method. The inactivated EP and FP were mixed with a chromatography salt buffer and an aluminum adjuvant according to the yield ratio of EP:FP harvested after purification, and two antigen dose groups of 0.5 μg and 1.5 μg were prepared.

[0444] (2) Female Balb / c mice aged 6 to 8 weeks were injected intraperitoneally with 500 μL of immunization per group (10 mice per group). PBS and aluminum adjuvant control groups were set up. Primary immunization was performed on day 0, booster immunization was performed on day 14, and blood was collected on day 28.

[0445] Example 9

[0446] This embodiment provides a method for detecting the titer of serum neutralizing antibodies, comprising the following steps:

[0447] (1) The serum samples obtained in Example 8 were diluted with diluent (MEM maintenance medium) at a ratio of 1:16 and added to column A of a 96-well plate at 100 μL / well. Two replicates were set up, and 50 μL of diluent was added to each well of columns B to H. The serum samples in column A were serially diluted 2-fold to column H, and 50 μL of the diluent was discarded after the last column was diluted.

[0448] (2) Dilute the neutralized virus to 100 CCID 50 / 50μL, draw 50μL vertically suspended dropwise into the diluted serum sample. Place the 96-well plate in a 37℃ incubator for 2 hours. Dilute the diluted neutralized toxin in a 10-fold gradient to 10CCID 50 / 50μL, 1CCID 50 / 50μL, 0.1CCID 50 / 50μL. Add 50μL of maintenance solution and 50μL of virus solution to each well (a total of 4 dilutions, namely 100, 10, 1, and 0.1 CCID 50 / 50μL), 8 replicate wells for each dilution, as a back titration plate, and place at 4℃.

[0449] (3) After neutralization, the digested RD cells were divided into 1×10 5 Plate the solution at a density of 1000 mL / min onto a neutralization plate and a back titration plate. Determine the neutralization result in 5 to 7 days.

[0450] The statistical results are as follows Figure 5 As shown. Humoral immunogenicity of isolated sera was evaluated using the EV-A71 national standard strain as the neutralizing toxin. Each point represents a single Balb / c mouse. Titers below 8 were obtained in the aluminum adjuvant and PBS inoculation groups, and the values ​​were assigned for plotting. One-way analysis of variance (ANOVA) was performed using the Sidak multiple comparison test.

[0451] The test results showed that 14 days after booster immunization, the serum neutralizing antibody titers of Balb / c mice immunized with high and low doses of EV-A71 TS+ / JS PX / 2019 virus strain were significantly higher than those of the serum after immunization with the same dose of EV-A71TS- / JS PX / 2019 virus strain.

[0452] Through cell culture virus technology, the present invention obtained the EV-A71 heat-stable strain (EV-A71TS+ / JS PX / 2019) and the EV-A71 heat-unstable strain (EV-A71TS- / JS PX / 2019). By determining the whole genome sequence of the two virus strains, the nucleotide and amino acid sites that affect the proliferation of the EV-A71 strain after treatment at 52°C were found. The mutation sites are different from the poliovirus heat stability-related sites reported in the existing literature, that is, they are not located in the VP1 hydrophobic pocket. The VP1 amino acids outside the hydrophobic pocket are not located in the heat-stable sites of other enteroviruses reported in the literature.

[0453] The two strains were further cultured in cell factories and purified, and the viral titers, particle yields, and humoral immunogenicity were compared and analyzed. The titer of the thermostable EV-A71 strain (EV-A71TS+ / JS PX / 2019) was comparable to that of the thermolabile strain, and its stability, viral particle yield, and humoral immunogenicity were superior to those of the thermolabile EV-A71 strain (EV-A71TS- / JSPX / 2019). Quantitative analysis of the virus particles after culture and purification in Vero cells showed that the two amino acid changes in the VP1 sequence resulted in higher particle yields in cell culture compared to the parent strain (the thermolabile strain).

[0454] This characteristic contrasts with the reduced viral yield seen in thermostable poliovirus strains after amino acid changes at multiple sites, facilitating their use as high-yield inactivated vaccine strains. This thermostable strain can be used as an inactivated vaccine strain or in constructing VLP vaccines based on the discovered thermostable sites. It also provides a theoretical and applied foundation for the study of sites related to yield, particle stability, and immunogenicity in other enteroviruses.

[0455] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A heat-stable enterovirus group A type 71 virus strain, characterized in that The nucleotide sequence is shown in SEQ ID NO. 20, the amino acid sequence is shown in SEQ ID NO. 21, and the cell phenotype is that it can stably grow on Vero cells after heat treatment at 52°C. The mutated amino acid sites are as follows: The VP1 protein has an alanine residue at amino acid position 44 and a valine residue at amino acid position 247; The 45th amino acid of 2A protein is alanine.

2. The heat-stable enterovirus group A type 71 virus strain according to claim 1, characterized in that The deposit number is CCTCC NO: V202233.

3. A method for purifying virus particles of heat-stable enterovirus group A type 71 virus strain, characterized in that: The steps include: The heat-stable enterovirus group A type 71 strain according to claim 1 or 2 is inoculated into Vero cells; When more than 90% of the cells become pathological, the virus is harvested and concentrated by ultrafiltration, sucrose-bottomed centrifugation, sucrose density gradient centrifugation, CsCl density gradient centrifugation, and desalting to harvest the virus particles.

4. The method for purifying virus particles of the heat-stable enterovirus group A type 71 virus strain according to claim 3, characterized in that: The method also includes a step of detecting the antigen content of the hollow particles and the solid particles.

5. The method for purifying virus particles of the heat-stable enterovirus group A type 71 virus strain according to claim 3, characterized in that: It also includes a step of inactivating viral particles.

6. Use of the heat-stable enterovirus group A type 71 virus particles obtained by the purification method according to any one of claims 3 to 5 as a candidate vaccine.

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