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Coxsackie A group 5 virus strain capable of effectively infecting adult mice

An effective infection and virus strain technology, applied in the direction of positive-sense single-stranded RNA virus, virus, virus/phage, etc., to achieve the effect of strong growth ability

Pending Publication Date: 2020-07-14
WUHAN INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 2. Meng Xiaodan et al. Research on the Epidemiology and Etiology of Hand, Foot and Mouth Disease in Xiangyang City from 2016 to 2017. Wuhan Institute of Biological Products, unpublished.

Method used

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  • Coxsackie A group 5 virus strain capable of effectively infecting adult mice
  • Coxsackie A group 5 virus strain capable of effectively infecting adult mice
  • Coxsackie A group 5 virus strain capable of effectively infecting adult mice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Embodiment 1: Breeding of CV-A5-M14 mouse adapted strain

[0092] Cultivate the CV-A5-3487R4 / XY / CHN / 2017 virus strain in RD cells (the original strain is not a monoclonal strain, so it needs to be monoclonal after selection), and measure the virus titers on the RD cells according to the Reed-Muench method degree is 1x10 7 TCID 50 / ml.

[0093] Ten day-old SPF Kunming mice were infected with the virus strain by intracranial injection, 20 μL / mouse. The brains of mice on the verge of death were taken, weighed, and a 10% (W / V) homogenate was prepared, and the supernatant was harvested by centrifugation, and stored at -20°C. Then the harvested virus was continued to infect day-old suckling mice according to the same infection method, and was repeated three times.

[0094] Sequentially infect five-day-old suckling mice, repeat twice; seven-day-old mice, repeat twice.

[0095] Infect the 9-day-old suckling mice with the virus harvested from the 7-day-old infected sucklin...

Embodiment 2

[0097] Embodiment 2: CV-A5-M14 virus strain purification

[0098] 1. Plaque purification of CV-A5-M14 virus clone

[0099] (1) The CV-A5-M14 virus solution was serially diluted 10 times, and the dilution range was 10 -1 ~10 -6 .

[0100] (2) Take a 6-well plate with RD cell confluence of 90%-95%, add 200 μL / well of diluted virus suspension, and incubate for 2 hours.

[0101] (3) After incubation, discard the virus solution. After the pre-configured 2X low-melting point agarose is melted, mix it with 2X MEM virus maintenance solution 1:1 and spread it in a 6-well plate, 2ml / well.

[0102] (4) Microscopically check the CPE condition of each well, pick a CPE-positive clone from the well corresponding to the maximum dilution, suspend the virus in 300 μL virus maintenance solution, and blow and mix well.

[0103] (5) Take 100 μL of the harvest solution for ten-fold dilution, and the dilution range is 10 -1 ~10 -3 , repeat the above steps for three consecutive plaque purifica...

Embodiment 3

[0109] Example 3: CV-A5-M14-611 and CV-A5-M14-111 Whole Genome Sequence Determination

[0110] 1. Viral RNA extraction

[0111] Use the Sangon Bioengineering (Shanghai) Co., Ltd. Column Viral RNA Extraction and Purification Kit (Product No.: B518667) to extract and purify viral RNA.

[0112] 2. cDNA synthesis by reverse transcription

[0113] The reverse transcription synthesis cDNA reaction system is as follows

[0114]

[0115] (1) Add the reagents into the PCR tube, mix well, place in the PCR instrument, react at 65°C for 5min, and then quickly place it on ice to quench.

[0116] (2) Add reagents to the above reaction solution, mix well and place in PCR instrument, 30°C, 15min; 42°C, 60min; 70°C, 15min.

[0117] 3. PCR amplification

[0118] (1) Amplification primers: CV-A5 full sequence determination and extension primers are shown in Table 1, three pairs of primers are used for PCR (1F5R, 6F8R, 9F10R), and others are primers for sequencing.

[0119] Table 1: CV-A5...

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Abstract

The invention relates to a coxsackie A group 5 type (CV-A5) virus strain capable of effectively infecting adult young mice. The virus strain is CV-A5-M14-611, and has a complete amino acid sequence shown as SEQ ID NO. 1. The invention also relates to an attenuated strain CV-A5-M14-111 of the CV-A5 virus strain, and the attenuated strain has a complete amino acid sequence shown as SEQ ID NO. 3. Theinvention also relates to application of the virus strain in preparation of an animal model for evaluating vaccines and / or medicines for treating hand, foot and mouth disease (HFMD) and application of the virus strain in preparation of a preparation for evaluating a protection effect of the vaccines for treating the HFMD.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Coxsackie group A type 5 virus strain capable of effectively infecting older mice. Background technique [0002] Hand, foot and mouth disease (HFMD) is a statutory Class C infectious disease caused by a variety of enterovirus serotypes. HFMD occurs mostly in children under the age of 5. Current research shows that EV-71, CV-A6, CV-A10, CV-A16, and CV-A5 are the main pathogens causing HFMD. Other enterovirus serotypes in the Coxsackievirus A and B group are also causative agents of HFMD outbreaks and shedding [1] . [0003] CV-A5 belongs to the genus Enterovirus of the family Picornaviridae, with a diameter of about 27-30nm. The virus particles are symmetrically spherical with 20 faces, and the core contains a single-stranded positive-strand RNA genome of about 7400 nucleotide residues. According to literature reports, CV-A5 commonly causes HFMD, herpetic angina (Her...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/04C12R1/93
CPCC12N7/00C12N2770/32021Y02A50/30
Inventor 申硕靳卫平吴杰卢佳麦健仪王泽鋆孟胜利
Owner WUHAN INST OF BIOLOGICAL PROD CO LTD
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