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

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

Example Embodiment

[0091] Example 1: Breeding of CV-A5-M14 mouse-adapted strains

[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 breeding), and measure the virus titer on RD cells according to the Reed-Muench method Degree is 1x10 7 TCID 50 / ml.

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

[0094] Infect five-day-old suckling mice in turn, and repeat twice; seven-day-old mice, repeat twice.

[0095] The virus harvested from 7-day-old infected suckling mice was infected with 9-day-old sucking mice in the same way,...

Example Embodiment

[0097] Example 2: Purification of CV-A5-M14 strain

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

[0099] (1) 10-fold serial dilution of CV-A5-M14 virus solution, the dilution range is 10 -1 ~10 -6 .

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

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

[0102] (4) Microscopically inspect the CPE status of each well, pick the CPE-positive clone from the well corresponding to the maximum dilution, and suspend the virus in 300 μL of virus maintenance solution by pipetting and mixing.

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

Example Embodiment

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

[0110] 1. Viral RNA extraction

[0111] Viral RNA was extracted and purified using a column-type viral RNA extraction and purification kit (Cat. No. B518667) from Sangon Bioengineering (Shanghai) Co.

[0112] 2. Synthesis of cDNA by reverse transcription

[0113] The reaction system for reverse transcription synthesis of cDNA is shown below

[0114]

[0115] (1) Add the reagents to the PCR tube, mix it evenly, put it in the PCR machine, react at 65°C for 5 minutes, and then quickly put it on ice for quenching.

[0116] (2) Add reagents to the above reaction solution, mix evenly, and place in a PCR apparatus at 30°C for 15 minutes; 42°C for 60 minutes; and 70°C for 15 minutes.

[0117] 3. PCR amplification

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

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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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