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Coxsackie virus A16 type mouse-adapted strain and application thereof

A technology of Coxsackie virus and mouse-adapted strains, which is applied in the fields of virology and molecular biology, and can solve problems such as safety and effectiveness evaluation methods

Active Publication Date: 2015-05-13
SINOVAC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, due to the species restriction of CA16 virus, which is only sensitive to the host and not to other animals, the strict safety and effectiveness evaluation methods required for CA16 virus vaccines and drugs before they go on the market have become a worldwide problem.

Method used

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  • Coxsackie virus A16 type mouse-adapted strain and application thereof
  • Coxsackie virus A16 type mouse-adapted strain and application thereof
  • Coxsackie virus A16 type mouse-adapted strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Isolation, cultivation and identification of embodiment 1CA16 virus strain

[0021] (1) Virus isolation and culture

[0022] The fecal samples of patients with positive CA16 virus PCR results were collected from the HFMD epidemic area in Zhejiang Province in 2010 (the cumulative number of HFMD cases reached 111,783). After treatment, they were inoculated onto African green monkey kidney passage cells (Vero), and cultured for three generations for virus isolation. , to obtain the CA16 virus strain.

[0023] (2) Virus identification

[0024] 1. RT-PCR detection

[0025] The CA16 genomic RNA of the strain was extracted, reverse-transcribed into cDNA, and identified using VP1 conserved region primers (upstream primer: 5'-TTGCAGACATGATTGACCAG-3', downstream primer: 5'-GAGTGATGGTTCAACACACA-3'). The results showed that There was a single band at 212bp in the isolated strain ( figure 1 ), it can be proved that the strain isolated from clinical specimens is CA16 virus.

[0...

Embodiment 2CA16

[0030] Preparation of embodiment 2CA16 mouse-adapted strain

[0031] Virus liquid: Infect RD cells with the above-mentioned CA16 virus strain, harvest the virus liquid after 7 days, freeze and thaw three times, and centrifuge to take the supernatant virus liquid for later use.

[0032] Animals: Purchase clean grade BALB / c pregnant mice, provide sufficient food and drinking water, clean the cage and replace the bedding every week.

[0033] Preparation and library construction of CA16 mouse-adapted strain: Inoculate the prepared CA16 virus solution into the brain of suckling mice after birth, with an inoculation volume of 30 μL / mouse (the virus titer is 6.12lg CCID50 / mL), and set normal suckling mice at the same time. control. The status of suckling mice was observed every day after virus inoculation. The brain tissue of the sick mice was collected and homogenized, and after centrifugation, the adapted strains were obtained by continuing passage for 4 times according to the ab...

Embodiment 3CA16

[0038] Application of embodiment 3CA16 mouse adaptation strain

[0039] Dilute the calibrated mouse-adapted strain to 20LD50 / 0.1mL, mix it with CA16 virus-immune sheep serum and human CA16 virus-infected convalescent serum at 37°C, after neutralization, inject 5-day-old suckling mice for preliminary evaluation of CA16 virus Protective effect of sera after immunization and human sera after natural infection. The results are shown in Table 1:

[0040] Table 1 Results of antivirus protection

[0041]

[0042] The results showed that the prepared mouse-adapted strain could effectively evaluate the protection of CA16 virus immune sera and naturally infected CA16 human sera. It can further lay the foundation for the study of Coxsackie A16 etiology, pathogenesis, screening of high-efficiency drugs and development of vaccines.

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Abstract

The invention provides a Coxsackie virus A16 type mouse-adapted strain and an application thereof. The Coxsackie virus A16 type mouse-adapted strain is mCA16V-1243 with a preservation number of CGMCCNo.7860. The mouse-adapted strain provides powerful tool for research of Coxsackie A16 type viral vaccines and screen of efficient antibacterial drugs and for the study of etiology and virus infection mechanisms.

Description

technical field [0001] The invention relates to the fields of virology and molecular biology, in particular to a mouse-adapted strain of Coxsackievirus A16 and application thereof. Background technique [0002] Hand, foot and mouth disease is a global infectious disease, and there are reports of the prevalence of this disease in most parts of the world. It was first reported in New Zealand in 1957, the Coxsackie virus was isolated for the first time in 1958, and the name HFMD was first proposed in 1959. The pathogen of hand, foot and mouth disease found in the early stage is mainly Cox A16 type. The report related to hand, foot and mouth disease and EV71 infection began in the early 1970s, and EV71 was first confirmed in the United States in 1972. Since then, EV71 infection and Cox A16 infection have appeared alternately, becoming the main pathogen of HFMD. It mostly occurs in children under 5 years old and can cause herpes on the hands, feet, mouth and other parts. A smal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A01K67/027A61K49/00C12R1/93
Inventor 李雅静薛晨宝王巍巍姜德玉张燕高强尹卫东
Owner SINOVAC BIOTECH