Building and evaluation of an animal model infected with a coxsackievirus A10 domesticated strain TA151R-1

A coxsackie virus, a technology for infecting animals, applied in the biological field, can solve the problems of less reported A10 virus strains, affect the efficiency of virus strains, and unstable passage, so as to achieve increased expression levels, high virus titers, and viral loads Increased effect

Inactive Publication Date: 2018-03-02
SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are many studies on Coxsackie virus, but there are few reports on the A10 virus strain
People such as Zhuang Zaicheng disclosed A10 virus strain M2014 in CN106661102A, but, this virus strain can only infect HEK293 cell, and can not infect other cell strains that are used for vaccine production, as, Vero cell, MRC-5 cell, MDCK cell As well as CHO cells, the highest virus titer can only reach 2.5×10 8 TCID 50 / mL, which largely limits the application of this virus strain
In our previous work, we screened the A10 virus strain TA151R, established the A10 suckling mouse infection model, and prepared the whole virus inactivated vaccine ("Protective Efficiencies of Formaldehyde-Inactivated Whole-Virus Vaccine and Antivirals in a Murine Model of Coxsackievirus A10Infection", Zhenjie Zhang et al., "Journal of Virology", Volume 91, 2017, e00333-17); However, in the follow-up research process, we found that the titer of TA151R virus strain decreased and the passage was unstable during the passage process The problem that affects the efficiency of the virus strain in practical application

Method used

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  • Building and evaluation of an animal model infected with a coxsackievirus A10 domesticated strain TA151R-1
  • Building and evaluation of an animal model infected with a coxsackievirus A10 domesticated strain TA151R-1
  • Building and evaluation of an animal model infected with a coxsackievirus A10 domesticated strain TA151R-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Isolation, Screening and Domestication of Coxsackievirus A10 Type Strain

[0043] The inventors carried out a lot of isolation and screening work in the early stage. In 2015, the Coxsackie virus A10 strain TA151R was isolated from the stool sample of a 4-year-old child in Shandong Province, and the infection was established using this strain. Animal model, prepared the whole virus inactivated vaccine, and studied the immune protection effect of the vaccine ("Protective Efficacies of Formaldehyde-Inactivated Whole-Virus Vaccine and Antivirals in a Murine Model of Coxsackievirus A10 Infection", Zhenjie Zhang et al., "Journal of Virology", Volume 91, 2017, e00333-17). In the follow-up research process, we found that TA151R would have the problems of decreased titer and unstable passage during the passage process, which seriously restricted the application of this strain; in order to solve this problem, we carried out domestication screening for TA151R.

[0044] ...

Embodiment 2

[0047] Example 2 Passage stability of high titer domesticated strain XH01-08

[0048] RD cells were used to continuously subculture the high-titer domesticated strain XH01-08 in Example 1. When the cells showed a cytopathic effect area of ​​more than 80%, they were repeatedly frozen and thawed 3 times, and the first-generation virus liquid was collected and assayed. Virus titer: The harvested first-generation virus was continuously passaged 30 times in the same way, and the virus was collected and titered at each generation, and the virus titer of different generations was compared. like figure 1 As shown, XH01, XH02, XH05, XH06, and XH08 showed different titer reduction or titer instability during the passage process, while XH03, XH04, and XH07 were more stable and could produce high titer of virus.

Embodiment 3

[0049] Example 3 Antiserum Test of Domesticated Strain XH03 / XH04 / XH07

[0050] 0.4% formaldehyde-inactivated TA151R, XH03, XH04, and XH07 antigens were mixed with complete Freund's adjuvant and incomplete Freund's adjuvant in a volume ratio of 1:1, and after ultrasonic emulsification, 6-week-old ICR mice (n= 6) Inject complete Freund's adjuvant and antigen emulsion 300ul intraperitoneally, and after an interval of 7 days, inject incomplete Freund's adjuvant and antigen emulsion 300ul intraperitoneally, and take peripheral blood from the orbital venous plexus of immunized mice 20 days later and centrifuge serum. After 2-fold serial dilution of the immune serum starting from 1:8, they were mixed with 100CCID50 (Cell culture infective dose 50%) of CVA10 respectively, placed at 37°C for 2 hours, and the mixture was added to a 96-well plate of monolayer RD cells (1 ×10 5 / hole), put CO 2 Cultivate in an incubator at 37°C for 48 hours, observe the CPE of the cells, and take the ...

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Abstract

A coxsackievirus A10 domesticated strain TA151R-1 high in tilter and stable in passage is disclosed. The virus strain can infect RD cells, HEK293 cells, Vero cells, MRC-5 cells, Hep-2 cells, WI-38 cells, and other cell lines, and can be used for preparing a univalent vaccine or a polyvalent vaccine. The prepared vaccine can protect a body from being harmed by the coxsackievirus, and can completelyprotect the body from attack by heterogenous viruses. An infected animal model can be built efficiently.

Description

Technical field: [0001] The present application relates to the field of biotechnology, in particular to the establishment and evaluation of an animal model infected with the domesticated Coxsackievirus A10 strain TA151R-1. Background technique: [0002] Hand, foot, and mouth disease (HFMD) is an intestinal infectious disease caused by a small RNA virus. The patients are mainly infants under 5 years old. It mainly causes mild clinical symptoms such as mucosal herpes and angina. Symptoms can also cause severe illness, leading to neurological diseases such as severe aseptic meningitis and acute flaccid paralysis. Since the epidemic in 2009, the incidence of Coxsackievirus A group 6 (A6) and 10 (A10) has increased year by year in the Western Pacific region and some countries in Europe, including Singapore, Thailand, Japan, Spain, France, Mainland China and Taiwan make it the main pathogen causing HFMD. As non-EV71 and non-A16, A6 and A10 are considered to be the main pathogens...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/76A61K49/00
CPCA61K35/76A61K49/0008
Inventor 张振杰史卫峰李娟董兆鹏
Owner SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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