Rabies virus attenuated strains as well as breeding method and application thereof

A technology of rabies virus and attenuated strains, applied in the field of virus vaccines, to achieve good and stable immunogenicity, low residual virulence, and clear background of virus species

Inactive Publication Date: 2015-04-29
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, China still lacks vaccine strains that meet WHO standards and can be used to prepare live attenuated vaccines

Method used

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  • Rabies virus attenuated strains as well as breeding method and application thereof
  • Rabies virus attenuated strains as well as breeding method and application thereof
  • Rabies virus attenuated strains as well as breeding method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, adaptive passage and plaque purification of rabies virus CTN-181 and CTN-181-SG strains

[0025] 1. Adaptation of rabies-fixed toxic human diploid cells to subculture and plaque purification

[0026] Rabies virus CTN strain was isolated from the brain tissue of rabies patients in Zibo, Shandong Province by the applicant, and the fixed strain CTN-M strain was obtained by passage in the brain of mice for 56 generations. Passage to the 80th generation, the virus seeds of the 80th generation were purified three times according to the neutral red plaque to obtain the CTN-181 strain.

[0027] 2. Submandibular gland passage of guinea pigs

[0028] Take the CTN-181 strain and inject several guinea pigs subcutaneously in the neck with the stock solution, each inoculate 1ml, take 2 guinea pigs on the second day after inoculation, and use CO 2 Put it to death, soak it in 3% Lysol water for 5 minutes, take out the guinea pig and put it on its back on double-layer ga...

Embodiment 2

[0031] Example 2, Screening of low residual neurovirulence clones

[0032] 1. Method

[0033] 1. Neurovirulence screening in the brain of 3-week-old mice

[0034] The CTN-1-(1-20) prepared above was quickly melted under running water, and the stock solution (10 0 ) do 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 6 10-fold serial dilutions, take 10 0 -10 -3 Four groups of dilute doses were inoculated into the brain of 6 3-week-old Kunming mice, 0.03ml / only, and another 6 mice were inoculated with PBS in the brain as a blank control. On the fourth day, observe whether there was non-specific death. Days, the incidence and death of mice were recorded, and the LD of the clone was calculated as the cumulative mortality of four dilutions 50 . take 10 -4 -10 -6 The diluted virus solution and the blank dilution solution were used for simultaneous plaque titration, and the PFU value was calculated.

[0035]2. Screening of neurotoxicity in the brain of 5-day-old, 10-day-old, an...

Embodiment 3

[0051] Embodiment 3, amplification, titration and determination of CTN-1-3, CTN-1-19 attenuated strain

[0052] 1. Amplification of virus species

[0053] After culturing BHK-21 cells into a dense monolayer, the virus seeds were inoculated into CTN-1-3 and CTN-1-19 strains at an infection rate of 0.1 MOI, and the cells were incubated at 37°C in 5% CO 2 Discard the culture medium after adsorption for 1 hour, rinse with sterilized PBS, add an appropriate amount of maintenance solution, transfer to 35°C to continue the culture, and collect the culture supernatant when the cell lesion reaches "+++", and centrifuge at 4000r / min for 10 minutes at 4°C. Remove cell debris within 1 minute, take the supernatant and add 10% calf serum, mix well, divide into small tubes, and freeze at -70°C for later use.

[0054] 2. Virus species titration

[0055] Take one of each of the frozen virus species, and make six 10-fold serial dilutions starting from 1:10 on the 24-well culture plate (10 -1...

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Abstract

The invention discloses a breeding method of rabies virus attenuated strains; the method comprises the following steps: using a CTN-181 strain homologous with a strain CTN-1-V for the production of rabies vaccine for Chinese people as a parent strain, and cloning and screening by virtue of in vitro cell line culture, animal in vivo tissue alternate passage and a plaque purification technology; screening neurovirulence in the brains of mice at different day ages so as to obtain two attenuated strains which are respectively named as CTN-1-3 and CTN-1-19; and carrying out amplification, titration, verification and whole genome sequencing to the attenuated strains so as to obtain a CTN-1-3 whole genome sequence as shown in SEQ ID NO:1 and a CTN-1-19 whole genome sequence as shown in SEQ ID NO:2. The attenuated strains disclosed by the invention are low in strain residual virulence, good in immunogenicity and good in genetic stability; the strains have reached or exceeded requirements of World Health Organization (WHO) on the safety and the validity of candidate strains; and the strains are suitable for producing oral live vaccine for rabies.

Description

technical field [0001] The invention belongs to the field of virus vaccines, in particular, a live attenuated rabies vaccine strain with high attenuation and good immunogenicity is selected by biological means. Background technique [0002] Rabies virus (RABV / RV) belongs to Rhabdoviridae (rhabdoviridae) genus Lyssavirus (lyssavirus), is not segmented single-stranded RNA enveloped virus. According to serotype and genotype, rabies virus can be divided into 4 serotypes and 7 genotypes. Rabies virus can cause rabies in humans and animals, and once onset, the fatality rate is almost 100%. my country is one of the countries where rabies is most prevalent in the world. In recent years, the national rabies death toll is about 2,000, ranking second in the world after India. [0003] Although vaccinating humans is an effective way to prevent and control the occurrence of human rabies, it cannot completely control and eliminate rabies, especially in developing countries with a large ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04A61K39/245A61P31/22
Inventor 俞永新石磊泰邹剑
Owner NAT INST FOR FOOD & DRUG CONTROL
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