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High-titer genotype I Japanese encephalitis virus strain and application thereof

A technology of Japanese encephalitis virus and Japanese encephalitis, which is applied in the direction of viruses, antiviral agents, viruses/bacteriophages, etc., can solve the problems of inability to prepare immune protection vaccines, lack of protection of genotype I viruses, and low growth titers. Advanced problems, to achieve the effect of strong protection, good safety, and high growth titer

Inactive Publication Date: 2016-05-04
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the vaccines for preventing porcine Japanese encephalitis are genotype Ⅲ virus vaccines. However, because the genotypes and antigenic epitopes of type I and type Ⅲ JEV are partially different, the genotype Ⅲ virus vaccines have no effect on some genotype Ⅰ viruses. Protection (see Xiao-LingPan, "Emergence of Genotype I of Japanese Encephalitis Virus as the Dominant Genotype in Asia", JOURNALOF VIROLOGY, Oct.2011, p.9847–9853 Vol.85, No.19)
[0004] Therefore, it is particularly important to prepare the genotype I porcine encephalitis Japanese encephalitis vaccine. However, due to the poor antigenicity and low growth titer of the existing genotype I type JEV, it is impossible to prepare a vaccine with better immune protection effect, such as, The highest protection rate of the JEVYADX201202 strain does not exceed 50%, which is difficult to apply in practice. This is also an important reason why there is no genotype Ⅰ porcine Japanese encephalitis vaccine on the market so far.

Method used

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  • High-titer genotype I Japanese encephalitis virus strain and application thereof
  • High-titer genotype I Japanese encephalitis virus strain and application thereof
  • High-titer genotype I Japanese encephalitis virus strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The isolation and identification of embodiment 1 strain of the present invention

[0041] 1. Isolation of virus

[0042] 1 Collection of samples

[0043] According to the seasonal regularity of JE prevalence in Sichuan and the characteristics of mosquito transmission, referring to the results of epidemiological surveys, use ultraviolet mosquito traps to collect mosquito samples from pig farms in various places from July to October: place mosquito traps before 7:00 every night , retrieved at 8 am the next day, took the mosquitoes out of the mosquito trap, put them into a sample bag marked with information, and placed them in a -20°C refrigerator for temporary storage. The brain tissue and cerebrospinal fluid of the stillborn fetuses collected were collected and stored in a -80°C ultra-low temperature freezer.

[0044] 2 Processing of samples

[0045] Mosquito sample processing: Take out the samples temporarily stored in the -20°C refrigerator, pick out other insects, ...

Embodiment 2

[0133] The preparation of embodiment 2 vaccines of the present invention

[0134] 1. Preparation method

[0135] The preservation number obtained in Example 1 is CCTCCNO:V201526 genotype I Japanese encephalitis virus SCYA201201 strain, prepare vaccine of the present invention according to the following method:

[0136] 1. Preparation of porcine Japanese encephalitis virus inactivated stock solution:

[0137] (1) Subculture BHK-21 cells according to the conventional method. After the cells cover 80% of the cell flask, discard the nutrient solution, wash the cells with PBS, inoculate the monolayer cells with porcine Japanese encephalitis virus, and discard after absorbing for 1 hour. Add the virus solution to the maintenance solution containing 2% calf serum and incubate at 37°C.

[0138] (2) When more than 80% of the cells have cytopathic changes (CPE), freeze and thaw three times, centrifuge at 12000r / m for 10min, collect the virus suspension, and store at -20°C.

[0139] (...

experiment example 2

[0161] Experimental example 2 Mice challenge protection experiment of vaccine of the present invention

[0162] 1. Experimental method

[0163] Select the female SPF level Kunming mouse of 3 weeks old, prepare the virus titer that is 10 respectively by the preparation method of embodiment 2 7 PFU / ml, 10 8 PFU / ml and 10 9 PFU / ml total 3 dilutions of the vaccine of the present invention, intraperitoneal injection of 0.3ml of the vaccine of the present invention and the commercial mouse brain inactivated vaccine (HW1 strain, gene type III, toxicity before inactivation ≥ 2.5 × 10 7.0 LD 50 / ml, purchased from Wuhan Zhongbo Biotechnology Co., Ltd.), set negative control and blank control at the same time, 7 days after the first immunization, the same dose was boosted once, and after 7 days of boosting immunization, each mouse except the blank control group was selected with strong poison ( JEVDX1 strain, genotype Ⅰ) was challenged with 100LD50 (25 μL) in the brain, observed con...

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Abstract

The invention relates to a high-titer genotype I Japanese encephalitis virus strain. The high-titer genotype I Japanese encephalitis virus strain is characterized in that the high-titer genotype I Japanese encephalitis virus strain is preserved in the China Center for Type Culture Collection with the preservation number of CCTCC NO: V201526. The invention further discloses a genotype I Japanese encephalitis vaccine. The genotype I Japanese encephalitis virus strain is high in growth titer and antigenicity, the valence of an antibody generated by the vaccine prepared with the virus strain is high, protective efficacy is high, safety performance is high, and clinical application prospects are broad.

Description

technical field [0001] The invention relates to a high-titer genotype I porcine Japanese encephalitis virus strain and its application. Background technique [0002] Japanese encephalitis is an important zoonotic disease caused by Japanese encephalitis virus (JEV). Pigs, cattle, horses and other animals are susceptible. Culex mosquitoes are the main vector of JEV, which mostly occurs in summer and autumn when there are more mosquitoes. Pigs are important storage hosts and amplification hosts of Japanese encephalitis virus. Japanese encephalitis-infected pigs are the main source of infection of Japanese encephalitis. Pigs are indispensable for the epidemic of the disease and are one of the main pathogens that cause reproductive disorders in sows , the lethality rate to pigs is not high, but causes serious loss to whole pig breeding. Mainly it can cause abortion, stillbirth or mummification of pregnant sows, acute inflammatory reaction or infertility of boars. [0003] JEV ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/12A61P31/14C12R1/93
CPCA61K39/12A61K2039/541A61K2039/545C12N7/00C12N2770/24121C12N2770/24134Y02A50/30
Inventor 曹三杰文心田伍锐岳辉喜黄小波文翼平袁磊刘瀚扬刘洪明田耕
Owner SICHUAN AGRI UNIV
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