Pseudorabies virus passage attenuated strain and application thereof

A pseudorabies virus, passaging and weakening technology, applied in the direction of viruses, antiviral agents, virus antigen components, etc., can solve the problems of high residual virulence, insufficient protective efficacy, enhancement and other problems

Active Publication Date: 2019-10-29
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, since 2011, a mutated PRV wild strain has re-emerged in many pig farms in my country. The virulence of this mutant strain is significantly enhanced compared with the traditional strain, and it can cause 100% death to pigs over 9 weeks old. Although the original vaccine can provide certain protection, its protective effect is seriously insufficient and cannot prevent the onset and shedding of the virus. Studies have shown that its main antigen genes gB, gC and gD have obvious mutations with traditional vaccine strains. In order to better prevent and control PRV mutations The harm of strains to pigs, the use of mutant strains to develop homologous vaccines is the key to the prevention and control of porcine pseudorabies in my country. The development of new vaccines for mutant strains usually uses methods such as artificial gene deletion or passage weakening. When the artificial gene is missing, if There are fewer deleted genes, there may be high residual toxicity, and the safety is not high enough
For example, the invention patent with the application number 201510388390.9 discloses that the LA-A strain obtained by artificially deleting the gE gene of the pseudorabies virus wild virus AH02LA strain is only safe for 4-week-old piglets, but it is still highly pathogenic to newborn piglets
When the artificial gene is deleted, if there are many missing genes, it may affect the cultivation and immune efficacy of the virus, and the artificial gene deletion has biosafety risks, and the safety evaluation of agricultural genetically modified organisms must be carried out, and it can be used only after passing the test

Method used

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  • Pseudorabies virus passage attenuated strain and application thereof
  • Pseudorabies virus passage attenuated strain and application thereof
  • Pseudorabies virus passage attenuated strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1. Acquisition and identification of natural attenuated strains of pseudorabies virus passage

[0021] 1. Blind transmission of pseudorabies virus PRV AH02LA strain

[0022] Primary chicken embryo fibroblasts (Chicken embryofibroblasts, CEF) were prepared by using 9-10-day-old SPF chicken embryos, and cultured in DMEM medium (Gibco) containing 10% newborn bovine serum for 24-48 hours at 37°C. Make a single layer and set aside. The virus used for subculture is pseudorabies virus (Pseudorabies virus, PRV) AH02LA strain, which is a virulent strain and highly homologous to the PRV mutant strains that have been popular in my country since 2011. This strain has been submitted to the Chinese Microbiological Culture Collection It was deposited by the General Microorganism Center of the Management Committee, the preservation number is CGMCC NO.10891, and the preservation date is June 16, 2015.

[0023] Inoculate 0.1 mL of PRV AH02LA strain F6 virus solution into 1 well ...

Embodiment 2

[0054] Embodiment two, the growth characteristic identification of PRV LA2017 strain

[0055] The F1, F18 and F35 generations of PRV LA2017 strain were inoculated into a six-well plate covered with monolayer CEF at an MOI of 0.004, and placed in 5% CO 2 After incubating in a constant temperature incubator at 37°C for 1 hour, aspirate the supernatant, wash with PBS buffer for 3 times, and replace with cell maintenance solution, 2 mL per well, in 5% CO 2 Culture was carried out at 37°C in a constant temperature incubator, and the virus content in the culture was measured at 0h, 6h, 12h, 24h, 36h, 48h and 72h after inoculation. The F5 generation of PRV AH02LA strain was set as the control, cultured and detected by the same method. Repeat 3 times. The cell maintenance medium is DMEM medium (Gibco) containing 3% (volume percent concentration) fetal bovine serum (FBS).

[0056] In addition, the growth characteristics of F1, F18 and F35 generations of PRV LA2017 strain on ST cells...

Embodiment 3

[0059]Example 3, the safety of PRV LA2017 strain on newborn piglets and pregnant sows

[0060] 1. Test material

[0061] 1.1 virus

[0062] F1 batch of PRV LA2017 strain 20170622, virus titer 10 8.5 TCID 50 / mL.

[0063] The F1 generation of PRV LA2017 strain is prepared by the following method: Inoculate CEF cells with pseudorabies virus LA2017 strain seed virus, adopt DMEM medium containing 3% (volume percentage concentration) fetal bovine serum (FBS), in 5% CO 2 Cultivate in a constant temperature incubator at 37°C for 36 hours, harvest the virus culture, centrifuge after repeated freezing and thawing, and take the supernatant to obtain the F1 generation virus liquid of PRV LA2017 strain.

[0064] 1.2 Diagnostic kits

[0065] PRV gE antibody diagnostic kit, PRV gB antibody diagnostic kit, porcine reproductive and respiratory syndrome virus (PRRSV) antibody diagnostic kit and swine fever virus (CSFV) antibody diagnostic kit, from Beijing IDES Yuanheng Biotechnology Ltd...

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Abstract

The invention provides a pseudorabies virus passage attenuated strain and an application thereof, and belongs to the field of vaccines of animal medicine. The pseudorabies virus passage attenuated strain is a pseudorabies virus LA2017 strain, and the preservation number is CGMCC No.18170. The invention also provides an application of the pseudorabies virus passage attenuated strain to preparationof pseudorabies vaccines, and a vaccine using the pseudorabies virus passage attenuated strain as an active component. The LA2017 strain is a natural deletion attenuated strain, the virulence is notably reduced, and when new-born piggies are inoculated with the LA2017 strain, no adverse reactions are caused. The strain is obtained by a passage attenuated method, so that the biological security risk is low. After weaned highly-susceptible piggies are subjected to primary inoculation of the vaccines prepared from the LA2017 strain for 7 days, 100% of protective effects can be achieved, and the immunization persistent period can achieve 5 months. The vaccines not only can prevent pathogenesis but also can prevent toxin expelling, and purification of pseudorabies virus variation strains can beextremely facilitated.

Description

technical field [0001] The invention belongs to the field of vaccines in animal medicine, and in particular relates to a pseudorabies virus passaging attenuated strain and application thereof. Background technique [0002] Pseudorabies is a common disease that occurs in many animals, and its pathogen is pseudorabies virus (Pseudorabies Virus, PRV). Domestic animals (including cattle, sheep, etc.), wild animals (including foxes and minks, etc.) Typical neurological symptoms. Pigs are the storage host of pseudorabies virus. Piglets usually have a high mortality rate after onset, while large pigs generally do not die, but they can be infected and carry and shed the virus for a long time. Pigs with different ages have different clinical characteristics of pseudorabies. Abortion often occurs in sows; newborn piglets have neurological symptoms and high mortality; fattening pigs mainly have respiratory symptoms, and adult pigs grow stagnant after being infected with pseudorabies ...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/08A61K39/245A61P31/22C12R1/93
CPCC12N7/00A61K39/12A61P31/22C12N2710/16721C12N2710/16734A61K2039/5254A61K2039/552
Inventor 王继春陈赛赛郭容利乔永峰王志胜许梦微郑亚婷刘娅梅张传健吕家轩
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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