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Method for high-efficiency amplification of Tembusu virus by chicken hepatocytes

A technology of tambusu virus and chicken liver cells is applied in the field of efficient amplification of tambusu virus by using chicken liver cells, which can solve the problems of restricting the healthy and sustainable development of the poultry industry, accompanied by neurological symptoms, decreased egg production and the like, Achieve good application value, increase virus titer, and save time.

Active Publication Date: 2019-02-01
YANGZHOU UNIV
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  • Claims
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Problems solved by technology

The main characteristics of poultry infected with TMUV are decreased or even stopped egg production, accompanied by neurological symptoms, and death in severe cases, which seriously restricts the healthy and sustainable development of the domestic poultry industry

Method used

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  • Method for high-efficiency amplification of Tembusu virus by chicken hepatocytes

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Embodiment

[0017] A method for efficiently amplifying Tembusu virus, comprising the following steps:

[0018] (1) Cell culture

[0019] Chicken hepatocytes were cultured in DMEM-F12 medium containing 10% fetal bovine serum, and DF-1 cells and BHK-21 cells were cultured in DMEM medium containing 5% fetal bovine serum. After the cells were covered with a single layer, they were washed once with PBS, digested with 0.25% trypsin, and passaged to 6-well or 96-well cell culture plates.

[0020] (2) Efficient amplification of Tambusu virus in chicken hepatocytes

[0021] Chicken hepatocytes, DF-1 cells and BHK-21 cells were inoculated into a 6-well plate for culture. When the confluence was 60% to 70%, the Tambusu virus AHRD2018 strain was inoculated into three kinds of cells, MOI was 0.01, and three kinds of cells were inoculated. Two replicates were made for each type of cells, cultured in 2ml medium containing 2% fetal bovine serum for 4 days, 200μl supernatant was collected regularly and ...

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Abstract

The invention relates to a method for high-efficiency amplification of Tembusu virus by chicken hepatocytes, wherein the method includes the following steps: (1) cell culture: culturing the chicken hepatocytes in a DMEM-F12 culture medium containing fetal bovine serum with the volume concentration of 10%, after the cells grow on a single layer fully, washing with PBS once, digesting with pancreatic enzymes with the mass concentration of 0.25%, and passaging to a cell culture plate; and (2) high-efficiency amplification of Tembusu virus in the chicken hepatocytes: inoculating the chicken hepatocytes into the cell culture plate and culturing, when the confluence degree is 60% to 70%, inoculating the chicken hepatocytes with the Tembusu virus AHRD2018 strain with MOI of 0.01, culturing in 2 ml of a culture medium containing fetal bovine serum with the volume concentration of 2% for 4 days, collecting 200 [mu]l of a supernatant at a fixed time and cryopreserving in an -80 DEG C refrigerator for standby application, and at the same time, supplementing 200 [mu]l of the culture medium containing fetal bovine serum with the volume concentration of 2% into each hole. The characteristic thatthe chicken hepatocytes can produce high titer virus more prematurely is utilized, and the Tembusu virus is amplified by the chicken hepatocytes having the virus replication rate significantly higherthan that of DF-1 cells and BHK-21 cells.

Description

technical field [0001] The invention relates to a method for efficiently amplifying Tambusu virus, in particular to a method for efficiently amplifying Tambusu virus by using chicken liver cells. Background technique [0002] Tembusu virus (TMUV) is a single-stranded positive-sense RNA virus that can cause a sharp decline in poultry egg production. The virus belongs to the family Flaviviridae, the genus Flavivirus, and the Entayavirus group of mosquito-borne viruses. The main characteristics of poultry infected with TMUV are decreased or even stopped egg production, accompanied by neurological symptoms, and death in severe cases, which seriously restricts the healthy and sustainable development of the domestic poultry industry. Although the use of TMUV vaccines has effectively reduced the prevalence and incidence of TMUV, the range of susceptible hosts of TMUV virus is expanding from ducks to geese and laying hens in recent years. The isolation of highly pathogenic TMUV st...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/02C12N5/071
CPCC12N5/067C12N7/00C12N2770/24151
Inventor 叶建强任丹李拓凡邵红霞秦爱建高巍张鑫宇
Owner YANGZHOU UNIV
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