Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (17): 3415-3426.doi: 10.3864/j.issn.0578-1752.2018.17.016

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Optimization of Cultivation Conditions for Reassortant Avian Influenza Virus H7N9 H7-Re1 Strain

LI Li1, DU XIN1, ZHANG LiNa1, YANG Liu1, GAO XiaoQing1, TANG DongXue1, ZHAO HaiYuan1, JIANG XiaoMei2, ZHANG TianShu3, LI JinXiang4   

  1. 1Jilin Guanjie Biotechnology co. LTD, Meihekou 135000, Jilin; 2The Xinjiang Uygur Autonomous Region Academy of Animal Science, Urumqi 830000; 3Animal Health Surveillance Institute of Xinjiang Uygur Autonomous Region, Urumqi 830000; 4 Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2017-04-18 Online:2018-09-01 Published:2018-09-01

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Abstract: 【Background】Outbreaks of highly pathogenic avian influenza outbreak caused huge economic loss and damage the environment health, current vaccination remains one of the main measures to control avian influenza in China, which requires a lot of safety, high efficiency and low cost of the avian influenza vaccine. Due to the limitations of raw material sources, complex process, individual differences, long cultivation period and difficulty in amplifying culture, the process of preparing avian influenza vaccine by chicken embryo method is deficient. However, it is gradually becoming a trend to use bioreactor to produce virus vaccine on a large scale.It can not only increase the output of units, but also achieve high density cell and high virus yield, and ensure the quality of products. Currently, China’s vaccine for the prevention and control of avian influenza is the reassortant avian influenza virus (H5+H7) divalent inactivated vaccine (H5N1 Re-8 strain +H7N9 H7-Re1 strain). The production capacity of the single tank of avian influenza inactivated vaccine is the maximum of 6 000 L.The supply of high viral antigen is one of the main factors influencing the production of high efficiency vaccine.【Objective】In order to provide stable and efficient production antigens, a kind of domestication tests were carried out. 【Method】The reassortant avian influenza virus H7N9 H7-Re1 was proliferated on MDCK cells and suspended MDCK cells. Virus titers were tested by comparing different harvest times, viral inoculation doses and concentrations of TPCK on MDCK cells which the reassortant avian influenza virus H7N9 H7-Re1 strain were cultured in. The best harvest time was determined, 64 hours, dosage 0.008% or the MOI 10-4 and TPCK-trypsin concentration 2 μg·ml-1. According to the determined optimal culture conditions, the virus titer of each generation was tested. 【Result】The results showed that when virus were extended up to the fifth generation, HA reached 1:256 and the content of every 1 ml virus reached 108.5 TCID50, and the content of every 0.1 ml virus reached 108.5 EID50. Therefore, it can be determined that the 5th generation virus is the best generation for producing virus. 【Conclusion】The reassortant avian influenza virus was optimized and tested on the suspension MDCK cells. The best harvest time of the H7N9 H7-Re1 strain on suspension MDCK cells was 48 h, MOI 10-4, and the best TPCK-trypsin concentration was 4-8 μg·ml-1. In the actual production, MDCK cells or suspended MDCK cells can be selected to expand the virus.

Key words: reassortant avian influenza virus, MDCK cell, suspended MDCK cells, virus titer

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