H9N2亚型猪源性流感病毒在小鼠肺微血管内皮细胞内最佳增殖条件的研究

doi:10.16431/j.cnki.1671-7236.2018.06.036

摘要/Abstract

摘要：

试验旨在确定H9N2亚型猪源性流感病毒（SIV）在小鼠肺微血管内皮细胞（PMVEC）中增殖的最佳条件。将PMVEC解冻、复苏、培养，取长成单层的PMVEC，在不同浓度TPCK-胰蛋白酶维持液（0.1、0.2、0.3、0.4、0.6、0.8、1.0 μg/mL）、不同H9N2亚型SIV（A/swine/HeBei/012/2008/（H9N2）接种剂量（1：10、1：100、1：1 000、1：10 000、1：100 000、1：1 000 000和1：10 000 000）及不同病毒吸附时间（0.5、1.0、2.0和3.0 h）条件下观察PMVEC形态变化，并测定细胞上清液中H9N2亚型SIV的HA滴度。在未加病毒的情况下，低于0.6 μg/mL的TPCK-胰蛋白酶对PMVEC的生长未造成任何影响，但随着TPCK-胰蛋白酶浓度的增加，PMVEC开始出现肿胀、变圆，甚至脱落；采用含有0.6 μg/mL TPCK-胰蛋白酶维持液将H9N2亚型SIV稀释为不同的浓度感染PMVEC，在0.3 μg/mL TPCK-胰蛋白酶维持液、10^-2病毒稀释倍数感染条件下48和72 h HA滴度分别为4.6log2和6.4log2；病毒吸附时间为2 h且中间震荡20 s的条件下H9N2 HA滴度最佳。结果表明，当TPCK-胰蛋白酶维持液浓度为0.3 μg/mL、病毒接种浓度为10^-2、吸附时间为2 h且中间震荡20 s时，H9N2亚型SIV在PMVEC中增殖最佳，达到6.8log2。

关键词: H9N2亚型; 猪流感病毒(SIV); 微血管内皮细胞(PMVEC); 增殖

Abstract:

This study was aimed to determine the optimal proliferation conditions for culturing of H9N2 subtype swine influenza virus(SIV) in mouse pulmonary microvascular endothelial cell (PMVEC).The PMVECs were defrosted,recovered and cultured,then the morphological changes of PMVECs were observed and the HA titer of H9N2 subtype SIV of the cell culture supernate was determined by hemagglutination test after action with different concentrations of TPCK-trypsin maintenance media (0.1,0.2,0.3,0.4,0.6,0.8 and 1.0 μg/mL),different doses of H9N2 subtype SIV (1:10,1:100,1:1 000,1:10 000,1:100 000,1:1 000 000 and 1:10 000 000) and different viral adsorption time (0.5,1.0,2.0 and 3.0 h) on single layer of PMVECs.When the dose of TPCK-trypsin was lower than 0.6 μg/mL,there was no effect on morphology structure of PMVEC uninfected with H9N2 subtype SIV.However,the PMVEC began to swell,round and exfoliate with the increase of the TPCK-trypsin concentration;PMVECs were infected with different dose of H9N2 subtype SIV diluted with 0.6 μg/mL TPCK-trypsin culture media,the viral titer were 4.6log2 and 6.4log2 on 48 and 72 h in 0.3 μg/mL cell culture maintenance medium and the dose of 10^-2 H9N2 subtype SIV;Meanwhile,the H9N2 subtype SIV titer was reached to peak value on condition of 2 h adsorption time and vibration 20 s after adsorption 1 h.When the TPCK-trypsin final concentration was 0.3 μg/mL,H9N2 subtype SIV dose of 10^-2,and 2 h viral adsorption and vibration 20 s in the middle of adsorption time,the H9N2 swine influenza virus had optimal proliferation in PMVEC cell,the viral titer was 6.8log2.

Key words: H9N2 subtype; SIV; PMVEC; proliferation

中图分类号:

S852.65

李珮瑶, 梁亭, 罗强, 王少华, 李军, 徐明举, 张瑞华, 徐彤. H9N2亚型猪源性流感病毒在小鼠肺微血管内皮细胞内最佳增殖条件的研究[J]. 《中国畜牧兽医》, 2018, 45(6): 1708-1714.

LI Peiyao, LIANG Ting, LUO Qiang, WANG Shaohua, LI Jun, XU Mingju, ZHANG Ruihua, XU Tong. Study on Optimal Proliferation Conditions of H9N2 Subtype Swine Influenza Virus in Mouse Pulmonary Microvascular Endothelial Cells[J]. , 2018, 45(6): 1708-1714.

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