Department of Virus and Microbiological Special diagnostics, National Influenza Center, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark.
Department of Clinical Microbiology, Rigshospitalet, Blegdamsvej 9, 2100 København Ø, Denmark.
J Clin Virol. 2018 May;102:1-6. doi: 10.1016/j.jcv.2018.02.005. Epub 2018 Feb 11.
Antiviral treatment of influenza virus infections can lead to drug resistance of virus. This study investigates a selection of mutations in the full genome of H3N2 influenza A virus isolated from a patient in treatment with oseltamivir.
Respiratory samples from a patient were collected before, during, and after antiviral treatment. Whole genome sequencing of the influenza virus by next generation sequencing, and low-frequency-variant analysis was performed. Neuraminidase-inhibition tests were performed with oseltamivir and zanamivir, and viruses were propagated in sial-transferase gene transfected Madin-Darby Canine Kidney cells.
A deletion at amino acid position 245-248 in the neuraminidase gene occurred after initiation of treatment with oseltamivir. The deleted virus had highly reduced inhibition against oseltamivir but was sensitive to zanamivir. Nine days after discontinuation of oseltamivir treatment the deleted H3N2 virus was still present in the patient. After three passages of the deleted virus in cell culture, the deletion was retained. Several variant mutations appeared in the other genes of the H3N2 virus, where most striking were two major out-of-frame deletions in the polymerase basic 2 (PB2) gene, indicating defective interfering-like viral RNA.
The viruses harboring the 245-248 deletion in the neuraminidase gene were still present after discontinuation of oseltamivir treatment and passages in cell cultures, indicating a potential risk for transmission of the deleted virus. Full genome deep sequencing was useful to reveal variant mutations that might be selected due to antiviral treatment, and defective interfering-like viral PB2 RNA in the respiratory samples was detected.
抗流感病毒治疗可导致病毒耐药。本研究对 1 例奥司他韦治疗患者的 H3N2 甲型流感病毒全基因组中选择的突变进行了研究。
采集抗病毒治疗前、治疗中和治疗后患者的呼吸道样本。采用下一代测序对流感病毒进行全基因组测序和低频变异分析。用奥司他韦和扎那米韦进行神经氨酸酶抑制试验，并在转染唾液酸转移酶基因的 Madin-Darby 犬肾细胞中繁殖病毒。
在开始奥司他韦治疗后，神经氨酸酶基因的 245-248 位氨基酸缺失。缺失病毒对奥司他韦的抑制作用显著降低，但对扎那米韦敏感。奥司他韦治疗停止 9 天后，患者体内仍存在缺失的 H3N2 病毒。在细胞培养中传代 3 次后，该缺失仍保留。H3N2 病毒的其他基因也出现了一些变异突变，其中最显著的是聚合酶碱性蛋白 2（PB2）基因中两个主要的框外缺失，提示存在缺陷干扰样病毒 RNA。
奥司他韦治疗停止和细胞培养传代后，仍存在携带神经氨酸酶基因 245-248 位缺失的病毒，表明该缺失病毒有传播的潜在风险。全基因组深度测序有助于揭示由于抗病毒治疗而可能选择的变异突变，并且在呼吸道样本中检测到缺陷干扰样病毒 PB2 RNA。
