Comparison of mutation patterns in full-genome A/H3N2 influenza sequences obtained directly from clinical samples and the same samples after a single MDCK passage.
Comparison of mutation patterns in full-genome A/H3N2 influenza sequences obtained directly from clinical samples and the same samples after a single MDCK passage.
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DOI：
10.1371/journal.pone.0079252
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Koay ES
中科院分区：
文献类型：
--
作者：
Lee HK;Tang JW;Kong DH;Loh TP;Chiang DK;Lam TT;Koay ES
人流感病毒可以有效地从临床样本中分离使用Madin-Darby犬肾（MDCK）细胞。然而，已知该过程在病毒适应该非人类细胞系时诱导病毒突变。我们进行了一项系统性研究，以记录在整个甲型流感/H3 N2基因组中观察到的MDCK诱导突变的模式。2009-2011年收集的77份临床样本被纳入研究。每个样本获得两个完整的流感病毒基因组：一个来自直接从临床样本获得的病毒，一个来自在MDCK细胞中培养的匹配分离株。比较从这些来源中获得的全基因组序列显示，77个分离株中有42%获得了至少一个MDCK诱导的突变。这些突变的存在或不存在与病毒载量或样本来源（住院患者与门诊患者）无关。值得注意的是，所有5个血凝素错义突变仅在血凝素1结构域观察到，特别是在病毒的受体结合位点和抗原位点内或附近。此外，23%的77个菌株经历了MDCK诱导的错义突变，D151 G/N，在神经氨酸酶段。已发现该突变与对神经氨酸酶抑制剂的药物敏感性降低和病毒受体与宿主细胞的结合效率增加有关。相比之下，直接从临床样本中获得的神经氨酸酶序列均不包含D151 G/N突变，这表明该突变可能是MDCK培养诱导变化的指标。这些D151突变可能混淆血凝抑制试验和神经氨酸酶抑制剂耐药结果的解释，当这些结果基于MDCK分离株时。这些分离株目前在世卫组织流感疫苗和耐药性监测项目中常规使用。因此，通过在进一步序列分析后仔细排除此类D151突变体，可以避免潜在的数据解释误判。
Human influenza viruses can be isolated efficiently from clinical samples using Madin-Darby canine kidney (MDCK) cells. However, this process is known to induce mutations in the virus as it adapts to this non-human cell-line. We performed a systematic study to record the pattern of MDCK-induced mutations observed across the whole influenza A/H3N2 genome. Seventy-seven clinical samples collected from 2009-2011 were included in the study. Two full influenza genomes were obtained for each sample: one from virus obtained directly from the clinical sample and one from the matching isolate cultured in MDCK cells. Comparison of the full-genome sequences obtained from each of these sources showed that 42% of the 77 isolates had acquired at least one MDCK-induced mutation. The presence or absence of these mutations was independent of viral load or sample origin (in-patients versus out-patients). Notably, all the five hemagglutinin missense mutations were observed at the hemaggutinin 1 domain only, particularly within or proximal to the receptor binding sites and antigenic site of the virus. Furthermore, 23% of the 77 isolates had undergone a MDCK-induced missense mutation, D151G/N, in the neuraminidase segment. This mutation has been found to be associated with reduced drug sensitivity towards the neuraminidase inhibitors and increased viral receptor binding efficiency to host cells. In contrast, none of the neuraminidase sequences obtained directly from the clinical samples contained the D151G/N mutation, suggesting that this mutation may be an indicator of MDCK culture-induced changes. These D151 mutations can confound the interpretation of the hemagglutination inhibition assay and neuraminidase inhibitor resistance results when these are based on MDCK isolates. Such isolates are currently in routine use in the WHO influenza vaccine and drug-resistance surveillance programs. Potential data interpretation miscalls can therefore be avoided by careful exclusion of such D151 mutants after further sequence analysis.
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作者：
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通讯作者：
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DOI：
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发表时间：
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