Gooskens Jairo, Zevenhoven-Dobbe Jessika C, Claas Eric C, Kroes Aloys C M, Posthuma Clara C
Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
PLoS One. 2014 Apr 3;9(4):e92970. doi: 10.1371/journal.pone.0092970. eCollection 2014.
The pandemic influenza A (H1N1) 2009 virus (pH1N1) contains novel gene segments of zoonotic origin that lack virulence and antiviral resistance markers. We aimed to evaluate the applicability and accuracy of mass spectrometry-based comparative sequence analysis (MSCSA) to detect genetic mutations associated with increased virulence or antiviral resistance in pH1N1. During the 2009 H1N1 pandemic, routine surveillance specimens and clinical antiviral resistance monitoring specimens were analyzed. Routine surveillance specimens obtained from 70 patients with pH1N1 infection were evaluated for mutations associated with increased virulence (PB1-F2, PB2 and NS1 genes) or antiviral resistance (neuraminidase gene, NA) using MSCSA and Sanger sequencing. MSCSA and Sanger sequencing results revealed a high concordance (nucleotides >99%, SNPs ∼ 94%). Virulence or resistance markers were not detected in routine surveillance specimens: all identified SNPs encoded for silent mutations or non-relevant amino acid substitutions. In a second study population, the presence of H275Y oseltamivir resistant virus was identified by real-time PCR in 19 of 35 clinical antiviral resistance monitoring specimens obtained from 4 immunocompromised patients with ≥ 14 days prolonged pH1N1 excretion. MSCSA detected H275Y in 24% (4/19) of positive specimens and Sanger sequencing in 89% (17/19). MSCSA only detected H275Y when the mutation was dominant in the analyzed specimens. In conclusion, MSCSA may be used as a rapid screening tool during molecular surveillance of pH1N1. The low sensitivity for the detection of H275Y mutation in mixed viral populations suggests that MSCSA is not suitable for antiviral resistance monitoring in the clinical setting.
2009年甲型H1N1大流行性流感病毒（pH1N1）包含来自动物源的新型基因片段，这些片段缺乏毒力和抗病毒耐药性标记。我们旨在评估基于质谱的比较序列分析（MSCSA）在检测与pH1N1毒力增加或抗病毒耐药性相关的基因突变方面的适用性和准确性。在2009年H1N1大流行期间，对常规监测标本和临床抗病毒耐药性监测标本进行了分析。使用MSCSA和桑格测序法，对从70例pH1N1感染患者获得的常规监测标本进行评估，以检测与毒力增加（PB1-F2、PB2和NS1基因）或抗病毒耐药性（神经氨酸酶基因，NA）相关的突变。MSCSA和桑格测序结果显示高度一致（核苷酸>99%，单核苷酸多态性~94%）。在常规监测标本中未检测到毒力或耐药性标记：所有鉴定出的单核苷酸多态性编码的都是沉默突变或不相关的氨基酸替换。在第二个研究人群中，通过实时聚合酶链反应在从4例免疫功能低下且pH1N1排泄延长≥14天的患者获得的35份临床抗病毒耐药性监测标本中的19份中鉴定出了H275Y奥司他韦耐药病毒。MSCSA在24%（4/19）的阳性标本中检测到H275Y，桑格测序法在89%（17/19）中检测到。只有当突变在分析标本中占主导地位时，MSCSA才检测到H275Y。总之，MSCSA可作为pH1N1分子监测期间的快速筛查工具。在混合病毒群体中检测H275Y突变的低灵敏度表明，MSCSA不适合在临床环境中进行抗病毒耐药性监测。
