Virus Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA.
Virus Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA; Battelle Memorial Institute, Atlanta, GA 30333, USA.
Antiviral Res. 2014 Jan;101:93-6. doi: 10.1016/j.antiviral.2013.11.001. Epub 2013 Nov 14.
Propagation of influenza A(H3N2) viruses in MDCK cells has been associated with the emergence of neuraminidase (NA) variants carrying a change at residue 151. In this study, the pyrosequencing assay revealed that ∼90% of A(H3N2) virus isolates analyzed (n=150) contained more than one amino acid variant (D/G/N) at position 151. Susceptibilities of the virus isolates to zanamivir and oseltamivir were assessed using the chemiluminescent and fluorescent NA inhibition (NI) assays. In the chemiluminescent assay, which utilizes NA-Star® substrate, up to 13-fold increase in zanamivir-IC50 was detected for isolates containing a high proportion (>50%) of the G151 NA variant. However, an increase in zanamivir-IC50s was not seen in the fluorescent assay, which uses MUNANA as substrate. To investigate this discrepancy, recombinant NAs (rNAs) were prepared and tested in both NI assays. Regardless of the assay used, the zanamivir-IC50 for the rNA G151 was much greater (>1500-fold) than that for rNA D151 wild-type. However, zanamivir resistance conferred by the G151 substitution was masked in preparations containing the D151 NA which had much greater activity, especially against MUNANA. In conclusion, the presence of NA D151G variants in cell culture-grown viruses interferes with drug susceptibility assessment and therefore measures need to be implemented to prevent their emergence.
甲型 H3N2 流感病毒在 MDCK 细胞中的传播与神经氨酸酶（NA）变体的出现有关，这些变体在残基 151 处发生了变化。在这项研究中，焦磷酸测序分析显示，分析的（n=150）约 90%的 A(H3N2)病毒分离株在位置 151 处含有不止一种氨基酸变体（D/G/N）。使用化学发光和荧光 NA 抑制（NI）测定法评估病毒分离株对扎那米韦和奥司他韦的敏感性。在利用 NA-Star®底物的化学发光测定中，对于含有高比例（>50%）G151 NA 变体的分离株，扎那米韦 IC50 增加了多达 13 倍。然而，在使用 MUNANA 作为底物的荧光测定中，未观察到扎那米韦 IC50 的增加。为了研究这种差异，制备了重组 NA（rNA）并在两种 NI 测定中进行了测试。无论使用哪种测定，rNA G151 的扎那米韦 IC50 都远远大于（>1500 倍）rNA D151 野生型。然而，在含有活性更高的 D151 NA 的制剂中，G151 取代赋予的扎那米韦耐药性被掩盖了，尤其是对 MUNANA。总之，在细胞培养中生长的病毒中存在 NA D151G 变体，会干扰药物敏感性评估，因此需要采取措施来防止其出现。
