Hurt Aeron C, Holien Jessica K, Barr Ian G
WHO Collaborating Centre for Reference and Research on Influenza, North Melbourne, Victoria 3051, Australia.
Antimicrob Agents Chemother. 2009 Oct;53(10):4433-40. doi: 10.1128/AAC.00334-09. Epub 2009 Aug 3.
To identify mutations that can arise in highly pathogenic A(H5N1) viruses under neuraminidase inhibitor selective pressure, two antigenically different strains were serially passaged with increasing levels of either oseltamivir or zanamivir. Under oseltamivir pressure, both A(H5N1) viruses developed a H274Y neuraminidase mutation, although in one strain the mutation occurred in combination with an I222M neuraminidase mutation. The H274Y neuraminidase mutation reduced oseltamivir susceptibility significantly (900- to 2,500-fold compared to the wild type). However the dual H274Y/I222M neuraminidase mutation had an even greater impact on resistance, with oseltamivir susceptibility reduced significantly further (8,000-fold compared to the wild type). A similar affect on oseltamivir susceptibility was observed when the dual H274Y/I222M mutations were introduced, by reverse genetics, into a recombinant seasonal human A(H1N1) virus and also when an alternative I222 substitution (I222V) was generated in combination with H274Y in A(H5N1) and A(H1N1) viruses. These viruses remained fully susceptible to zanamivir but demonstrated reduced susceptibility to peramivir. Following passage of the A(H5N1) viruses in the presence of zanamivir, the strains developed a D198G neuraminidase mutation, which reduced susceptibility to both zanamivir and oseltamivir, and also an E119G neuraminidase mutation, which demonstrated significantly reduced zanamivir susceptibility (1,400-fold compared to the wild type). Mutations in hemagglutinin residues implicated in receptor binding were also detected in many of the resistant strains. This study identified the mutations that can arise in A(H5N1) under either oseltamivir or zanamivir selective pressure and the potential for dual neuraminidase mutations to result in dramatically reduced drug susceptibility.
为了确定在神经氨酸酶抑制剂选择压力下高致病性A(H5N1)病毒可能出现的突变，两种抗原性不同的毒株分别在不断增加的奥司他韦或扎那米韦水平下连续传代。在奥司他韦压力下，两种A(H5N1)病毒均出现了H274Y神经氨酸酶突变，不过在一个毒株中，该突变与I222M神经氨酸酶突变同时发生。H274Y神经氨酸酶突变显著降低了对奥司他韦的敏感性（与野生型相比降低了900至2500倍）。然而，双重H274Y/I222M神经氨酸酶突变对耐药性的影响更大，奥司他韦敏感性进一步显著降低（与野生型相比降低了8000倍）。当通过反向遗传学将双重H274Y/I222M突变引入重组季节性人类A(H1N1)病毒时，以及当在A(H5N1)和A(H1N1)病毒中与H274Y同时产生另一种I222替代突变（I222V）时，观察到对奥司他韦敏感性有类似影响。这些病毒对扎那米韦仍完全敏感，但对帕拉米韦的敏感性降低。在扎那米韦存在的情况下对A(H5N1)病毒进行传代后，这些毒株出现了D198G神经氨酸酶突变，该突变降低了对扎那米韦和奥司他韦的敏感性，还出现了E型119G神经氨酸酶突变，该突变显示对扎那米韦的敏感性显著降低（与野生型相比降低了1400倍）。在许多耐药毒株中还检测到了血凝素残基中与受体结合相关的突变。本研究确定了在奥司他韦或扎那米韦选择压力下A(H5N1)可能出现的突变，以及双重神经氨酸酶突变导致药物敏感性显著降低的可能性。
