Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, Tennessee 38105-2794, USA.
J Virol. 2010 Aug;84(16):8042-50. doi: 10.1128/JVI.00689-10. Epub 2010 Jun 2.
The fitness of oseltamivir-resistant highly pathogenic H5N1 influenza viruses has important clinical implications. We generated recombinant human A/Vietnam/1203/04 (VN; clade 1) and A/Turkey/15/06 (TK; clade 2.2) influenza viruses containing the H274Y neuraminidase (NA) mutation, which confers resistance to NA inhibitors, and compared the fitness levels of the wild-type (WT) and resistant virus pairs in ferrets. The VN-H274Y and VN-WT viruses replicated to similar titers in the upper respiratory tract (URT) and caused comparable disease signs, and none of the animals survived. On days 1 to 3 postinoculation, disease signs caused by oseltamivir-resistant TK-H274Y virus were milder than those caused by TK-WT virus, and all animals survived. We then studied fitness by using a novel approach. We coinoculated ferrets with different ratios of oseltamivir-resistant and -sensitive H5N1 viruses and measured the proportion of clones in day-6 nasal washes that contained the H274Y NA mutation. Although the proportion of VN-H274Y clones increased consistently, that of TK-H274Y virus decreased. Mutations within NA catalytic (R292K) and framework (E119A/K, I222L, H274L, and N294S) sites or near the NA enzyme active site (V116I, I117T/V, Q136H, K150N, and A250T) emerged spontaneously (without drug pressure) in both pairs of viruses. The NA substitutions I254V and E276A could exert a compensatory effect on the fitness of VN-H274Y and TK-H274Y viruses. NA enzymatic function was reduced in both drug-resistant H5N1 viruses. These results show that the H274Y NA mutation affects the fitness of two H5N1 influenza viruses differently. Our novel method of assessing viral fitness accounts for both virus-host interactions and virus-virus interactions within the host.
对具有抗奥司他韦高致病性 H5N1 流感病毒的适应性具有重要的临床意义。我们生成了含有神经氨酸酶（NA）突变 H274Y 的重组人 A/Vietnam/1203/04（VN；1 类）和 A/Turkey/15/06（TK；2.2 类）流感病毒，该突变赋予了对 NA 抑制剂的抗性，并在雪貂中比较了野生型（WT）和耐药病毒对的适应性水平。VN-H274Y 和 VN-WT 病毒在上呼吸道（URT）中复制到相似的滴度，并引起相当的疾病症状，并且没有动物存活。在接种后 1 至 3 天，耐奥司他韦的 TK-H274Y 病毒引起的疾病症状比 TK-WT 病毒引起的疾病症状轻，所有动物均存活。然后，我们使用一种新方法研究了适应性。我们用不同比例的耐奥司他韦和敏感的 H5N1 病毒共同接种雪貂，并测量第 6 天鼻冲洗液中含有 H274Y NA 突变的克隆比例。尽管 VN-H274Y 克隆的比例持续增加，但 TK-H274Y 病毒的比例却下降了。NA 催化（R292K）和框架（E119A/K、I222L、H274L 和 N294S）位点或 NA 酶活性位点附近（V116I、I117T/V、Q136H、K150N 和 A250T）的突变自发出现（没有药物压力）在两种病毒对中。NA 取代 I254V 和 E276A 可以对 VN-H274Y 和 TK-H274Y 病毒的适应性产生补偿作用。两种耐药性 H5N1 病毒的 NA 酶功能均降低。这些结果表明，H274Y NA 突变对两种 H5N1 流感病毒的适应性有不同的影响。我们评估病毒适应性的新方法既考虑了病毒-宿主相互作用，也考虑了宿主内病毒-病毒相互作用。
