Division of Infectious Diseases, Oregon Health and Science University and Department of Veterans Affairs Medical Center, Portland, OR 97239, USA.
J Clin Virol. 2011 Apr;50(4):287-91. doi: 10.1016/j.jcv.2011.01.004. Epub 2011 Feb 3.
Cytomegalovirus UL54 DNA polymerase mutations observed in clinical specimens are of diagnostic significance if confirmed to affect antiviral drug susceptibility.
Validate an updated recombinant phenotyping method to determine the degree of drug resistance conferred by previously uncharacterized UL54 sequence variants, in comparison with known resistance-related mutations.
Bacterial artificial chromosome clones of viral DNA were mutagenized by recombination, transfected to produce live virus and phenotyped by standardized reporter-based yield reduction assays.
Sixteen recombinant viruses were constructed, representing baseline sequences, known resistance-related mutations and amino acid changes of unproven significance from clinical specimens. Phenotypes of baseline strains and known mutants were comparable to results from prior methods and helped to resolve some published inconsistencies. Mutations F412L, F412S, L545W were newly confirmed to confer ganciclovir and cidofovir resistance, while Q578H conferred ganciclovir and foscarnet resistance with borderline cidofovir resistance. Some foscarnet-resistant mutants were appreciably growth-retarded.
Results add to known exonuclease domain mutations that confer ganciclovir-cidofovir cross-resistance, polymerase domain mutations that confer foscarnet resistance with variably decreased ganciclovir/cidofovir susceptibility, and increase the list of sequence variants with no measurable impact on drug susceptibility. The phenotypic diversity of similar UL54 genotypic variants complicates the interpretation of genotypic resistance testing. Technical improvements are facilitating the phenotyping of remaining unknown sequence variants.
如果确认临床标本中观察到的巨细胞病毒 UL54 DNA 聚合酶突变会影响抗病毒药物敏感性，则具有诊断意义。
验证一种更新的重组表型测定方法，以确定以前未表征的 UL54 序列变异对药物耐药性的影响程度，并与已知的耐药相关突变进行比较。
通过重组使病毒 DNA 的细菌人工染色体克隆发生突变，转染以产生活病毒，并通过标准化基于报告基因的产量减少测定进行表型测定。
构建了 16 种重组病毒，代表基线序列、已知耐药相关突变以及临床标本中未证实有意义的氨基酸变化。基线株和已知突变体的表型与先前方法的结果相当，有助于解决一些已发表的不一致问题。F412L、F412S 和 L545W 突变新确认赋予更昔洛韦和西多福韦耐药性，而 Q578H 赋予更昔洛韦和膦甲酸钠耐药性，西多福韦耐药性具有边缘性。一些膦甲酸钠耐药突变体的生长明显受到抑制。
结果增加了已知的外切酶结构域突变，这些突变赋予更昔洛韦-西多福韦交叉耐药性，聚合酶结构域突变赋予膦甲酸钠耐药性，同时更昔洛韦/西多福韦敏感性降低，增加了对药物敏感性无明显影响的序列变异列表。类似 UL54 基因型变异的表型多样性使基因型耐药检测的解释变得复杂。技术改进正在促进剩余未知序列变异的表型测定。
