Clinical Pathology, Istituto Giannina Gaslini, Genova, Italy.
J Clin Virol. 2012 May;54(1):48-55. doi: 10.1016/j.jcv.2012.01.006. Epub 2012 Feb 1.
Human cytomegalovirus (HCMV) is an opportunistic pathogen especially for immuno-suppressed subjects that might develop pharmacological resistance in patients undergoing prolonged antiviral treatment. Ganciclovir (GCV) is the drug used as first choice therapy in affected children and a GCV-resistant phenotype is mainly linked to mutations of the viral protein kinase UL97.
Here a new quantitative pyrosequence (PSQ) method is presented that allows detection and quantification of the viral species carrying the more frequent UL97 mutations responsible for GCV resistance in clinical samples (>80% of known cases).
The system has been validated using two independent approaches (cloning and sequencing of UL-97 gene fragments and real-time PCR) and clinical samples derived from 3 pediatric patients.
The UL97 pyrosequencing analysis has indicated a significant increase of mutant viruses carrying the H520Q and C592G mutations. In particular, the H520Q viral mutation, known to increase GCV resistance (IC50=10) increased around 5 times during hospitalization. In addition, C592G (known to have IC50=2.9) also increased 3 times.
PSQ is a quick, cheap, high throughput and sensitive analysis method to detect GCV-associated resistance mutation useful to follow antiviral therapy in perinatal CMV-infection as well as in immune-suppressed patients.
人巨细胞病毒（HCMV）是一种机会性病原体，特别是对于免疫抑制的患者，这些患者在接受长期抗病毒治疗时可能会产生药物耐药性。更昔洛韦（GCV）是用于受影响儿童的首选治疗药物，而 GCV 耐药表型主要与病毒蛋白激酶 UL97 的突变有关。
本文提出了一种新的定量焦磷酸测序（PSQ）方法，该方法可用于检测和定量临床样本中导致 GCV 耐药的常见 UL97 突变的病毒株（>80%的已知病例）。
该系统已通过两种独立的方法（克隆和测序 UL-97 基因片段和实时 PCR）以及来自 3 名儿科患者的临床样本进行了验证。
UL97 焦磷酸测序分析表明，携带导致 GCV 耐药的 H520Q 和 C592G 突变的突变病毒显著增加。特别是，已知增加 GCV 耐药性的 H520Q 病毒突变（IC50=10）在住院期间增加了约 5 倍。此外，C592G（已知 IC50=2.9）也增加了 3 倍。
PSQ 是一种快速、廉价、高通量且敏感的分析方法，可用于检测 GCV 相关耐药突变，有助于监测围产期 CMV 感染以及免疫抑制患者的抗病毒治疗。
