Schalkwijk Hanna Helena, Gillemot Sarah, Frobert Emilie, Morfin Florence, Ducastelle Sophie, Conrad Anne, Fiten Pierre, Opdenakker Ghislain, Snoeck Robert, Andrei Graciela
Molecular Structural and Translational Virology Research Group, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium.
Virpath Unit, CIRI, Inserm U1111, CNRS, UMR5308, ENS Lyon, Université Claude Bernard Lyon 1 and Virology Unit, Institut des Agents Infectieux, Groupement Hospitalier Nord, Hospices Civils de Lyon, 69004 Lyon, France.
Viruses. 2025 Jul 9;17(7):962. doi: 10.3390/v17070962.
Herpes simplex virus 2 (HSV-2) remains a significant cause of morbidity and mortality in immunocompromised individuals, despite the availability of effective antivirals. Infections caused by drug-resistant isolates are an emerging concern among these patients. Understanding evolutionary aspects of HSV-2 resistance is crucial for designing improved therapeutic strategies. Here, we characterized 11 HSV-2 isolates recovered from various body sites of a single immunocompromised patient suffering from a primary HSV-2 infection unresponsive to acyclovir and foscarnet. The isolates were analyzed phenotypically and genotypically (Sanger sequencing of viral thymidine kinase and DNA polymerase genes). Viral clone isolations, deep sequencing, viral growth kinetics, and dual infection competition assays were performed retrospectively to assess viral heterogeneity and fitness. Sanger sequencing identified mixed populations of DNA polymerase mutant variants. Viral clones were plaque-purified and genotyped, revealing 17 DNA polymerase mutations (K533E, A606V, C625R, R628C, A724V, S725G, S729N, I731F, Q732R, M789T/K, Y823C, V842M, R847C, F923L, T934A, and R964H) associated with acyclovir and foscarnet resistance. Deep-sequencing of the DNA polymerase detected drug-resistant variants ranging between 1 and 95%, although the first two isolates had a wild-type DNA polymerase. Some mutants showed reduced fitness, evidenced by (i) the frequency of variants identified by deep-sequencing not correlating with the proportion of mutants found by plaque-purification, (ii) loss of the variants upon passaging in cell culture, or (iii) reduced frequencies in competition assays. This study reveals the rapid evolution of heterogeneous drug-resistant HSV-2 populations under antiviral therapy, highlighting the need for alternative treatment options and resistance surveillance, especially in severe infections.
尽管有有效的抗病毒药物，但单纯疱疹病毒2型（HSV - 2）仍然是免疫功能低下个体发病和死亡的重要原因。耐药菌株引起的感染是这些患者中一个新出现的问题。了解HSV - 2耐药性的进化方面对于设计改进的治疗策略至关重要。在此，我们对从一名患有原发性HSV - 2感染且对阿昔洛韦和膦甲酸钠无反应的免疫功能低下患者的不同身体部位分离出的11株HSV - 2菌株进行了特征分析。对这些菌株进行了表型和基因型分析（病毒胸苷激酶和DNA聚合酶基因的桑格测序）。回顾性地进行了病毒克隆分离、深度测序、病毒生长动力学和双重感染竞争试验，以评估病毒的异质性和适应性。桑格测序鉴定出DNA聚合酶突变变体的混合群体。对病毒克隆进行了噬斑纯化和基因分型，发现了17种与阿昔洛韦和膦甲酸钠耐药相关的DNA聚合酶突变（K533E、A606V、C625R、R628C、A724V、S725G、S729N、I731F、Q732R、M789T/K、Y823C _、V842M、R847C、F923L、T934A和R964H）。DNA聚合酶的深度测序检测到耐药变体的比例在1%至95%之间，尽管前两个分离株具有野生型DNA聚合酶。一些突变体显示出适应性降低，这表现为：（i）深度测序鉴定出的变体频率与噬斑纯化发现的突变体比例不相关；（ii）在细胞培养传代时变体丢失；或（iii）在竞争试验中频率降低。这项研究揭示了抗病毒治疗下异质性耐药HSV - 2群体的快速进化，突出了需要替代治疗方案和耐药性监测，特别是在严重感染中。
