Scholarlink

Background <p>Human herpesvirus 6A (HHV-6A) is a member of the betaherpesvirus family and is associated with neurotropic diseases. Despite its clinical significance, HHV-6A research has been hampered by challenges in the generation of recombinant viruses. Although bacterial artificial chromosome (BAC) systems and well-established mutagenesis techniques are available for HHV-6A, its tendency towards latency and slow viral replication pose inherent challenges to reconstituting infectious virus. Virus reconstitution has been achieved by only a few laboratories worldwide and remains a hurdle for HHV-6A research.</p> Methods <p>We addressed these key bottlenecks of HHV-6A reconstitution by systematically refining nucleofection and stimulation conditions. Using a reporter virus, we improved cell preparation, implemented a dimethyl sulfoxide (DMSO) treatment, removed contaminating DNA by exonuclease V digestion, optimized the cell recovery after nucleofection, and assessed novel stimulation strategies that accelerate virus replication.</p> Results <p>In this study, we established a cost-effective and robust method for HHV-6A reconstitution. Combining DMSO and Exonuclease V pretreatment with an optimized recovery after nucleofection resulted in an increased transfection efficiency of up to 30%. Selected stimuli promoted lytic replication and facilitated the recovery of infectious virus. Combining IOX2 + hydrocortisone exceeded all other stimuli, reducing the reconstitution time to two weeks. Our optimized protocol has proven to be highly reproducible across multiple laboratories, different mutant viruses, instruments, and operators, ensuring reliability and broad applicability. It also allowed us to generate a novel reporter virus that shed light on the replication kinetics of the virus.</p> Discussion <p>This efficient HHV-6A reconstitution protocol addresses long-standing challenges, offering a widely adoptable method that simplifies recombinant virus generation and enhances future research into viral gene functions and infection mechanisms.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Enhanced virus reconstitution of human herpesvirus 6A (HHV-6A)

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年10月20日

查看全文
  • Jana Reich,
  • Dilan Serdar,
  • Jenny M. Ospina,
  • Yingnan Cheng,
  • Ann-Christin Weißmann,
  • Roosmarijn van der Woude,
  • Georg Beythien,
  • Melissa L. Whyte,
  • Annie Gravel,
  • Thomas Hennig,
  • Louis Flamand,
  • Amy Hudson,
  • Benedikt B. Kaufer

摘要

Background

Human herpesvirus 6A (HHV-6A) is a member of the betaherpesvirus family and is associated with neurotropic diseases. Despite its clinical significance, HHV-6A research has been hampered by challenges in the generation of recombinant viruses. Although bacterial artificial chromosome (BAC) systems and well-established mutagenesis techniques are available for HHV-6A, its tendency towards latency and slow viral replication pose inherent challenges to reconstituting infectious virus. Virus reconstitution has been achieved by only a few laboratories worldwide and remains a hurdle for HHV-6A research.

Methods

We addressed these key bottlenecks of HHV-6A reconstitution by systematically refining nucleofection and stimulation conditions. Using a reporter virus, we improved cell preparation, implemented a dimethyl sulfoxide (DMSO) treatment, removed contaminating DNA by exonuclease V digestion, optimized the cell recovery after nucleofection, and assessed novel stimulation strategies that accelerate virus replication.

Results

In this study, we established a cost-effective and robust method for HHV-6A reconstitution. Combining DMSO and Exonuclease V pretreatment with an optimized recovery after nucleofection resulted in an increased transfection efficiency of up to 30%. Selected stimuli promoted lytic replication and facilitated the recovery of infectious virus. Combining IOX2 + hydrocortisone exceeded all other stimuli, reducing the reconstitution time to two weeks. Our optimized protocol has proven to be highly reproducible across multiple laboratories, different mutant viruses, instruments, and operators, ensuring reliability and broad applicability. It also allowed us to generate a novel reporter virus that shed light on the replication kinetics of the virus.

Discussion

This efficient HHV-6A reconstitution protocol addresses long-standing challenges, offering a widely adoptable method that simplifies recombinant virus generation and enhances future research into viral gene functions and infection mechanisms.