Boutolleau David, Duros Caroline, Bonnafous Pascale, Caïola Delphine, Karras Alexandre, Castro Nathalie De, Ouachée Marie, Narcy Philippe, Gueudin Marie, Agut Henri, Gautheret-Dejean Agnès
Laboratoire de Virologie, UPRES EA 2387, Groupe Hospitalier Pitié-Salpêtrière, Paris, France.
J Clin Virol. 2006 Mar;35(3):257-63. doi: 10.1016/j.jcv.2005.08.002. Epub 2005 Sep 23.
Human herpesvirus 6 (HHV-6) isolates are classified into two variants, termed HHV-6A and HHV-6B, on the basis of distinct genetic, antigenic and biological characteristics, but the specific pathogenicity of each variant remains poorly understood.
To design a rapid, sensitive and specific real-time variant-specific PCR (VS-PCR) method to differentiate both variants in biological specimens.
The VS-PCR was adapted from a real-time PCR assay, based on TaqMan technology, previously developed for the genome quantitation of both HHV-6 variants [Gautheret-Dejean A, Manichanh C, Thien-Ah-Koon F, Fillet AM, Mangeney N, Vidaud M, et al. Development of a real-time polymerase chain reaction assay for the diagnosis of human herpesvirus-6 infection and application to bone marrow transplant patients. J Virol Meth 2002;100:27-35], a consensual reverse primer (Taq2) being changed into two variant-specific primers named H6A and H6B. This method was applied to a large set of biological specimens obtained in different pathological contexts.
The sensitivity threshold was about 10 copies/well for HHV-6A-specific PCR (PCR-A) and 1 copy/well for HHV-6B-specific PCR (PCR-B). Both assays showed a linear dynamic range from 10 to 100,000 copies of HHV-6 DNA. Regarding the specificity and the capacity of discrimination of each assay, one variant could be detected and identified in the presence of more than 1000 times higher concentrations of the other variant in virus mixtures. The comparison of the results obtained with this VS-PCR with those previously obtained with a classic PCR method allowed us to validate our new technique on a wide panel of biological samples, including numerous patients with severe HHV-6-related symptoms. The high prevalence of HHV-6B was confirmed in healthy individuals and immunocompromised patients. HHV-6A was identified in distinct samples from several patients exhibiting neurological disorders.
We developed a new VS-PCR assay, able to differentiate HHV-6A and HHV-6B in biological samples, even in the case of mixed infections. Our study confirms the wide prevalence of HHV-6B and highlights the potential greater neuropathogenic role of HHV-6A in immunocompromised patients and young infants.
人疱疹病毒6型（HHV-6）分离株根据不同的遗传、抗原和生物学特性分为两个变种，即HHV-6A和HHV-6B，但每个变种的具体致病性仍知之甚少。
设计一种快速、灵敏且特异的实时变种特异性PCR（VS-PCR）方法，以区分生物标本中的这两个变种。
VS-PCR改编自一种基于TaqMan技术的实时PCR检测方法，该方法先前用于定量检测HHV-6两个变种的基因组[Gautheret-Dejean A, Manichanh C, Thien-Ah-Koon F, Fillet AM, Mangeney N, Vidaud M, 等。开发一种用于诊断人疱疹病毒6型感染的实时聚合酶链反应检测方法及其在骨髓移植患者中的应用。病毒学方法杂志2002；100：27-35]，将通用反向引物（Taq2）改为两个变种特异性引物，分别命名为H6A和H6B。该方法应用于从不同病理背景下获取的大量生物标本。
HHV-6A特异性PCR（PCR-A）的灵敏度阈值约为10拷贝/孔，HHV-6B特异性PCR（PCR-B）的灵敏度阈值为1拷贝/孔。两种检测方法的线性动态范围均为10至100,000拷贝的HHV-6 DNA。关于每种检测方法的特异性和区分能力，在病毒混合物中存在浓度比另一个变种高1000倍以上的情况下，仍可检测和鉴定出一个变种。将该VS-PCR获得的结果与先前用经典PCR方法获得的结果进行比较，使我们能够在包括众多有严重HHV-6相关症状患者的大量生物样本上验证我们的新技术。健康个体和免疫功能低下患者中HHV-6B的高流行率得到证实。在几名患有神经系统疾病患者的不同样本中鉴定出了HHV-6A。
我们开发了一种新的VS-PCR检测方法，能够区分生物样本中的HHV-6A和HHV-6B，即使在混合感染的情况下也是如此。我们的研究证实了HHV-6B的广泛流行，并突出了HHV-6A在免疫功能低下患者和幼儿中可能更大的神经致病作用。
