Grayeli A B, Palmer P, Tran Ba Huy P, Soudant J, Sterkers O, Lebon P, Ferrary E
INSERM U.426, Faculté Xavier Bichat, Université Paris 7, France.
J Clin Microbiol. 2000 Jul;38(7):2655-60. doi: 10.1128/JCM.38.7.2655-2660.2000.
Otosclerosis is a localized bone dystrophy of unknown etiology mainly involving the stapes. The hypothesis of a persistent infection by the measles virus was based on the inconstant detection of the virus by various methods, including reverse transcription-PCR (RT-PCR) of patients' stapes samples. The aim of this work was to investigate the presence of the measles virus in stapedial otosclerosis foci by different sensitive methods. Pathologic stapes samples were obtained from 35 patients suffering from otosclerosis. Measles virus detection was performed by (i) cocultures of Vero cells and primary cell cultures of bone samples (n = 7), (ii) immunofluorescence study of these cocultures (n = 3), and (iii) RT-PCR on RNA directly obtained from fresh frozen samples (n = 28) and on RNA extracted from the primary cell cultures (n = 2). Viral genomic regions coding for N (nucleoprotein) and M (matrix) proteins were separately amplified. PCR sensitivity was optimized on the measles virus Edmonston strain. Glyceraldehyde-3-phosphate dehydrogenase mRNA was used as a marker of total RNA recovery. PCR products were tested by Southern blot hybridization technique to improve sensitivity and specificity. PCRs amplifying the M and the N protein genes were able to detect the control measles virus RNA at titers as low as 0.1 and 0.01 50% tissue culture infective dose, respectively. With these highly sensitive methods, we could not evidence the presence of the measles virus in any of our bone samples or primary bone cell cultures. Our results do not confirm the hypothesis of persistent measles virus infection in otosclerosis.
耳硬化症是一种病因不明的局限性骨营养不良，主要累及镫骨。麻疹病毒持续感染的假说基于通过多种方法（包括对患者镫骨样本进行逆转录 - 聚合酶链反应（RT - PCR））对该病毒的不恒定检测。这项工作的目的是通过不同的敏感方法研究镫骨耳硬化病灶中麻疹病毒的存在情况。从35例耳硬化症患者获取病理镫骨样本。通过以下方法进行麻疹病毒检测：（i）将Vero细胞与骨样本的原代细胞培养物共培养（n = 7），（ii）对这些共培养物进行免疫荧光研究（n = 3），以及（iii）对直接从新鲜冷冻样本中获得的RNA（n = 28）和从原代细胞培养物中提取的RNA（n = 2）进行RT - PCR。分别扩增编码N（核蛋白）和M（基质）蛋白的病毒基因组区域。在麻疹病毒埃德蒙斯顿株上优化PCR敏感性。使用甘油醛 - 3 - 磷酸脱氢酶mRNA作为总RNA回收的标志物。通过Southern印迹杂交技术检测PCR产物以提高敏感性和特异性。扩增M和N蛋白基因的PCR能够分别检测到低至0.1和0.01 50%组织培养感染剂量的对照麻疹病毒RNA。使用这些高度敏感的方法，我们在任何骨样本或原代骨细胞培养物中均未证实麻疹病毒的存在。我们的结果不支持耳硬化症中存在麻疹病毒持续感染的假说。
