No evidence of measles virus in stapes samples from patients with otosclerosis

Abstract

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.

FIG. 1

Representative stapes bone cell culture at confluence.

FIG. 2

RT-PCR and Southern blot sensitivity assessments. (A) RT-PCR amplification of the measles virus nucleoprotein (N protein) and matrix protein (M protein) in serial dilution of an Edmonston strain measles virus solution of known titer. PCR products underwent electrophoresis on a 2% agarose gel containing ethidium bromide and were visualized under UV light. The expected lengths in base pairs are indicated for each PCR product. The corresponding titers are indicated in TCID₅₀. (B) Southern blot membranes containing N protein gene first-step PCR and M protein gene PCR products from serial dilution of the control measles virus solution. Corresponding titers are indicated in TCID₅₀ for each lane. Note that for protein M, Southern blot detection was positive at 0.1 TCID₅₀, while the PCR was negative at the same titer.

FIG. 3

Measles virus RT-PCR and Southern blot assessment of 10 stapes samples from patients with otosclerosis. Patient numbers are indicated above each lane (patients 17 to 26). The expected lengths in base pairs are indicated for each PCR product. Electrophoresis on agarose gels containing RT-PCR products of the measles virus nucleocapsid protein (N protein) gene after the first-step amplification and matrix protein (M protein) gene after a single-step amplification are shown for 10 representative patients. Two positive controls (+) were used at 1 and 0.1 TCID₅₀ titers for the RT-PCR and the Southern blot assays. Their detection was as expected according to the sensitivity assessment. Lanes (—), negative controls. A human osteoblastic cell line (SaOS-2) RNA was used as positive control for GAPDH assessment. Southern blot membranes for N protein gene PCR product after the first-step amplification and M protein gene PCR product are represented under the corresponding agarose gels. The GAPDH RT-PCR assessment was used as a control for total RNA recovery. Agarose gels containing the GAPDH RT-PCR products for the same samples are shown in the lower part of the figure.