Molecular detection of measles virus in primary cell cultures of otosclerotic tissue.
CONCLUSIONS
Primary cell cultures were established from otosclerotic/otospongiotic footplate bone particles. Although this procedure is time-consuming, the quality and quantity of RNA isolated from these cells were much higher in comparison with the direct isolation of RNA from footplate bone samples and the preparation was more suitable for the detection of measles virus (MeV) RNA.


OBJECTIVE
Morphological and biochemical investigations suggest that persistent MeV infection participates in the development of otosclerotic foci. However, this hypothesis is controversial because the detection of MeV in otosclerotic foci is inconsistent since the results are dependent on the presence and stage of foci in the investigated bone particles. Unfortunately, this cannot be confirmed before investigation. To study the presence of the MeV by different techniques in otosclerotic foci, stapes footplate fragments were collected during stapedectomy from patients suffering from clinical otosclerosis.


METHODS
MeV-specific RT-PCR was performed on total RNA isolated directly from four fresh frozen footplate bone fragments and from the cells of 16 primary cultures of otosclerotic tissue samples. In order to rescue persisting MeV, the primary footplate cells were cocultured with MeV permissive B95a cells.


RESULTS
MeV was not detected in RNA from fresh frozen otosclerotic materials, but analysis of the RNA from 5 of the 16 primary cell cultures showed MeV-positive results. Nucleotide sequencing of a 317 bp MeV-specific RT-PCR fragment confirmed the presence of the MeV RNA genome. Here, we report the first determination of MeV sequences in total RNA isolated from primary cells cultured from otosclerotic tissue. Persisting MeV in primary footplate cells could not be recovered by coculturing with B95a cells.
Gantumur
Tsagaan
T
Department of Otorhinolaryngology, Klinikum rechts der Isar, Head and Neck Surgery, Technical University of Munich, Ismaningerstr. 22, 81675, Munich, Germany.
Niedermeyer
Hans P
HP
Neubert
Wolfgang J
WJ
Arnold
Wolfgang
W
D000328:Adult; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D018929:Cell Culture Techniques; D005260:Female; D016679:Genome, Viral; D006801:Humans; D008297:Male; D008459:Measles virus / Q000235:genetics*; D008875:Middle Aged; D010040:Otosclerosis / Q000601:surgery / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013199:Stapes / Q000821:virology*; D013201:Stapes Surgery; D014776:Virus Cultivation
D012367:RNA, Viral