Typing of rabies virus isolates by DNA enzyme immunoassay.

Background: Alternatives to antigenic typing are needed for epidemiologic surveys of the rabies virus associated with translocated coyotes and foxes, especially in areas where a closely related rabies virus is transmitted by striped skunks.

Objectives: We developed and evaluated two enzyme based typing methods for rabies virus. The products of a reverse transcription-polymerase chain reaction (RT/PCR) of the nucleoprotein gene were hybridized to type specific probes and detected by enzyme assay after immobilization on microtiter plates.

Study Design: We tested RT/PCR products of 27 rabies isolates by two different DNA enzyme immunoassays (DEIA) and evaluated the quality of the results from the corresponding nucleotide sequence of the samples.

Results: Using a set of two probes, one of the DEIAs correctly identified 26/27 samples as variants of rabies virus associated with either skunks, foxes, or coyotes. The identity of one fox rabies sample was unresolved by this assay. The second DEIA correctly identified 24/27 samples as variants of rabies virus associated with either skunks, foxes, or coyotes. This assay did not resolve the identity of two fox rabies samples, and misidentified one fox rabies sample as a skunk rabies sample.

Conclusions: DEIA can be used for epidemiologic studies of variants of rabies virus associated with skunks, foxes, and coyotes. Both DEIA methods were effective when typing probes recognized changes at a minimum of two nucleotide positions between variants, but only one assay method was sufficiently stringent to detect a single base pair mismatch. The inherent mutability of RNA viruses must be considered when designing and evaluating typing methods.