Typing of rabies virus isolates by DNA enzyme immunoassay

Background: Alternatives to antigenic typing are needed for epidemiologic s urveys 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 typ ing methods for rabies virus. The products of a reverse transcription-polym erase chain reaction (RT/PCR) of the nucleoprotein gene were hybridized to type specific probes and detected by enzyme assay after immobilization on m icrotiter plates. Study design: We tested RT/PCR products of 27 rabies isol ates by two different DNA enzyme immunoassays (DEIA) and evaluated the qual ity of the results from the corresponding nucleotide sequence of the sample s. Results: Using a set of two probes, one of the DEIAs correctly identifie d 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 assa y 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 u sed for epidemiologic studies of variants of rabies virus associated with s kunks, foxes, and coyotes. Both DEIA methods were effective when typing pro bes recognized changes at a minimum of two nucleotide positions between var iants, but only one assay method was sufficiently stringent to detect a sin gle base pair mismatch. The inherent mutability of RNA viruses must be cons idered when designing and evaluating typing methods. (C) 1999 Published by Elsevier Science B.V. All rights reserved.