Screening of active lyssavirus infection in wild bat populations by viral RNA detection on oropharyngeal swabs

Abstract

Brain analysis cannot be used for the investigation of active lyssavirus infection in healthy bats because most bat species are protected by conservation directives. Consequently, serology remains the only tool for performing virological studies on natural bat populations; however, the presence of antibodies merely reflects past exposure to the virus and is not a valid marker of active infection. This work describes a new nested reverse transcription (RT)-PCR technique specifically designed for the detection of the European bat virus 1 on oropharyngeal swabs obtained from bats but also able to amplify RNA from the remaining rabies-related lyssaviruses in brain samples. The technique was successfully used for surveillance of a serotine bat (Eptesicus serotinus) colony involved in a case of human exposure, in which 15 out of 71 oropharyngeal swabs were positive. Lyssavirus infection was detected on 13 oropharyngeal swabs but in only 5 brains out of the 34 animals from which simultaneous brain and oropharyngeal samples had been taken. The lyssavirus involved could be rapidly identified by automatic sequencing of the RT-PCR products obtained from 14 brains and three bat oropharyngeal swabs. In conclusion, RT-PCR using oropharyngeal swabs will permit screening of wild bat populations for active lyssavirus infection, for research or epidemiological purposes, in line not only with conservation policies but also in a more efficient manner than classical detection techniques used on the brain.

FIG. 1

Primer sequences and mismatches with the different rabies-related lyssaviruses: RABV (genotype 1), LBV (genotype 2), MOKV (genotype 3), DUVV (DVHV) (genotype 4), EBV1a (subtype a, genotype 5), EBV1b (subtype b, genotype 5), EBV2a (subtype a, genotype 6), EBV2b (subtype b, genotype 6). Sequences of primers LISEBL1F, LISEBL1R, LISEBL2F, and LISEBL2R are shown.

FIG. 2

RT-PCR results for bat samples. The upper band (323 bp) is the internal control band. The lower band (117 bp) is the lyssavirus-specific band. Lanes 1, 2, 4 to 9, and 11 are lyssavirus negative, lane 10 is lyssavirus positive, lane 3 has presence of enzyme inhibitors, and lanes 12 and 13 are negative and positive controls (with no internal control).

FIG. 3

Results of the PCR test on all rabies-related lyssaviruses. Lanes 1 and 2, LBV; lanes 3 to 5, MOKV; lane 6, DUVV; lanes 7 and 11, EBV1; lanes 8 and 9, EBV2; lane 10, ABV; lane 12, RABV (CVS strain); lane 13, negative control; lane 14, molecular size marker (123 bp; Life Technologies, Gaithersburg, Md.). No internal control was included in this assay.