Differentiation of the seven major lyssavirus species by oligonucleotide microarray

Abstract

An oligonucleotide microarray, LyssaChip, has been developed and verified as a highly specific diagnostic tool for differentiation of the 7 major lyssavirus species. As with conventional typing microarray methods, the LyssaChip relies on sequence differences in the 371-nucleotide region coding for the nucleoprotein. This region was amplified using nested reverse transcription-PCR primers that bind to the 7 major lyssaviruses. The LyssaChip includes 57 pairs of species typing and corresponding control oligonucleotide probes (oligoprobes) immobilized on glass slides, and it can analyze 12 samples on a single slide within 8 h. Analysis of 111 clinical brain specimens (65 from animals with suspected rabies submitted to the laboratory and 46 of butchered dog brain tissues collected from restaurants) showed that the chip method was 100% sensitive and highly consistent with the "gold standard," a fluorescent antibody test (FAT). The chip method could detect rabies virus in highly decayed brain tissues, whereas the FAT did not, and therefore the chip test may be more applicable to highly decayed brain tissues than the FAT. LyssaChip may provide a convenient and inexpensive alternative for diagnosis and differentiation of rabies and rabies-related diseases.

Fig 1

The clock dial-like layout of the oligoprobe array printed on the glass slide and its hybridization profile of the 7 major lyssavirus species. (A) Each microarray has a capacity for 210 (15 × 14) probes. The lyssavirus species typing oligoprobes (numbers 1 to 50) and positive- and negative-control probes were spotted in duplicate on each microarray, except for probe 21, which was spotted in triplicate. The HEX-labeled oligomarker was spotted 8 times: 4 at the center and 4 at the vertices. Probe spot numbers: 1 to 6, RABV; 7 to 13, LBV; 14 to 21, MOKV; 22 to 27, DUVV; 28 to 33, EBLV-1; 34 to 43, EBLV-2; 44 to 50: ABLV; 51 to 53, positive control; 54 to 56, negative control; 57, HEX-labeled oligomarker. Each slide contained 12 copies of the clock dial-like microarrays. (B) Hybridization of the 7 species of lyssaviruses using the LyssaChip. N, negative control; 1, RABV; 2, LBV; 3, MOKV; 4, DUVV; 5, EBLV-1; 6, EBLV-2; 7, ABLV.

Fig 2

Sensitivity of detection by LyssaChip. The detection of serially diluted template by the LyssaChip showed high sensitivity in detecting all 7 viruses, and EBLV-2 in particular.

Fig 3

Sequence analysis of probe 33 (boxed). Twenty-two sequences of the entire 1,353-bp lyssavirus N gene were obtained from GenBank and aligned by using MegAlign software (version 7.0; LaserGene) with ClustalW. The region corresponding to oligoprobe 33 is shown from the 3′ end (left) to 5′ end (right). (A) The top line is the antisense sequence of probe 33 (70 nt). Sequence alignment revealed that EBLV-1 is divided into 2 lineages, EBLV-1a and EBLV-1b. Probe 33 belongs to EBLV-1a, with 98.6% homology to EBLV-1a and 97.1% to EBLV-1b. There was a mismatched nucleotide with the EBLV-1b sequence at position 13 from the 3′ end of the probe. (B) LyssaChip detection profile of the EBLV-1 control (EBLV-1b) and blinded sample 5 (EBLV-1a).