DIAGNOSIS
The identification of Negri bodies was the major diagnostic criterion until 1958, when the FAT was developed. The FAT has become the recommended procedure because it is fast, inexpensive and reliable when performed in a competent laboratory with high-quality reagents.
Virus isolation is a routine back- up procedure, providing unambiguous identification of the aetiologic agent. The mouse inoculation test (MIT) is still in used in some countries. As the MIT yields delayed results (between 7 and 20 days), it was replaced in the 1980s by the rabies tissue culture infection test (RTCIT), employing murine neuroblastoma cells.Laboratory diagnosis of rabies has been standardized through several expert committees under the supervision of the World Health Organization (WHO)1-12’13), and the World Organisation for Animal Health (OIE)(14). The main procedures for the routine diagnosis of rabies consist of FAT and RTCIT. These techniques are expensive and not suited for the analysis of large series of samples, for example from field epidemiological surveys. For large numbers of samples, an enzyme- linked immunosorbent assay (ELISA) (the rapid rabies enzyme immunodiagnosis, or RREID) was described. It was also more reliable than other tests when performed on autolysed specimens. However, the RREID is no longer commercially available. Another ELISA, named WELYSSA, which uses mouse monoclonal antibodies (MAbs) to capture the antigen (viral nucleoprotein), has been described recently1-15). In a diagnostic setting, this test is intended for use in tandem with the FAT, which remains the reference technique. WELYSSA exhibits a good sensitivity and specificity for the lyssaviruses circulating in Europe.
Molecular techniques for the detection of rabies and rabies-related lyssaviruses usually use reverse transcription polymerase chain reaction (RT- PCR) methods targeting the viral nucleoprotein gene.
However, another region of the lyssavirus viral genome, the polymerase gene, can be used for detection and phylogenetic analysis. This gene encompasses several highly conserved nucleotide blocks (as block III)(16). A new reverse transcription hemi-nested polymerase chain reaction (RT-hnPCR) protocol employing this region was recently developed. Other techniques such as nucleic acid sequence- based amplification (NASBA) or loop-mediated isothermal amplification (LAMP) techniques have also been proposed but never tested in the European epidemiological context. These tests can be performed on necropsy specimens but also on filter (FTA) paper technology. This was shown to be useful for the storage, transport, collection and subsequent molecular analysis of viral rabies RNA, facilitating epidemiological investigations in the field(17).Specific RT-PCR were also developed for field studies investigating the circulation in EBLV in Europe. More recently, a single, closed-tube, non-nested RT-PCR with TaqMan technology that distinguishes between classical rabies virus (genotype 1) and European bat lyssaviruses 1 and 2 (genotypes 5 and 6) in real time was developed. This TaqMan assay is done on only a single tube and for all reactions is rapid, sensitive and specific, and allows for the genotyping of unknown isolates concomitant with the RT-PCR(18).
Conventional seroneutralization methods were also widely used for surveys of EBLV circulation in the natural environment where bats roost(19), but this technique is expensive and time-consuming. Recently, glycoprotein (G-protein) cDNA from RABV (challenge virus standard- 11) and EBLV1 and EBLV2 were cloned and co-expressed with human immunodeficiency virus (HIV) or murine leukemia virus (MLV) gag-pol and packageable green fluorescent protein (GFP) or luciferase reporter genes in human cells. The harvested lentiviral (HIV) vector provided high-titre pseudotype stocks, which were shown to be suitable for neutralization assays.
These pseudotypes have two major advantages over live- virus neutralization tests: i) they can be handled in low- biohazard-level laboratories; and ii) the use of reporter genes such as GFP or beta-galactosidase will allow the assay to be undertaken at low cost in laboratories worldwide. This constitutes a robust microassay applicable to surveillance studies(20).Seroneutralization (the rapid fluorescent focus inhibition test (RFFIT) or fluorescent antibody virus neutralization test (FAVN)) can be used to monitor rabies antibodies in a wild animal population, for example after vaccination campaigns. A virus neutralizing test using an indirect immunoperoxidase technique (VNT- IIP) for rabies has been developed for the titration of dog and cat serum samples in Japan. The VNT- IIP has the advantage that results obtained can be viewed by the naked eye. But this is still expensive and time-consuming. Recently, ELISA to titrate rabies antibodies in vaccinated wild and domestic carnivores have been described with varying sensitivity and specificity. Therefore, further evaluation organized by the Community Reference Institute for rabies serology (Anses Nancy, France) in the frame of international movements of pets(21) is needed before ELISA can be recommended for routine use and replace seroneutralization in Europe.