Genetic characteristics of field and attenuated rabies viruses ... - Evira

(cDNA), followed by the amplification of the cDNA by PCR (Tordo et al., 1995, 1996).
The RT-PCR procedure consists of the following steps: total RNA extraction,
cDNA synthesis with random or specific primers, amplification of the cDNA with specific
primers, and visualization of the results with horizontal electrophoresis in agarose gel
containing ethidium bromide observed under UV light (Heaton et al., 1999).
The RT-PCR is widely used for rabies diagnosis, and different parts of the genome
can be targeted for this reason, but in most cases the N gene is utilized (Kulonen and
Boldina, 1993; Bourhy et al., 1999; Ito et al., 2003; Losa-Rubio et al., 2005). A rapid RT-
PCR technique was developed for the detection of the classical rabies virus (genotype 1)
and the rabies related EBLVs (genotypes 5 and 6), and also to distinguish between the
six established rabies and rabies-related virus genotypes (Black et al., 2000, 2002). The
PCR can also be applied to detect the rabies virus genome in formalin-fixed paraffinembedded
brain tissue (Kulonen et al., 1999) and for the intravitam diagnosis of rabies in
humans by testing saliva and cerebrospinal fluid (Crepin et al., 1998). The Real-time
PCR is a quantitative technique which allows the detection of an increase in the amount
of DNA (cDNA) during amplification. It is currently used for the ante- and post-mortem
diagnosis of rabies and the discrimination of the Lyssavirus genotypes (Wakeley et al.,
2005; Nagaraj et al., 2006; Saengseesom et al., 2007).
1.4.3. Virus isolation
The mouse inoculation test (MIT) was one of the first diagnostic tests for rabies.
Laboratory mice are inoculated intracerebrally or subcutaneously with the supernatant of
the sample suspension. The inoculated mice must be observed for up to 30 days after
inoculation. Death during the first 48 hours after inoculation must be considered as nonspecific;
all the animals dead after this period must be dissected and brain samples must
be tested for rabies by the FAT (Koprowski, 1996). This method can be used for testing
the brain and salivary gland suspensions, as well as the saliva samples, for the presence
of live rabies virus (Adeiga and Audu, 1996; Delpietro et al., 2001).
The cell culture inoculation test has already replaced the MIT in many countries
and implies the isolation of rabies virus in a cell culture monolayer with visualization by
the FAT. A number of cell lines have been selected and tested: cow brain cells (CB3),
cerebral and cerebellar grey matter cells of mice (MBC-2, MBC-3), chicken embryo
fibroblasts (CEF), murine, feline and human glial cultures, human monocytic U937 and
THP-1 cells, murine macrophage IC-21, murine monocytic WEHI-3BD- and PU5-1R
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