In Western Europe, most cases of rabies in humans or pets are imported and may be caused by any species within the Genus Lyssavirus. It is assumed that most lyssaviruses can cause the rabies syndrome in humans and other mammals. The other lyssavirus species are mainly maintained in bats and have a more restricted distribution: DUVV, LBV, MOKV, SHIBV, and IKOV have been detected exclusively in Africa, EBLV-1, -2 and BBLV in Europe, ABLV in Australia, and ARAV, KHUV, IRKV, and WCBV in Asia. The classic rabies virus (RABV) has a worldwide distribution and uses carnivores and bats as main reservoir.
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WCBV and IKOV do not cross-react serologically with any of the two phylogroups. Phylogroup 2 includes LBV, MOKV, and SHIBV. Phylogroup 1 includes RABV, DUVV, EBLV-1, EBLV-2, ABLV, ARAV, KHUV, and IRKV. The genus is subdivided into phylogroups 1 and 2. Ikoma virus (IKOV) and Bokeloh bat lyssavirus (BBLV) await classification in the genus. More recently, Aravan virus (ARAV), Khujand virus (KHUV), Irkut virus (IRKV), Shimoni bat virus (SHIBV), and West Caucasian bat virus (WCBV) were also added. Traditionally, these include Rabies virus (RABV), Lagos bat virus (LBV), Mokola virus (MOKV), Duvenhage virus (DUVV), European bat lyssaviruses-1 and -2 (EBLV-1 and EBLV-2), and Australian bat lyssavirus (ABLV). So far, 12 species have been classified in the genus Lyssavirus. Rabies is a fatal viral encephalitis that results from infection with negative strand RNA-viruses belonging to the genus Lyssavirus, family Rhabdoviridae, order Mononegavirales. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The limit of detection was ≤1 50% tissue culture infectious dose. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test.
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In silico sequence alignment showed a functional match with the remaining lyssavirus species. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species.