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

denaturation at 94 ° C for 1 minute, primer annealing at 55 ° C for 1 minute, and
polymerization at 72 ° C for 1 minute were done. The products were run in 2% agarose gel
and results were visualized using ethidium bromide (papers II, III, and IV).
3.5.4. Nucleotide sequencing
Before sequencing, the PCR products were purified by using the “MicroSpin S-
400HR” columns (Amersham Pharmacia Biotech, USA). The BigDye Terminator Cycle
Sequencing kit was used according to the manufacturer’s instructions; the sequencing
run was carried out using the 16-capillary sequencer ABI3100 Genetic Analyzer (Applied
Biosystems, Foster City, CA, USA) at the Institute of Biotechnology, University of
Helsinki, and at the Evira Virology Unit. The sequencing results were edited by using the
Staden Package v.2003.0-beta (Staden et al., 2000).
3.5.5. Entire genome sequencing of the vaccine strain RV-97
The RV-97 strain used herein was obtained from the live, attenuated, oral antirabies
vaccine intended for the immunization of wild carnivores (“Sinrab”, FGI “ARRIAH”).
After 1 passage in BHK-21 cell culture (clone C13), the strain was used for further
genetic studies. The entire genome sequence of the vaccine strain RV-97 was obtained
by joining 16 fragments (Fig. 7). The 3’- and 5’- termini of the genome were also obtained
using the universal primer END_Oligo (paper V).
Figure 7. Schematic location of the primer pairs within the viral genome.
Designations on the figure: N – nucleoprotein; P – phosphoprotein; M – matrix
protein; G – glycoprotein; L – RNA-dependent RNA polymerase; Ψ – G-L
intergenic region (pseudogene).
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