Georgia
Veterinary Scholar
Program

Recombinant rabies virus with mutation of the phosphorylated serine to alanine reverted to its virulent genotype in vivo.

Previously, a recombinant rabies virus (L16SA) was constructed by mutating the phosphorylated serine to alanine on the nucleoprotein. Consequently the virus yield in cell culture was 10,000 times less than that for the wild-type virus L16. Furthermore, L16SA did not revert when cultured in cells for 15 passages. If this virus behaves similarly in vivo, it could be developed as an attenuated rabies virus vaccine. To determine the virulence of the recombinant virus L16SA in vivo, seven groups of mice (5 per group) were used. Three groups of mice were infected with different dosages (7.5x104, 7.5x105, 7.5x106 ffu) of L16SA. Another three groups of mice were infected with different dosages (7.5x104, 7.5x105, 7.5x106 ffu) of L16, and one group remained uninfected as a control. The mice were observed twice daily over fourteen days for clinical signs or death. Brain samples were collected for reverse transcription and polymerase chain reaction (RT-PCR) using rabies virus specific primers. Direct sequencing of the PCR products was performed to determine if the mutant virus had reverted in vivo. Infected animals developed rabies and died 3 to 13 days after infection. By 14 days after infection, 94% of the mice infected with L16 had died. In contrast, 60% of the mice infected with L16SA had died. The LD50 calculated for L16 is less than 7.5 x 104 ffu whereas the LD50 for L16SA is 3.75 x 105 ffu. The recombinant L16SA virus is about ten times more attenuated in vivo than L16, which is less than that observed in vitro cell culture. Direct sequencing of the PCR products revealed that the recombinant L16SA virus did revert to its virulent genotype indicating instability of the single point mutation. Multiple mutations may increase the stability of the recombinant virus.