Genetic stability of the CVS-31 standard sample of Lyssavirus rabies after successive passages in mice: mutations in phosphoprotein and glycoprotein genes

The Lyssavirus rabies (RABV) is a negative RNA virus of approximately 12kb that encodes five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M or M2), glycoprotein (G), and RNA polymerase (L). The aim of the present study was to identify the stability of the P and G genes from the standard sample CVS-31 RABV. Initially, 10% suspensions of the sample were prepared. Groups of 6 Swiss Webster mice were inoculated with this suspension intracerebrally, and after the animals' death, a new suspension was prepared with central nervous system (CNS) of these animals, and then inoculated into a new group of mice. This process was repeated for 10 successive virus passages. The CNS pool in each of the passages was subjected to RNA extraction, reverse transcription followed by polymerase chain reaction (RT-PCR) with specific primers for the P and G genes. The amplicons were sequenced and analyzed phylogenetically. Non-synonymous mutations were identified throughout the fourth and sixth successive passage. The identified mutations occurred in the regions of amino acids P233, G20, and G249, which may be regions with unknown or low activity in the proteins studied. Although the mutations identified in this study were not maintained in subsequent passages, it can be concluded that less than 10 passages in mice were sufficient to mutate the RABV genome. However, the mutations did not generate significant changes in the virus, supporting the stability documented in the literature and likely this stability remains in vitro , given the absence of immunological pressure from the animal.