2.8 Phylogenetic and Recombination analyses
Phylogenetic analysis was performed using MEGA 7(Kumar et al., 2004). Multiple alignments were carried out using CLUSTAL X version 1.83. A phylogenetic tree was constructed using the neighbor-joining method and distances were determined using the Kimura two-parameter method (Ohshima et al., 2009; Whelan et al., 2001). The putative recombinant sequence and its parents were identified using two different recombination detection programs, namely RDP4 and Simplot software packages. RDP4 is composed of the following 8 separate programs and enables automated analyses of a nucleotide sequence alignment using all of the programs concomitantly: RDP, Chimaera, BootScan, 3Seq, GENECONV, MaxChi, SIScan and LARD (Martin et al., 2015 Ohshima et al., 2009). Simplot uses conventional parameter settings.
RESULTS
Histopathologic examination
An 8-month-old female cub who was paralyzed was euthanized and sent for pathological examination. The most significant lesion at autopsy was a 5cm round cavity in the center of the lion’s brain, which was covered by a connective tissue capsule (Figure 1). The histopathological analyses of the brain showed typical encephalitis with glial cell proliferation, neuronal atrophy and necrosis, and encephalomalacia. The cerebellum showed nerve cell necrosis and capillary congestion. The spinal cord and limb nerves showed vesicular degeneration and partial necrosis, respectively (Figure 2).