FIG 1.

In vivo passage of SAFV-3 in neonatal mice. (A) Schematic diagram showing the serial in vivo passage of SAFV-3. Within 24 h of birth, neonatal BALB/c mice (n = 5) were inoculated intracerebrally at 10⁴ CCID₅₀s/10 μl with SAFV-3 (AM strain) collected from a patient with aseptic meningitis. On day 3 p.i., five cerebella were collected from neonatal mice and 10% tissue homogenates were prepared. After centrifugation, 10 μl of the supernatant (termed 1Cb) was inoculated intracerebrally into the next group of neonatal mice within 24 h of their birth. This process was conducted a total of five times. After propagation of the virus from the supernatant of 5Cb in LLC-MK₂ cells, the culture supernatant, called the AM-5Cb strain, was stored as stock virus. (B) Amino acid substitutions occurring during the in vivo serial passages. These passaged strains acquired three sequential amino acid substitutions in viral structural proteins, VP2-160, VP2-239, and VP3-62. The three amino acid substitutions were VP2-H160R, VP2-Q239R, and VP3-K62M. (C) Viral RNA copy number during the serial passage of SAFV-3 in the mouse cerebellum. The number of viral RNA copies was determined by real-time RT-PCR and is expressed relative to the number of mouse beta-actin RNA copies. (**, P < 0.01; ***, P < 0.001; one-way ANOVA).