Article Figures & Data
Figures
- Fig. 1.
Viral binding to heparin. Radiolabeled SV was applied to heparin-Sepharose and eluted with a 50 to 500 mM NaCl gradient. Elution at a higher NaCl concentration indicates stronger binding to heparin. Eluted virus is intact and fully infectious. (Five other viruses are not shown; see Table 2 for results.)
- Fig. 2.
Viral binding to cultured cells. Radiolabeled SV was allowed to bind to CHO or glycosaminoglycan-deficient pgsA-745 cells at 4°C for 2 h. The mean binding ± standard deviation for three replicates is shown. The data along the x axis are taken from Table 2.
- Fig. 3.
Kinetics of viral clearance. Mice were injected intravenously with a 100-μl bolus of purified virus. Serum was collected, and titers were determined by a plaque assay with BHK cells at various times postinjection; V/V 0 indicates the fraction of virus remaining. Each point represents the mean ± standard deviation for three mice. Curves were fitted by nonlinear regression as described in Materials and Methods. Symbols: ●, K76T; ○, K230M; ▾, N62D; ▿, K159E; ■, K76E; □, K76N; ⧫, R157H; ◊, Toto 1101.
- Fig. 4.
Serum titers. (A) CD-1 pups (2 days old) were infected subcutaneously with 1,000 PFU of virus, and serum titers were assessed at various times by a plaque assay (mean for three mice at each time point; error bars are not shown). All LP viruses retained their LP phenotype. A two-way analysis of variance showed a significant (P, <0.0001) effect of viral strain on titer, and titers of Toto 1101 were lower than those of all seven mutant viruses when compared by Scheffe's test (P, <0.05). (B) Plotting of peak titer (mean ± standard deviation) versus elution from heparin-Sepharose failed to indicate a simple correlation between virus replication in vivo and the strength of binding to heparin.
- Fig. 5.
Survival curves. Litters of 2-day-old CD-1 pups were infected subcutaneously with 1,000 PFU of virus, and survival was monitored for 15 days. R157H, K230M, N62D, and K76E produced significantly greater mortality than Toto 1101. Survival curves were compared pairwise against Toto 1101 by use of the log rank test with a threshold for significance (P) of <0.0071; the Bonferonni correction for multiple comparisons was used.
Tables
- Table 1.
Sequencing of LP isolates from infected mice
Virus Coding change(s) in E2a Parental virus Mouse serum sourceb 11D2 R157H Toto 1101 Adult IFN-α/β receptor knockout 11E2 D5V, K76E Toto 1101 Adult IFN-α/β receptor knockout 11G1 K159E, T161Kc Toto 1101 Adult IFN-α/β receptor knockout 105 K76N, T308S AR339 Adult μMT (antibody deficient) 106 K230M AR339 Adult μMT (antibody deficient) 4A1 N62D Toto 1101 Neonatal CD-1 a All LP mutants had at least one amino acid change in E2 but none in E1 or 6K.
b Mice were infected subcutaneously, except for μMT mice, which were infected intracerebrally.
c Sequencing of the parental Toto 1101 virus, which had been passaged several times in BHK cells, showed that the parental population contained some viruses with the T161K mutation.
- Table 2.
Recombinant viruses
Virus Plaque diam (mm) with: Heparin-Sepharose elution (mM NaCl) ELISA reactivityb of E2 MAb: Agar Agarose R6 G5 202 Toto 1101 1.3 ± 0.4a 3.3 ± 0.7 323 + + + R157H 2.5 ± 0.7 2.8 ± 0.6 289 + + + N62D 2.0 ± 0.7 2.2 ± 0.6 276 − + + K159E 2.8 ± 0.8 2.6 ± 0.7 261 − + + K230M 2.4 ± 0.8 2.6 ± 0.7 202 + + + K76N 2.3 ± 0.9 2.5 ± 0.7 142 + + + K76T 2.3 ± 0.8 2.6 ± 0.8 97 + + + K76E 3.1 ± 0.8 2.5 ± 0.8 66 + + + a The plaque size (mean ± standard deviation) of Toto 1101 under agar was significantly smaller (P, <0.05) than those of all other viruses under agar and was also significantly smaller than that of Toto 1101 under agarose (Kruskal-Wallis analysis of variance ranks followed by Dunn's multiple-comparison test).
b +, reactivity; −, no reactivity.
- Table 3.
Organ distribution of radiolabeled virus 30 min after intravenous injection
Organ Mean % of total counts injected ± SD (n = 3) for: Toto 1101 K159E K76E Liver 53.0 ± 4.7 21.8 ± 0.9 12.0 ± 2.8 Spleen 4.1 ± 0.8 12.6 ± 7.9 10.8 ± 8.6 Kidneys 0.53 ± 0.02 0.89 ± 0.13 0.70 ± 0.08 Lungs 0.24 ± 0.05 0.47 ± 0.19 0.50 ± 0.24 Brain 0.05 ± 0.03 0.09 ± 0.01 0.08 ± 0.01
