Engineering Responses to Amino Acid Substitutions in the VP0- and VP3-Coding Regions of PanAsia-1 Strains of Foot-and-Mouth Disease Virus Serotype O

FIG 1. Effects of amino acid difference at position 2079 in the VP0 coding region of PanAsia-1 strains on phenotypic properties of the indicated FMDVs. rHN/TAR6-VP0, rHN/FJ9-VP0, and their seven site-directed mutants were selected to be tested by three different plaque assays on BHK-21 cells and virulence comparison in suckling mice (means ± the SD are indicated; ANOVA results are indicated [*, P < 0.05; **, P < 0.01; ***, P < 0.001, if necessary]). (A) The plaques formed by the Y2079-encoding viruses (■) were distinctly larger than those formed by the 2079H-encoding viruses (□) on BHK-21 cells. At least 50 plaques were analyzed for each virus. (B) The Y2079-eoncoding viruses (●, ▲, ■, ◆, and ж) showed higher virulence in suckling mice than the 2079H-encoding viruses (□, ◇, ○, and △). A total of 20 animals were injected with each virus supernatant (about 100 PFU/200 μl per mouse). (C) The Y2079-encoding viruses (■) displayed a moderate decrease in acid-induced inactivation at acidic pH 6.9 compared to that of the 2079H-encoding viruses (□). The infectivity (%) of each diluted virus (0.5 MOI) at different pH values was calculated as the percentage of PFU by comparison with that obtained…

FIG 2. Effect of amino acid replacements at position 3174 in the VP3 coding region of the specific FMDVs for heparin affinity in BHK-21 cells. The infection of BHK-21 cells with rHN, rHN/FJ9-VP0, rHN/FJ9-VP423, and their seven site-directed mutants was examined by the RGD-containing peptide VR-17 inhibition and heparin binding assays (see Materials and Methods). O/Fujian/CHA/5/99 tc and rHN/TAR6-VP0 were used as negative and positive controls separately. The results from these two plaque assays demonstrated that the remaining plaques of each virus on BHK-21 cells seemed to have been reduced by the addition of heparin (1 mg/ml, right) rather than the preincubation of the RGD-containing peptide VR-17 (1 mM, left). The high affinity of these ten rescued viruses for heparin was not affected by four uncharged amino acid replacements at position 3174 in VP3.

FIG 3. Location of isolate-specific amino acid residues in VP0 and VP3 of the icosahedral capsid of the selected PanAsia-1 strains of FMDV serotype O. The ribbon protein diagram represents 1 of the 60 protomeric subunits, plus one additional VP3 from a neighboring protomer. The structural proteins VP1 (missing 211 to 213 residues), VP2 (missing 1 to 4 residues), VP3, and VP4 (missing 1 to 14 and 41 to 64 residues) are highlighted as red, blue, green, and yellow (internal), respectively. The individual amino acid residues of VP1 to VP3 (listed in Table S1) are labeled as the space-filling atomic models. The target residues (left) of enlarged areas (right) are marked in pink font. The atomic-level interaction of isolate-specific amino acid residues was visualized, and the minimal side chain-side chain distances measured of critical amino acid residues were 4.71 Å between 2079H (Nτ) and R1145 (Nω) of O/Fujian/CHA/9/99 tc, 4.68 Å between L2080Q (Cδ) and S1144A (Cβ), 5.20 Å between E2136G (N) and L1192 (Cγ) of O/Tibet/CHA/6/99 tc, 5.63 Å between D2133N (Cβ) and L1192 (Cδ), 5.78 Å between 3060G (Cα) and K2134 (Cε) of the SPV [small plaque-cloned virus] of O/JPN/2000 strain, 4.42 Å between…