These findings indicate the existence of alternative RGD-independent pathways for FMDV entry into cell. In the present study we report that two viruses harboring alternative receptor binding sites (RDD
or RSD) were generated after short-term passage of an FMDV field isolate (Asia1/JS/CHA/05) in different environments. The non-RGD receptor recognition motifs were stably maintained during subsequent passage in cell culture. To study the ability of an RDD-containing FMD viral genome to accommodate substitution in receptor learn more binding site and non-RGD viruses to cause disease in susceptible animals, we assembled an RDD-containing FMDV (Asia1/JSp1c8) full-length cDNA clone and derived mutant clones harboring RGD or RSD motif with a single amino acid substitution (RDD→RGD, RDD→RSD) in the receptor binding site. Following transfection of BSR/T7 cell with three full-length plasmids, the resulting viruses were examined for BIIB057 supplier their infectious potential in-vitro and in-vivo. Results Sequence analysis of Asia1/JS/CHA/05 and its derivatives Deduced amino acid
sequence analysis of the 1D-encoding region showed that Asia1/JS/CHA/05 had a consensus RGD triplet at position 143-145 of VP1, while Asia1/JSp1c8 obtained an alternative RDD triplet at this position. However, careful inspection of the electropherograms from the Asia1/JSM4 VP1 gene sequencing reactions revealed the presence of two selleck inhibitor genetic subpopulations, one with RGD and the other with RSD at receptor binding site. To further investigate the genetic heterogeneity within these samples, 10 biological clones containing VP1 genes of each Asia1/JS/CHA/05, Asia1/JSp1c8 and Asia1/JSM4 were sequenced. Sclareol The 10 clones obtained from each of the Asia1/JS/CHA/05 and Asia1/JSp1c8 viruses respectively encode RGD and RDD tripeptide at position 143-145 of VP1. For Asia1/JSM4, four clones encoded RSD and six clones maintain the RGD motif
at the same position. These results were identical to the amino acid sequence analysis performed by direct sequencing of PCR amplicons. Additionally, amino acid sequence analysis of the capsid-coding regions of Asia1/JS/CHA/05, Asia1/JSp1c8, and Asia1/JSM4 showed that Asia1/JSp1c8 had seven amino acid substitutions in the capsid region (1 in 1A, 3 in 1B, 1 in 1C and 3 in 1D; Table 1) compared with Asia1/JS/CHA/05 and Asia1/JSM4. Table 1 Comparison of the P1 amino acid sequence of Asia1/JS/CHA/05, Asia1/JS/p1c8, and Asia1/JSM4 Capsid region Amino acid residue position a Asia1/JS/CHA/05 Asia1/JSM4 Asia1/JS/p1c8 1A 73 S S N 1B 107 I I V 132 E E K 134 D D G 1C 133 T T A 1D 144 G G/S D 154 N N S 202 K K E a Amino acid residues are numbered from the amino terminus to the carboxyl terminus. Single letter amino acid code is used.