The Pseudoknot Region of the 5′ Untranslated Region Is a Determinant of Viral Tropism and Virulence of Foot-and-Mouth Disease Virus

Figures

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  FIG 1. The FMDV O/GD/CHA/2015 strain belongs to the O/ME-SA/PanAsia lineage. Phylogenetic analysis of the VP1 sequences of FMDV O/GD/CHA/2015 and the other 42 FMDV isolates was carried out. ClustalX 1.83 and MEGA 6.06 software were used for construction of the tree. The analyses were carried out using the neighbor-joining method and the Kimura two-parameter nucleotide substitution model in MEGA 6.06 (www.megasoftware.net). The number of bootstrap replicates was set at 1,000. All other parameters were default values. Partial PK region deletions found in the viral genome of the FMDV strains are marked in blue.

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  FIG 2. A PK region deletion in the serotype O FMDV genome occurred naturally in pigs, and all the porcinophilic Cathay topotype FMDVs also included a partial PK region deletion. (A) The 86-nt deletion in the PK region identified in O/GD/CHA/2015 after comparison to other PanAsia FMDV strains reported previously in China. The red lines indicate the deleted region in O/GD/CHA/2015. (B) Schematic representation showing the 86-nt deletion in the PK region of O/GD/CHA/2015 compared to 70 other published serotype O FMDVs isolated in Asian countries available in the NCBI database. (C) Comparison of the 3A protein sequences of Cathay topotype FMDV strains, O/GD/CHA/2015, and the other O/ME-SA/PanAsia lineage viruses isolated in China. The red box indicates the previously reported porcinophilic Cathay topotype FMDV strains with 10 amino acids in the 3A protein. The blue box indicates a 15-amino-acid deletion in the 3A protein of the FMDV OMIII strain. The sequences of 3A proteins of O/GD/CHA/2015 and O/CHA/7/2011 are marked in blue.

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  FIG 3. O/GD/CHA/2015 had no pathogenic ability in cattle. (A) Ten pigs were challenged by intramuscular injection with 107 TCID50/animal of O/GD/CHA/2015 (with a deletion in the PK region) or O/CHA/7/2011 (with the complete PK region). Clinical signs were monitored daily and viremia was determined at 1, 3, 5, 7, and 9 dpc. (B and C) The cattle were challenged by intradermal inoculation with 107 TCID50/animal (B) or 108 TCID50/animal (C) of O/GD/CHA/2015 or O/CHA/7/2011. Clinical signs were monitored daily and viremia was determined at 1, 3, 5, 7, 9, 15, 20, 25, and 30 dpc. Viral RNAs in the bovine OPF collected at 15, 20, 25, and 30 dpc were measured using qPCR. The left y axes represent the viral RNA amounts (viremia and OP fluid), and the right y axes represent the clinical scores (the gray bar graphs indicate the recorded clinical scores at different days postchallenge). The x axes represent the day postchallenge. (D) Comparison of the 5′ UTR region sequences of O/GD/CHA/2015 obtained from the tissue specimens, the propagated O/GD/CHA/2015 in BHK-21 cells, the O/GD/CHA/2015 recovered from the challenged pigs, and the O/CHA/7/2011 strain (a schematic representation).

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  FIG 4. Construction and rescue of two recombinant viruses, rO-WT and rO-DPKs86. (A) Schematic representation showing the constructs of pO/CHA/7/2011 and pO/CHA/7/2011-Δ86. (B) Plaque assays of rO-WT and rO-DPKs86 on BHK-21 cells and the determined virus titers at the indicated time points.

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  FIG 5. rO-DPKs86 showed significantly decreased infective ability for bovine cells and cattle compared with rO-WT. (A) Amounts of viral RNA in rO-WT- or rO-DPKs86-infected BHK-21 cells at 4, 8, 12, 16, 20, and 24 hpi (left panel); one-step growth curves for rO-WT and rO-DPKs86 in BHK-21 cells (middle panel); viral CPE caused by rO-WT and rO-DPKs86 in BHK-21 cells at 16 hpi (right panel). (B) Plaque morphology caused by rO-WT or rO-DPKs86 in PK-15 cells (upper panel); viral RNA levels and virus loads in rO-WT- or rO-DPKs86-infected PK-15 cells at 4, 8, 12, 16, 20, and 24 hpi (middle panel); viral CPE caused by rO-WT and rO-DPKs86 in PK-15 cells at 20 hpi (lower panel). (C) Plaque morphology caused by rO-WT or rO-DPKs86 in BTY cells (upper panel); viral RNA levels and virus loads in rO-WT- or rO-DPKs86-infected BTY cells at 4, 8, 12, 16, 20, and 24 hpi (middle panel); viral CPE caused by rO-WT and rO-DPKs86 in BTY cells at 24 hpi (lower panel). (D) Ten pigs were challenged by intramuscular injection with 107 TCID50/animal of rO-WT or rO-DPKs86. Clinical signs were monitored daily and viremia was determined at 1, 3, 5, 7, and 9 dpc. The left y axes represent the viral RNA levels (log10RNA…

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  FIG 6. rO-DPKs43 showed significantly decreased infective ability in cattle compared to rO-WT. (A) Structures of genetically engineered virus genomes of rO-WT and rO-DPKs43. (B) Plaque assays, virus titers, and the CPE observation of rO-WT and rO-DPKs43 in BHK-21 cells. (C) Plaque morphology and CPE caused by rO-WT or rO-DPKs43 in PK-15 cells. (D) Plaque morphology and CPE caused by rO-WT or rO-DPKs43 in BTY cells. (E) Ten pigs were challenged by intramuscular injection with 107 TCID50/animal of rO-WT or rO-DPKs43. Clinical signs were monitored daily, and viremia was determined at 1, 3, 5, 7, and 9 dpc. The left y axes represent the viral RNA levels (log10RNA copies/200 μl), and the right y axes represent the clinical scores (the gray bar graphs indicate the recorded clinical scores at different days postchallenge). The x axes represent the day postchallenge. (F) Ten cattle were challenged by intradermal inoculation with 108 TCID50/animal of rO-WT or rO-DPKs43. Clinical signs were monitored daily and viremia was determined at 1, 3, 5, 7, 9, 15, 20, 25, and 30 dpc. Viral RNAs in the bovine OPF collected at 15, 20, 25, and 30 dpc were measured.