Genetic Determinants of Altered Virulence of Type O Foot-and-Mouth Disease Virus

Figures

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  FIG 1. A 70-nt deletion in the S fragment within the 5′ UTR and an amino acid insertion in Lpro occurred naturally in several O/SEA/Mya-98 FMDV strains. (A) Alignment of the 5´ UTR sequences of O/SEA/Mya-98 lineage FMDV strains. A 70-nt deletion in the S fragment within the 5´ UTR was identified in O/Mya98/JX/2010, HKN/20/2010, O/GSLX/2010, HKN/18/2010, HKN/6/2010, HKN/4/2010, HKN/1/2010, and HKN/19/2010 after comparison with other O/SEA/Mya-98 FMDV strains. (B) Alignment of the O/SEA/Mya-98 lineage FMDV polyprotein amino acid sequences available in GenBank. A leucine/valine insertion at position 10 of Lpro which coexisted with the 70-nt deletion in the S fragment was identified in O/Mya98/JX/2010, HKN/20/2010, and O/GSLX/2010 strains.

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  FIG 2. Strain O/Mya98/JX/2010 was not pathogenic in cattle. (A) Ten cattle were challenged by intradermal inoculation with 107 TCID50/animal of O/Mya98/JX/2010 or O/BY/CHA/2010. The clinical signs were monitored daily, and viral RNA levels in the blood were measured at 1, 3, 5, 7, 9, 11, 13, and 15 days postchallenge (dpc). The viral RNAs in the bovine submandibular lymph nodes and tonsil tissues were measured using qPCR at 15 dpc. RNAemia is defined as the detection of FMDV RNA in blood samples of challenged animals. The x axes show the day postchallenge. The left y axes represent the viral RNA amounts in the blood and tissues; the right y axes represent the clinical scores at different days postchallenge (gray bars). (B) Ten pigs were inoculated intramuscularly with 107 TCID50 of O/Mya98/JX/2010 or O/BY/CHA/2010. The clinical signs and viral RNAs in the blood and organ tissues accessible by the virus were determined as described above to evaluate the infection status of the viruses in pigs.

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  FIG 3. Construction and rescue of four recombinant viruses. (A) Schematic representation showing the constructs of prO, pr-D70, pr-L10, and pr-D70-L10. (B) Plaque assays of rO, rO-D70, rO-L10, and rO-D70-L10 on BHK-21 cells. (C) Alignment of the 5′-UTR region sequences of the four recombinant viruses, O/Mya98/JX/2010, and O/BY/CHA/2010, as well as another O/SEA/Mya-98 lineage FMDV strain, O/GZ/CHA/2010. (D) Alignment of the polyprotein sequences of the four recombinant viruses and O/Mya98/JX/2010, O/BY/CHA/2010, and O/GZ/CHA/2010. A solid black rectangle indicates the leucine insertion in Lpro.

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  FIG 4. rO-D70-L10 showed significantly decreased infective ability for bovine cells compared to that of rO. One-step growth curves are shown for the four recombinant viruses in BHK-21 (A), IBRS-2 (B), PK-15 (C), and BTY (D) cells. The cells were infected with each virus at a multiplicity of infection (MOI) of 0.5 and maintained at 37°C. Samples of supernatants were harvested at the indicated times, and viral titers were measured.

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  FIG 5. rO-D70-L10 showed slightly reduced pathogenicity in pigs compared with rO. Four groups of pigs, with five pigs in each group, were inoculated intramuscularly with 107 TCID50 of rO, rO-D70, rO-L10, and rO-D70-L10, respectively. Clinical signs were monitored daily, and viral RNA levels in the blood were measured at 1, 3, 5, 7, 9, 11, 13, and 15 dpc. The viral RNAs in the submandibular lymph nodes and tonsil tissues were measured using qPCR at 15 dpc. RNAemia is defined as the detection of FMDV RNA in blood samples of challenged animals. The x axes show the days postchallenge. The left y axes represent the viral RNA amounts in the blood and tissues, and the right y axes represent the clinical scores at different days postchallenge (gray bars).

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  FIG 6. rO-D70-L10 did not cause clinical manifestations in cattle. (A) Twenty cattle were challenged by intradermal inoculation with 107 TCID50/animal of rO, rO-D70, rO-L10, or rO-D70-L10. The clinical signs were monitored daily, and viral RNA levels were measured at 1, 3, 5, 7, 9, 11, 13, and 15 dpc. The viral RNAs in the submandibular lymph nodes and tonsil tissues were measured using qPCR at 15 dpc. (B) Five cattle were challenged by intradermal inoculation with 108 TCID50/animal of rO-D70-L10. Clinical signs and viral RNAs in the blood and submandibular lymph nodes and tonsil tissues were determined as described above. RNAemia is defined as the detection of FMDV RNA in blood samples of challenged animals. The x axes show the days postchallenge. The left y axes represent the viral RNA amounts in the blood and tissues; and the right y axes represent the clinical scores at different days postchallenge (gray bars).