Adaption of FMDV Asia-1 to Suspension Culture: Cell Resistance Is Overcome by Virus Capsid Alterations

Figures

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  Figure 1. Storage of FMDV A24 Cruzeiro and Asia-1 Shamir in different culture media for 24 h. Equal amounts of virus were incubated in serum-free Cellvento™ BHK200 media, Cellvento™ BHK200 supplemented with 5% fetal bovine serum (FBS), and Minimum Essential Medium Eagle (MEM) supplemented with 5% FBS. Every four hours, one aliquot was titrated to determine remaining infectivity. All experiments were independently performed three times.

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  Figure 2. Incubation of FMDV A24 Cruzeiro, O1 Manisa and Asia-1 Shamir in PBS solutions of different pH for 30 min. Equal amounts of virus were incubated at pH 7.5, 7.0, 6.8, 6.5 and 5.5 for 30 min. The solutions were neutralized and remaining infectivity was determined by titration. Values on the y-axis represent the reduction in titer compared to virus incubated at pH 7.5. All experiments were independently performed three times.

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  Figure 3. Different NH4Cl sensitivity of FMDV serotypes A and Asia-1. BHK monolayers as well as suspension cells were treated with 50 mM or 25 mM ammonium chloride or left untreated. BHK179 were infected with FMDV Asia-1 Shamir and A24 Cruzeiro (original), and BHK-2P were infected with the adapted virus strain A24-2P. After 24 h of incubation, the remaining infectivity was determined by titration. Values on the y-axis represent the reduction in virus yield compared to untreated cells. Experiments were performed in duplicates three times independently. Significance code: *** p < 0.001.

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  Figure 4. Detection of FMDV antigen in adherent BHK164 after treatment with supernatant from transfected BHK-2P. Adherent BHK164 were incubated with stock virus, clarified supernatant from transfected BHK-2P, or viral RNA for 4 h at 37 °C, then fixed and stained with serum from FMDV-infected cattle (green staining). Cell nuclei are shown in blue (a) Cells infected with A24 Cruzeiro and Asia-1 Shamir stock virus as positive controls (PC); (b) Cells incubated with supernatant from transfected BHK-2P; (c) Cells incubated with viral RNA alone (no transfection reagent) as negative controls (NC).

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  Figure 5. Infectivity after attachment of A24 Cruzeiro, Asia-1 Shamir and adapted Asia-1 isolates to BHK179 or BHK-2P. The viruses were incubated either with the adherent cell line BHK179 or the suspension cell line BHK-2P for 15 min on ice to prevent internalization. Cells were washed to remove unbound virus and titrated. The y-axis shows the difference in titer between the BHK-2P and the BHK179 cell preparation. Experiments were performed in duplicates and repeated for a total of three times. Significance code: *** p < 0.001.

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  Figure 6. Capsid maps of the outer surface molecules of FMDV. The capsid map was created with VIPER [20] using FMDV O1/BFS/1860 and A10/Argentina/61 as templates: (a) overview of the exposed residues on the outside of the virus capsid, color-coded for VP1 (blue), VP2 (green) and VP3 (red); (b) zoom on the location where mutations were found in #3 Asia-1, #8 Asia-1 and #9 Asia-1; (c) crystallographic structure of an FMDV capsid pentamer. The VP1 residues 83, 109 and 110 are highlighted. All three mutations are in close spatial proximity to each other and to the fivefold symmetry axis. Residue 110 was mutated in all three viruses, and it appears that this exchange was only possible in combination with a second exchange: either Q108R (in #3 Asia-1 and #8 Asia-1) or T83A (in #9 Asia-1).

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  Figure 7. Serum neutralization tests revealed an altered neutralization profile of the adapted viruses. Asia-1 Shamir and the adapted Asia-1 isolates #3, #8 and #9 were incubated with serum from experimentally infected (“RD460”) or vaccinated (“PC2/99”) cattle. Experiments were performed in duplicates and repeated for a total of three times. Significance code: *** p < 0.001.