Foot-and-Mouth Disease Virus Interserotypic Recombination in Superinfected Carrier Cattle

Figure 1. Study schematic and results overview. (A) A total of 12 cattle were infected with FMDV A24 Cruzeiro by intra-nasopharyngeal deposition on study day 0. On day 21 (eight cattle), or day 35 (four cattle), the animals were superinfected with FMDV O1 Manisa using the same route and dosage. Samples of oropharyngeal fluid (OPF) were obtained using a probang cup, starting at 10 or 14 days after superinfection and continued biweekly through to the termination of the studies at 32 or 35 days after superinfection. (B) Illumina sequencing of OPF samples revealed that distinct samples contained the parental viruses alone or combined with other viruses, including inter-serotypic recombinant viruses.

Figure 2. Serial FMDV sample sequencing analysis: Animal #1914. Foot-and-mouth disease viruses isolated from oropharyngeal fluid (OPF) samples were sequenced and analyzed. (A) Locations (x-axis) and counts (y-axis) of chimeric reads identified in OPF samples for coding regions 2A–3D; included sample timepoints ranged from 35 dpiA/14 dpiO through 45 dpiA/24 dpiO. Asterisk (*) denotes multiple paired chimeric reads confirmed; as multiple breakpoints may exist on individual recombinant genomes, the total number of inferred recombinants is also noted. (B) Alignment of majority viruses identified at each time point in OPF samples, coding regions 2A–3D. The 5′ regions of recombinant viruses up to 2A were fully FMDV-O-derived. Serotype A is the reference for the alignment. Infection with serotype O occurred at 21 dpiA/0 dpiO. An FMDV-O reference sequence has been included for comparison. Abbreviations: dpi—days post-infection; Rec.—recombinant; ref.—reference. (C) Relative composition of viruses identified in each sample.

Figure 3. Serial FMDV sample sequencing analysis: Animal #1929. Foot-and-mouth disease viruses isolated from oropharyngeal fluid (OPF) samples were sequenced and analyzed. (A) Locations (x-axis) and counts (y-axis) of chimeric reads identified in OPF samples for coding regions; included sample timepoints ranged from 31 dpiA/10 dpiO through 52 dpiA/31 dpiO. Asterisk (*) denotes multiple paired chimeric reads confirmed; as multiple breakpoints may exist on individual recombinant genomes, the total number of inferred recombinants is also noted. (B) Alignment of majority viruses identified at each time point in OPF samples, coding regions 2A–3D. The 5′ regions of recombinant viruses up to 2A were fully FMDV-O-derived. Serotype A is the reference for the alignment. Infection with serotype O occurred at 21 dpiA/0 dpiO. An O reference sequence has been included for comparison. Abbreviations: dpi—days post-infection; Rec.—recombinant; ref.—reference. (C) Relative composition of viruses identified in each sample.

Figure 4. Serial FMDV sample sequencing analysis: Animal #1930. Foot-and-mouth disease viruses isolated from oropharyngeal fluid (OPF) samples were sequenced and analyzed. (A) Locations (x-axis) and counts (y-axis) of chimeric reads identified in OPF samples for coding regions 2A–3D; included sample timepoints ranged from 31 dpiA/10 dpiO through 42 dpiA/21 dpiO. Asterisk (*) denotes multiple paired chimeric reads confirmed; as multiple breakpoints may exist on individual recombinant genomes, the total number of inferred recombinants is also noted. (B) Alignment of majority viruses identified at each time point in OPF samples, coding regions 2A–3D. The 5′ regions of recombinant viruses up to 2A were fully FMDV-O-derived. Serotype A is the reference for the alignment. Infection with serotype O occurred at 21 dpiA/0 dpiO. An FMDV-O reference sequence has been included for comparison. Abbreviations: dpi—days post-infection; Rec.—recombinant; ref.—reference. (C) Relative composition of viruses identified in each sample.

Figure 5. Serial FMDV sample sequencing analysis: Animal #1932. Foot-and-mouth disease viruses isolated from oropharyngeal fluid (OPF) samples were sequenced and analyzed. (A) Locations (x-axis) and counts (y-axis) of chimeric reads identified in OPF samples for coding regions 2A–3D; included sample timepoints ranged from 31 dpiA/10 dpiO through 42 dpiA/21 dpiO. Asterisk (*) denotes multiple paired chimeric reads confirmed; as multiple breakpoints may exist on individual recombinant genomes, the total number of inferred recombinants is also noted. (B) Alignment of majority viruses identified at each time point in OPF samples, coding regions 2A–3D. The 5′ regions of recombinant viruses up to 2A were fully FMDV-O-derived. Serotype A is the reference for the alignment. Infection with serotype O occurred at 21 dpiA/0 dpiO. An FMDV-O reference sequence has been included for comparison. Abbreviations: dpi—days post-infection; Rec.—recombinant; ref.—reference. (C) Relative composition of viruses identified in each sample.

Figure 6. Serial FMDV sample sequencing analysis: Animal #1938. Foot-and-mouth disease viruses isolated from oropharyngeal fluid (OPF) samples were sequenced and analyzed. (A) Locations (x-axis) and counts (y-axis) of chimeric reads identified in the 45 dpiA/24 dpiO OPF sample for coding regions 2A–3D. Asterisk (*) denotes multiple paired chimeric reads confirmed. (B) Alignment of majority viruses identified at each time point in OPF samples, coding regions 2A–3D; OPF sampled 14–24 dpiA/-(21-11) dpiO are not shown, but were identical to 31 dpiA/-4 dpiO within the 2A–3D region. The 5′ regions of the recombinant virus up to 2A was fully FMDV-O-derived. Serotype A is the reference for the alignment. Infection with serotype O occurred at 35 dpiA/0 dpiO. An FMDV-O reference sequence has been included for comparison. Abbreviations: dpi—days post-infection; Rec.—recombinant; ref.—reference. (C) Relative composition of viruses identified in each sample.

Figure 7. In vitro characterization of recombinant FMDV. FMDV isolated from oropharyngeal fluid sampled from animal #1914 at 35 dpiA/14 dpiO (#1914_35d in plots) was compared to serotypes A and O inoculum viruses and non-recombinant FMDV-A virus isolated from animal #1914 at 49 dpiA/28 dpiO (#1914_49d in plots). (A) Violin plots of plaque sizes measured after 24 h growth in LFBK-αvβ6 cells. Black and colored dotted lines indicate median size and quartiles, respectively. (B) Sample images of plaque morphology. (C) Growth rates of plaque-purified isolates, grown in LFBK-αvβ6 cells. (D) Sucrose-gradient-spectrography measures of specific infectivity (viral particle (VP) per plaque-forming unit (PFU) of plaque-purified viruses.