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

Abstract

Viral recombination contributes to the emergence of novel strains with the potential for altered host range, transmissibility, virulence, and immune evasion. For foot-and-mouth disease virus (FMDV), cell culture experiments and phylogenetic analyses of field samples have demonstrated the occurrence of recombination. However, the frequency of recombination and associated virus-host interactions within an infected host have not been determined. We have previously reported the detection of interserotypic recombinant FMDVs in oropharyngeal fluid (OPF) samples of 42% (5/12) of heterologously superinfected FMDV carrier cattle. The present investigation consists of a detailed analysis of the virus populations in these samples including identification and characterization of additional interserotypic minority recombinants. In every animal in which recombination was detected, recombinant viruses were identified in the OPF at the earliest sampling point after superinfection. Some recombinants remained dominant until the end of the experiment, whereas others were outcompeted by parental strains. Genomic analysis of detected recombinants suggests host immune pressure as a major driver of recombinant emergence as all recombinants had capsid-coding regions derived from the superinfecting virus to which the animals did not have detectable antibodies at the time of infection. In vitro analysis of a plaque-purified recombinant virus demonstrated a growth rate comparable to its parental precursors, and measurement of its specific infectivity suggested that the recombinant virus incurred no penalty in packaging its new chimeric genome. These findings have important implications for the potential role of persistently infected carriers in FMDV ecology and the emergence of novel strains.

Keywords: FMD; FMDV; aphthovirus; coinfection; foot-and-mouth disease virus; pathogenesis; persistent infection; recombination.

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 dpi_A/14 dpi_O through 45 dpi_A/24 dpi_O. 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 dpi_A/0 dpi_O. 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 dpi_A/10 dpi_O through 52 dpi_A/31 dpi_O. 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 dpi_A/0 dpi_O. 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 dpi_A/10 dpi_O through 42 dpi_A/21 dpi_O. 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 dpi_A/0 dpi_O. 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 dpi_A/10 dpi_O through 42 dpi_A/21 dpi_O. 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 dpi_A/0 dpi_O. 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 dpi_A/24 dpi_O 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 dpi_A/-(21-11) dpi_O are not shown, but were identical to 31 dpi_A/-4 dpi_O 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 dpi_A/0 dpi_O. 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 dpi_A/14 dpi_O (#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 dpi_A/28 dpi_O (#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.