Simultaneous and Staggered Foot-and-Mouth Disease Virus Coinfection of Cattle

Abstract

Foot-and-mouth disease (FMD) field studies have suggested the occurrence of simultaneous infection of individual hosts by multiple virus strains; however, the pathogenesis of foot-and-mouth disease virus (FMDV) coinfections is largely unknown. In the current study, cattle were experimentally exposed to two FMDV strains of different serotypes (O and A). One cohort was simultaneously infected with both viruses, while additional cohorts were initially infected with FMDV A and subsequently superinfected with FMDV O after 21 or 35 days. Coinfections were confirmed during acute infection, with both viruses concurrently detected in blood, lesions, and secretions. Staggered exposures resulted in overlapping infections as convalescent animals with persistent subclinical FMDV infection were superinfected with a heterologous virus. Staggering virus exposure by 21 days conferred clinical protection in six of eight cattle, which were subclinically infected following the heterologous virus exposure. This effect was transient, as all animals superinfected at 35 days post-initial infection developed fulminant FMD. The majority of cattle maintained persistent infection with one of the two viruses while clearing the other. Analysis of viral genomes confirmed interserotypic recombination events within 10 days in the upper respiratory tract of five superinfected animals from which the dominant genomes contained the capsid coding regions of the O virus and nonstructural coding regions of the A virus. In contrast, there were no dominant recombinant genomes detected in samples from simultaneously coinfected cattle. These findings inculpate persistently infected carriers as potential FMDV mixing vessels in which novel strains may rapidly emerge through superinfection and recombination. IMPORTANCE Foot-and-mouth disease (FMD) is a viral infection of livestock of critical socioeconomic importance. Field studies from areas of endemic FMD suggest that animals can be simultaneously infected by more than one distinct variant of FMD virus (FMDV), potentially resulting in emergence of novel viral strains through recombination. However, there has been limited investigation of the mechanisms of in vivo FMDV coinfections under controlled experimental conditions. Our findings confirmed that cattle could be simultaneously infected by two distinct serotypes of FMDV, with different outcomes associated with the timing of exposure to the two different viruses. Additionally, dominant interserotypic recombinant FMDVs were discovered in multiple samples from the upper respiratory tracts of five superinfected animals, emphasizing the potential importance of persistently infected FMDV carriers as sources of novel FMDV strains.

Keywords: FMD; FMDV; cattle; coinfection; foot-and-mouth disease; foot-and-mouth disease virus; pathogenesis; persistence; virus.

FIG 1

In vivo experimental design. The in vivo study included four groups of four cattle each. Group 1 was infected with a mixed inoculum containing FMDV A24 and FMDV O1M on day 0 and monitored through 35 days. Group 2 was infected with FMDV A24 on day 0, superinfected with FMDV O1M on day 21, and monitored through a total of 49 days (28 days post-FMDV O1M infection). Group 3 was infected with FMDV A24 on day 0, superinfected with FMDV O1M on day 21, and monitored through a total of 56 days (35 days post-FMDV O1M infection). Group 4 was infected with FMDV A24 on day 0, superinfected with FMDV O1M on day 35, and monitored through a total of 70 days (35 days post-FMDV O1M infection).

FIG 2

Strain-specific detection of FMDV A24 and O1M in antemortem samples from group 1. Group 1 cattle were infected with a mixed (FMDV A24 and O1M) inoculum on day 0 and monitored through 35 days. Colored bars in the chart represent the duration (days) of detection of FMDV RNA from A24 (blue) and O1M (red) by strain-specific qRT-PCR in serum and nasal swabs for each of 4 cattle. Purple bars represent presence of FMDV vesicles (based on clinical examinations performed from 0 to 10 days postinfection). FMDV detection in oropharyngeal fluid (OPF) samples was performed by strain-specific qRT-PCR and virus isolation (VI). For OPF samples, blue (FMDV A24) and red (FMDV O1M) bars indicate that samples were positive by both qRT-PCR and VI, with strain-specific RNA detection in unpassaged OPF as well as VI supernatants. Virus identities in VI supernatants were confirmed by FMDV sequence analysis by NGS. Light blue (FMDV A24) and pink (FMDV O1M) bars indicate that OPF samples were positive by strain-specific qRT-PCR but negative for the corresponding virus strain by VI. DPI, days post-dual infection. Numbered days correspond to days on which samples were collected.

FIG 3

Strain-specific detection of FMDV A24 and O1M in vesicular lesions of acutely coinfected cattle. The figure illustrates virus identity in vesicular lesions sampled during acute infection of the 4 cattle in group 1, which had been infected with a mixed inoculum on day 0. Sample analysis was done by NGS, with reads assembled to references A24 Cruzeiro (GenBank accession no. AY593768) and O1 Manisa (GenBank accession no. AY593823). Colored circles represent vesicular lesions caused by FMDV O1M (red) or A24 (blue) on the feet or in the oral cavity. Overlapping circles of both colors indicate that both viruses were detected in one sample. Each animal was sampled twice during the clinical phase of infection. Sampled vesicles do not represent all vesicles observed in the animals. *, two distinct vesicles were harvested from the dental pad and tongue on day 3 from animal 1907. Both vesicles contained genomes of both viruses.

FIG 4

Strain-specific detection of FMDV A24 and O1M in antemortem samples from groups 2 and 3. Group 2 (A) and group 3 (B) cattle were infected with FMDV A24 on day 0, superinfected with FMDV O1M on day 21 (red arrow), and monitored through 28 (group 2) or 35 (group 3) days after the FMDV O1M challenge. Colored bars in the chart represent the duration (days) of detection of FMDV RNA from A24 (blue) and O1M (red) by strain-specific qRT-PCR in serum and nasal swabs for each of 4 cattle per group. Purple bars represent presence of FMDV vesicles (based on clinical examinations performed from 0 to 10 days postinfection and superinfection). FMDV detection in oropharyngeal fluid (OPF) samples was performed by strain-specific qRT-PCR and virus isolation (VI). For OPF samples, blue (FMDV A24) and red (FMDV O1M) bars indicate that samples were positive by both qRT-PCR and VI, with strain-specific RNA detection in unpassaged OPF as well as VI supernatants. Light blue bars indicate that OPF samples were positive by strain-specific qRT-PCR (FMDV A24), but negative for the corresponding virus strain by VI. Virus identities in VI supernatants were confirmed by FMDV sequence analysis by NGS. Green bars indicate the presence of recombinant FMDVs with serotype O capsid coding regions and serotype A nonstructural coding regions. DPI, days post-initial infection. Numbered days correspond to days on which samples were collected.

FIG 5

Alignments of dominant O1M-A24 recombinants. Consensus-level recombinant viruses from oropharyngeal fluid samples obtained from 5 different cattle aligned to (a) FMDV O1M and (b) FMDV A24 reference sequences. Each recombinant virus was the dominant virus in the sample on the indicated days post-initial inoculation (dpi). In each alignment, horizontal rows colored light gray indicate identity with the reference sequence, while black banding indicates dissimilarity. *, the consensus sequence of the animal 1930 31-dpi sample, though estimated to be dominant (Table 3), could not be resolved.

FIG 6

Strain-specific detection of FMDV A24 and O1M in antemortem samples from group 4. Group 4 cattle were infected with FMDV A24 on day 0, superinfected with FMDV O1M on day 35 (red arrow), and monitored through 35 days after the FMDV O1M challenge. Colored bars in the chart represent the duration (days) of detection of FMDV RNA from A24 (blue) and O1M (red) by strain-specific qRT-PCR in serum and nasal swabs for each of 4 cattle. Purple bars represent the presence of FMDV vesicles (based on clinical examinations performed from 0 to 10 days postinfection). FMDV detection in oropharyngeal fluid (OPF) samples was performed by strain-specific qRT-PCR and virus isolation (VI). For OPF samples, blue (FMDV A24) and red (FMDV O1M) bars indicate that samples were positive by both qRT-PCR and VI, with strain-specific RNA detection in unpassaged OPF as well as VI supernatants. Virus identities in VI supernatants were confirmed by FMDV sequence analysis by NGS. Light blue (FMDV A24) and pink (FMDV O1M) bars indicate that OPF samples were positive by strain-specific qRT-PCR, but negative for the corresponding virus strain by VI. DPI, days post-initial infection. Numbered days correspond to days on which samples were collected.

FIG 7

Neutralizing anti-FMDV titers in serum. Neutralizing titers were measured against FMDV A24 and FMDV O1M in serum samples obtained on the first and last study days as well as on the day of the superinfection of study groups 2, 3, and 4. All animals had seroconverted against both viruses by the termination of the studies. No animals had cross-reacting seroreactivity against FMDV O1M prior to FMDV O1M infection. Animals of study groups 2 and 4 had significantly higher titers against FMDV A24 compared to FMDV O1M on the final study day (***, P = 0.007; **, P = 0.004). LOD, limit of detection.