The pathogenesis of persistent foot-and-mouth disease virus (FMDV) infection was investigated in 46 cattle that were either naive or had been vaccinated using a recombinant, adenovirus-vectored vaccine 2 weeks before challenge. The prevalence of FMDV persistence was similar in both groups (62% in vaccinated cattle, 67% in nonvaccinated cattle), despite vaccinated cattle having been protected from clinical disease. Analysis of antemortem infection dynamics demonstrated that the subclinical divergence between FMDV carriers and animals that cleared the infection had occurred by 10 days postinfection (dpi) in vaccinated cattle and by 21 dpi in nonvaccinated animals. The anatomic distribution of virus in subclinically infected, vaccinated cattle was restricted to the pharynx throughout both the early and the persistent phases of infection. In nonvaccinated cattle, systemically disseminated virus was cleared from peripheral sites by 10 dpi, while virus selectively persisted within the nasopharynx of a subset of animals. The quantities of viral RNA shed in oropharyngeal fluid during FMDV persistence were similar in vaccinated and nonvaccinated cattle. FMDV structural and nonstructural proteins were localized to follicle-associated epithelium of the dorsal soft palate and dorsal nasopharynx in persistently infected cattle. Host transcriptome analysis of tissue samples processed by laser capture microdissection indicated suppression of antiviral host factors (interferon regulatory factor 7, CXCL10 [gamma interferon-inducible protein 10], gamma interferon, and lambda interferon) in association with persistent FMDV. In contrast, during the transitional phase of infection, the level of expression of IFN-λ mRNA was higher in follicle-associated epithelium of animals that had cleared the infection. This work provides novel insights into the intricate mechanisms of FMDV persistence and contributes to further understanding of this critical aspect of FMDV pathogenesis.IMPORTANCE The existence of a prolonged, asymptomatic carrier state is a political impediment for control and potential eradication of foot-and-mouth disease (FMD). When FMD outbreaks occur, they are often extinguished by massive depopulation of livestock due to the fear that some animals may have undiagnosed subclinical infection, despite uncertainty over the biological relevance of FMD virus (FMDV) persistence. The work described here elucidates aspects of the FMDV carrier state in cattle which may facilitate identification and/or abrogation of asymptomatic FMDV infection. The divergence between animals that clear infection and those that develop persistent infection was demonstrated to occur earlier than previously established. The host antiviral response in tissues maintaining persistent FMDV was downregulated, whereas upregulation of IFN-λ mRNA was found in the epithelium of cattle that had recently cleared the infection. This suggests that the clearing of FMDV infection is associated with an enhanced mucosal antiviral response, whereas FMDV persistence...

To characterize the early events of foot-and-mouth disease virus (FMDV) infection in cattle subsequent to simulated natural exposure, 16 steers were aerosol inoculated with FMDV and euthanized at various times. Samples were collected from each steer antemortem (serum, nasal swabs, and oral swabs) and postmortem (up to 40 tissues per animal) and screened for FMDV by virus isolation and for FMDV RNA by real-time reverse transcription polymerase chain reaction. Tissues that tested positive for FMDV or viral RNA were examined by immunohistochemistry and multichannel immunofluorescence microscopy. In previremic steers, FMDV was most consistently localized to nasopharyngeal tissues, thereby indicating this region as the most important site of primary viral replication. The earliest site of microscopic localization of FMDV antigens was the lymphoid follicle-associated epithelium of the pharyngeal mucosa-associated lymphoid tissue of the nasopharynx at 6 hours postaerosolization. At early time points after aerosol inoculation, viral antigens colocalized with cytokeratin-positive pharyngeal epithelial cells; intraepithelial FMDV-negative, MHCII/CD11c-double-positive dendritic cells were present in close proximity to FMDV-positive cells. Onset of viremia coincided with marked increase of viral loads in pulmonary tissues and with substantial decrease of viral detection in nasopharyngeal tissues. These data indicate that subsequent to aerogenous exposure to FMDV, the temporally defined critical pathogenesis events involve (1) primary replication in epithelial cells of the pharyngeal mucosa-associated lymphoid tissue crypts and (2) subsequent widespread replication in pneumocytes in the lungs, which coincides with (3) the establishment of sustained viremia.

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Foot-and-mouth disease (FMD) is a worldwide problem limiting the trade of animals and their products from affected countries. The rapid isolation, serotyping, and vaccine matching of FMD virus from disease outbreaks is critical for enabling the implementation of effective vaccination programs and to stop the spread of infection during outbreaks. Some primary cells have been shown to be highly susceptible to most strains of FMD virus (FMDV) but are difficult and expensive to prepare and maintain. Since the ␣ V ␤ 6 integrin is a principal receptor for FMDV, we transduced a bovine kidney cell line to stably express both the ␣ V and ␤ 6 bovine integrin subunits. This stable cell line (LFBK-␣ V ␤ 6 ) showed ␤ 6 expression and enhanced susceptibility to FMDV infection for >100 cell passages. LFBK-␣ V ␤ 6 cells were highly sensitive for detecting all serotypes of FMDV from experimentally infected animals, including the porcinophilic FMDV strain O/TAW/97. In comparison to other cell types that are currently used for virus isolation, LFBK-␣ V ␤ 6 cells were more effective at detecting FMDV in clinical samples, supporting their use as a more sensitive tool for virus isolation.F oot-and-mouth disease virus (FMDV) is a severe economic concern for meat-producing nations since the trade of animal products is limited in the countries where the virus is present. The rapid spread of the virus among susceptible animals results in severe morbidity and, in some cases, death, especially in young animals (reviewed in reference 1). Infection or vaccination with one of the seven different serotypes does not confer cross-protection to other serotypes or even to some subtypes of the same serotype. Vaccines for FMDV are widely used to prevent clinical disease, but since vaccines are serotype and subtype specific, the virus(es) causing outbreaks must be isolated and serologically characterized for vaccine matching prior to selecting the appropriate vaccine antigen to be used in vaccine formulations (reviewed in reference 2).Although molecular techniques, such as PCR coupled with genomic sequencing, can be used in samples containing enough virus to rapidly identify the virus serotype and its relationship to other FMDV strains, appropriate vaccine prediction requires virus growth in cell culture to carry out neutralization tests using reference sera. Inefficient recovery of virus from animal samples can delay diagnosis and vaccine selection, hampering the rapid implementation of control measures; thus, virus isolation protocols are designed for maximum sensitivity. Some primary cells, such as bovine thyroid (BTY), are highly susceptible to a wide range of FMDV serotypes (3), but they are difficult and costly to prepare and lose FMDV susceptibility after multiple passages (4). Primary lamb kidney (LK) cells are also very sensitive to FMDV, and unlike BTY cells, LK cells maintain their sensitivity to FMDV infection after cryopreservation (5). Immortalized cell lines (e.g., baby hamster kidney fibroblasts and porcine kidney epithelial cells), ...