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Identification of FMDV infection sites and application of Laser Micro-Dissection together with real-time PCR

Background: Although the pathogenesis of foot-and-mouth disease (FMD) has been extensively investigated previously, relatively little data are available about the localisation of FMD virus (FMDV) in vivo. Accordingly, the aim was to increase our understanding of the localisation of FMDV and the early events in infection. To this end, the cells in the ruminant upper respiratory tract infected with FMDV were identified, during acute and persistent infection. Laser Microdissection (LMD) linked with real-time PCR (LMD/qRT-PCR) were employed to estimate levels of FMDV genomes at specific sites during infection.

Results: During the acute phase of FMD, infection was detected in the epithelium of tongue, foot and pharyngeal tissues. FMDV detection was very strong in the germinativum layer and concentrated in the cytoplasm of the cell. In the spinosum layer, the FMDV detection is more diffuse and observed throughout this layer of the lesion. No positive signal was found in the corneum layer. Mucus gland and pharyngeal lymphoid nodules nearby the epithelium were also positive for FMDV.

During the persistent stage, except in the epithelial cells of the soft palate and nasopharynx, FMDV was also found in the germinativum layer of the epithelium of the dorsal side soft palate and of the nasopharynx. The virus was also present in the lymph nodules and mucus glands of the dorsal soft palate and nasopharynx.

The LMD/qRT-PCR assay generated quantitative data on viral RNA levels in different cellular layers of epithelium during the infection, and allowed identification of the early localisation sites of the virus during the establishment of infection.. The highest levels of FMDV were detected within the stratum spinosum during the acute stage of disease. Within the foot epithelia, the first site of infection was the stratum basale, which is on the basement membrane; within the soft palate the primary cell type of infection is the spinosum cells of the dorsal surface.

These data provide the essential basis for the understanding of development of FMD pathogenesis and carrier status, and provide for future detailed analyses oof infected and carrier animals.

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Reported by

Pirbright Laboratory, Ash Road
GU24 ONF Pirbright
United Kingdom
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