A Consistent Orally-Infected Hamster Model for Enterovirus A71 Encephalomyelitis Demonstrates Squamous Lesions in the Paws, Skin and Oral Cavity Reminiscent of Hand-Foot-and-Mouth Disease

Abstract

Enterovirus A71 (EV-A71) causes self-limiting, hand-foot-and-mouth disease (HFMD) that may rarely be complicated by encephalomyelitis. Person-to-person transmission is usually by fecal-oral or oral-oral routes. To study viral replication sites in the oral cavity and other tissues, and to gain further insights into virus shedding and neuropathogenesis, we developed a consistent, orally-infected, 2-week-old hamster model of HFMD and EV-A71 encephalomyelitis. Tissues from orally-infected, 2-week-old hamsters were studied by light microscopy, immunohistochemistry and in situ hybridization to detect viral antigens and RNA, respectively, and by virus titration. Hamsters developed the disease and died after 4–8 days post infection; LD50 was 25 CCID50. Macroscopic cutaneous lesions around the oral cavity and paws were observed. Squamous epithelium in the lip, oral cavity, paw, skin, and esophagus, showed multiple small inflammatory foci around squamous cells that demonstrated viral antigens/RNA. Neurons (brainstem, spinal cord, sensory ganglia), acinar cells (salivary gland, lacrimal gland), lymphoid cells (lymph node, spleen), and muscle fibres (skeletal, cardiac and smooth muscles), liver and gastric epithelium also showed varying amounts of viral antigens/RNA. Intestinal epithelium, Peyer’s patches, thymus, pancreas, lung and kidney were negative. Virus was isolated from oral washes, feces, brain, spinal cord, skeletal muscle, serum, and other tissues. Our animal model should be useful to study squamous epitheliotropism, neuropathogenesis, oral/fecal shedding in EV-A71 infection, person-to-person transmission, and to test anti-viral drugs and vaccines.

Squamous epitheliotropism of Enterovirus A71 in human epidermis and oral mucosa

“…In EV-A71-infected hamsters, multiple, focal inflammatory skin lesions were found on footpads/paws, lips and other parts of the skin following oral infection11. Viral antigens/RNA were also localized within the cytoplasm of epidermal squamous cells found in these areas, as well as squamous cells of the oral and oesophageal mucosa.…”

Modelling person-to-person transmission in an Enterovirus A71 orally infected hamster model of hand-foot-and-mouth disease and encephalomyelitis

“…A mouse-adapted EV-A71 strain (MAVS), which has previously been shown to consistently orally infect 2-week-old hamsters, was grown and titrated in Vero cells as previously described. 27 …”

“…To determine the amount of live virus excretion, oral washes and feces were collected daily from four groups of index and contact animals, as described previously. 27 Samples were immediately frozen and stored at −80 °C for subsequent virus isolation. In addition, oral wash and fecal samples from four index animals at 4 dpi and from contact animals at eight days post-exposure were titrated for viral titers.…”

“…Approximately 20 standard tissue cross-section, paraffin-embedded blocks were prepared as previously described; tissues sampled included tissues from the oral cavity, brain, spinal cord, internal organs, hind limb muscle, paws and skin. 27 From the 5th group of animals, instead of fixing tissues, we colleted fresh organs/tissues including samples from the brain, spinal cord, stomach and hind limb muscle and immediately froze the tissues at −80 °C for subsequent virus titration along with the collected sera.…”

“…This model demonstrates oral shedding and fecal excretion of virus and squamous cell infection in the paws, skin and oral cavity. 27 In this study, we investigated the potential of this model to model person-to-person transmission of EV-A71 via oral and fecal viral shedding and to help identify the factors that might influence transmission.…”