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.
Hand-foot-and-mouth disease is a self-limiting paediatric infectious disease commonly caused by Enterovirus A71 (Genus: Enterovirus, Family: Picornaviridae). Typical lesions in and around the hands, feet, oral cavity and other places may rarely be complicated by acute flaccid paralysis and acute encephalomyelitis. Although virus is readily cultured from skin vesicles and oral secretions, the cellular target/s of Enterovirus A71 in human skin and oral mucosa are unknown. In Enterovirus A71-infected human skin and oral mucosa organotypic cultures derived from the prepuce and lip biopsies, focal viral antigens and viral RNA were localized to cytoplasm of epidermal and mucosal squamous cells as early as 2 days post-infection. Viral antigens/RNA were associated with cytoplasmic vacuolation and cellular necrosis. Infected primary prepuce epidermal keratinocyte cultures showed cytopathic effects with concomitant detection of viral antigens from 2 days post-infection. Supernatant and/or tissue homogenates from prepuce skin organotypic cultures and primary prepuce keratinocyte cultures showed viral titres consistent with active viral replication. Our data strongly support Enterovirus A71 squamous epitheliotropism in the human epidermis and oral mucosa, and suggest that these organs are important primary and/or secondary viral replication sites that contribute significantly to oral and cutaneous viral shedding resulting in person-to-person transmission, and viraemia, which could lead to neuroinvasion.
Coxsackievirus A16 (CV-A16) is one of the major causes of mild and self-limiting hand-foot-and-mouth disease (HFMD) in young children, which may occasionally leads to serious neurological complications. In this study, we had developed a novel, consistent, orally infected CV-A16 HFMD hamster model with encephalomyelitis. Four groups of 7-day-old hamsters in a kinetic study were orally infected with mouse-adapted CV-A16 strains and sacrificed at 1-4 days post infection (dpi), respectively. Tissues were studied by light microscopy, immunohistochemistry to detect viral antigens, in situ hybridization to detect viral RNA, and by viral titration. In a separate transmission experiment, orally infected index hamsters were housed together with contact hamsters to investigate oral and fecal viral shedding by virus culture and reverse transcription polymerase chain reaction (RT-PCR). At severe infection/death endpoints, index and contact hamster infection were also histopathologically analyzed. In the kinetic study, infected hamsters developed signs of infection at 4 dpi. Viral antigens/RNA were localized to brainstem (medulla/pons; reticular formation and motor trigeminal nucleus) and spinal cord anterior horn neurons, oral squamous epithelia and epidermis from 3 to 4 dpi. Salivary and lacrimal glands, myocardium, brown adipose tissue, intestinal smooth muscle, and skeletal muscle infection was also demonstrated. Viremia at 1 dpi and increasing viral titers in various tissues were observed from 2 dpi. In the transmission study, all contact hamsters developed disease 3-5 days later than index hamsters, but demonstrated similar histopathological findings at endpoint. Viral culture and RT-PCR positive oral washes and feces confirmed viral shedding. Our hamster model, orally infected by the natural route for human infection, confirmed CV-A16 neurotropism and demonstrated squamous epitheliotropism reminiscent of HFMD, attributes not found in other animal models. It should be useful to investigate neuropathogenesis, model person-to-person transmission, and for testing antiviral drugs and vaccines.
Archival oral tissues comprising 51 squamous cell carcinomas, 18 non-malignant lesions and 7 normal mucosa samples were investigated for human herpesvirus-6 (HHV-6)-encoded antigens and HHV-6 DNA. The virus-specific antigens were detected by an immunohistochemical method using monoclonal antibodies. Two further techniques used for HHV-6 DNA detection included the polymerase chain reaction (PCR) with virus-specific primers and in situ hybridization using digoxigenin-labelled oligonucleotides specific for HHV-6A and HHV-6B genotypes. A high proportion (79-80%) of the squamous cell carcinomas were positive for HHV-6 with the various detection methods. In cases of lichen planus and leukoplakia a high prevalence rate (67-100%) was noted with in situ hybridization and immunohistochemical techniques but a lower proportion (22-33%) was detected with the PCR method. All 7 normal tissues tested were negative for HHV-6. The HHV-6 variant B was found in 60% of the oral carcinoma tissues analysed. The study demonstrates the frequent presence of HHV-6 in neoplastic and non-malignant lesions of the oral cavity. While the role of HHV-6 in oral mucosal tissues remains to be determined, the in vitro tumorigenic potential of the virus suggests a possible role in the etiopathogenesis of oral lesions.
Enterovirus A71 (EV-A71) causes hand-foot-and-mouth disease (HFMD), which may be complicated by fatal encephalomyelitis. Although fecal-oral or oral-oral routes are important in person-to-person transmission, how viral shedding and exposure may predispose individuals to infection remains unknown. We investigated person-to-person transmission by using a model of HFMD and encephalomyelitis based on EV-A71 oral infection of 2-week-old hamsters. Animals (index animals) infected with 104 50% cell culture infective doses of virus uniformly developed severe disease four days post-infection (dpi), whereas littermate contacts developed severe disease after six to seven days of exposure to index animals. Virus was detected in oral washes and feces at 3-4 dpi in index animals and at three to eight days after exposure to index animals in littermate contact animals. In a second experiment, non-littermate contact animals exposed for 8 or 12 h to index animals developed the disease six and four days post-exposure, respectively. Tissues from killed index and contact animals, studied by light microscopy, immunohistochemistry and in situ hybridization, exhibited mild inflammatory lesions and/or viral antigens/RNA in the squamous epithelia of the oral cavity, tongue, paws, skin, esophagus, gastric epithelium, salivary glands, lacrimal glands, central nervous system neurons, muscles (skeletal, cardiac and smooth muscles) and liver. Orally shed viruses were probably derived from infected oral mucosa and salivary glands, whereas fecal viruses may have derived from these sites as well as from esophageal and gastric epithelia. Asymptomatic seroconversion in exposed mother hamsters was demonstrated. Our hamster model should be useful in studying person-to-person EV-A71 transmission and how drugs and vaccines may interrupt transmission.