Wednesday, December 14, 2016

Current Hand, Foot & Mouth Disease Outbreaks. How Can We Study It?

Recently, news headlines have stirred panic among the American public depicting a “highly contagious viral infection” as a threat to not only children, but now adults as well. Localized infections of Hand, Foot and Mouth Disease (HFMD) arose on university campuses in Illinois and Florida this past summer and the virus has been sporadically in the news since then. Symptoms of the common infection caused by Coxsackievirus A16 include fever, sore throat, painful sores around and inside the mouth that could become ulcers and a skin rash with red spots that could develop into blisters on the surface of hands and feet, knees, elbows, buttocks or genitalia.[1],[2],[10] Although the disease is usually benign, Coxsackievirus A16, though more commonly Enterovirus 71, pathogenesis has potential to induce fatalities. Coxsackievirus A16 caused the death of a 15-month-old boy in 2005 and a 2000 Enterovirus A71 outbreak in Singapore resulted in several deaths.[3], [4] Enterovirus A71 virus targets epithelial cells and the walls of the gastrointestinal tract, but secondary infections occasionally attack the nervous system leading to more severe consequences.[5] Persistent infections, although rare, can cause a variety of complications, including aspetic meningitis, brain-stem encephalitis, poliomyelitis-like paralysis, pleurodynia, myocarditis, and death.[5] 

For a fairly common disease among children, little is known about how to prevent Enterovirus A71 infection. In a paper released earlier this year, Win Kyaw Phyu and colleagues progressed a small animal model to study the disease, highlighting a potential vaccine development against the more virulent HFMD.[6] The non-enveloped, single-stranded, (+)-RNA virus is transmitted through fecal-oral and oral-oral routes, most commonly in pediatric populations, though current outbreaks are affect young adult and adult populations. Enterovirus A71 probably reaches neurons through viremia, establishing an infection that can cause encephalomyelitis.[7] Previous studies have used a mouse model to describe retrograde axonal viral transport from peripheral nerves to infect neurons in the central nervous system (CNS), but these models were based on direct inoculum injected into the spinal cord or muscle tissue instead of the natural route of transmission.[8] Here, Phyu and colleagues further establish a hamster model as suitable for Enterovirus A71 infection through oral infection and highlights potential protective qualities of a mouse-adapted virus.[6][9]

The experiment was performed on 2-week-old Syrian golden hamsters, which demonstrated consistent infection by oral-route, with an Enterovirus A71 mouse-adapted virus (EV-A71 MAV) at a 50% lethal dose (LD50) calculated using the method of Reed & Muench (25 CCID50).[6] 6 groups of 6 hamsters were orally infected with PBS containing 105, 104, 103, 102, 10, and 1 CCID50 of inoculum, examining every 3 hours for 14 days for signs of infections including weight loss, humped posture, ruffled fur and hind limb paralysis.[6] 17 animals were found with limb paralysis and were euthanized. 7 had died overnight and were excluded in analysis of tissue samples. After 14 days, the skin was separated from the carcasses and cross-sections of the oral-cavity, brain, spinal cord, internal organs, limb muscles and paws were taken for analysis. A second experiment infected 8, 2-week-old hamsters with 104 CCID50 for correlation with pathological findings by analyzing serum and solid organs in addition to skin and cross-sections.[6]Tissue sections were stained with hematoxylin and eosin for light microscopy and then additionally prepared for immunohistochemistry (IHC) and in situ hybridization (ISH).

Results from the LD50 experiment determined survival of the hamsters was dose-dependent with complete fatality of all animals infected with 102 CCID50 or higher between 3 to 8 days post infection, displaying clear signs of the disease (Figure 1).[6] These animals also showed macroscopic skin ulcers/leisons around the nose, lip and paw. 16.7% of animals died in the low 10 CCID50 group with all surviving animals not displaying any signs of illness and only one animal in the 1 CCID50 group developed limb paralysis on the 10th day after infection, recovering 4 days afterwards.[6] In all animals, IHC and ISH displayed strong positive signals indicating small foci of viral antigens/RNA in the epithelium covering parts of the oral cavity, paws, limbs, head, neck, chest, and even the hair follicle germline. Most importantly, the oral infection stimulated viral replication in neuronal bodies in the brainstem (62% of animals), spinal cord anterior horn (90% of animals) and in sensory ganglia (90% of animals) as depicted by the presence of viral antigens and RNA via IHC and ISH positive signals (Figure 2).[6]  
Phyu and colleagues further expanded on previous knowledge of EV-A71 by inducing an epithelial and neuronal infection in hamsters via oral routes.  Identifying viral particles in oral cavities and skin lesions confirms the transmission of the virus and how person-to-person contact is likely to generate outbreaks, like the one currently being observed in the US. The study also determined the absence of viral particles in the gastrointestinal tract signifying that viral replication in these areas are insufficient to cause transmission of the virus and consistent with human oral tests for the virus are more likely to be positive than rectal swabs.[6] Phyu and colleagues also suggest that EV-A71 presence in that retrograde axonal transport of the virus to CNS neurons through viremia is important in pathogenesis, consistent with previous studies.[6],[8] This hamster model was also found to be more susceptible to infection of EV-A71 after oral infection of MAV.[6]Other mice models have not established a consistent infection of EV-A71, but inoculum with EV-A71 and MAV establishes a persistent infection of HFMD for study.[6]Researchers involved hypothesize that the susceptibility stems from immune system immaturity and availability of viral receptors.[6]  

The importance of this model revolves around the benign nature of HFMD. HFMD doesn’t cause severe complications unless the infection becomes persistent. Establishment of a consistent disease in a small animal model allows researchers to analyze viral pathogenesis, assess why the virus has preference for neurons upon secondary infection, and how the virus is able to do this. Insight into viral mechanisms generates new ways to attack the virus with anti viral drugs and even a vaccine. Although this study outlined and confirmed previous knowledge, I do not believe they did enough original work. Perhaps they should have further isolated the virus in neuronal tissues, such as the spinal cord and brain stem, to isolate viral proteins and attempted a subunit vaccine. The researchers successfully established that the virus was dose dependent and produced a persistent infection in the presence of MAV, but this model had already been done before. If they had attempted to inoculate some hamsters at birth with some of EV-A71’s proteins in a third experiment, they would have not only confirmed that the hamster model using MAV was an efficient way to study the virus, but also an effective way to derive and test vaccines. As someone who works in a child-care facility, I know all too well that once one toddler has HFMD, the entire center will start breaking out in tiny blisters. It is a painful disease for pediatric victims and can have severe consequences if it is able to infect the nervous system. Further studies should be conducted to work against this virus and develop a vaccine, especially since it is highly common and can be virulent.

[3] Chan et. al. (2003). Epidemic Hand, Foot and Mouth Disease by Human enterovirus 71, Singapore. Emerging infectious Disease. 9. 1.
[4] Wang, Chung-Yi M.D.; Li Lu, Frank M.D.; Wu, Mei-Hwan M.D., Ph.D.; Lee, Chin-Yun M.D., Ph.D.; Huang, Li-Min M.D., Ph.D. (2004) Fatal Coxsackievirus A16 Infection. Pediatric Infectious Disease Journal. 23. 3. 275-276.
[5] Palacios, G & Oberste, MS. (2005) Enteroviruses as agents of emerging infectious diseases. Journal of Neurovirology. 11. 424-433.
[6] Phyu, Ong & Wong. (2016). 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. PLoS ONE 11(1).
[7] Liu LD, Zhao H, Zhang Y, Wang J, Che Y, Dong C, et al. (2011) Neonatal rhesus monkey is a potential animal model for studying pathogenesis of EV71 infection. Virology; 412:91–100.
[8] Ong KC, Wong KT. (2015) Understanding Enterovirus 71 neuropathogenesis and its impact on other neurotropic enteroviruses. Brain Pathology; 25:614–624.
[9] Ch'ng WC, Stanbridge EJ, Wong KT, Ong KC, Yusoff K, Shafee N. Immunization with recombinant enterovirus 71 viral capsid protein 1 fragment stimulated antibody responses in hamsters. Virol J 2012; 9:155
[10] Pictures of HFMD and further information from Medicine Net: http://www.medicinenet.com/hand-foot-and-mouth_syndrome/article.htm

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