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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