Immunocompetent and Immunodeficient Mouse Models for Enterovirus 71 Pathogenesis and Therapy

Abstract

Enterovirus 71 (EV71) is a global health threat. Children infected with EV71 could develop hand-foot-and-mouth disease (HFMD), encephalitis, paralysis, pulmonary edema, and death. At present, no effective treatment for EV71 is available. We reviewed here various mouse models for EV71 pathogenesis and therapy. Earlier studies relied on the use of mouse-adapted EV71 strains. To avoid artificial mutations arising de novo during the serial passages, recent studies used EV71 clinical isolates without adaptation. Several human receptors for EV71 were shown to facilitate viral entry in cell culture. However, in vivo infection with human SCARB2 receptor transgenic mice appeared to be more limited to certain strains and genotypes of EV71. Efficacy of oral infection in these transgenic models is extremely low. Intriguingly, despite the lack of human receptors, immunodeficient neonatal mouse models can still be infected with EV71 clinical isolates via oral or intraperitoneal routes. Crossbreeding between SCARB2 transgenic and stat1 knockout mice generated a more sensitive and user-friendly hybrid mouse model. Infected hybrid mice developed a higher incidence and earlier onset of CNS disease and death. Different pathogenesis profiles were observed in models deficient in various arms of innate or humoral immunity. These models are being actively used for antiviral research.

Full text

viruses
Review
Immunocompetent and Immunodeficient Mouse
Models for Enterovirus 71 Pathogenesis and Therapy
Chiaho Shih 1, *, Chun-Che Liao 1, Ya-Shu Chang 1, Szu-Yao Wu 1, Chih-Shin Chang 1,2 and
An-Ting Liou 1
1
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan; jfliao@ibms.sinica.edu.tw (C.-C.L.);
daph7516@gmail.com (Y.-S.C.); guguma126@yahoo.com.tw (S.-Y.W.); sony721108@hotmail.com (C.-S.C.);
sh3333@msn.com (A.-T.L.)
2Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 11221, Taiwan
*Correspondence: cshih@ibms.sinica.edu.tw; Tel.: +886-2-2652-3996; Fax: +886-2-2652-3597
Received: 1 November 2018; Accepted: 26 November 2018; Published: 28 November 2018


Abstract:
Enterovirus 71 (EV71) is a global health threat. Children infected with EV71 could
develop hand-foot-and-mouth disease (HFMD), encephalitis, paralysis, pulmonary edema, and death.
At present, no effective treatment for EV71 is available. We reviewed here various mouse models
for EV71 pathogenesis and therapy. Earlier studies relied on the use of mouse-adapted EV71 strains.
To avoid artificial mutations arising de novo during the serial passages, recent studies used EV71
clinical isolates without adaptation. Several human receptors for EV71 were shown to facilitate
viral entry in cell culture. However,
in vivo
infection with human SCARB2 receptor transgenic mice
appeared to be more limited to certain strains and genotypes of EV71. Efficacy of oral infection
in these transgenic models is extremely low. Intriguingly, despite the lack of human receptors,
immunodeficient neonatal mouse models can still be infected with EV71 clinical isolates via oral or
intraperitoneal routes. Crossbreeding between SCARB2 transgenic and stat1 knockout mice generated
a more sensitive and user-friendly hybrid mouse model. Infected hybrid mice developed a higher
incidence and earlier onset of CNS disease and death. Different pathogenesis profiles were observed
in models deficient in various arms of innate or humoral immunity. These models are being actively
used for antiviral research.
Keywords: Enterovirus 71; EV71; EV-A71; animal models; pathogenesis; therapy
1. Introduction
Enterovirus 71 (EV71 or EV-A71) was first isolated from the stool of encephalitis patients in
California in 1969 [
1
]. It is a positive-strand RNA virus, belonging to the Picornaviridae family [
2
,
3
].
The genomic structure of EV71 is as outlined in Figure 1. Similar to poliovirus and other plus-strand
RNA viruses, it contains an IRES element, which drives the translation of a polyprotein from a 7.4 kb
genomic mRNA template. Upon protease cleavage, this polyprotein precursor can then convert into
various smaller-size structural and non-structural proteins, leading to capsid assembly. As the focus in
this review is about pathogenesis and animal models, readers interested in the EV71 replication cycle
are advised to consult other reviews in literature.
Viruses 2018,10, 674; doi:10.3390/v10120674 www.mdpi.com/journal/viruses

Viruses 2018,10, 674 2 of 20
Viruses 2018, 10, x FOR PEER REVIEW 2 of 18
Figure 1. The genomic organization of EV71. The plus-strand RNA of EV71 genome encodes a single
polyprotein precursor. Upon proteolytic processing by virus-encoded proteases 2A and 3C, this
polyprotein precursor converts into several smaller mature products, including 4 structural proteins
(VP1-VP4 capsid protein) and 7 non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). 3B encodes
a VPg protein, which is covalently bound to the 5′ end of the RNA genome. 3D encodes RNA-
dependent RNA polymerase. IRES: internal ribosome entry site. This cartoon illustration is based on
gt B5 clinical isolates in Taiwan [4,5]. However, it is representative of the general genomic structure
of all genotypes isolated elsewhere.
Adults infected with EV71 could develop subclinical or cold-like syndrome. However, children
younger than five years of age could develop hand-foot-and-mouth disease (HFMD) and herpangina.
In severe cases, EV71-infected children could develop aseptic meningitis, encephalitis or myocarditis
[6]. In a long-term follow-up study, EV71-infected patients with CNS involvement and/or
cardiopulmonary failure was associated with delayed neurodevelopment and reduced cognitive
functioning [7]. Unfortunately, some patients were still not saved due to pulmonary edema and/or
heart failure [8].
Although EV71 was first discovered in the US decades ago, recent major outbreaks occurred
frequently in the Asia-Pacific region, including Taiwan, Singapore, China, Malaysia, and Japan [6,9–
12]. China alone reported 1894 deaths between 2008 and 2011 [12]. Sporadic cases were also reported
from Europe [13,14], Russia [15] and Australia [16], indicating that EV71 is a global health threat. Like
poliovirus in the last century, EV71 is emerging rapidly as another pathogenic picornavirus for
children in this century.
Intravenous immunoglobulin (IVIG) is a therapeutic strategy for EV71-associcated brainstem
encephalitis [17]. However, an antibody-dependent enhancement (ADE) phenomenon was observed
in EV71-infected patients [18–21], suggesting that IVIG may not be the most adequate treatment in
clinical practice. Milrinone, a phosphodiesterase inhibitor, was used to treat patients with EV-71
induced pulmonary edema (PE). While Milrinone is not an antiviral per se, mortality was
significantly decreased in the Milrinone-treated group, suggesting that it could be a useful
therapeutic drug for EV71-associated PE [22].
To investigate the pathogenesis and therapy for EV71 infection, we will need both in vitro and
in vivo models of EV71 infection. In this review, we cataloged here from the literature a total of 16
cell lines (Table 1) and 22 animal models (Table 2), which had been tested for EV71 infection and
pathogenesis. We compare below their respective advantages, limitations, and caveats in using these
models.
EV71 RNA ≈ 7.4 kb
Figure 1.
The genomic organization of EV71. The plus-strand RNA of EV71 genome encodes a
single polyprotein precursor. Upon proteolytic processing by virus-encoded proteases 2A and 3C,
this polyprotein precursor converts into several smaller mature products, including 4 structural proteins
(VP1-VP4 capsid protein) and 7 non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). 3B encodes a
VPg protein, which is covalently bound to the 5
0
end of the RNA genome. 3D encodes RNA-dependent
RNA polymerase. IRES: internal ribosome entry site. This cartoon illustration is based on gt B5 clinical
isolates in Taiwan [
4
,
5
]. However, it is representative of the general genomic structure of all genotypes
isolated elsewhere.
Adults infected with EV71 could develop subclinical or cold-like syndrome. However, children
younger than five years of age could develop hand-foot-and-mouth disease (HFMD) and herpangina.
In severe cases, EV71-infected children could develop aseptic meningitis,
encephalitis or myocarditis [6]
.
In a long-term follow-up study, EV71-infected patients with CNS involvement and/or cardiopulmonary
failure was associated with delayed neurodevelopment and reduced cognitive functioning [
7
].
Unfortunately, some patients were still not saved due to pulmonary edema and/or heart failure [8].
Although EV71 was first discovered in the US decades ago, recent major outbreaks occurred
frequently in the Asia-Pacific region, including Taiwan, Singapore, China, Malaysia, and Japan [
6
,
9
–
12
].
China alone reported 1894 deaths between 2008 and 2011 [
12
]. Sporadic cases were also reported
from Europe [
13
,
14
], Russia [
15
] and Australia [
16
], indicating that EV71 is a global health threat.
Like poliovirus in the last century, EV71 is emerging rapidly as another pathogenic picornavirus for
children in this century.
Intravenous immunoglobulin (IVIG) is a therapeutic strategy for EV71-associcated brainstem
encephalitis [17]. However, an antibody-dependent enhancement (ADE) phenomenon was observed
in EV71-infected patients [
18
–
21
], suggesting that IVIG may not be the most adequate treatment in
clinical practice. Milrinone, a phosphodiesterase inhibitor, was used to treat patients with EV-71
induced pulmonary edema (PE). While Milrinone is not an antiviral per se, mortality was significantly
decreased in the Milrinone-treated group, suggesting that it could be a useful therapeutic drug for
EV71-associated PE [22].
To investigate the pathogenesis and therapy for EV71 infection, we will need both
in vitro
and
in vivo
models of EV71 infection. In this review, we cataloged here from the literature a total of
16 cell lines (Table 1) and 22 animal models (Table 2), which had been tested for EV71 infection
and pathogenesis. We compare below their respective advantages, limitations, and caveats in using
these models.

Viruses 2018,10, 674 3 of 20
Table 1. Summary of 16 cell lines used for EV71 in vitro infection.
Cell Line Species Tissue Origin EV71 Genotype CPE #References
RD
Human
Rhabdomyosarcoma A, B, C, D +[23–30]
SK-N-SH Neuroblastoma C2, D +[24,27]
SH-SY5Y Neuroblastoma C4 +[31,32]
NSC-34 motor neuron cell line B2, B4 & C2 −[33]
SF268 glioblastoma * +[34]
Neuro-2A Mouse Neuroblastoma D +[24,35]
HT29
Human
intestinal epithelial cell B4, C +[25,26,28]
Caco-2 colorectal carcinoma B, C2 +[27,29]
C2BBel
mature intestinal epithelial cell (derived from Caco-2)
C+[28]
HeLa Human cervical carcinoma EV71:BS (Accession NO KF514878), C4 +[35,36]
HEK293T Human embryonic kidney EV71:BS (Accession NO KF514878), C4 +[35,37]
THP-1 Human monocytic cell line C2, C4 [19,37]
human PBMC C2 [19]
Vero Monkey African green monkey kidney A, EV71:BS (Accession NO KF514878) +[30,35]
COS-7 African green monkey kidney (SV40 transformed) EV71:BS (Accession NO KF514878) +[35]
NIH/3T3 Mouse mouse embryonic fibroblast mouse-adapted strain (EV:TLLm) +[35]
* information not available. #CPE, cytopathic effect. Genotype D is a C4 subgenotype.

Viruses 2018,10, 674 4 of 20
Table 2. Summary of 22 animal models used for EV71 in vivo infection.
Mouse Strain Host Age @Route Genotype Titer/Mouse Phenotype Virus-Replicating Tissue Reference
Balb/C
1-day-old i.p. B4 1×104PFU limb paralysis & death intestine [38]
2-day-old
i.p./i.c.
C4 103TCID50 * brain, liver and intestine [39]
5-day-old i.p. C4 (mouse adapted strain) 104TCID50 limb paralysis & death spleen, heart, liver, muscle and brain [40]
7-day-old
i.m.
i.p.
i.c.
B3 (CHO cell adapted strain;
CHO-26M) (mouse adapted strain;
MP-26M)
103TCID50 death
muscle (CHO-26M) blood, spleen,
heart, liver, muscle and brain
(MP-26M)
[41]
9-day-old i.p. C4 104.5 TCID50 limb paralysis
skeletal muscle, spinal cord, brainstem,
lung, and jejunum [24]
ICR
1-day-old oral C (mouse adapted strain) 106–107PFU paralysis & skin lesion skin, intestine and spinal cord [42]
1-day-old i.p. C4 (Fuyang-0805a) 2×105TCID50 none muscles, intestines, lungs, and brain [43]
7-day-old oral C (mouse adapted strain M2) 5×106PFU death * [44]
7-day-old i.p. C2 (mouse adapted strain MP4) 2.5 ×106PFU death brain, spinal cord, lung and muscle [21]
3-day-old i.p. C2 (clinical isolate) 4×106PFU death * [45]
7-day-old oral C2 (mouse adapted strain MP4) 5×105PFU death * [45]
12–14-day-old i.p. C (mouse adapted strain M2) 5×104–105PFU paralysis & death * [46]
14-day-old i.p. B3 (mouse adapted strain) 100 CCID50 paralysis brain and muscle [47]
14-day- &
28-day-old
i.p.
i.m.
oral
s.c.
B3 (mouse adapted strain) 105CCID50 paralysis mainly in CNS [48]
C57BL/6 9-day-old oral C (mouse adapted strain M2) 3×107PFU * * [44]
C57BL/6J 14-day-old i.p. C (mouse adapted strain M2) 3×105PFU hunched posture,
paralysis & death brain [49]
PSGL-1 Tg 10-day-old i.p. C4 (clinical isolate) 108TCID50 no disease onset brain, spinal cord and skeletal muscle [50]
C4 (mouse adapted strain MP10) 5×106TCID50 hindlimb paralysis *

Viruses 2018,10, 674 5 of 20
Table 2. Cont.
Mouse Strain Host Age @Route Genotype Titer/Mouse Phenotype Virus-Replicating Tissue Reference
hSCARB2 Tg 3-week-old
i.v. C (Isehara strain) 102–6 TCID50
ataxia, paralysis & death
CNS [51]
i.c. A, B and C 102–6 TCID50
i.p. C (Isehara strain) 103–6 TCID50
oral C (Isehara strain) 106–7 TCID50
pEF-1α-hSCARB2 1-day-old s.c. B 1×107PFU
HFMD-like syndrome &
paralysis CNS, intestine, muscle and skin [52]
7-day-old s.c. C 3×104PFU
AG129 younger than
2-week-old
i.p. B4 5×105–107PFU limb paralysis & death CNS [53]
oral 106–107PFU
AG129 10-week-old i.p. B2 (mouse adapted strain) 1.3 ×105
TCID50/mL limb paralysis & death ND [54]
A129
G129 7-day-old i.p. B5 (clinical isolates) 108PFU limb paralysis & death ND [4]
stat-1 KO 7-day-old i.p. B5 (clinical isolates) 108PFU limb paralysis & death CNS [4]
pSCARB2x
stat-1 KO
younger than
2-week-old i.p. C2 (clinical isolates) 106–108PFU [5]
NOD/SCID
3-day-old oral B5 (clinical isolates) 108PFU hair loss, skin lesion,
limb paralysis & death muscle and spleen [4]
7-day-old i.p. 107–108PFU
3–4 week-old i.v./i.c. B1 (mouse adapted strain) 106.5 CCID50 skeletal muscle and heart [55]
IP10 KO 14-day-old i.p. C (mouse adapted strain M2) 3×105PFU The death rate was
higher than C57BL/6J ND [49]
* information not available. @The age that mice infected with EV71.

Viruses 2018,10, 674 6 of 20
2. In Vitro Propagation Systems
The most common cell line for EV71 infection in culture is the human RD cell line of
rhabdomyosarcoma origin [
23
]. This RD cell line can produce a high yield of virus particles and thus
is often used for amplification of EV71 in animal experiments [
4
] and studies on viral structure [
56
].
In addition to RD cells, various cell lines used in poliovirus infection, such as HeLa and HEK293,
are also susceptible to EV71 infection and transfection [
36
,
37
]. These cell lines were used as a platform
to investigate the function of viral protein. From clinical samples, EV71 protein can be identified in
brainstem [
6
,
57
], and thus EV71 is considered as a neurotropic virus [
1
]. Several neuronal cell lines,
including SK-N-SH, NSC-34 and SF268, had been used in a number of studies, such as dissecting the
viral life cycle, host response to EV71 [33], phylogenetic analysis [24], and anti-viral drug testing [34].
In natural infection, EV71 is transmitted through an oral-fecal route [
58
]. Some previous studies
therefore focused on the GI tract immune responses to viral infection and replication kinetics in the
intestinal epithelial cells [25,26].
Using the Jurkat cell line and an expression cloning strategy by panning, Nishimura et al.
identified human P-selectin glycoprotein ligand-1 (PSGL-1; CD162) as a functional receptor on
leukocytes for EV71 viral entry [
59
]. However, PSGL-1 cannot be the only receptor for EV71,
since PSGL-1-independent infection in non-leukocytes was also observed. Yamayoshi et al. reported
that human scavenger receptor class B, member 2 (SCARB2, also known as lysosomal integral
membrane protein II or CD36b like-2) is another receptor for EV71 [
60
]. The exogenous expression of
human SCARB2 converted non-permissive cell lines to instead support EV71 propagation and develop
cytopathic effects. EV71 infection can be interfered by the antibody to SCARB2 or by soluble SCARB2.
Several alternative receptors for EV71 have also been reported [61–64].
In addition to Jurkat cells, THP-1 and human PBMC had been used to study the innate immunity
in EV71 infection [
37
]. Many other cell lines contributed to the studies on EV71 molecular virology,
virus-host interactions, or drug screening (Table 1). So far, the human rhabdomyosarcoma cell
line RD appeared to be the most commonly used gold standard in literature, in part due to
its high yield production of different EV71 genotypes [
65
]. As more and more laboratories are
using the same RD cell line (e.g., from ATCC), it would be easier to cross-compare results from
different laboratories. Since these cell line models are limited one way or the other in their resemblance
to human natural infection, an alternative approach is needed to validate the findings from the cell
culture system. In this regard, animal modeling can provide more complete and convincing evidence
for our understanding on EV71 in vivo infection and pathogenesis.
3. In Vivo Animal Models
Experimental results learned from the cell culture system need to be validated in animal models
and human clinical trials. Table 2summarizes different EV71 mouse models reported in literature
so far. Overall, one thing in common in these different models is their frequent use of limb paralysis
and mortality for scoring disease severity. This is in part due to the fact that paralysis and death
are more apparent, objective, and convenient phenotypes than other parameters, such as ataxia,
or pulmonary edema. Another common feature is the requirement of a neonatal host age for
productive
in vivo
infection, in particular with clinical isolates. Although the exact mechanism for this
young age requirement remains unclear, it is technically demanding and not user-friendly, when the
newborns are too small and fragile to handle experimentally. When tissue cytokine/chemokine
profiles were examined in these different EV71 mouse models, they were often found to share
some inflammatory cytokines in common, such as IL1
β
, IL6, IL10, IP10, MCP-1, IFN-
α
, and IFN-
γ
,
etc. [
4
,
52
,
53
]. For neuropathogenesis models, the distribution of viral antigens is also quite similar
among different models, which mimics human cases with fatal encephalomyelitis. For example,
inflammation and viral antigens in human cases were observed mainly in the spinal cord, brainstem,
hypothalamus, cerebellar dentate nucleus, and cerebrum [
66
]. In the stat-1 KO model, VP1 was strongly
expressed throughout the entire spinal cord, including the anterior horn and the gray matter [
4
].

Viruses 2018,10, 674 7 of 20
Furthermore, VP1 in the pons of the brainstem, but not in the cerebral cortex, supports the concept
of retrograde axonal transport from the peripheral nerves to the spinal cord ascending to the CNS.
This result is supported from the mouse models infected with mouse-adapted EV71 strains [
67
,
68
] and
poliovirus [69].
As summarized above, there is a high degree of similarity among EV71-infected human patients
and mouse models. However, there are also important differences among these mouse models,
which will need to be discussed individually.
3.1. Mouse Models Dependent on Mouse-Adapted EV71 Strains
In general, adult humans are resistant to EV71 infection and pathogenesis. Similarly, adult mice
cannot be easily infected with EV71 clinical isolates. To solve this problem, researchers developed
mouse-adapted strains of EV71, such as MP4, in an ICR mouse model [
21
,
27
,
44
–
46
]. MP4 was generated
through i.p. injection of the parental EV71 TW/4643/98 into one-day-old ICR mice. Adapted EV71
recovered from the brain tissue was re-used to repeat the serial injection-adaptation cycles.
EV71 recovered from the fourth passage was designated as MP4 [
27
]. Comparing to its parental
EV71 isolates, MP4 is more cytotoxic with a larger plaque size. Because EV71 is a RNA virus
with a quasi-species nature, mouse-adapted strains tend to accumulate many artificial mutations
from the serial passages. One legitimate concern is that these adaptive mutations cannot really be
found in human natural infection. In addition to MP4, mouse adapted strains were also generated
in the immunodeficient NOD/SCID mouse model [
55
]. Comparison of the amino acid sequences
between clinical isolates and mouse adapted strains revealed major differences in structural proteins,
such as VP1,
VP3, and protease 2A [
55
]. Therefore, it always remains uncertain whether results
obtained from using the adapted strain of EV71 can be automatically extended to human natural
infection with the clinical isolates.
3.2. Immunodeficient Mouse Models
Host immune system plays a critical role against viral infection [
70
]. Immunodeficient mice,
even without bearing any human receptors for viral entry, can support infection with EV71
clinical isolates, instead of relying on mouse-adapted strains. Innate immunity, such as IFNs, is required
for protection from EV71 infection and pathogenesis [
71
,
72
]. AG129 mice, which is deficient in
both
α
/
β
and
γ
interferon receptors, developed limb paralysis and death, when infected with a
non-mouse-adapted EV71 via i.p. or oral routes. Viral protein was detected in CNS in both i.p.
and oral infection [
53
]. Like mouse AG129, mouse A129 (deficient in
α
/
β
interferon receptor) can
also be infected with mouse-adapted strain of EV71 [
54
]. Moreover, mouse G129 deficient in
γ
interferon receptor developed limb paralysis via an i.p. route with clinical isolates of EV71 [
4
] (Table 2).
Another host factor stat-1 is a key transcription factor in IFN signaling. The stat-1 knockout (KO) mice
can be successfully infected with both genotype B and C clinical isolates of EV71 [4,5]. Infected stat-1
KO mice developed hindlimb paralysis with abundant viral protein in the CNS system, supporting an
important role of IFN signaling in protecting CNS from EV71 infection and pathogenesis.
In addition to innate immunity, humoral immunity also plays an important role in EV71 infection
and pathogenesis. For example, injection with an EV71-specific neutralizing antibody before or
after infection, can strongly reduce lethality and tissue viral loads of a mouse-adapted strain M2
in a B cell-deficient C57BL/6-derived mouse model [
44
]. In another example, NOD/SCID mice
deficient in T and B lymphocytes can be infected with a mouse-adapted strain of EV71 [
55
]. However,
since mouse-adapted EV71 can infect even immunocompetent mice [
27
], it was unclear if the missing
T and B lymphocytes in the NOD/SCID mouse model were really important for protection from
mouse-adapted EV71 [
55
]. This issue was addressed in one study when NOD/SCID mice were
inoculated with EV71 clinical isolates and developed limb paralysis via either an i.p. or oral route.
Although limb paralysis has been used as a common marker for scoring EV71 pathogenesis in
mouse modeling, acute flaccid paralysis does not occur as frequently as hand-foot-and-mouth

Viruses 2018,10, 674 8 of 20
disease (HFMD) in human natural infection. Interestingly, in the NOD/SCID model infected with
clinical isolates, an HFMD-like phenotype with skin rash was observed, for the first time in mouse
models [
4
]. Spleen atrophy was found in some EV-71 infected children [
57
,
73
], and was also noted
in this NOD/SCID model infected with clinical isolates [
4
]. Moreover, strong VP1 staining and
severe inflammation were apparent in the paralyzed muscle. On the other hand, no viral protein
and pathology was ever detected in the CNS in EV71-infected NOD/SCID mice. It is worth
mentioning here that in this NOD/SCID model, kinetic analysis in a time course experiment revealed
more delayed clearance of non-infectious viral RNA relative to the clearance of infectious viral
particles [
4
]. Furthermore, the inflammatory IL-23/IL-17 axis appeared to be activated in the infected
NOD/SCID model. It remains to be seen whether these intriguing phenomena are idiosyncratic to the
NOD/SCID model, or they can be generalized to other mouse models or human patients.
Interferon-gamma-inducible protein-10 (IP-10) is a highly expressed chemokine in
EV71-patients [
74
]. IP-10 knock-out mice infected with mouse-adapted EV71 exhibited a higher
mortality rate than the wild type control mice, suggesting a protective role of IP-10 in EV71 infection
and pathogenesis [49].
While the immunodeficient models are very useful for studying pathogenesis and evaluating
efficacies of drug and vaccine candidates, it is not always a good system for testing immune modulators
in therapeutic research. In addition, it is not as direct a platform for vaccine research either, since it can
only be used for comparing potencies of anti-EV71 neutralizing antibodies raised against different
vaccine candidates.
3.3. Transgenic Mouse Models
Recently, many cellular receptors have been proposed for EV71 viral entry
in vitro
, including
hSCARB2 (human scavenger receptor class B) [
60
], PSGL1 (P-selectin glycoprotein ligand 1 [
59
],
annexin II [
61
], and nucleolin [
62
]. These receptors facilitated EV71 infection in non-susceptible
cell lines, such as mouse fibroblast L929. However, transgenic (Tg) mice expressing human PSGL-1
failed to support infection with EV71 clinical isolates [
50
]. Among these reported cellular receptors,
only human SCARB2 (hSCARB2) has so far been shown to support EV71 infection in vivo.
There are two different hSCARB2 Tg mouse models in literature (Table 3) [
51
,
52
]. In the
EF-1a-hSCARB2 model, EF-1a promoter is used to drive hSCARB2 expression in the C57B/6 mouse
background [
52
]. In the SC2-hSCARB2 model, the native hSCARB2 promoter in a human SCARB2
BAC clone drives the transgene expression in the C57B/6 mouse background [
51
]. Both models
can be infected with EV71, develop limb paralysis, and detect viral protein in multiple tissues,
including muscle (muscle-tropism) and the CNS system (neurotropism). On the other hand, these two
models cannot be easily compared with each other due to a number of differences (Table 3): (1) virus
strains and genotypes (B4, B5, C2, C4 vs. Isehara); (2) host age at virus inoculation (1- or 7-day-old
vs. 3-wk-old); (3) virus dose (PFU vs. TCID
50
); (4) inoculation route (s.c. vs. i.c., i.v., i.p.); (5) assays
for readout (individual score of paralysis & death vs. combined score of ataxia, paralysis, and death).
In addition to the Isehara strain (gt C2), other genotypes of EV71 were tested in the SC2-hSCARB2
model [
51
]. However, since the non-Tg wild type mouse control was included only in the study using
the Isehara strain, we listed here in Table 3only the results based on the Isehara strain.

Viruses 2018,10, 674 9 of 20
Table 3. Comparisons between human SCARB2-transgenic and wild type non-transgenic mice in EV71 infection and pathogenesis.
Mouse Strains Promoter Virus Strain
(Genotype) Host Age Virus Dose Inoculation
Route
% Earlier symptom **
(Tg vs. non-Tg)
% Limb Paralysis
(Tg vs. non-Tg)
% Death
(Tg vs. non-Tg) References
pEF-1α- hSCARB2
ubiquitous
EF-1
Promoter
E59 (B4) 1-day-old 3×104PFU s.c. 100 vs. 57.1 18.8 vs. 14.2 0 vs. 0
[52]
N-2838 (B5) 100 vs.75 44.4 vs. 12.5 0 vs. 0
5476 (C2) 7-day-old 107PFU s.c. Nd @100 vs. 57.1 100 vs. 0
N3340 (C4) Nd @100 vs. 100 100 vs. 0
pSC2- hSCARB2 native
promoter Isehara (C2) 3-wk-old 106TCID50
i.c. N/A #100 vs. 0 *
[51]
i.v. N/A #89 vs. 0 *
i.p. N/A #39 vs. 0 *
oral N/A #5 vs. 0 *
@: No disease detected; #: No information available in the original paper; * combined score of ataxia, limb paralysis, and death; ** Earlier symptoms include hair loss and scurf.

Viruses 2018,10, 674 10 of 20
In the pEF-1a-hSCARB2 transgenic mouse model, limb paralysis rate cannot be differentiated
between Tg and non-Tg control mice, when EV71 gt B4 or gt C4 are used (Table 3) [
52
]. However,
mortality rates between Tg and non-Tg are different for gt C4; and the incidences of earlier symptoms
are different between Tg and non-Tg, when gt B4 was used. Despite the limitation in host age and
susceptibility to certain EV71 genotypes in this pEF-1a-hSCARB2 transgenic model, it had been used
in evaluating the efficacy of EV71 vaccine or adjuvant [
75
]. The Isehara strain behaved like a strain
with a high degree of virulence. For other viral isolates, such as BrCr, SK-EV006, and C7, much higher
viral doses were required for developing neurological symptoms in the hSCARB2-Tg10 mouse model.
In fact, even subcutaneous infection of the neonatal non-Tg control mice with the Isehara strain caused
paralysis [
51
]. In addition to the CNS, viral antigens were detected in skeletal muscle of both non-Tg
and Tg mice. In this regard, the hSCARB2 Tg mice are similar to the NOD/SCID mice in supporting
the replication of muscle-tropic EV71.
Neither model of these hSCARB2 Tg mice has so far been shown to support efficient oral
(intragastric) infection, which is a major route for transmission in children [
51
,
52
]. Before these
models are shown to be susceptible to a broader source of different clinical isolates and genotypes, it is
a caveat to carefully choose the best EV71 strains in the experimental design based on the hSCARB2
transgenic model.
3.4. A Hybrid Mouse Model
As discussed above, both hSCARB2 receptor and immune deficiency can contribute to efficient
EV71 infection and pathogenesis. Therefore, it is possible that a combination of a transgenic hSCARB2
and host immune deficiency might further enhance infection efficiency. Indeed, a new hybrid
mouse model was generated by cross-breeding hSCARB2 Tg and stat-1 KO mice. Comparing to
its parental hSCARB2 Tg or stat-1 KO mice, this hybrid mouse model is more user-friendly.
For example, at 2-week-old age, it can still be i.p. infected with different genotypes of EV71 at
1000-fold lower titer (pfu) than the parental mice [
5
]. EV71-infected hybrid mice exhibited high
density of viral protein in the CNS, such as mid-brain and spinal cord. Like the stat-1 KO model,
although EV71-infected hybrid mice developed limb paralysis, neither viral RNA nor protein was ever
detected in the muscle. It suggests that paralysis originated solely from the CNS injury, rather than
myositis and muscle destruction. This new hybrid (hSCARB2 Tg/stat-1 KO) model could serve as a
user-friendly platform for evaluating drug or vaccine efficacy [5].
Although this hybrid mouse model is a far more sensitive system for studying EV71-associated
neuropathogenesis and antiviral therapy, it is deficient in interferon signaling. Therefore, it is not a
good model for studying the therapeutic potential of immune modulators.
4. Pathogenesis Mechanism
Disease manifestations in EV-71 infected children are very diverse, ranging from subclinical infection,
common cold-like syndromes, hand-foot-and-mouth disease, to uncomplicated brainstem encephalitis,
severe dysregulated autonomic nerve system, fatal pulmonary edema, and cardiopulmonary collapse.
To date, while neurological disorders can be modeled in stat-1 KO mice, hybrid mice, and hSCARB2
Tg mice, pulmonary edema and cardiopulmonary failure remain to be recapitulated in EV71
mouse models. Because viral protein has never been detected in patients’ cardiomyocytes, it is generally
believed that cardiopulmonary failure is caused indirectly by the dysregulated autonomic nerve system
in the infected brainstem. Depending on the combination of both viral and host factors, current EV71
animal models could develop a wide variety of pathogenesis similar to human infection. The viral
factors include the infectious dose of EV71, genotypes, mutations, and possibly, sequence heterogeneity
(quasispecies). The host factors include the innate and adaptive immunity, routes of virus inoculation,
and the critical host age receiving viral infection.

Viruses 2018,10, 674 11 of 20
4.1. Viral Determinants of Virulence
In the case of viral factors, both viral load and sequence variations of different EV71 isolates
could significantly contribute to different degrees of virulence and clinical outcome. In the 1970s,
EV71 epidemics occurred in North America and Europe (genotype A, B1, and B2) and showed a
higher percentage of paralysis than the dermatological symptoms [
76
,
77
]. In contrast, clinical reports
in Asia-Pacific after the 1980s (other than genotype A, B1, and B2) observed both dermatological
and neurological symptoms [
3
,
21
,
45
]. Comparison of these results suggested that differences in
ethnic groups or/and genotypes of EV71 may have different sequelae. Sequence analysis identified
some nucleotide or amino acid differences between fatal and non-fatal cases [
78
–
80
]. For example,
it has been shown that point mutations at amino acid 145 of VP1 or amino acid 149 of VP2 could
determine whether neonatal mice can or cannot be successfully infected with a mouse-adapted EV71
strain [
55
,
81
]. In addition to the coding sequences, adaptive mutations at the IRES region also affected
the virulence in mice [
82
]. Finally, tissue tropism could also be associated with viral sequence variations.
Sequence comparisons of different EV71 isolated from respiratory, gastrointestinal, CNS, and blood
specimens, revealed nucleotide differences at three different positions. Further study showed that
amino acid 97 of VP1 was critical for neurotropism [
83
]. Both amino acid 97 and amino acid 145 of the
capsid protein VP1 were further investigated by two different research groups for their contribution
to pathogenesis in hSCARB2 Tg and non-Tg mouse models, respectively [
84
,
85
]. VP1 mutant L97R
was thought to be more virulent, because it acquired better binding to heparan sulfate on the target
cells [
85
]. Heparan sulfate is a common attachment receptor for many viruses, including EV71 [
63
].
In contrast, VP1 mutant 145G was thought to be less neurovirulent than VP1 mutant 145E, due to
its better binding to heparan sulfate than mutant 145E [
84
]. It was proposed that VP1-145G virus is
preferentially adsorbed by heparan sulfate attachment receptors on an excessive number of non-target
cells during circulation
in vivo
, leading to trapping and attenuation of mutant VP1-145G. In summary,
it would have to be resolved in the future, whether heparan sulfate binding property of EV71 capsid
protein VP1 and in vivo viral virulence, are positively or negatively correlated.
4.2. Host Factors
In this section, we will focus our scope on only those host factors which were investigated in
animal models. In one previous study [
4
], mouse models with different immunogenetic backgrounds
were i.p. infected with the same clinical isolates of EV71, and different disease outcomes and viral
tropisms were observed. For example, in the NOD/SCID mouse model infected with an EV71 clinical
isolate (gt B5), EV71 was predominantly muscle tropic, with severe atrophy and inflammation in the
paralyzed hindlimb muscle. In contrast, in the stat-1 KO and the hybrid mouse models, EV71 behaved
neurotropic with VP1 signals in both brain and spinal cord [4,5]. In the stat-1 KO model, VP1 protein
was often enriched in the Purkinje layer of cerebellar cortex, pons, brain stem,
and spinal cord.
IBA-1+ microglia cells were found to be amplified, indicating a high degree of neuronal injury.
In the hybrid mouse model, Nissl staining of spinal cord sections revealed abnormal neurons with
extensive vaccuoles. Unlike the NOD/SCID model, neither VP1 signal nor inflammation was ever
detected in the paralyzed muscle in stat-1 KO or hybrid mouse models. Since stat-1 is an upstream
transcription factor important for interferon signaling, stat-1 or interferon deficient CNS appeared
to favor neurotropic EV71. By contrast, limb muscles deficient in T and B cells appeared to favor
muscle-tropic EV71.
In human clinical cases, since EV71 infection in Europe and Asia-Pacific had different clinical features,
the difference in their respective ethnic (and immunogenetic) backgrounds (Caucasian vs. Asian)
could be responsible for their different disease manifestations [
6
,
76
,
86
]. Cytotoxic T-lymphocyte
antigen-4 (CTLA-4) is an important regulator of T-cell cytotoxicity and tolerance. The polymorphism
of CTLA-4 was found to be associated with encephalitis in EV71-infected children [
87
]. Other genetic
polymorphisms, such as human leukocyte antigen-A33 (HLA-A33) [
88
], tumor necrosis factor-
α
(TNF-
α
), and interferon-
α
receptor 1 (IFNAR1) [
89
,
90
], were also found to be associated with

Viruses 2018,10, 674 12 of 20
disease severity in EV71 patients. In summary, both viral and host factors are important for
EV71 immunopathogenesis.
5. Therapy
EV71 vaccination should be an effective way to protect children from infection. While inactivated
whole virus of EV71 appears to be a good vaccine candidate [
91
–
94
], it is unclear if it can be used
for all age groups of children. Therapeutics against EV71 remains an urgent need. We summarized
here in Table 4a list of candidate therapeutics, which had been tested in patients or animal models.
We categorized these candidate compounds into four different groups according to their approaches.
Group 1 relied on passive immunization with anti-EV71 neutralizing sera raised against
recombinant VP1 protein, virus-like particles (VLP), formaldehyde-inactivated whole virus, and IVIG
from human source [ref cited in Table 4]. Although IVIG were used in clinical practice [
17
],
the possibility of antibody-dependent enhancement of EV71 must be seriously considered [
19
–
21
].
Another important issue is whether these immune sera or antibodies can efficiently and broadly
cross protect against EV71 of different genotypes. Mouse monoclonal antibodies (mAb) need to be
humanized and re-tested in animal models in the future. Immune escape variants could be selected
during mAb treatment.
Group 2 includes immune modulators, such as interferon (IFN-
α
) or TLR agonists. Pre-treatment
with Poly (I:C) or co-treatment with mouse IFN and EV71, can reduce the death rate in EV71-infected
mice [
45
]. However, some of these studies in Group 2 are prophylactic rather than therapeutic.
Oral intake of GS-9620 (a TLR7 agonist) inhibited viral replication and increased the survival rate of
ICR mice infected with a mouse-adapted strain MP10 [
95
]. In clinical trials, IFN-
α
can decrease the
fever and viral load in mild HFMD patients [
72
,
96
]. Altogether, activation of IFN signaling could be a
potential therapy for EV71 infection.
Group 3 includes anti-inflammation treatments, such as anti-IL6 Ab, and complement
inhibitors [
38
,
97
]. Although compounds in Group 3 may not have any direct antiviral effect, they can
help reduce inflammatory injury by excessive cytokines.
Group 4 includes various compounds, whose mechanisms either remain unclear at present, or do
not belong to the above Group 1–3 [
22
,
98
–
101
]. For example, Milrinone is supposed to help maintain
the level of catecholamine [
22
]. As a side note, it is worth mentioning that disinfectants against EV71
have also been investigated. While disinfectants have no therapeutic value, solutions of chlorine
dioxide (ClO2) [102] and methylene blue (photosensitizer) [103] can inactivate EV71.

Viruses 2018,10, 674 13 of 20
Table 4. Therapeutic compounds tested in animal models and human patients.
Therapeutic
Approaches Drug Candidates Drug Delivery Methods Human or
Animal Model
Host Age at Virus
Inoculation
Virus
Inoculation
EV71 Clinical Isolates *
or Adapted Strains #
Virus
Genotype Ref.
GROUP 1
Passive
immunization
bispecific anti-EV71 &
CA16 Ab Bs(scFv) i.p. injection at 24 h after inoculation BALB/c 1-day-old i.c. EV71/pSVA-MP4 #B3 [104]
VP1-specific mAb i.p. injection at 3, 24, and 48 h after
inoculation SCARB2-Tg 1-day & 7-day-old s.c. E59 & 5746-TW98 * B4 & C2 [105]
human plasma or IVIG in vitro
pre-mix with EV71 at 37
◦
C for 1 h
ICR 2-day-old i.p. KM593929 * C4 [106]
human IVIG 3 i.p. injections at 4 h, 1- and 2-days after
inoculation Kunming mice 7-day-old i.p. AH08/06 * C4 [107]
EV71-specific Ab
i.p. injection 1 day before and 1 day after
(ICR), or 2 and 4 days after inoculation
(C57BL/6)
ICR & C57BL/6 7-day & 9-day-old oral M2 #C2 [44]
adult immune sera i.p. injection at 1hr before and 24 h after
inoculation young gerbils 21-day-old i.p. or i.m. strain 58301 * C4 [108]
human IVIG
i.v. infusion at a dosage of 1 g/kg/day for
2 days human patients <2-yr-old
natural infection
N/A N/A [17]
GROUP 2
Immune
modulators
Poly (I:C) i.p. injection at 12 h before inoculation ICR 3-day-old i.p. or oral MP4 #C2 [45]
Ad-IFN-a one intranasal shot within 12 h
post-inoculation BALB/c 6-day-old i.p. strain 41 * B4 [71]
antagomir -146a i.p. injection before or after virus
inoculation C57BL/6 7-day-old i.p. mEV71 #B2 [109]
GS-9620 oral uptake at 2, 26, 50 h post-inoculation ICR 10-day-old i.p. MP10 #C4 [95]
GROUP 3
Anti-inflammation
anti-IL6 Ab i.p. co-injection of Ab and virus (day 0) BALB/c 1-day-old i.p. strain 41 * B4 [38]
complement inhibitor
(CR2-crry) treatment post-inoculation ICR 7-day-old i.c. BrCr * A [97]
GROUP 4
Others
Retro-2cycl i.p. injection following EV71 inoculation BALB/c <1-day-old i.c. KJ508817 * C4 [98]
Pleconaril daily i.p. injection for 5 days ICR 1-day-old i.p. BrCr * A [99]
andrographolide
sulfonate
i.p. injection once daily until 10 days
post-inoculation ICR 7-day-old i.p. BJ09/07 #C4 [100]
adoptive transfer of
macrophage
i.p. injection with adult macrophage at 1
day post-inoculation ICR 10-day-old i.p. MP10 #C4 [101]
Milrinone i.v. injection within 2–6 h after admission
at dosage 0.35–0.55 mg/kg/min for 72 h. human patients <2-yr-old
natural infection
N/A N/A [22]
i.p.: intraperitoneal; i.v.: intravenous; i.c.: intracranial; i.m.: intra-muscular; s.c.: subcutaneous; N/A: information not available in the original paper; * clinical isolates;
#
mouse-adapted strain.

Viruses 2018,10, 674 14 of 20
6. Conclusions
In 2018, there are many issues that need to be solved in the modeling of EV71 infection and
pathogenesis. First, while the immune deficient NOD/SCID mice offer an efficient model for oral
infection with EV71 clinical isolates, one remaining challenge is to establish an oral infection system in
an immune-competent mouse model. Second, another mystery is the requirement of a neonatal age
for productive EV71 infection and pathogenesis. It is widely speculated that some kind of age-related
immune maturation could confer the resistance to pediatric infections, such as EV71. However, if so,
the exact nature of such an age-dependent arm of the immune system remains elusive. It cannot be
excluded that a putative EV71 receptor could be expressed in muscle or nerve only transiently at the
neonatal age, but rapidly turned off at an older age during mouse development. Third, there is an
urgent need for FDA-approved antivirals against the frequent EV71 epidemics in Asia and worldwide.
Lessons that we learned from EV71 should be useful for other picornaviruses closely related to EV71,
such as the most recent D68 virus that emerged in North America a few years ago.
Funding:
The authors were supported by Institute of Biomedical Sciences, Academia Sinica and the EID Program,
Ministry of Science and Technology, Taiwan (MOST 106-2320-B-001- 010, MOST 106-0210-01-15-02, MOST
107-2321-B-001-028, and MOST 107-0210-01-19-01).
Acknowledgments:
We thank the collaborations from Ya-Yen Yu, Chi-Yung Lin, and Jen-Shiou Lin at Changhua
Christian Hospital, Changhua, Taiwan.
Conflicts of Interest: We declare no competing financial interests.
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