- Open Access
Animal models of enterovirus 71 infection: applications and limitations
© Wang and Yu; licensee BioMed Central Ltd. 2014
Received: 31 December 2013
Accepted: 11 April 2014
Published: 17 April 2014
Human enterovirus 71 (EV71) has emerged as a neuroinvasive virus that is responsible for several outbreaks in the Asia-Pacific region over the past 15 years. Appropriate animal models are needed to understand EV71 neuropathogenesis better and to facilitate the development of effective vaccines and drugs. Non-human primate models have been used to characterize and evaluate the neurovirulence of EV71 after the early outbreaks in late 1990s. However, these models were not suitable for assessing the neurovirulence level of the virus and were associated with ethical and economic difficulties in terms of broad application. Several strategies have been applied to develop mouse models of EV71 infection, including strategies that employ virus adaption and immunodeficient hosts. Although these mouse models do not closely mimic human disease, they have been applied to determine the pathogenesis of and treatment and prevention of the disease. EV71 receptor-transgenic mouse models have recently been developed and have significantly advanced our understanding of the biological features of the virus and the host-parasite interactions. Overall, each of these models has advantages and disadvantages, and these models are differentially suited for studies of EV71 pathogenesis and/or the pre-clinical testing of antiviral drugs and vaccines. In this paper, we review the characteristics, applications and limitation of these EV71 animal models, including non-human primate and mouse models.
Since the effective control of poliovirus, enterovirus 71 (EV71) has been regarded as the most important neurotropic enterovirus. EV71 belongs to the species Human enterovirus A of the genus Enterovirus within the family Picornaviridae and is the major pathogen of hand-foot-mouth disease (HFMD). EV71 was first described in 1974 after it was isolated from patients in California and from then on, EV71 infection has been reported in at least 12 small and large outbreaks throughout the world [1–4]. Two subsequent outbreaks occurred in Malaysia and Taiwan in the late 1990s, and these outbreaks mainly affected young children and were characteristically associated with severe neurological complications and high rates of fatality [3, 5]. Since then, the Asia-Pacific region, including Taiwan, Malaysia, Vietnam, Singapore, Japan and China, has experienced more frequent large-scale EV71-associated HFMD epidemics.
EV71 infection is generally mild and self-limited, but occasionally such infection leads to central nervous system (CNS) infections that include aseptic meningitis, brainstem encephalitis and acute flaccid paralysis [6, 7]. Fatal EV71 cases develop and progress rapidly and are typically associated with neural complications, pulmonary edema and function collapse, which make clinical management challenging [8, 9]. The precise mechanisms of EV71-mediate disease, particularly the neuropathogenesis, are still not fully understood because suitable and relevant animal models have not been established.
Similar to poliovirus, EV71 has a limited host range; humans are the only known natural host. Early studies showed that the members of Picornaviridae display distinct pathogenicities in newborn mice for which neither poliovirus nor echovirus are pathogenic; coxsackie A viruses were found to cause generalized myositis, and coxsackie B viruses were found to induce myocarditis [10, 11]. Soon after the discovery of EV71, experimental EV71 infections were reported in neonatal mice [12, 13] and cynomolgus monkeys . However, EV71 experimental infection models were not established until the severe outbreaks in the Asia-Pacific region around the late 1990s [6, 15].
Enterovirus 71 animal models for pathogenesis studies
Clinical manifestation of animals and viral replication sites
Clinical isolates and prototype BrCr
The monkeys developed neurological manifestations including both pyramidal and extrapyramidal tract signs, such as flaccid paralysis, tremor and ataxia. The virus replicated in the spinal cord, brainstem, cerebella and cerebrum. EV71 had a wider neurotropism than that of polioviruses.
i.v./105.5 to 107 TCID50
Rhesus monkey, adult and neonate
Clinical isolate (FY-23, C4 genotype)
i.c., i.v./106.5 CCID50
The infected adult monkeys developed CNS infection and neuron impairment with extra-neuronal pathological changes confined to the lung tissues and not the pancreas and spleen, where high viral loads were detected.
i.t./ 104.5 CCID50
In neonate monkeys, HFMD-liked papules and vesicles were found on the limbs and in the mouth after intratracheal infection. However, the typical neurological complication was not observed. High viral titers were transiently detected in the brown adipose tissue, skeletal muscle and CNS.
oral/106.5 CCID50 twice
Mice - immunocompetent
ICR, 1- to 14-day-old
Mouse-adapted EV71 strain (MP4, C2 genotype)
oral/5 × 106 pfu
The mice developed rear limb paralysis (with massive and widespread necrotizing myositis) and neuropathologies (with neuronal loss and apoptosis) in the spinal cord and brainstem before death. The spinal cord, brain and muscle were the major organs for virus replication in the late phase of infection. Retrograde axonal transport in neurons might represent the major transmission route of EV71 in mice.
i.m./5 × 103 pfu
i.c./5 × 104 pfu
BALB/c, 1- to 7-day-old
Mouse-adapted strain (MP-26 M)
i.c. /3.4 × 104 TCID50
The mice developed limb paralysis followed by death after MP-26 M infection. Skeletal muscle displayed severe necrotizing myositis and contained a high viral load. Viruses were also isolated from the blood, hearts, livers, spleens and brains of infected mice. The VP1 mutation (G145E) alone was sufficient to increase the virulence of the virus in mice.
i.m./3.4 × 103 TCID50
i.p./3.4 × 104 TCID50
Mouse-adapted strain (MAVs, B3 genotype)
i.c., i.p., s.c., oral/105 CCID50
The mice developed paralysis followed by death after i.c., i.p., s.c. and i.m. administration but not after oral inoculation of the virus. The highest viral titers were detected in the skeletal muscle, spleen and spinal cord. The virus might enter the CNS via peripheral motor nerves after skeletal muscle infection.
i.m./3 × 105 CCID50
Mouse muscle-adapted strain (Fuyang-0805a, C4 genotype)
The Fuyang-0805a strain showed strong myotropism and induced severe necrotizing myositis in both skeletal and cardiac muscles. The virus was detected in the muscle, heart and intestines.
Mice - immunodeficiency
NOD/SCID, 3- to 4-week old
NOD/SCID mouse-adapted strain
The infected mice showed paralysis of the hind limbs. Viral RNA was first detected in the CNS and serum and then high copy numbers were detected in the heart, skeletal muscle and spinal cord.
(EV71(NOD/SCID), B1 genotype)
A129 and AG129 mouse-adapted strain (B2 genotype)
i.p./5.2 × 104 TCID50
The infected AG129 mice but not A129 mice developed limb paralysis, eye irritation, loss of balance and control of movements and exhibited high mortality.
Clinical isolate (5865/SIN/00009, B4 genotype)
The infected mice displayed progressive limb paralysis before death. The virus accumulated in the CNS and resulted in massive damage in the limb muscles, brainstem and anterior horn of spinal cord. Low viral particles in the limbs after oral inoculation indicated that the paralysis was a consequence of EV71 neuroinvasion.
Clinical isolate (EV71/58301, C4 genotype)
The infected animals developed neurological disorders such as hind limb paralysis, slowness, and ataxia before death. Significantly high viral titers were detected in the spinal cord, brainstem and skeletal muscle.
Clinical isolates C4 genotype and Mouse muscle-adapted strain (Fuyang-0805a, C4 genotype)
The transgenic mice were only susceptible to a mouse muscle-adapted EV71 strain and not the EV71 clinical isolates and exhibited severe symptoms that were comparable to those of the wild-type mice upon EV71 infection. High viral titers were detected in the muscle, spinal cord and brain after mouse-adapted virus infection. This study concluded that human PSGL-1 alone was not sufficient to provoke the infectivity of EV71 in mice.
SCARB2, 1- to 14-day-old
Clinical isolates, C2, C4, B4 and B5 genotypes and CA16
s.c./3 × 104 to 106 pfu
EV71 B genotypes were capable of inducing HFMD-like lesions and neurological disease with limb paralysis in 1-day-old but not transgenic mice older than 2 weeks of age. In contrast, 7- and 14-day-old but not 21-day-old transgenic mice were more susceptible to the C genotypes of EV71 and coxsackievirus A16 and exhibited more severe CNS diseases, limb paralysis and death than did the non-transgenic mice.
Clinical isolates (Isehara/Japan/99, C genotype)
i.c., i.v., i.p., oral/ 104 to 106 TCID50
The transgenic mice displayed ataxia, paralysis and death. The CNS, including the spinal cord, brainstem, cerebellum, cerebrum, hypothalamus and thalamus, was the major replication site for the virus.
Clinical features of EV71 infection in humans
Like other types of enteroviral infections, EV71 infection may cause persistent fever, diarrhea, rashes, aseptic meningitis and encephalitis, usually without life-threatening manifestations [3, 16]. The majority of infected children have asymptomatic and self-limiting infections. Mild cases are characterized by upper respiratory tract infection and cutaneous diseases such as HFMD and herpangina. The illness is characterized by 3–4 days of fever and the development of a vesicular enanthem on the hands, feet and buttocks and herpangina that involves popular lesions on the mucosa of the anterior pillars of Fauces . However, potentially life-threatening neurological and systemic complications, such as brain stem encephalitis, acute flaccid paralysis, autonomic nervous system dysregulation and pulmonary edema, are of the greatest clinical and public concern [3, 17–20].
Animal models used for the study of EV71 tropism and pathogenesis
a. Non-human primate models
Early studies showed that non-human primates, including cynomolgus, rhesus and green monkeys, are susceptible to EV71 infection [12, 14, 21]. Non-human primate models have been used to evaluate and characterize the neurovirulence of EV71 since the outbreaks of the late 1990s. Similar to the neurological manifestations in humans, cynomolgus monkeys display both pyramidal tract signs (flaccid paralysis) and extrapyramidal tract signs (including tremor and ataxia) with a broad viral antigen distribution that involves the spinal cord, brainstem, cerebellar cortex, dentate nuclei and cerebrum following intraspinal and intravenous inoculation of EV71 [22, 23]. The neuropathological features are highly consistent with those observed in humans with severe EV71 encephalitis at autopsy [5, 18, 24–26], which is indicative of the similarity of the susceptibilities of human and cynomolgus monkey CNS tissues to EV71. EV71 exhibits a wider neurotropism than does poliovirus in cynomolgus monkeys, and wild-type strains, including those isolated from patients with fatal encephalitis or hand, foot, and mouth disease (HFMD), exhibit no marked differences with respect to neurovirulence after infection; thus, this monkey species may not be suitable for assessing the neurovirulence level of the virus [22, 23]. Additionally, the clinical manifestations in cynomolgus monkeys are not correlated with those in patients. The monkeys do not manifest cutaneous lesions or develop pulmonary edema, although they have brainstem lesions [22, 23]. Pulmonary edema has been reported to be frequently associated with fatal EV71 infection in children and is considered to be related to the damage to the brainstem [4, 5, 27, 28].
Cynomolgus monkeys have been used to identify the molecular determinants of EV71. EV71 mutant strains derived from the prototype BrCr strain, which contains mutations in the 5′ non-translated region (NTR), 3D polymerase and 3′ NTR, exhibit attenuated neurovirulence with a limited viral spread in the CNSs of monkeys . Furthermore, this attenuated EV71 strain may be an effective vaccine as it is able to induce a broad spectrum of neutralizing antibody responses against different genotypes of EV71 .
Zhang et al. reported another non-human primate model involving adult rhesus monkeys. After inoculation with an EV71 clinical isolate (EV71/FY-23) intracerebrally, intravenously, orally (in drinking water) or intratracheally, monkeys develop CNS infections and neuronal impairment with extra-neuronal pathological changes that are confined to the lung tissues (i.e., cellular infiltration and tissue damage) and are not present in the pancreas and spleen where high viral loads are detected. Additionally, the infected-animals do not develop vesicular lesions on the skin and exhibit neither reduced muscle tension in the limbs nor typical neurological symptoms. These results suggest that, in addition to neurotropism, EV71 also elicits respiratory tract tropism in rhesus monkeys; these observations contrast with those based on cynomolgus monkeys and mice. This adult rhesus monkey model presents with all of the infectious and pathogenic processes of a systemic EV71 infection and reveals the clinical manifestations, kinetics of viremia, viral loads in the neuronal and extra-neuronal tissues, immune responses and histopathological changes. Interestingly, intracerebral inoculation induces pulmonary edema and hemorrhages around the hilum of the lung in half of the monkeys. Although it is not known whether the pulmonary edema results from a viral cytolytic effect in the lungs or is a consequence of CNS damage and inflammatory reactions, this adult rhesus monkey model is the first system to show that human EV71 can induce pulmonary edema in a non-natural host.
Hand, foot, and mouth disease-like papules and vesicles are formed on the limbs and in the mouths of 1- to 1.5-month-old neonatal rhesus monkeys after EV71 infection through the intratracheal route . Both the brown adipose tissue and skeletal muscle of the infected monkeys contain large amounts of virus, which suggests that these tissues are the primary target sites of viral replication. Similar to that observed in adult rhesus monkeys, the major inflammatory response is found in the tracheas and lungs of the neonates. In contrast to the cynomolgus monkeys, the typical neurological complications, including flaccid paralysis and ataxia, are not observed in the neonates, although both the medulla oblongata and thalamus contain very high virus titers and display neuropathological lesions. Additionally, the monkeys do not display the clinical symptoms (i.e., serious encephalomyelitis and respiratory failure) that are frequently present in severe human cases. Neonatal rhesus monkeys seem to be a relevant model for EV71-induced HFMD. Subsequent studies have shown that this neonatal monkey model was feasible for evaluating the efficacy of an inactivated EV71 vaccine .
b. Mouse models
b.1. Mouse-adaptation models
In agreement with early studies [12, 13], we confirmed that neonatal mice are susceptible to an EV71 clinical isolate (EV71/Tainan/4643/98, GenBank accession number AF304458) . In the course of the development of a mouse model for EV71, we realized that the mice exhibited an age-dependent susceptibility to EV71 infection. Mice older than 14 days of age were resistant to infection with EV71 clinical isolates regardless of the inoculation route. Specifically, neither symptoms nor mortality were observed in the 3- to 6-week-old BALB/c, C3H/HeN or ICR mice after inoculation with the EV7/4643 strain (4.7 × 109 pfu/ml; via through intracranial, intravenous, intraperitoneal, or oral routes; Y. F. Wang and C. K. Yu, unpublished observation). This age-dependent susceptibility to EV71 has been subsequently confirmed in numerous mouse models [34–37].
To increase the virulence of EV71 in mice and to develop an oral infection model that is suitable for the study of the pathogenesis of this disease and for vaccine development, particularly live attenuated vaccines, we generated a mouse–adapted EV71 strain, i.e., EV71/MP4, after four serial passages of the parental strain EV71/4643 in mice. Strain MP4 (5 × 106 PFU/mouse) was capable of orally infecting 7-day-old mice and resulted in rear limb paralysis (with massive and widespread necrotizing myositis) and neuropathology (with neuronal loss and apoptosis) in the spinal cord and brainstem prior to the death of the infected mice [6, 15]. Subsequent study  and our unpublished observations demonstrated that the mouse-adapted EV71/MP4 strain displayed strong neurotropism and was capable of consistently infecting and producing diseases that included brain infection, flaccid paralysis, pulmonary dysfunction and death in mice up to 14 days of age following intraperitoneal, intramuscular, intracranial, or oral inoculation. Furthermore, by observing the lag time of the disease progression between the distal and proximal viral inoculations via the use of colchicine, a fast axonal transport inhibitor, we demonstrated that retrograde axonal transport in neuron cells, but not hematogenous transport, might be the major transmission route of EV71 in mice . This assumption has been supported by other studies that have used both mice [34, 39] and rhesus monkeys .
This mouse-adapted EV71 model also allowed us to discover that type I interferon is an essential innate defense mechanism that controls EV71 infection in mice  and that EV71 per se inhibited the type I IFN system via the 3C protease while provoking a proinflammatory cytokine response .
The mouse-adaption approach has been extensively adopted by other investigators to develop infectious models. Chua et al. selected a mouse-adapted strain, i.e., MP-26 M, after six passages of a cell-adapted clinical isolate (CHO-adapted EV71-26 M) in newborn BALB/c mice via intracranial inoculation. After intramuscular, intracerebral or intraperitoneal challenge of the EV71/MP-26 M, both 1- and 7-day-old BALB/c mice developed limb paralysis with high tissue viral loads in the skeletal muscle. Interestingly, this study again demonstrated that the VP1 mutation (G145E) alone was sufficient to increase the virulence of this virus in mice. Our related studies revealed that the substitution of three of the nucleotides of EV71/MP4 in the 5′-NTR (C158U), VP1 (G145E), and VP2 (K149M) regions were responsible for the increased viral infectivity in vitro and the mouse virulence [6, 42, 43]. These results agree with those of Arita et al. and Chua et al..
Similarly, Ong et al. observed that EV71 could induce encephalomyelitis in 2-week-old but not 4-week-old ICR mice after inoculation with a mouse-adapted strain that was isolated from 1-day-old mice after serial passage of the virus in the brain tissue. The mouse-adapted EV71 strain was capable of infecting mice through both oral and parenteral routes and was capable of entering the CNS via peripheral motor nerves.
Wang et al. isolated a mouse muscle-adapted EV71 strain named Fuyang-0805a after four passages of the parental EV71 strain in skeletal muscle. The Fuyang-0805a strain exhibited strong myotropism and induced severe necrotizing myositis in both skeletal and cardiac muscles and induced intestinitis in 1-day-old ICR mice after intraperitoneal infection. There were many mutations in the genome of the muscle-adapted virus, including VP1 (E145Q), which had previously been reported by Chua et al. and Huang et al..
Arita et al. took another approach to generating mouse-adapted EV71 strains in adult immunodeficient mice, which theoretically involved less selective pressure on the virus. The EV71(NOD/SCID) strain was isolated after three passages of the parental virus in the brains of 3-week-old NOD/SCID mice and caused paralysis in 3- to 4-week-old NOD/SCID mice. The adapted virus contained a single amino acid substitution in the VP1 region of EV71 (G145E) that was found to be essential for the mouse-adapted phenotype in NOD/SCID mice. Likewise, Caine et al. generated mouse-adapted EV71 strains in adult immunodeficient A129 (α/β IFN receptor deficient) and AG129 (both α/β and γ IFN receptor deficient) mice. The resulting virus was highly lethal to 10-week-old AG129 mice and exhibited 100% lethality; the animals developed clinical symptoms that included limb paralysis, eye irritation and loss of balance before death. It would be worthwhile to know whether this adapted virus also acquired increased virulence for immunocompetent mice.
To overcome the bias of the natural tropism of mouse-adapted/muscle-adapted strains, Khong et al. successfully infected 2-week-old AG129 mice with a non-mouse adapted EV71 strain (a clinical isolate termed 5865/SIN/00009). In these immunodeficient mice, the virus exhibited strong neurotropism and induced neurological manifestations after intraperitoneal and oral routes of inoculation. Basically, this model exhibited features that closely resemble those of the murine models that had previously been reported, including clinical manifestations, tissue tropism, and histopathological changes. Collectively, the defect of the IFN system may debilitate the application of this model for the study of disease mechanisms, as alpha IFN is an essential innate defense mechanism for controlling EV71 in mice , and defects in beta IFN signaling (type II IFN) may alter the EV71-induced immunopathogenesis observed in immunocompetent hosts .
Yao et al. experimentally infected 21-day-old gerbils (Meriones unguiculatus) with an EV71 clinical isolate (EV71/58301, C4 genotype) via intraperitoneal inoculation. The infected animals developed neurological disorders and histopathological abnormalities that were similar to those that have been reported in the mouse models.
b.2. Transgenic mouse models
Viral receptors determine the host ranges and tissue-specific tropisms of enterovirus . Two human receptors for EV71 were identified in 2009: the human P-selectin glycoprotein ligand-1 (PSGL-1, CD162) , and the human scavenger receptor class B, member 2 (SCARB2) . PSGL-1 is a sialomucin membrane protein that is expressed exclusively by myeloid and lymphoid white blood cells and platelets and has a major role in the early stages of inflammation. SCARB2 is also known as lysosomal integral membrane protein II or CD36b-like-2. SCARB2 is localized mainly to lysosomes and is widely expressed in many human tissues and cell types. Additionally, sialyated glycans  and annexin II  have also been reported to be candidate receptors for EV71. Given the success of poliovirus receptor transgenic mice in studies of poliovirus pathogenesis and vaccine efficacy, transgenic mice expressing EV71 receptors have been actively developed.
Liu et al. established a transgenic mouse line that expresses the human PSGL-1 gene. However, these animals are only susceptible to a mouse muscle-adapted EV71 strain and not to EV71 clinical isolates, and these animals exhibited severe symptoms that are comparable to those of wild-type mice upon EV71 infection. Given that PSGL-1 is expressed by the dendritic cells in lymph nodes and macrophages in the intestinal mucosa , which are the primary sites of EV71 replication, it has been speculated that human PSGL-1 might act as a cofactor in the early stage of EV71 infection and that human PSGL-1 alone is not sufficient to provoke the infectivity of EV71 in mice. Indeed, clinical isolates have consistently exhibited low binding affinities for PSGL-1.
Lin et al. described a human SCARB2 transgenic mouse line in which the molecule is expressed ubiquitously. In these human SCRB2 transgenic mice, EV71 B genotypes are associated with HFMD-like lesions (i.e., visible hair loss and scurf) and neurological disease that include limb paralysis in 1-day-old mice but not in mice older than 2 weeks of age. In contrast, 7- and 14-day-old but not 21-day-old human SCARB2 transgenic mice are more susceptible to the C genotypes of EV71 and coxsackievirus A16, which are more likely to produce severe CNS diseases, limb paralysis and death in these mice compared to non-transgenic mice. Overall, the human SCARB2 transgenic mice have features that are similar to those of the wild-type mouse models that have previously been reported, including age-dependent susceptibility (only until 2 weeks of age), primarily viral replication sites in the muscle and CNS and pathological changes.
Fujii et al. generated transgenic mice that express the human SCARB2 with an expression profile that is similar to that of humans (i.e., CNS neurons, lung pneumocytes, hepatocytes and intestinal epithelium). The human SCARB2 transgenic mice older than 6 weeks of age are susceptible to infection by EV71 clinical isolates and coxsackievirus A16, which use SCARB2 as a receptor after intracranial, intravenous, intraperitoneal, and intra-gastric inoculations. The mice display EV71 neurotropism, neuropathology and clinical features (i.e., ataxia, paralysis and death) that are similar to those displayed by humans, monkeys and wild-type mice. The non-neuronal tissues do not contain EV71 antigens or exhibit pathological changes, such as pulmonary edema and cutaneous lesions. However, EV71 antigens and cutaneous lesions have been noted in 1-day-old transgenic mice after infection.
Pulmonary edema and the subsequent rapid onset of cardiopulmonary failure are hallmarks of EV71-induced mortality . Unfortunately, none of the mouse models, either the transgenic or non-transgenic models, exhibit pulmonary edema, although CNS neuropathology, muscular lesions, paralysis and death occur consistently following neuroinvasion. The absence of pulmonary edema limits the applicability of these murine models to the study of the disease mechanism of EV71.
Several reasons may explain why pulmonary edema is absent in EV71-infected mice. First, EV71-infected mice display a cytokine profile that is distinct from that of human patients, and the human cytokine profile may be essential for the formation of pulmonary edemas . Second, the nucleus tractus solitarii (NTS), an area known to contribute to the development of pulmonary edema in rats following injury [57, 58], is not the CNS target of EV71 infection in mice. Third, over-activation of the sympathetic nervous system may be required for the development of classic pulmonary edema symptoms, which may be absent in the mouse models .
Animal models for testing vaccines and therapeutics
Both live attenuated and inactivated whole virus vaccines have achieved successful control of poliovirus . Thus, it is logical to believe that EV71, which is similar to poliovirus, can also be controlled with by vaccination. The fact that passive transfer of specific antiserum provides protection against EV71 lethal challenge clearly illustrates not only the important role of humoral immunity in the control of EV71 but also the feasibility of vaccination . Several EV71 vaccine candidates, including live-attenuated virus , inactivated whole virus [33, 54, 61–64], recombinant viral protein [65–67], virus-like particles and DNA vaccines [66, 68, 69], have been developed, and their immunogenicity and efficiency have been evaluated in animal models.
Although they are not the natural hosts for human EV71, both mice and rhesus monkeys have demonstrated the same immunogenicity (in terms of neutralization of antibody titers and specific CD8+ T cell responses) elicited by EV71 vaccine candidates . Furthermore, it is technically feasible to assess the efficiency of inactivated vaccines in both animal species using distinct indicators, i.e., survival rates (mice), pathology (monkeys) and tissue viral loads (monkeys).
Given the age-dependent susceptibility to EV71 of laboratory mice, the EV71 vaccine candidates have primarily been tested using maternal immunization in which the dams are immunized before or during pregnancy, and the newborn pups receive lethal challenges after delivery. The efficacy of a formalin-inactivated whole-virus vaccine was first demonstrated in this setting as it prolonged the survival of pups after EV71 lethal challenge . Subsequently, this strategy had been applied to the testing of other vaccine formulations [70–72].
Using an oral infection model, for the first time, we proved the concept that live vaccines are feasible for protection against EV71 . In our hands, oral infection (intra-gastric challenge with mouse-adapted EV71 strains) only worked in mice that were 7 days old or younger. Despite this short time frame for immunization, we were able to demonstrate that 1-day-old mice were tolerant of an avirulent EV71 strain and mounted both systemic and mucosal antibody responses with neutralizing activities. More importantly, live EV71 active immunization at 1 day of age reduced the mortality of the mice following lethal challenge at 7 days of age. The same strategy had been applied to the testing of the efficacy of other live and inactivated EV71 vaccine candidates  and to examination of the therapeutic potential of a neutralizing antibody based on neonatal human SCARB2 transgenic mice .
Our related study showed that whole body exposures to EV71 viral suspension are an effective and less stressful alternative for inducing the lethal infection of newborn mice via the oral route . In laboratory mice, fecal-oral transmission of EV71 between infected and non-infected littermates occurs after close contact [6, 15]. Systemic infection of neonate rhesus monkeys has also been noted following intranasal inoculation with stool specimens from EV71-infected monkeys .
Non-human primates have also been used to test the immunogenicity and immunoprotection of potential EV71 vaccine candidates [32, 61, 68]. Macaque monkeys develop both specific humoral and cellular immune responses to both an inactivated EV71 vaccine and an EV71 virus-like particle (VLP) vaccine . However, challenge experiments have not been performed to evaluate the efficacies of these vaccine candidates. Chen et al. reported that neonatal (1.5-month-old) rhesus monkeys that are intramuscularly immunized with a formalin-inactivated, alum-based EV71 vaccine exhibit repressed viral-induced inflammatory reactions in the CNS and reduced IL-6, IFN-γ, and TNF-α production. Adult rhesus monkeys that are immunized with the inactivated vaccine also exhibit good humoral and cellular immune responses and reduced pathologies and tissue viral loads upon live virus challenge .
Antibody-related reactions, i.e., antibody-dependent enhanced infection [76, 77] and antibody-mediated cytotoxicity , may represent a safety concern for vaccine development. Ch’ng et al. demonstrated that the absence of EV71-neutralizing antibodies in the cerebrospinal fluid of vaccine-immunized and EV71-challenged monkeys, which implies that antibody-mediated responses might not be an issue, at least in monkeys.
EV71 mouse models had been employed to test the anti-EV71 activities of drugs that were originally developed against human rhinoviruses and polioviruses, including pleconaril [79, 80], ribavirin [34, 79, 81] and rupintrivir . The efficacies of interferon-α [40, 83] and interfering RNAs (siRNAs)  in inhibiting viral replication have also been examined in this setting. Generally, these anti-viral interventions exhibit certain protective or therapeutic effects when given either before or after EV71 infection that include reductions in morality and tissue virus loads and the alleviation of lesions. As susceptibility to EV71 is age dependent, only neonatal mice can be effectively infected. Thus, if an adult model were available, increasing the dosage and the number of treatments would improve the efficacy of the antiviral drugs.
Lactoferrin is a glycoprotein that is present in external secretions of mammals, such as breast milk, tears, saliva and mucous secretion . Studies have demonstrated that lactoferrin can inhibit EV71 infection by blocking the absorption or receptor-mediated binding of the virus to the target cells [86, 87]. Chen et al. generated a transgenic mouse that expressed the porcine lactoferrin in milk. Interestingly, four-day-old lectoferrin transgenic mice are resistant to EV71 lethal challenge. This finding raises the possibility of developing transgenic cows or goats for the mass production of lactoferrin-enriched milks and provides an evidence base that encourages breast-feeding in humans.
None of the monkey or mouse EV71 models that have been developed thus far recapitulate all of the aspects of the human disease; some of these models lack face validity (i.e., resemblance to human symptoms) and others lack construct validity (i.e., similarity to the underlying cause of the human disease). The limited studies that have been conducted in non-human primates have shown a diversity of EV71 infections in different monkey species in terms of tissue tropisms, primary viral replication sites and disease manifestations. In general, the non-human primate models are suitable for studying the course of EV71 systemic infection. Indeed, these studies using monkey models have advanced our knowledge of the transmission modes and routes, clinical manifestations, pathologies, viral distributions, and immune responses related to EV71 infection, and these factors are impossible to directly observe in human patients. Additionally, monkeys seem to be better suited than mice for evaluating the immunogenicity of EV71 vaccine candidates and are feasible for use in vaccine challenge tests that use indicators other than those used in the mouse models.
It is still unknown whether monkey EV71 neurovirulence tests will be valuable as quantitative and sensitive methods for detecting neurovirulent variants in live attenuated vaccine products because all of the EV71 genotypes and genogroups that have been tested are neurovirulent to cynomolgus monkeys. Furthermore, monkey systems would most likely be replaced by EV71 receptor-transgenic mice model when such models become available.
No adult mouse model with full validity has been established so far. Although adaptation would increase the mouse virulence of the EV71 clinical isolates, the mouse-adapted viruses themselves are unable to infect immunocompetent mice beyond weanling age. Although older immunodeficient mice (i.e., young adults) are susceptible to the adapted viruses derived from immunodeficient hosts, pulmonary edema has never been observed. Moreover, the strong myotropism of the mouse-adapted virus would definitely alter the role of virus-related neuropathology in the disease mechanism. Despite these shortcomings, neonatal mouse models with mouse-adapted viruses have contributed significantly to the study of EV71 infection  and vaccine development . More effort should be applied to creating adult mouse models with non-mouse adapted strains. These types of model will provide better systems with longer age time frames for the induction of the disease and/or immunization.
Human PSGL-1 transgenic mice are far from practical. PSGL-1 is not expressed in neurons, which are the cellular target of EV71; thus, these transgenic mice only acquiring a transient infection of the gastrointestinal tract. Young adult human SCARB2 transgenic mice (up to 6 weeks of age) seem to be more vulnerable to the virus; however, EV71-infected mice do not express the complete set of human symptoms nor do they exhibit a disease mechanism that is similar to the underlying cause of the human disease. In theory, the identification of all of the human EV71 receptors and co-receptors and the generation of transgenic mice that express all of these receptors are crucial to EV71 research and vaccine development. The transgenic system represents a promising approach to create relevant models of EV71.
The increased size and frequency of EV71 outbreaks that have occurred over the past 16 years in the Asia-Pacific region have caused serious public health concerns. Fortunately, an inactivated EV71 vaccine has already undergone a phase III clinical trial in China . Although an effective vaccine is expected in the near future, many questions regarding the nature and host response of the virus must be resolved, as these factors may compromise the safety of the vaccine. These issues include viral evolution and recombination [91, 92] and antibody-mediated reactions [76–78]. Appropriate animal models with full validity are indispensable for answering these questions and providing a better system for vaccine and drug development.
- Stanway G, Brown F, Christian P, Hovi T, Hyypia T, King AMQ, Knowles NJ, Lemon SM, Minor PD, Pallansch MA, Palmenberg AC, Skern T: Family Picornaviridae. Virus Taxonomy Eighth Report of the International Committee on Taxonomy of Viruses. Edited by: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. 2005, San Diego: Elsevier Academic Press, 757-778.Google Scholar
- Schmidt NJ, Lennette EH, Ho HH: An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis. 1974, 129: 304-309. 10.1093/infdis/129.3.304.View ArticlePubMedGoogle Scholar
- Ho M, Chen ER, Hsu KH, Twu SJ, Chen KT, Tsai SF, Wang JR, Shih SR: An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N Engl J Med. 1999, 341: 929-935. 10.1056/NEJM199909233411301.View ArticlePubMedGoogle Scholar
- Landry ML, Fonseca SN, Cohen S, Bogue CW: Fatal enterovirus type 71 infection: rapid detection and diagnostic pitfalls. Pediatr Infect Dis J. 1995, 14: 1095-1100. 10.1097/00006454-199512000-00013.View ArticlePubMedGoogle Scholar
- Chan LG, Parashar UD, Lye MS, Ong FG, Zaki SR, Alexander JP, Ho KK, Han LL, Pallansch MA, Suleiman AB, Jegathesan M, Anderson LJ: Deaths of children during an outbreak of hand, foot, and mouth disease in sarawak, malaysia: clinical and pathological characteristics of the disease. For the Outbreak Study Group. Clin Infect Dis. 2000, 31: 678-683. 10.1086/314032.View ArticlePubMedGoogle Scholar
- Wang YF, Chou CT, Lei HY, Liu CC, Wang SM, Yan JJ, Su IJ, Wang JR, Yeh TM, Chen SH, Yu CK: A mouse-adapted enterovirus 71 strain causes neurological disease in mice after oral infection. J Virol. 2004, 78: 7916-7924. 10.1128/JVI.78.15.7916-7924.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- McMinn PC: An overview of the evolution of enterovirus 71 and its clinical and public health significance. FEMS Microbiol Rev. 2002, 26: 91-107. 10.1111/j.1574-6976.2002.tb00601.x.View ArticlePubMedGoogle Scholar
- Lin MT, Wang JK, Lu FL, Wu ET, Yeh SJ, Lee WL, Wu JM, Wu MH: Heart rate variability monitoring in the detection of central nervous system complications in children with enterovirus infection. J Crit Care. 2006, 21: 280-286. 10.1016/j.jcrc.2006.02.005.View ArticlePubMedGoogle Scholar
- Liu L, Zhao H, Zhang Y, Wang J, Che Y, Dong C, Zhang X, Na R, Shi H, Jiang L, Wang L, Xie Z, Cui P, Xiong X, Liao Y, Zhao S, Gao J, Tang D, Li Q: Neonatal rhesus monkey is a potential animal model for studying pathogenesis of EV71 infection. Virology. 2011, 412: 91-100. 10.1016/j.virol.2010.12.058.View ArticlePubMedGoogle Scholar
- Hsiung GD: Chapter 14: Picornaviridae. Hsiung’s Diagnostic Virology. Edited by: Hsiung GD, Fong CKY, Landry ML. 1994, Yale University Press: New Haven, 119-140. 4Google Scholar
- Melnick JL: The discovery of the enteroviruses and the classification of poliovirus among them. Biologicals. 1993, 21: 305-309. 10.1006/biol.1993.1088.View ArticlePubMedGoogle Scholar
- Chumakov M, Voroshilova M, Shindarov L, Lavrova I, Gracheva L, Koroleva G, Vasilenko S, Brodvarova I, Nikolova M, Gyurova S, Gacheva M, Mitov G, Ninov N, Tsylka E, Robinson I, Frolova M, Bashkirtsev V, Martiyanova L, Rodin V: Enterovirus 71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria. Arch Virol. 1979, 60: 329-340. 10.1007/BF01317504.View ArticlePubMedGoogle Scholar
- Zheng ZM, Zhang JH, Zhu WP, He PJ: Isolation of enterovirus type 71 from the vesicle fluid of an adult patient with hand-foot-mouth disease in China. Virol Sin. 1989, 4: 375-382.Google Scholar
- Hashimoto I, Hagiwara A, Kodama H: Neurovirulence in cynomolgus monkeys of enterovirus 71 isolated from a patient with hand, foot and mouth disease. Arch Virol. 1978, 56: 257-261. 10.1007/BF01317855.View ArticlePubMedGoogle Scholar
- Chen YC, Yu CK, Wang YF, Liu CC, Su IJ, Lei HY: A murine oral enterovirus 71 infection model with central nervous system involvement. J Gen Virol. 2004, 85: 69-77. 10.1099/vir.0.19423-0.View ArticlePubMedGoogle Scholar
- Wang SM, Lei HY, Yu CK, Wang JR, Su IJ, Liu CC: Acute chemokine response in the blood and cerebrospinal fluid of children with enterovirus 71-associated brainstem encephalitis. J Infect Dis. 2008, 198: 1002-1006. 10.1086/591462.View ArticlePubMedGoogle Scholar
- Wang SM, Lei HY, Liu CC: Cytokine immunopathogenesis of enterovirus 71 brain stem encephalitis. Clin Dev Immunol. 2012, 2012: 876241-PubMed CentralPubMedGoogle Scholar
- Wang SM, Liu CC, Tseng HW, Wang JR, Huang CC, Chen YJ, Yang YJ, Lin SJ, Yeh TF: Clinical spectrum of enterovirus 71 infection in children in southern Taiwan, with an emphasis on neurological complications. Clin Infect Dis. 1999, 29: 184-190. 10.1086/520149.View ArticlePubMedGoogle Scholar
- Huang CC, Liu CC, Chang YC, Chen CY, Wang ST, Yeh TF: Neurologic complications in children with enterovirus 71 infection. N Engl J Med. 1999, 341: 936-942. 10.1056/NEJM199909233411302.View ArticlePubMedGoogle Scholar
- Lin TY, Chang LY, Hsia SH, Huang YC, Chiu CH, Hsueh C, Shih SR, Liu CC, Wu MH: The 1998 enterovirus 71 outbreak in Taiwan: pathogenesis and management. Clin Infect Dis. 2002, 34 (Suppl 2): S52-S57.View ArticlePubMedGoogle Scholar
- Hashimoto I, Hagiwara A: Pathogenicity of a poliomyelitis-like disease in monkeys infected orally with enterovirus 71: a model for human infection. Neuropathol Appl Neurobiol. 1982, 8: 149-156. 10.1111/j.1365-2990.1982.tb00269.x.View ArticlePubMedGoogle Scholar
- Nagata N, Shimizu H, Ami Y, Tano Y, Harashima A, Suzaki Y, Sato Y, Miyamura T, Sata T, Iwasaki T: Pyramidal and extrapyramidal involvement in experimental infection of cynomolgus monkeys with enterovirus 71. J Med Virol. 2002, 67: 207-216. 10.1002/jmv.2209.View ArticlePubMedGoogle Scholar
- Nagata N, Iwasaki T, Ami Y, Tano Y, Harashima A, Suzaki Y, Sato Y, Hasegawa H, Sata T, Miyamura T, Shimizu H: Differential localization of neurons susceptible to enterovirus 71 and poliovirus type 1 in the central nervous system of cynomolgus monkeys after intravenous inoculation. J Gen Virol. 2004, 85: 2981-2989. 10.1099/vir.0.79883-0.View ArticlePubMedGoogle Scholar
- Lum LC, Wong KT, Lam SK, Chua KB, Goh AY: Neurogenic pulmonary oedema and enterovirus 71 encephalomyelitis. Lancet. 1998, 352: 1391-View ArticlePubMedGoogle Scholar
- Wong KT, Lum LC, Lam SK: Enterovirus 71 infection and neurologic complications. N Engl J Med. 2000, 342: 356-358.View ArticlePubMedGoogle Scholar
- Shieh WJ, Jung SM, Hsueh C, Kuo TT, Mounts A, Parashar U, Yang CF, Guarner J, Ksiazek TG, Dawson J, Goldsmith C, Chang GJ, Oberste SM, Pallansch MA, Anderson LJ, Zaki SR: Pathologic studies of fatal cases in outbreak of hand, foot, and mouth disease, Taiwan. Emerg Infect Dis. 2001, 7: 146-148. 10.3201/eid0701.700146.PubMed CentralView ArticlePubMedGoogle Scholar
- Chang LY, Lin TY, Hsu KH, Huang YC, Lin KL, Hsueh C, Shih SR, Ning HC, Hwang MS, Wang HS, Lee CY: Clinical features and risk factors of pulmonary oedema after enterovirus-71-related hand, foot, and mouth disease. Lancet. 1999, 354: 1682-1686. 10.1016/S0140-6736(99)04434-7.View ArticlePubMedGoogle Scholar
- Ho M: Enterovirus 71: the virus, its infections and outbreaks. J Microbiol Immunol Infect. 2000, 33: 205-216.PubMedGoogle Scholar
- Arita M, Shimizu H, Nagata N, Ami Y, Suzaki Y, Sata T, Iwasaki T, Miyamura T: Temperature-sensitive mutants of enterovirus 71 show attenuation in cynomolgus monkeys. J Gen Virol. 2005, 86: 1391-1401. 10.1099/vir.0.80784-0.View ArticlePubMedGoogle Scholar
- Arita M, Nagata N, Iwata N, Ami Y, Suzaki Y, Mizuta K, Iwasaki T, Sata T, Wakita T, Shimizu H: An attenuated strain of enterovirus 71 belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in cynomolgus monkeys. J Virol. 2007, 81: 9386-9395. 10.1128/JVI.02856-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang Y, Cui W, Liu L, Wang J, Zhao H, Liao Y, Na R, Dong C, Wang L, Xie Z, Gao J, Cui P, Zhang X, Li Q: Pathogenesis study of enterovirus 71 infection in rhesus monkeys. Lab Invest. 2011, 91: 1337-1350. 10.1038/labinvest.2011.82.View ArticlePubMedGoogle Scholar
- Chen H, Zhang Y, Yang E, Liu L, Che Y, Wang J, Zhao H, Tang D, Dong C, Yang L, Shen D, Wang X, Liao Y, Wang L, Na R, Liang Y, Li Q: The effect of enterovirus 71 immunization on neuropathogenesis and protein expression profiles in the thalamus of infected rhesus neonates. Virology. 2012, 432: 417-426. 10.1016/j.virol.2012.06.026.View ArticlePubMedGoogle Scholar
- Yu CK, Chen CC, Chen CL, Wang JR, Liu CC, Yan JJ, Su IJ: Neutralizing antibody provided protection against enterovirus type 71 lethal challenge in neonatal mice. J Biomed Sci. 2000, 7: 523-528. 10.1007/BF02253368.View ArticlePubMedGoogle Scholar
- Khong WX, Yan B, Yeo H, Tan EL, Lee JJ, Ng JK, Chow VT, Alonso S: A non-mouse-adapted enterovirus 71 (EV71) strain exhibits neurotropism, causing neurological manifestations in a novel mouse model of EV71 infection. J Virol. 2012, 86: 2121-2131. 10.1128/JVI.06103-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Chua BH, Phuektes P, Sanders SA, Nicholls PK, McMinn PC: The molecular basis of mouse adaptation by human enterovirus 71. J Gen Virol. 2008, 89: 1622-1632. 10.1099/vir.0.83676-0.View ArticlePubMedGoogle Scholar
- Arita M, Ami Y, Wakita T, Shimizu H: Cooperative effect of the attenuation determinants derived from poliovirus sabin 1 strain is essential for attenuation of enterovirus 71 in the NOD/SCID mouse infection model. J Virol. 2008, 82: 1787-1797. 10.1128/JVI.01798-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Fujii K, Nagata N, Sato Y, Ong KC, Wong KT, Yamayoshi S, Shimanuki M, Shitara H, Taya C, Koike S: Transgenic mouse model for the study of enterovirus 71 neuropathogenesis. Proc Natl Acad Sci U S A. 2013, 110: 14753-14758. 10.1073/pnas.1217563110.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen CS, Yao YC, Lin SC, Lee YP, Wang YF, Wang JR, Liu CC, Lei HY, Yu CK: Retrograde axonal transport: a major transmission route of enterovirus 71 in mice. J Virol. 2007, 81: 8996-9003. 10.1128/JVI.00236-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Ong KC, Badmanathan M, Devi S, Leong KL, Cardosa MJ, Wong KT: Pathologic characterization of a murine model of human enterovirus 71 encephalomyelitis. J Neuropathol Exp Neurol. 2008, 67: 532-542. 10.1097/NEN.0b013e31817713e7.View ArticlePubMedGoogle Scholar
- Liu ML, Lee YP, Wang YF, Lei HY, Liu CC, Wang SM, Su IJ, Wang JR, Yeh TM, Chen SH, Yu CK: Type I interferons protect mice against enterovirus 71 infection. J Gen Virol. 2005, 86: 3263-3269. 10.1099/vir.0.81195-0.View ArticlePubMedGoogle Scholar
- Lee YP, Wang YF, Wang JR, Huang SW, Yu CK: Enterovirus 71 blocks selectively type I interferon production through the 3C viral protein in mice. J Med Virol. 2012, 84: 1779-1789. 10.1002/jmv.23377.View ArticlePubMedGoogle Scholar
- Yeh MT, Wang SW, Yu CK, Lin KH, Lei HY, Su IJ, Wang JR: A single nucleotide in stem loop II of 5′-untranslated region contributes to virulence of enterovirus 71 in mice. PLoS One. 2011, 6: e27082-10.1371/journal.pone.0027082.PubMed CentralView ArticlePubMedGoogle Scholar
- Huang SW, Wang YF, Yu CK, Su IJ, Wang JR: Mutations in VP2 and VP1 capsid proteins increase infectivity and mouse lethality of enterovirus 71 by virus binding and RNA accumulation enhancement. Virology. 2012, 422: 132-143. 10.1016/j.virol.2011.10.015.View ArticlePubMedGoogle Scholar
- Wang W, Duo J, Liu J, Ma C, Zhang L, Wei Q, Qin C: A mouse muscle-adapted enterovirus 71 strain with increased virulence in mice. Microbes Infect. 2011, 13: 862-870. 10.1016/j.micinf.2011.04.004.View ArticlePubMedGoogle Scholar
- Caine EA, Partidos CD, Santangelo JD, Osorio JE: Adaptation of enterovirus 71 to adult interferon deficient mice. PLoS One. 2013, 8: e59501-10.1371/journal.pone.0059501.PubMed CentralView ArticlePubMedGoogle Scholar
- Yao PP, Qian L, Xia Y, Xu F, Yang ZN, Xie RH, Li X, Liang WF, Huang XX, Zhu ZY, Zhu HP: Enterovirus 71-induced neurological disorders in young gerbils. Meriones unguiculatus: development and application of a neurological disease model. PLoS One. 2012, 7: e51996-10.1371/journal.pone.0051996.PubMed CentralView ArticlePubMedGoogle Scholar
- JM B: Receptors. The Picornavirus. Edited by: Ehrenfeld E, Domingo E, Roos RP. 2010, Washington, DC: ASM Press, 73-86.Google Scholar
- Nishimura Y, Shimojima M, Tano Y, Miyamura T, Wakita T, Shimizu H: Human P-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71. Nat Med. 2009, 15: 794-797. 10.1038/nm.1961.View ArticlePubMedGoogle Scholar
- Yamayoshi S, Yamashita Y, Li J, Hanagata N, Minowa T, Takemura T, Koike S: Scavenger receptor B2 is a cellular receptor for enterovirus 71. Nat Med. 2009, 15: 798-801. 10.1038/nm.1992.View ArticlePubMedGoogle Scholar
- Yang B, Chuang H, Yang KD: Sialylated glycans as receptor and inhibitor of enterovirus 71 infection to DLD-1 intestinal cells. Virol J. 2009, 6: 141-10.1186/1743-422X-6-141.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang SL, Chou YT, Wu CN, Ho MS: Annexin II binds to capsid protein VP1 of enterovirus 71 and enhances viral infectivity. J Virol. 2011, 85: 11809-11820. 10.1128/JVI.00297-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu J, Dong W, Quan X, Ma C, Qin C, Zhang L: Transgenic expression of human P-selectin glycoprotein ligand-1 is not sufficient for enterovirus 71 infection in mice. Arch Virol. 2012, 157: 539-543. 10.1007/s00705-011-1198-2.View ArticlePubMedGoogle Scholar
- Laszik Z, Jansen PJ, Cummings RD, Tedder TF, McEver RP, Moore KL: P-selectin glycoprotein ligand-1 is broadly expressed in cells of myeloid, lymphoid, and dendritic lineage and in some nonhematopoietic cells. Blood. 1996, 88: 3010-3021.PubMedGoogle Scholar
- Lin YW, Yu SL, Shao HY, Lin HY, Liu CC, Hsiao KN, Chitra E, Tsou YL, Chang HW, Sia C, Chong P, Chow YH: Human SCARB2 transgenic mice as an infectious animal model for enterovirus 71. PLoS One. 2013, 8: e57591-10.1371/journal.pone.0057591.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu CC, Tseng HW, Wang SM, Wang JR, Su IJ: An outbreak of enterovirus 71 infection in Taiwan, 1998: epidemiologic and clinical manifestations. J Clin Virol. 2000, 17: 23-30. 10.1016/S1386-6532(00)00068-8.View ArticlePubMedGoogle Scholar
- Wang SM, Lei HY, Su LY, Wu JM, Yu CK, Wang JR, Liu CC: Cerebrospinal fluid cytokines in enterovirus 71 brain stem encephalitis and echovirus meningitis infections of varying severity. Clin Microbiol Infect. 2007, 13: 677-682. 10.1111/j.1469-0691.2007.01729.x.View ArticlePubMedGoogle Scholar
- Feng GG, Nishiwaki K, Kondo H, Shimada Y, Ishikawa N: Inhibition of fibrin-induced neurogenic pulmonary edema by previous unilateral left-vagotomy correlates with increased levels of brain nitric oxide synthase in the nucleus tractus solitarii of rats. Auton Neurosci. 2002, 102: 1-7. 10.1016/S1566-0702(01)00394-0.View ArticlePubMedGoogle Scholar
- Talman WT, Perrone MH, Reis DJ: Acute hypertension after the local injection of kainic acid into the nucleus tractus solitarii of rats. Circ Res. 1981, 48: 292-298. 10.1161/01.RES.48.2.292.View ArticlePubMedGoogle Scholar
- Huang SW, Lee YP, Hung YT, Lin CH, Chuang JI, Lei HY, Su IJ, Yu CK: Exogenous interleukin-6, interleukin-13, and interferon-gamma provoke pulmonary abnormality with mild edema in enterovirus 71-infected mice. Respir Res. 2011, 12: 147-10.1186/1465-9921-12-147.PubMed CentralView ArticlePubMedGoogle Scholar
- Pliaka V, Kyriakopoulou Z, Markoulatos P: Risks associated with the use of live-attenuated vaccine poliovirus strains and the strategies for control and eradication of paralytic poliomyelitis. Expert Rev Vaccines. 2012, 11: 609-628. 10.1586/erv.12.28.View ArticlePubMedGoogle Scholar
- Dong C, Liu L, Zhao H, Wang J, Liao Y, Zhang X, Na R, Liang Y, Wang L, Li Q: Immunoprotection elicited by an enterovirus type 71 experimental inactivated vaccine in mice and rhesus monkeys. Vaccine. 2011, 29: 6269-6275. 10.1016/j.vaccine.2011.06.044.View ArticlePubMedGoogle Scholar
- Chen CW, Lee YP, Wang YF, Yu CK: Formaldehyde-inactivated human enterovirus 71 vaccine is compatible for co-immunization with a commercial pentavalent vaccine. Vaccine. 2011, 29: 2772-2776. 10.1016/j.vaccine.2011.01.094.View ArticlePubMedGoogle Scholar
- Bek EJ, Hussain KM, Phuektes P, Kok CC, Gao Q, Cai F, Gao Z, McMinn PC: Formalin-inactivated vaccine provokes cross-protective immunity in a mouse model of human enterovirus 71 infection. Vaccine. 2011, 29: 4829-4838. 10.1016/j.vaccine.2011.04.070.View ArticlePubMedGoogle Scholar
- Ong KC, Devi S, Cardosa MJ, Wong KT: Formaldehyde-inactivated whole-virus vaccine protects a murine model of enterovirus 71 encephalomyelitis against disease. J Virol. 2010, 84: 661-665. 10.1128/JVI.00999-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang M, Jiang S, Wang Y: Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice. Biochem Biophys Res Commun. 2013, 430: 387-393. 10.1016/j.bbrc.2012.11.035.View ArticlePubMedGoogle Scholar
- Ch’ng WC, Saw WT, Yusoff K, Shafee N: Immunogenicity of a truncated enterovirus 71 VP1 protein fused to a Newcastle disease virus nucleocapsid protein fragment in mice. Acta Virol. 2011, 55: 227-233. 10.4149/av_2011_03_227.View ArticlePubMedGoogle Scholar
- Premanand B, Prabakaran M, Kiener TK, Kwang J: Recombinant baculovirus associated with bilosomes as an oral vaccine candidate against HEV71 infection in mice. PLoS One. 2013, 8: e55536-10.1371/journal.pone.0055536.PubMed CentralView ArticlePubMedGoogle Scholar
- Lin YL, Yu CI, Hu YC, Tsai TJ, Kuo YC, Chi WK, Lin AN, Chiang BL: Enterovirus type 71 neutralizing antibodies in the serum of macaque monkeys immunized with EV71 virus-like particles. Vaccine. 2012, 30: 1305-1312. 10.1016/j.vaccine.2011.12.081.View ArticlePubMedGoogle Scholar
- Tung WS, Bakar SA, Sekawi Z, Rosli R: DNA vaccine constructs against enterovirus 71 elicit immune response in mice. Genet Vaccines Ther. 2007, 5: 6-10.1186/1479-0556-5-6.PubMed CentralView ArticlePubMedGoogle Scholar
- Wu CN, Lin YC, Fann C, Liao NS, Shih SR, Ho MS: Protection against lethal enterovirus 71 infection in newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine. 2001, 20: 895-904. 10.1016/S0264-410X(01)00385-1.View ArticlePubMedGoogle Scholar
- Liu JN, Wang W, Duo JY, Hao Y, Ma CM, Li WB, Lin SZ, Gao XZ, Liu XL, Xu YF, Xu WB, Qin C, Zhang LF: Combined peptides of human enterovirus 71 protect against virus infection in mice. Vaccine. 2010, 28: 7444-7451. 10.1016/j.vaccine.2010.08.080.View ArticlePubMedGoogle Scholar
- Chiu CH, Chu C, He CC, Lin TY: Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71. Microbes Infect. 2006, 8: 1671-1678. 10.1016/j.micinf.2006.01.021.View ArticlePubMedGoogle Scholar
- Wu TC, Wang YF, Lee YP, Wang JR, Liu CC, Wang SM, Lei HY, Su IJ, Yu CK: Immunity to avirulent enterovirus 71 and coxsackie A16 virus protects against enterovirus 71 infection in mice. J Virol. 2007, 81: 10310-10315. 10.1128/JVI.00372-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Chang HW, Lin YW, Ho HM, Lin MH, Liu CC, Shao HY, Chong P, Sia C, Chow YH: Protective efficacy of VP1-specific neutralizing antibody associated with a reduction of viral load and pro-inflammatory cytokines in human SCARB2-transgenic mice. PLoS One. 2013, 8: e69858-10.1371/journal.pone.0069858.PubMed CentralView ArticlePubMedGoogle Scholar
- Wong TW, Huang HJ, Wang YF, Lee YP, Huang CC, Yu CK: Methylene blue-mediated photodynamic inactivation as a novel disinfectant of enterovirus 71. J Antimicrob Chemother. 2010, 65: 2176-2182. 10.1093/jac/dkq301.View ArticlePubMedGoogle Scholar
- Han JF, Cao RY, Deng YQ, Tian X, Jiang T, Qin ED, Qin CF: Antibody dependent enhancement infection of enterovirus 71 in vitro and in vivo. Virol J. 2011, 8: 106-10.1186/1743-422X-8-106.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen IC, Wang SM, Yu CK, Liu CC: Subneutralizing antibodies to enterovirus 71 induce antibody-dependent enhancement of infection in newborn mice. Med Microbiol Immunol. 2013, 202: 259-265. 10.1007/s00430-013-0289-y.View ArticlePubMedGoogle Scholar
- Ch’ng WC, Stanbridge EJ, Ong KC, Wong KT, Yusoff K, Shafee N: Partial protection against enterovirus 71 (EV71) infection in a mouse model immunized with recombinant Newcastle disease virus capsids displaying the EV71 VP1 fragment. J Med Virol. 2011, 83: 1783-1791. 10.1002/jmv.22198.View ArticlePubMedGoogle Scholar
- Rotbart HA, Webster AD: Treatment of potentially life-threatening enterovirus infections with pleconaril. Clin Infect Dis. 2001, 32: 228-235. 10.1086/318452.View ArticlePubMedGoogle Scholar
- Zhang G, Zhou F, Gu B, Ding C, Feng D, Xie F, Wang J, Zhang C, Cao Q, Deng Y, Hu W, Yao K: In vitro and in vivo evaluation of ribavirin and pleconaril antiviral activity against enterovirus 71 infection. Arch Virol. 2012, 157 (4): 669-679. 10.1007/s00705-011-1222-6.View ArticlePubMedGoogle Scholar
- Li ZH, Li CM, Ling P, Shen FH, Chen SH, Liu CC, Yu CK, Chen SH: Ribavirin reduces mortality in enterovirus 71-infected mice by decreasing viral replication. J Infect Dis. 2008, 197: 854-857. 10.1086/527326.View ArticlePubMedGoogle Scholar
- Zhang X, Song Z, Qin B, Chen L, Hu Y, Yuan Z: Rupintrivir is a promising candidate for treating severe cases of enterovirus-71 infection: evaluation of antiviral efficacy in a murine infection model. Antiviral Res. 2013, 97: 264-269. 10.1016/j.antiviral.2012.12.029.View ArticlePubMedGoogle Scholar
- Hung HC, Wang HC, Shih SR, Teng IF, Tseng CP, Hsu JT: Synergistic inhibition of enterovirus 71 replication by interferon and rupintrivir. J Infect Dis. 2011, 203: 1784-1790. 10.1093/infdis/jir174.View ArticlePubMedGoogle Scholar
- Tan EL, Tan TM, Tak Kwong Chow V, Poh CL: Inhibition of enterovirus 71 in virus-infected mice by RNA interference. Mol Ther. 2007, 15: 1931-1938. 10.1038/sj.mt.6300287.View ArticlePubMedGoogle Scholar
- Brock JH: The physiology of lactoferrin. Biochem Cell Biol. 2002, 80: 1-6. 10.1139/o01-212.View ArticlePubMedGoogle Scholar
- Lin TY, Chu C, Chiu CH: Lactoferrin inhibits enterovirus 71 infection of human embryonal rhabdomyosarcoma cells in vitro. J Infect Dis. 2002, 186: 1161-1164. 10.1086/343809.View ArticlePubMedGoogle Scholar
- Weng TY, Chen LC, Shyu HW, Chen SH, Wang JR, Yu CK, Lei HY, Yeh TM: Lactoferrin inhibits enterovirus 71 infection by binding to VP1 protein and host cells. Antiviral Res. 2005, 67: 31-37. 10.1016/j.antiviral.2005.03.005.View ArticlePubMedGoogle Scholar
- Chen HL, Wang LC, Chang CH, Yen CC, Cheng WT, Wu SC, Hung CM, Kuo MF, Chen CM: Recombinant porcine lactoferrin expressed in the milk of transgenic mice protects neonatal mice from a lethal challenge with enterovirus type 71. Vaccine. 2008, 26: 891-898. 10.1016/j.vaccine.2007.12.013.View ArticlePubMedGoogle Scholar
- McMinn PC: Recent advances in the molecular epidemiology and control of human enterovirus 71 infection. Curr Opin Virol. 2012, 2: 199-205. 10.1016/j.coviro.2012.02.009.View ArticlePubMedGoogle Scholar
- Zhu FC, Meng FY, Li JX, Li XL, Mao QY, Tao H, Zhang YT, Yao X, Chu K, Chen QH, Hu YM, Wu X, Liu P, Zhu LY, Gao F, Jin H, Chen YJ, Dong YY, Liang YC, Shi NM, Ge HM, Liu L, Chen SG, Ai X, Zhang ZY, Ji YG, Luo FJ, Chen XQ, Zhang Y, Zhu LW, Liang ZL, Shen XL: Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial. Lancet. 2013, 381: 2024-2032. 10.1016/S0140-6736(13)61049-1.View ArticlePubMedGoogle Scholar
- Huang SW, Hsu YW, Smith DJ, Kiang D, Tsai HP, Lin KH, Wang SM, Liu CC, Su IJ, Wang JR: Reemergence of enterovirus 71 in 2008 in taiwan: dynamics of genetic and antigenic evolution from 1998 to 2008. J Clin Microbiol. 2009, 47: 3653-3662. 10.1128/JCM.00630-09.PubMed CentralView ArticlePubMedGoogle Scholar
- McWilliam Leitch EC, Cabrerizo M, Cardosa J, Harvala H, Ivanova OE, Koike S, Kroes AC, Lukashev A, Perera D, Roivainen M, Susi P, Trallero G, Evans DJ, Simmonds P: The association of recombination events in the founding and emergence of subgenogroup evolutionary lineages of human enterovirus 71. J Virol. 2012, 86: 2676-2685. 10.1128/JVI.06065-11.PubMed CentralView ArticlePubMedGoogle Scholar
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