Investigations on the immunopathology of enterovirus 71 4

6.2.6 Fatal-causing strains induced greater cytotoxicity in vitro To elucidate the cellular mechanisms underlying the increased virulence of S10 and S41 in AG129 mice, a plaque formation

6.2.6 Fatal-causing strains induced greater cytotoxicity in vitro

To elucidate the cellular mechanisms underlying the increased virulence of S10 and S41 in AG129 mice, a plaque formation assay was first conducted to evaluate the cytotoxicity of

each EV71 strain in vitro Human RD cells were infected with the four EV71 virus strains for

a period of nine days before staining with crystal violet The area of plaques was then calculated

Results indicated that S10 and S41 formed larger plaques as compared to C2 and MS strains (Figure 6.6A and B) Since plaques are areas of clearing induced by infection, it appeared that S10 and S41 infection were more effective at infecting and lysing RD cells as compared

to the other two strains

To further investigate the mechanisms that account for the difference in the ability to trigger cell death, a cell viability assay was performed Minimal cellular cytotoxicity was observed for all cells infected with either EV71 strains for the first 24 hours PI(Fig 6.7A) At 48 hours

PI, some cellular cytotoxicity was observed for the cells infected with MS (88%) and C2 (75%) Strains, whereas it was more pronouncedfor the cells infected with S10- (50%) and S41- (51%) At 72 hours PI, the difference between infection of RD cells with fatal and non-fatal strains was further amplified by an additional decrease in cell viability for S10- (41%) and S41- (47%) infected cells

One possible hypothesis that may explain the greater cellular cytotoxicity observed with S10- and S41-infected cells is that these two strains could have a greater replication rate in RD cells thereby leading to greater cell death over time, which would also correlate with the

greater plaque area observed (Fig 6.6) Figure 6.7B shows the viral yield of infectious particles recovered from the culture supernatant for the four different strains measured at various time points by plaque assay A one-way ANOVA revealed that there was no significant difference between the virus production rate of the four virus strains in RD cells at

the P < 0.05 level Hence, greater cell death caused by S10 and S41 cannot be explained by a

higher rate of virion production, suggesting that other viral virulence mechanisms might play

a role in the induction of host cell death

Figure 6.6 In vitro plaque formation assay (A) Plaques formed in RD cell

monolayer after infection with non-infected control, MS Strain, C2 Strain, S10 and S41 at a MOI of 0.025 for a period of nine day Red arrows indicate cell boundary separating two plaques (B) Areas of plaques (mm2) were measured using grid paper and tabulated The observations are representative of two independent experiments Data were expressed as mean ± SEM

B

A

Figure 6.7 In vitro analysis of EV71 strain virulence RD cells were infected with the four

different strains of EV71 at an MOI of 0.025 (A) Following incubation with XTT for 4 hours, absorbance at 450 nm was then measured at the indicated time points Cell viability at each time point was expressed as a percentage of the absorbance measured for the non-infected cells (B) Quantitative analysis of virus yield were done by plaque assays on the culture supernatants obtained from various time points PI Data are expressed as log fold change over time Results are representative of two independent experiments

6.2.7 Comparative genomic analysis of EV71 strains

To identify the specific sequences of more virulent EV71 strains, multiple sequence alignment of the 4 complete EV71 protein-coding sequences were performed according to genomic sequence acquired from GenBank

The ORF of EV71 encodes a polyprotein of 2,193 amino acids Analysis of the amino acid sequences indicated that differences between fatal and non-fatal causing strains were not limited to one of the specific viral proteins, but a total of 8 amino acid residue differences in VP3, 2A, 2C, 3A, 3C, 3D regions (Table 6.2) In VP3 a proline is found at position 329, in the fatal strains whereas a leucine is present in the non-fatal strains In 2A protein, an aspartic acid in fatal strains versus an asparagine in non-fatal strains is found at position 919 Another difference was observed in protein 3A, at position 1502 with a valine in the fatal strains and a methionine in the non-fatal strains Most of the amino acid differences were located in the 3C region, at positions 1627, 1706 and 1730 with isoleucine, valine and methionine respectively

in the fatal strains; and threonine, isoleucine and glutamic acid in the non-fatal strains Two more differences were identified in the 3D polymerase region Cysteine and alanine were present at positions 1844 and 2167, respectively for the fatal strains, while histidine and threonine were sequenced at these respective positions for the non-fatal strains instead

Table 6.2 Amino acid substitutions in fatal and non-fatal causing EV71 clinical isolates

Position Region Fatal Non-Fatal Protein function

6.3 Discussion

For EV71, the overall virulence phenotype is probably best measured by the ability of a virus strain to cause neurologic complications in its human host following infection by the oral-fecal route (Weng et al, 2010) This phenotype reflects sequential steps in infection including replication in the gastrointestinal tract; invasion of the CNS and intrinsic neurovirulence

(Pallansch MA, 2001; Shieh et al, 2001; Yang et al, 2009) A number of clinical studies have

demonstrated that these parameters may vary independently for different EV71 strains, as a wide range of clinical manifestations and outcomes have been observed in EV71 infected patients (Shimizu et al, 1999; Bible et al, 2007) What defines the clinical outcome is poorly understood, although several hypotheses of risk factors of both host and virus origin were proposed to be involved in the wide-ranging clinical phenotypes displayed by infected individuals

In the present study, we have shown that infection with different strains of EV71 resulted in different clinical outcomes and manifestations in AG129 mice Severe disease in S10- and S41-infected mice was correlated with the presence of lesions in the CNS and severe, unresolved myositis in the skeletal muscles Mice infected with neural and severe muscle lesion-inducing strains exhibited limb paralysis and died as early as day 5 PI Further tropism analysis of the infected animals showed that fatal-causing strains were highly neurotropic, while the less virulent, non-fatal EV71 strains were transiently present in most organs systemically but did not reach the host’s brain throughout the infection Thus, limb paralysis observed in S41- and S10-infected mice before death was likely to be caused by the distinct, stereotypic spread of the virus to the brain, resulting in CNS damage that impaired motor function although the contribution of myositis could not be entirely excluded Similar observations were also made in several other animal models including mouse model using

mouse-adapted strains of EV71 and cynomolgus monkeys (Wang et al, 2004; Arita et al,

2005) In these mouse models of EV71 encephalomyelitis and the AG129 mouse model reproted herein, the distribution of virus infiltration and inflammation was similar to that seen

in human encephalomyelities, suggesting that motors pathways are importatnt for viral

transmission into and within the CNS (Wang et al, 2004; Ong et al, 2008) Based on these

observations, we hypothesized that neurovirulence is possibly the predominant virulence factor to cause limb paralysis and death in EV71 infection

Our hypothesis is further supported by the observations with C2-infected mice Despite high quantities of virus infectious particles found in the muscle and spine of C2-infected animals, only mild inflammation was observed in the skeletal muscle during late stage of infection (day 12 post-infection) Consequently, the animals remained asymptomatic throughout the infection suggesting that mice infected with C2 virus did not experience any severe disease causing pathology throughout the course of infection While we could not entirely rule out the contribution of myositis to EV71-induced disease manifestations, our results suggest that the spread of EV71 from the CNS through the neuronal pathway, rather than from the muscle through the haematogenous pathway, is likely to be the primary reason for the neurological manifestations and for the morbidity and mortality of AG129 mice This discrepancy in viral tropisms between the different EV71 strains remains to be further investigated but we speculate that the ability to bind to a specific receptor at the surface of neuronal cells could be different between EV71 strains

As EV71 infections for our study were carried out in the immunocompromised AG129 mouse model, caution must be taken when interpreting immune responses Despite the lack

of an interferon response, AG129 mice were reported to be able to mount a normal antibody

response although with a bias towards the IgG1 subclass (van den Broek et al, 1995)

Nonetheless, analyses here encompasses the comparison of immune responses between mice infected with the different EV71 strains instead of determining the absolute immune responses In general, no significant difference in adaptive immune responses was evoked between animals infected with different strains of EV71 Despite a diminished CD19+ B cell response in mice infected with the non-fatal strains, the downstream systemic IgG and IgM responses were not in concordance in these mice IgG and IgM responses were in fact greater

in non-fatal strains We therefore concluded that the adaptive immune response may not play

a role in determining clinical outcomes observed in mice infected with the different strains

In contrast, the levels of pro-inflammatory cytokine, IL-6 showed a greater potential in discriminating severe from non-severe clinical outcomes in the EV71 infected AG129 mice Previously, high levels of IL-6, IL-10, IFN-! and TNF-" in the serum and cerebral spinal

fluid from EV71-infected patients have been consistently reported (Lin et al, 2002; Wang et

al, 2003; 2007) IL-6 and TNF-" levels were elevated in patients with encephalitis and pulmonary edema (Lin et al, 2002) In addition, exogenous IL-6 and IFN-! treatment was reported to induce mild pulmonary edema in EV71-infected mice (Huang et al, 2011),

whereas the abrogation of endogenous IL-6 functions improved survival rates and clinical scores of S41-infected neonates (Chapter 3) Collectively, these results support that IL-6 represent a critical host factor that plays a role in the virulence of EV71 infection in both human and mice

Besides enhanced mouse lethality possibly via increased neurotropism and IL-6 production, our data from plaque formation assays demonstrated that S10 and S41 exhibited heightened

virulence in vitro compared to the other two strains A cell viability assay on RD cells also

showed that S10 and S41 caused a greater cytopathic effect, resulting in much greater cell death over time This correlated with the amount of damage observed in the CNS and the limb muscles for both S10- and S41-infected mice Greater virulence appeared to depend on the ability of the virus to cause cytopathic damage However, the virus infectious particle yield of the four strains were similar, thus indicating that increased cellular death was not the result of more infectious progeny being produced and released into the supernatant

The ability of EV71 to initiate apoptosis in cultured cells has been widely documented Apoptosis is likely induced by several non-structural proteins, such as 2A and 3C, that resulted in cleavage of eIF4G and stimulation factor-64 (CstF-64) complex, respectively

(Kuo et al, 2002; Weng et al, 2009) One explanation to the phenomenon mentioned earlier is

that akin to PV, cell destruction is not required for release of infectious EV71 virions and that interaction between virus and host factors alone may be sufficient to trigger the cytopathic

effect by initiating apoptosis (Morrison et al, 1994) Interestingly, we have also identified

differences in the 2Apro and 3Cpro coding regions between fatal and non-fatal strains A mutation in these two proteases in fatal strains could therefore result in better affinity and interaction with host factors such as eIF4G, leading to increased apoptosis of the infected cells

Mutations in other structural and non-structural proteins apart from 2A and 3C regions might also change viral infectivity and virulence Multiple sequence alignments of the 4 virus strains used in this study revealed that amino acid differences were also seen in the VP3, 3A

and 3D regions The viral capsid VP3 is responsible for receptor binding (Plevka et al, 2012),

while 3A and 3D both play critical roles in the replication and life cycle of the virus

(Solomon et al, 2010) Molecular genetic approaches such as reverse engineering and

site-directed mutagenesis may help to indentify the genetic determinants that contribute to EV71 virulence

Our present study demonstrated that two-week old immunodeficient AG129 mice are naturally susceptible to EV71 infection with clinical isolates with limited lab passages With

no adaptation necessary, genotypic data derived from this mouse model are more likely to identify relevant viral virulence determinants in contrast to the findings generated with a mouse-adapted virus Furthermore, shorter experimental time would be required to evaluate the strain-specific virulence in the AG129 mouse model compared to the mouse-adapted models, since infection of AG129 mice do not require prior animal adaptation of virus populations Unlike the neonatal mouse model, which development of severe skeletal muscle myositis is solely accounted for their clinical manifestations, the distribution of virus infiltration and inflammation in EV71-infectd AG129 mice was similar to that seen in human encephalomyelitis Our results thus suggest that AG129 mouse model is likely to be more suitable for the investigations of EV71 virulence and pathogenesis due to its ability to recapitulate some of the CNS infection events However, the defect in IFN signaling may lead to some compensatory changes in the pattern of the immune responses, which implies that immunopathogenesis findings derived from this model may not accurately reflect that seen in immunocompetent patients, nor may it be of good predictive value for any IFN-related antiviral therapeutic approach Finally, the potential drawback owing to the restriction

to infection of mice in the first two weeks of life should also be acknowledged, as this would limit the time permissive for the experimental study of EV71

Understanding the mechanisms of virulence of EV71 is crucial not only to develop improved treatment options for clinical care, but also as a predictor of the severity of disease In this

study, we have developed a robust platform for the in vivo characterization of EV71

pathogenesis, particularly viral mutations that lead to various functional differences Upon infection with various EV71 clinical isolates, AG129 mice developed differential clinical outcomes ranging from asymptomatic to limb paralysis and death, demonstrating their potential use for the evaluation of the virulence profiles of EV71 strains Combined with the use of molecular manipulation techniques such as reverse genetic engineering and directed mutagenesis, this small animal model might prove to be tremendously useful for the identification of critical genetic determinant of EV71 virulence, its pathogenesis and serve as

an indicator for disease severity for future outbreaks

Chapter 7 Conclusion and future work

Over the past 12 years, one of the etiological agents of HFMD, EV71, has caused major and regular epidemics across the Western-Pacific region It is now believed to be the most important threat to the world among the enteroviruses, now that poliovirus has been eradicated in most parts of the world through vaccination Children below 5 years of age constitute the population at risk with clinical manifestations ranging from typical HFMD i.e mild exanthema to severe neurological manifestations With no effective antiviral or vaccine

available, EV71-associated infections have become a major public health concern

7.1 Role of Interleukin-6 in the immunopathogenesis of EV71 infection

The pathophysiology of the infection is highly complex and although a large body of knowledge has accumulated over the last decade, several important aspects remain to be fully understood As for many encephalitis-inducing virus, widespread, uncontrolled inflammation

of the CNS has long been proposed as the underlying pathogenic mechanism of associated neurological dysregulation (Lin et al, 2003; Shekhar et al, 2005) Studies on EV71 patients have shown that EV71-induced inflammation occurs predominantly in the grey matter of the spinal cord, medulla oblongata, and to a lesser extent of the hypothalamus, subthalamic and cerebrum region ( Lum et al, 1998; Shen et al, 1999; Huang et al, 1999;

EV71-Hsueh et al, 2000; Wong et al, 2000; Wong et al, 2008) Typically, EV71-induced

encephalitis is associated with widespread histopathological changes in the CNS including perivascular cuffing by mononuclear cells, variable oedema, neuronophagia and extensive

amount of microglia nodules in specific regions (Yang et al, 2009) However, what triggers

this massive inflammation remain elusive, although several observations and findings made from EV71-infected patients suggested that cytokine storm may contribute primarily to the

neuronal dysfunction, which represents the often-lethal stage of EV71 disease (Lin et al, 2002; Wang et al, 2006)

To address the salient question in EV71 research, the study herein aimed to address the hypothesis and was the first to confirm the detrimental role of a pleiotropic cytokine, IL-6 in EV71 immunopathogenesis Two points are worth noting in this study First, we have noticed

a steady, persistent increase of systemic IL-6 in both EV71-infected neonatal Balb/c mice and two-week old AG129 mice In the immunocompromised AG129 mice, a higher IL-6 level is associated with mice infected with lethal EV71 strains as opposed to those infected with the less virulent, asymptomatic strains of EV71 Importantly, the difference is apparent

as early as day 4 PI, suggesting that distinctively high serum IL-6 levels may represent a potential biomarker for the early identification of disease severity

Owing to the diverse clinical outcomes following EV71 infection, the ability to check the level of a cytokine (or cytokine profile) that would forewarn the clinicians regarding the likelihood of severe diseases development would be invaluable Our observation that serum IL-6 levels correlate accurately with disease manifestations and/or disease outcomes in our animal models strongly suggests its potential to be used as biomarker for evaluating and predicting disease severity and be tested in outbreaks of varying populations and viral strains

A methodical approach encompassing high-throughput screening is likely to produce a number of potentially informative biomarkers including, but not limited to IL-6, that might have implications in future clinical settings

Second, we have shown that IL-6 plays a fundamental role in the pathophysiology of EV71 infection We showed in the neonate mouse model for EV71 infection that sustained high levels of IL-6 induced upon viral infection are detrimental to the host, leading to severe tissue damage, and eventually death Furthermore, we demonstrated that the administration of anti-IL-6 neutralizing antibodies after the onset of the clinical symptoms successfully improved survival rate and clinical score of the infected animals Collectively, these data suggest a new paradigm where IL-6 might play a critical role in the immunopathogenesis of EV71

Cytokine inhibitors have been previously developed as therapeutic armamentarium for treating autoimmune and infectious diseases including rheumatoid arthritis, asthma and

tuberculosis (Murdaca et al, 2011; Emery & Buch, 2012; Walsh, 2012) The demonstrated

beneficial effect of IL-6 blockade during EV71 infection suggests that anti-IL-6 neutralizing antibodies might represent an interesting therapeutic approach to limit disease progression and improve disease outcome in children infected with EV71 While anti-IL-6 antibody treatment alone can prevent disease manifestation up to a certain degree, a combined therapy consisting a mix concoction of cytokine inhibitors might synergistically control the overwhelming inflammation induced by EV71 infection, and could be more effective in dampening the disease This hypothesis was indirectly supported by a previous study where co-administration with exogenous IL-6, IL-13 and IFN-! induced mild pulmonary oedema and exacerbate pulmonary abnormality of EV71-infected mice while the administration of IL-13 alone failed to do so (Huang et al, 2011) Holistic approaches such as combination

therapy that target the signaling cascades as a whole might thus be potentially more efficacious than anti-IL-6 treatment alone due to the simultaneous inhibition of multiple effectors

One limitation of the present study is the use of neonatal mice as animal model IL-6 functions mainly as the inducer of acute-phase response and T- and B-cell stimulation Given the immaturity of the adaptive immune system in mouse neonates, the actual role of IL-6 in EV71 pathogenesis in humans may not be as critical as it is in the neonate mouse model It would thus be important and necessary to evaluate the role of this pro-inflammatory mediator and assess the effect of IL-6 neutralization in an immunologically mature animal model as it becomes available

Finally, a better understanding of the fundamental mechanisms and the engaged inflammatory signal-transduction pathways of the cytokines production would shed light on EV71-induced hyper-inflammatory syndrome Future studies should therefore attempt to identify the crucial signaling-mediators and their mechanism of action in the immunopathogenesis of EV71 and in particular, clarify the balance between adaptive and maladaptive effects of these molecules The interaction of EV71 with immunocytes may induce production of the inflammatory cytokines involved in brain encephalitis or pulmonary oedema (Patel and Bergelson, 2009) Forthcoming studies should also try to characterize the immunophenotype in EV71 more precisely and the elucidation of the roles of various target cells in relation to pathology Such studies are a prerequisite for the development of new treatment strategies in EV71, which target inflammatory and immunopathogenic mechanisms

in this virus infection

7.2 Role of DC in EV71 infection

DCs are crucial for the generation and the regulation of adaptive immunity As DCs have a pivotal role in marshalling immune responses, it is not surprising that viruses have evolved ways to target DCs thereby facilitating viral dissemination and allowing evasion of antiviral immunity In this study, we characterized the interaction between EV71 and DCs, the critical component of both innate and adaptive immunity It was demonstrated that the productive infection of BMDCs resulted in the release of high levels of IL-6 with other pro-inflammatory cytokines such as IL-10 and IL-12 We found that EV71 infects and replicates effectively within BMDCs but suppress their maturation and activation, thereby undermining the ability of the infected DCs to prime robust antiviral immune responses against the pathogen

The mechanisms underlying the functional disarray of BMDC activation and maturation is currently unknown, although we have demonstrated in this study that it involves the inability

to up-regulate co-stimulatory molecules including CD40, CD80 and MHC class II Future studies should therefore be focused on elucidating the mechanisms of the interactions of EV71 with DCs, the relative contributions and significance of various cellular receptors as well as the signaling pathways that contribute to the functional defects in DC maturation Further understanding of DC’s role in this aspect should shed light on EV71 pathogenesis, which would in turn facilitate the development of more effective intervention against EV71 infection and might aid in the development of novel strategies for the design of vaccines against EV71 infection

In addition, we have shown that EV71-infected BMDCs successfully induced the regulation of chemokine receptor CCR7 that resulted in enhanced the migration capability of

up-the immunocytes in vivo Enteroviruses use the enteric tract as an entry route via the oral fecal route of transmission before spreading to the mesenteric lymph node (MLN) (Pallansch

MA, 2001) In view of its ability to enhance DC migration, one could speculate that the virus hijacks the resident DC equivalents in the gastrointestinal tract as an initial Trojan horse to reach the MLNs and subsequently infects other susceptible cells that travel out of the lymph nodes into the CNS The Trojan horse strategy is not uncommon within enteric viruses, and could be potentially studied with the aid of bioimaging techniques that allows the

simultaneous tracking of virus and DC in vivo (Lekkerkerker et al, 2006; Rima & Duprex,

2011)

One of the enigmatic features of DC biology is the complexity of its subsets (Gordon & Taylor, 2005) The various subsets of DCs are highly diversified in their anatomical distribution, immunological function and cell-surface marker expression Such properties will likely affect their susceptibility to the virus and their capacity to disseminate viral

infection Owing to the low distribution of DCs in vivo, BMDCs were used in this study to model the immunological function of DCs in vitro However, these cells might not fully mimic the characteristics of the DC subsets that are involved in EV71 infection in vivo due to

their different tissue origins Therefore, it is important to examine EV71 infection and

transmission with the bona fide DC subsets in vivo One critical prospect will be to determine

if resident DCs in the mucosal gastrointestinal tract can be productively infected by EV71 and whether these cells can promote local spread of viruses in the gut mucosa Elucidating

the interactions of EV71 with DCs in vivo will be vital for uncovering the contribution of

DCs to the pathogenesis of EV71

Finally, similar approaches employed in this study should be extended to various target cells

of EV71, including T lymphocyte, monocyte, macrophage and B lymphocyte The inflammatory cytokine and chemokine responses of immune cells could be determined upon confirming their susceptibility to the virus In addition, comparison of virus yield and replication efficacy between EV71-infected immunocytes would provide key insights into the putative cellular source of EV71 reservoir and hence, contribute to the understanding of EV71-induced immunopathogenesis

pro-7.3 Development of a novel mouse model for EV71 infection

The lack of suitable small animal model of EV71 infection has severely impeded the progress

of deciphering the mechanisms underlying EV71 pathogenesis, as well as the advancement

of vaccine and therapeutic development In the past few years, substantial progresses have been made to identify host cells susceptible to EV71 infection, and this has led to the

development of in vitro models in primary and secondary human cell cultures On the other hand, animal models offer obvious advantages compared to in vitro systems as they can

mirror the cellular complexity that occur in the human host, making them more relevant than

in vitro settings for which very limited cell type interactions can be reproduced

Using an immunocompromised mouse strain, we have recently established a young adult mouse model that is susceptible to infection with non-mouse-adapted EV71 strains as this would allow the unbiased study of (some aspects of) EV71 pathogenesis as well as the evaluation of EV71 vaccine and drug candidates We showed that two-week-old and younger immunodeficient AG129 mice, which lack type I and II interferon receptors, are susceptible

to infection with a non-mouse-adapted EV71 strain via both the intraperitoneal and oral routes of inoculation The infected mice displayed progressive limb paralysis prior to death with virus accumulation in the CNS that is accompanied by massive damage in the limb muscles, brainstem, and anterior horn areas

Currently, there is no effective pharmaceutical compound against EV71, and treatment of severe cases is limited to supportive management e.g administering Milrinone, an inotropic

agent, to prevent cardiopulmonary failure (Wang et al, 2006) One critical obstacle to the

development of effective antiviral against EV71 lies in the lack of appropriate animal models

for the screening and testing of drug candidates One-day-old mouse neonates were found permissive to high infectious doses of EV71 but are not practical for anti-EV71 drug or vaccine testing with a susceptibility window limited to a couple of days after birth (Lee & Chang, 2010) The addition of the AG129 mouse model to the repertoire thus offers new opportunities for the convenient screening and testing of drug candidates as it circumvents the shortcoming of limited susceptibility window Furthermore, because this mouse model recapitulates some of the neurological features of the disease, it allows evaluate the efficacy

of a wider spectrum of drugs, for example, antagonist to cell factors that promote viral transmission to the CNS With the caveat of the absence of IFN signaling pathways, this immunocompromised mouse model nevertheless represents one step further towards the development of a relevant small animal model of EV71 infection

Lastly, at least five types of human cellular receptors specific to EV71 such as PSGL-1 and

SCARB2 have been recently identified separately (Lin et al, 2009; Nishimura et al, 2009; Yamayoshi et al, 2009; Yang et al, 2009; 2011) Since the endogenous mouse PSGL-1 and SCARB2 are not recognized by EV71 (Nishimura et al, 2009; Yamayoshi et al, 2009),

transgenic mice expressing known human EV71 receptors appear to be the next generation of

mouse models of EV71 However, in vitro studies have shown that EV71 is able to use more

than one receptor to infect various cell types Thus, it is anticipated that the expression of a single human receptor candidate is unlikely to allow the recapitulation of the entire EV71 pathogenesis and may bias the viral tropism It is also possible that a single receptor may not

be sufficient to confer increased disease susceptibility compared to the non-transgenic models Consistently, enhancement of infection of a transgenic mouse expressing human PSGL-1 was only observed with a mouse-adapted EV71 strain, but not with the non-adapted

clinical isolate (Liu et al, 2011) Thus the development of a transgenic mouse model that

expresses known human EV71 receptors in their native tissue distribution patterns would be ideally suited for the advancement of EV71-related research

7.4 Investigations on EV71 virulent determinants in the AG129 mouse model

The molecular and genetic factors specifically associated with EV71 virulence are still poorly understood Unlike most laboratory experiments, studies of the pathogenesis in the clinical settings are often confounded by many experimental variables such as differences in study designs and viral diagnostic capabilities, making direct comparison of epidemiological data implausible In this study, we attempted to dissect further the precise determinants of viral virulence, primarily by the use of our newly developed AG129 mouse model

In this study, four clinical isolates with varying clinical manifestations and outcomes in humans were used to infect two-week-old AG129 mice Our results showed that mice displayed clinical symptoms and outcomes that were relatively similar to those observed in humans Mice infected with fatal strains displayed limb paralysis before death Together with viral presence in the brain, histopathological analyses revealed that neurological damage in the CNS of infected mice were indicators of morbidity and mortality, although skeletal muscle myositis was also observed High levels of pro-inflammatory IL-6 were observed in AG129 mice infected with fatal strains, consistent with clinical observations in infected humans However, a greater viral virulence does not seem to correlate with greater replication ability in fatal strains Differences in amino acid sequences between fatal and non-fatal strains were found in the VP3, 2A, 3A, 3C and 3D regions and these differences could be important in determining viral virulence

The fatal strains S10 and S41 induced greater rate of cell death compared to the non-fatal MS and C2 strains Several studies have detailed the mechanism of EV71 induced apoptosis It

was reported that viral non-structural protein 2Apro and 3Cpro, interact directly with the host’s eIF4G and CstF-64 complex, which in turn initiate the caspase activated apoptosis pathway

(Kuo et al, 2002; Li et al, 2002) It will be interesting to delineate the apoptotic pathway

induced by the fatal and non-fatal strains of EV71, respectively, in order to address their differential rates in causing cell death Typical apoptosis assays such as caspase assay,

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and annexin

V assay can be carried out following cell infection with the various EV71 strains, and the relative expression of the major cellular apoptotic markers can be quantify in order to infer their relative roles in EV71 virulence Since we have shown earlier that virulent EV71 strains are highly neurotropic, verification of the above observations in human neuronal cells will be necessary

In addition, a full transcriptomic or proteomic comparative study of various susceptible cells infected with different strains of EV71 to study host responses after infection could be a way

to understand the mechanisms behind the virulence of fatal strains Proteomes of RD cells after infection with fatal S41 and non-fatal CVA16 were compared and revealed differential

expression levels for 16 proteins (Lee et al, 2011) EV71 is able to efficiently shutdown host

translational machinery and down-regulate host proteins responsible for combating viral replication The identification of RNA or proteins that have differential expression levels between cells infected with fatal or non-fatal strains of EV71 could reveal important determinants for virulence

Finally, as of all screening studies, final validations with a separate technique are crucial in establishing the role of genetic determinants as virulence factors and to associate specific

genetic variants with their phenotypes Reverse genetic methods such as site-directed mutagenesis can be carried out in the regions where amino acid differences set the fatal and non-fatal strains apart This will allow us to determine whether these mutations can play a role in determining virulence of EV71 strains For instance, a point mutation can be carried out at the 2A region from aspartic acid (present in fatal strains) to asparagine (present in non-fatal strains) If this mutation results in substantial decrease in morbidity and mortality upon infection in mice, this would mean that this amino acid position is important for determining virulence Likewise, site-directed mutagenesis at this region from asparagine to aspartic acid

in non-fatal strains can be carried out as well to further confirm the importance of this position in virulence

Based on evidences from our current study, AG129 mice infected with EV71 developed flaccid limb paralysis owing to a combination of skeletal muscle myositis and infection of the brainstem and anterior horns of the spinal cord Infectivity studies show that the skeletal muscle appears to be a major primary virus replication sites for EV71 in these mice Although the contribution of myositis cannot be excluded, myositis alone is unlikely to be the primary cause of death in the EV71-infected AG129 mice since C2 strain was found to infect the skeletal muscle tissue without causing clinically apparent infection Furthermore, there does not appear to be any inflammatory lesions in the muscle or spinal cord of C2-infected AG129 mice To further distinguish the respective role of myositis and CNS infection in morbidity and mortality, different inoculation routes could be applied to determine the actual transmission route of EV71 in AG129 mice Mice could be differentially infected via the intramuscular route and intracranial route If myositis were the primary cause

of morbidity and mortality, the intracranially infected mice would not manifest any clinical

symptoms early after inoculation, since time would be required for the virus spread to limb skeletal muscle Likewise, a longer lag time would be required for mice infected via the intramuscular route to exhibit limb paralysis if CNS infection were the primary cause of clinical manifestation,

The inherent complexity of EV71 lies in its ability to infect various cell types in various tissues and simultaneously exert its pathogenicity by manipulating the host immune system Virus dissemination within the host from its initial site of infection (gastrointestinal tract) to the CNS remains one of the pressing questions that remain to be addressed The relative roles of various immune mediators and immunocytes in EV71 pathogenesis are also salient

questions that await answers The challenging development of relevant in vivo models will

certainly be instrumental in this quest, and in the successful identification of effective therapeutics

Finally, although geographically confined to the Asia-Pacific region, worldwide spread of EV71 strains is likely to occur rather sooner than later due to increased rates of travel and population mobility Thus, rich developed western nations who do not view EV71 as a priority should acknowledge this possibility, and provide incentives for the pharmaceutical industry to devote resources to address this important public health concern

Chapter 8 References

Abe, Y., Horiuchi, A., Miyake, M & Kimura, S (1994) Anti-cytokine nature of natural human immunoglobulin: one possible mechanism of the clinical effect of intravenous

immunoglobulin therapy Immunological reviews, 139, 5–19

Adkins, B., Leclerc, C & Marshall-Clarke, S (2004) Neonatal adaptive immunity comes of

age Nature reviews immunology, 4, 553–564

Ang, L.W., Koh, B.K., Chan, K.P., Chua, L.T., James, L & Goh, K.T (2009) Epidemiology

and control of hand, foot and mouth disease in Singapore, 2001-2007 Annals of the

academy of medicine, Singapore, 38, 106–112

Arita, M., Ami, Y., Wakita, T & Shimizu, H (2008) 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 Journal of virology, 82, 1787–

1797

Arita, M., Nagata, N., Iwata, N., Ami, Y., Suzaki, Y., Mizuta, K., Iwasaki, T., Sata, T., Wakita, T & Shimizu, H (2007) An attenuated strain of enterovirus 71 belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in

cynomolgus monkeys Journal of virology, 81, 9386–9395

Arita, M., Shimizu, H., Nagata, N., Ami, Y., Suzaki, Y., Sata, T., Iwasaki, T & Miyamura,

T (2005) Temperature-sensitive mutants of enterovirus 71 show attenuation in

cynomolgus monkeys Journal of general virology, 86, 1391–1401

Arita, M., Wakita, T & Shimizu, H (2008) Characterization of pharmacologically active

compounds that inhibit poliovirus and enterovirus 71 infectivity Journal of general

virology, 89, 2518–2530

Austyn, J.M., Kupiec-Weglinski, J.W., Hankins, D.F & Morris, P.J (1988) Migration patterns of dendritic cells in the mouse Homing to T cell-dependent areas of spleen, and

binding within marginal zone The Journal of experimental medicine, 167, 646–651

Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y.J., Pulendran, B &

Palucka, K (2000) Immunobiology of dendritic cells Annual Review of Immunology, 18,

767–811

Barnard, D.L., Hubbard, V.D & Smee, D.F, Sidwell R.W., Watson K.G., Tucker S.P & Reece, P.A (2004) In Vitro Activity of Expanded-Spectrum Pyridazinyl Oxime Ethers Related to Pirodavir: Novel Capsid-Binding Inhibitors with Potent Antipicornavirus

Activity Antimicrobial agents Chemother, 48, 1766-1772

Barrios, C., Brawand, P., Berney, M., Brandt, C., Lambert, P.H & Siegrist, C.A (1996) Neonatal and early life immune responses to various forms of vaccine antigens qualitatively differ from adult responses: predominance of a Th2-biased pattern which

persists after adult boosting European journal of immunology, 26, 1489–1496

Basavappa, R., Syed, R., Flore, O., Icenogle, J.P., Filman, D.J & Hogle, J.M (1994) Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: structure of

the empty capsid assembly intermediate at 2.9 A resolution Protein science, 3, 1651–

1669

Beck, Z., Prohászka, Z & Füst, G (2008) Traitors of the immune system-enhancing antibodies in HIV infection: their possible implication in HIV vaccine development

Vaccine, 26, 3078–3085

Bible, J.M., Pantelidis, P., Chan, P.K.S & Tong, C.Y.W (2007) Genetic evolution of

enterovirus 71: epidemiological and pathological implications Reviews in medical

virology, 17, 371–379

Bland, J.M & Altman, D.G (2000) The odds ratio British Medical Journal, 320, 1468

Blomberg, J., Lycke, E., Ahlfors, K., Johnsson, T., Wolontis, S & Zeipel, von, G (1974) Letter: New enterovirus type associated with epidemic of aseptic meningitis and-or hand,

foot, and mouth disease Lancet, 2, 112

Brockman, M.A., Kwon, D.S., Tighe, D.P., Pavlik, D.F., Rosato, P.C., Sela, J., Porichis, F.,

Le Gall, S., Waring, M.T., Moss, K., Jessen, H., Pereyra, F., Kavanagh, D.G., Walker, B.D & Kaufmann, D.E (2009) IL-10 is up-regulated in multiple cell types during

viremic HIV infection and reversibly inhibits virus-specific T cells Blood, 114, 346–

356

Brown, B.A & Pallansch, M.A (1995) Complete nucleotide sequence of enterovirus 71 is

distinct from poliovirus Virus research, 39, 195–205

Brown, B.A., Oberste, M.S., Alexander, J.P., Kennett, M.L & Pallansch, M.A (1999) Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to

1998 Journal of virology, 73, 9969–9975

Calandra, T., Bernhagen, J & Mitchell, R (1994) The macrophage is an important and

previously unrecognized source of macrophage migration inhibitory factor Journal of

experimental medicine, 179, 1895-1902

Cardosa, M.J., Krishnan, S., Tio, P.H., Perera, D & Wong, S.C (1999) Isolation of subgenus

B adenovirus during a fatal outbreak of enterovirus 71-associated hand, foot, and mouth

disease in Sibu, Sarawak Lancet, 354, 987–991

Cardosa, M.J., Perera, D., Brown, B.A., Cheon, D., Chan, H.M., Chan, K.P., Cho, H & McMinn, P (2003) Molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region: comparative analysis of the VP1 and VP4 genes

Emerging infectious diseases, 9, 461–468

Caux, C., Vanbervliet, B., Massacrier, C., Dezutter-Dambuyant, C., de Saint-Vis, B., Jacquet, C., Yoneda, K., Imamura, S., Schmitt, D., & Banchereau J (1996) CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell

pathways in response to GM-CSF+TNF alpha Journal of experimental medicine, 184,

695–706

Chan, K.P., Goh, K.T., Chong, C.Y., Teo, E.S., Lau, G & Ling, A.E (2003) Epidemic hand,

foot and mouth disease caused by human enterovirus 71, Singapore Emerging infectious

diseases, 9, 78–85

Chan, L.G., Parashar, U.D., Lye, M.S., Ong, F.G., Zaki, S.R., Alexander, J.P., Ho, K.K., Han, L.L., Pallansch, M.A., Suleiman, A.B., Jegathesan, M & Anderson, L.J (2000) 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 Clinical infectious diseases, 31, 678–683

Chang, G.-H., Lin, L., Luo, Y.-J., Cai, L.-J., Wu, X.-Y., Xu, H.-M & Zhu, Q.-Y (2010)

Sequence analysis of six enterovirus 71 strains with different virulences in humans Virus

research, 151, 66–73

Chang, L., King, C., Hsu, K & Ning, H (2002) Risk Factors of Enterovirus 71 Infection and Associated Hand, Foot, and Mouth Disease/Herpangina in Children During an Epidemic

in Taiwan Pediatrics 109, e88

Chang, L.-Y., Chang, I.-S., Chen, W.-J., Huang, Y.-C., Chen, G.-W., Shih, S.-R., Juang, L., Shih, H.-M., Hsiung, C.A., Lin, T.-Y & Huang, L.-M (2008) HLA-A33 is associated

J.-with susceptibility to enterovirus 71 infection Pediatrics, 122, 1271–1276

Chang, L.-Y., Hsia, S.-H., Wu, C.-T., Huang, Y.-C., Lin, K.-L., Fang, T.-Y & Lin, T.-Y (2004) Outcome of enterovirus 71 infections with or without stage-based management:

1998 to 2002 The Pediatric infectious disease journal, 23, 327–332

Chang, L.-Y., Tsao, K.-C., Hsia, S.-H., Shih, S.-R., Huang, C.-G., Chan, W.-K., Hsu, K.-H., Fang, T.-Y., Huang, Y.-C & Lin, T.-Y (2004) Transmission and clinical features of

enterovirus 71 infections in household contacts in Taiwan JAMA, 291, 222–227

Chang, S.-C., Lin, J.-Y., Lo, L.Y.-C., Li, M.-L & Shih, S.-R (2004) Diverse apoptotic

pathways in enterovirus 71-infected cells Journal of neurovirology, 10, 338–349

Chen, C.-S., Yao, Y.-C., Lin, S.-C., Lee, Y.-P., Wang, Y.-F., Wang, J.-R., Liu, C.-C., Lei, H.-Y & Yu, C.-K (2007) Retrograde axonal transport: a major transmission route of

enterovirus 71 in mice Journal of virology, 81, 8996–9003

Chen, C.Y., Chang, Y.C., Huang, C.C., Lui, C.C., Lee, K.W & Huang, S.C (2001) Acute flaccid paralysis in infants and young children with enterovirus 71 infection: MR

imaging findings and clinical correlates American journal of neuroradiology, 22, 200–

205

Chen, H & Schifferli, D (2007) Comparison of a fimbrial versus an autotransporter display

system for viral epitopes on an attenuated Salmonella vaccine vector Vaccine, 25,

1626-1633

Chen, H., Wang, L., Chang, C., Yen, C., Cheng, W., Wu, S., Hung, C., Kuo, M & Chen, C (2008) Recombinant porcine lactoferrin expressed in the milk of transgenic mice protects

neonatal mice from a lethal challenge with enterovirus type 71 Vaccine, 26, 891-898

Chen, H.-F., Chang, M.-H., Chiang, B.-L & Jeng, S.-T (2006) Oral immunization of mice

using transgenic tomato fruit expressing VP1 protein from enterovirus 71 Vaccine, 24,

2944–2951

Chen, K.-T., Chang, H.-L., Wang, S.-T., Cheng, Y.-T & Yang, J.-Y (2007) Epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in Taiwan,

1998-2005 Pediatrics, 120, e244–52

Chen, L & Yeh, T (2009) Enterovirus 71 Infection of Human Immune Cells Induces the

Production of Proinflammatory Cytokines Journal of biomed laboratory science, 21, 83

Chen, T.-C., Chang, H.-Y., Lin, P.-F., Chern, J.-H., Hsu, J.T.-A., Chang, C.-Y & Shih, S.-R (2009) Novel antiviral agent DTriP-22 targets RNA-dependent RNA polymerase of

enterovirus 71 Antimicrobial agents and chemotherapy, 53, 2740–2747

Chen, T.-C., Liu, S.-C., Huang, P.-N., Chang, H.-Y., Chern, J.-H & Shih, S.-R (2008)

Antiviral activity of pyridyl imidazolidinones against enterovirus 71 variants Journal of

biomedical science, 15, 291–300

Chen, Y.-C., Yu, C.-K., Wang, Y.-F., Liu, C.-C., Su, I.-J & Lei, H.-Y (2004) A murine oral

enterovirus 71 infection model with central nervous system involvement The Journal of

general virology, 85, 69–77

Chiang, L.-C., Ng, L.-T., Cheng, P.-W., Chiang, W & Lin, C.-C (2005) Antiviral activities

of extracts and selected pure constituents of Ocimum basilicum Clinical and

experimental pharmacology & physiology, 32, 811–816

Chiu, C.H., Chu, C., He, C.-C & Lin, T.-Y (2006) Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica

serovar Typhimurium expressing VP1 of enterovirus 71 Microbes and infection, 8,

1671–1678

Choi, H.J., Lim, C.H., Song, J.H., Baek, S.H & Kwon, D.H (2009) Antiviral activity of

raoulic acid from Raoulia australis against Picornaviruses Phytomedicine, 16, 35–39

Chua, B.H., Phuektes, P., Sanders, S.A., Nicholls, P.K & McMinn, P.C (2008) The

molecular basis of mouse adaptation by human enterovirus 71 The Journal of general

virology, 89, 1622–1632

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 (1979) Enterovirus 71 isolated from cases of epidemic poliomyelitis-like

disease in Bulgaria Archives of virology, 60, 329–340

Chung, P.W., Huang, Y.C., Chang, L.Y., Lin, T.Y & Ning, H.C (2001) Duration of

enterovirus shedding in stool Journal of microbiology, immunology, and infection, 34,

167–170

Clay, C.C., Rodrigues, D.S., Ho, Y.S., Fallert, B.A., Janatpour, K., Reinhart, T.A & Esser,

U (2007) Neuroinvasion of fluorescein-positive monocytes in acute simian

immunodeficiency virus infection Journal of virology, 81, 12040–12048

clinicaltrials.gov A Clinical Trial for Inactivated Vaccine (Vero Cell) Against EV71 in

http://clinicaltrials.gov/ct2/show/study/NCT01313715 [Accessed July 25, 2011]

clinicaltrials.gov A Phase II, Safety and Efficacy Study of Inactivated EV 71 Vaccine

(Human Diploid Cell, KMB-17) in Chinese Infants Available at: http://clinicaltrials.gov/ct2/show/NCT01512706?term=EV71&rank=4 [Accessed March

13, 2012]

clinicatrials.gov A Study to Evaluate the Safety and Immunogenicity of EV71 Vaccine

Available at: http://clinicaltrials.gov/ct2/show/NCT01268787?term=EV71&rank=8 [Accessed March 13, 2012]

Cornell, C.T., Kiosses, W.B., Harkins, S & Whitton, J.L (2006) Inhibition of protein

trafficking by coxsackievirus b3: multiple viral proteins target a single organelle Journal

Couper, K.N., Blount, D.G & Riley, E.M (2008) IL-10: the master regulator of immunity to

infection Journal of immunology, 180, 5771–5777

Cunningham, A.L., Donaghy, H., Harman, A.N., Kim, M & Turville, S.G (2010)

Manipulation of dendritic cell function by viruses Current opinion in microbiology, 13,

Golgi, thereby inhibiting protein trafficking through the Golgi The Journal of biological

chemistry, 281, 14144–14150

De Palma, A.M., Pürstinger, G., Wimmer, E., Patick, A.K., Andries, K., Rombaut, B., De Clercq, E & Neyts, J (2008) Potential use of antiviral agents in polio eradication

Emerging infectious diseases, 14, 545–551

Dieu, M.C., Vanbervliet, B., Vicari, A., Bridon, J.M., Oldham, E., Aït-Yahia, S., Briere, F., Zlotnik, A., Lebecque, S & Caux, C (1998) Selective recruitment of immature and

mature dendritic cells by distinct chemokines expressed in different anatomic sites The

Journal of experimental medicine, 188, 373–386

Dodd, D.A., Giddings, T.H & Kirkegaard, K (2001) Poliovirus 3A protein limits

interleukin-6 (IL-6), IL-8, and beta interferon secretion during viral infection Journal of

virology, 75, 8158–8165

Douche-Aourik, F., Berlier, W., Féasson, L., Bourlet, T., Harrath, R., Omar, S., Grattard, F., Denis, C & Pozzetto, B (2003) Detection of enterovirus in human skeletal muscle from patients with chronic inflammatory muscle disease or fibromyalgia and healthy subjects

Journal of medical virology, 71, 540–547

Drake, J.W & Holland, J.J (1999) Mutation rates among RNA viruses Proceedings of the

National Academy of Sciences, 96, 13910–13913

Eberle, K.E., Nguyen, V.T & Freistadt, M.S (1995) Low levels of poliovirus replication in

primary human monocytes: possible interactions with lymphocytes Archives of virology,

Nature, 314, 548–550

Fauquet, C (2005) Virus taxonomy: classification and nomenclature of viruses: eighth report

of the International Committee on the Taxonomy of Viruses International Union of Microbiological Societies Virology Division

Fearon, D.T & Locksley, R.M (1996) The instructive role of innate immunity in the

acquired immune response Science, 272, 50–53

Foo, D., Alonso, S., Phoon, M., Ramachandran, N., Chow, V & Poh, C (2007) Identification of neutralizing linear epitopes from the VP1 capsid protein of Enterovirus

71 using synthetic peptides Virus research, 125, 61–68

Foo, D., Ang, R., Alonso, S., Chow, V., Quak, S & Poh, C (2007) Identification of immunodominant VP1 linear epitope of enterovirus 71 (EV71) using synthetic peptides

for detecting human anti-EV71 IgG antibodies in western blots Clinical microbiology

and infection 14, 286-288

Foo, D.G.W., Alonso, S., Tak Kwong Chow, V & Poh, C.L (2007) Passive protection against lethal enterovirus 71 infection in newborn mice by neutralizing antibodies

elicited by a synthetic peptide Microbes and infection, 9, 1299–1306

Fu, Y.-C., Chi, C.-S., Jan, S.-L., Wang, T.-M., Chen, P.-Y., Chang, Y., Chou, G., Lin, C.-C., Hwang, B & Hsu, S.-L (2003) Pulmonary edema of enterovirus 71 encephalomyelitis is

associated with left ventricular failure: implications for treatment Pediatric

pulmonology, 35, 263–268

Fu, Y.C., Chi, C.S., Chiu, Y.T., Hsu, S.L., Hwang, B., Jan, S.L., Chen, P.Y., Huang, F.L &

Chang, Y (2004) Cardiac complications of enterovirus rhombencephalitis Archives of

disease in childhood, 89, 368–373

Gijbels, K., Brocke, S., Abrams, J.S & Steinman, L (1995) Administration of neutralizing antibodies to interleukin-6 (IL-6) reduces experimental autoimmune encephalomyelitis and is associated with elevated levels of IL-6 bioactivity in central nervous system and

circulation Molecular medicine, 1, 795–805

Gilbert, G.L., Dickson, K.E., Waters, M.J., Kennett, M.L., Land, S.A & Sneddon, M (1988) Outbreak of enterovirus 71 infection in Victoria, Australia, with a high incidence of

neurologic involvement The Pediatric infectious disease journal, 7, 484–488

Girndt, M & Kohler, H (2003) Interleukin-10 (IL-10): an update on its relevance for

cardiovascular risk Nephrology, dialysis, transplantation, 18, 1976–1979

Gitlin, L., Stone, J.K., Andino, R (2005) Poliovirus escape from RNA interference: short

interfering RNA-target recognition and implications for therapeutic approaches Journal

of Virology 79, 1027-1035

Gordon, S & Taylor, P.R (2005) Monocyte and macrophage heterogeneity Nature Reviews

Immunology, 5, 953–964

Gromeier, M., Mueller, S., Solecki, D., Bossert, B., Bernhardt, G & Wimmer, E (1997)

Determinants of poliovirus neurovirulence Journal of neurovirology, 3, 35–8

Hagiwara, A., Tagaya, I & Yoneyama, T (1978) Epidemic of hand, foot and mouth disease

associated with enterovirus 71 infection Intervirology, 9, 60–63

Halim, S & Ramsingh, A.I (2000) A point mutation in VP1 of coxsackievirus B4 alters

antigenicity Virology, 269, 86–94

Han, J.-F., Cao, R.-Y., Deng, Y.-Q., Tian, X., Jiang, T., Qin, E.-D & Qin, C.-F (2011) Antibody dependent enhancement infection of enterovirus 71 in vitro and in vivo

Virology journal, 8, 106

Hashimoto, I & Hagiwara, A (1982) Pathogenicity of a poliomyelitis-like disease in monkeys infected orally with enterovirus 71: a model for human infection

Neuropathology and applied neurobiology, 8, 149–156

Hashimoto, I & Hagiwara, A (1983) Comparative studies on the neurovirulence of temperature-sensitive and temperature-resistant viruses of enterovirus 71 in monkeys

Acta neuropathologica, 60, 266–270

Hashimoto, I., Hagiwara, A & Kodama, H (1978) Neurovirulence in cynomolgus monkeys

of enterovirus 71 isolated from a patient with hand, foot and mouth disease Archives of

virology, 56, 257–261

Hasko, G (2001) Receptor-mediated interaction between the sympathetic nervous system and

immune system in inflammation Neurochemistry Research, 26, 1039-1044

Hawkes, R (1964) Enhancement of infectivity of arboviruses by specific antisera produced

in domestic fowls Australian Journal of Experimental Biology and Medical Science, 42,

465-482

Heremans, H., Dillen, C., Put, W., Van Damme, J & Billiau, A (1992) Protective effect of anti-interleukin (IL)-6 antibody against endotoxin, associated with paradoxically

increased IL-6 levels European journal of immunology, 22, 2395–2401

Herrero, L.J., Lee, C.S.M., Hurrelbrink, R.J., Chua, B.H., Chua, K.B & McMinn, P.C (2003) Molecular epidemiology of enterovirus 71 in peninsular Malaysia, 1997-2000

Archives of virology, 148, 1369–1385

Hintzen, G., Ohl, L., del Rio, M.-L., Rodriguez-Barbosa, J.-I., Pabst, O., Kocks, J.R., Krege, J., Hardtke, S & Förster, R (2006) Induction of tolerance to innocuous inhaled antigen relies on a CCR7-dependent dendritic cell-mediated antigen transport to the bronchial

lymph node Journal of immunology, 177, 7346–7354

Ho, M., Chen, E.R., Hsu, K.H., Twu, S.J., Chen, K.T., Tsai, S.F., Wang, J.-R & Shih, S.R

(1999) An epidemic of enterovirus 71 infection in Taiwan New England journal of

medicine, 341, 929–935

Ho, S.Y., Chua, S.Q., Foo, D.G.W., Locht, C., Chow, V.T., Poh, C.L & Alonso, S (2008)

Highly attenuated Bordetella pertussis strain BPZE1 as a potential live vehicle for

delivery of heterologous vaccine candidates Infection and immunity, 76, 111–119

Hoffmann, J.A., Kafatos, F.C., Janeway, C.A & Ezekowitz, R.A (1999) Phylogenetic

perspectives in innate immunity Science, 284, 1313–1318

Hogle, J.M., Chow, M & Filman, D.J (1985) Three-dimensional structure of poliovirus at

2.9 A resolution Science, 229, 1358–1365

Hsueh, C., Jung, S.M., Shih, S.R., Kuo, T.T., Shieh, W.J., Zaki, S., Lin, T.Y., Chang, L.Y., Ning, H.C & Yen, D.C (2000) Acute encephalomyelitis during an outbreak of enterovirus type 71 infection in Taiwan: report of an autopsy case with pathologic,

immunofluorescence, and molecular studies Modern pathology, 13, 1200–1205

Huang, C.C., Liu, C.-C., Chang, Y.C., Chen, C.Y., Wang, S.T & Yeh, T.F (1999)

Neurologic complications in children with enterovirus 71 infection New England

Hung, H.-C., Chen, T.-C., Fang, M.-Y., Yen, K.-J., Shih, S.-R., Hsu, J.T.-A & Tseng, C.-P (2010) Inhibition of enterovirus 71 replication and the viral 3D polymerase by

aurintricarboxylic acid Journal of antimicrobial chemotherapy, 65, 676–683

Hühn, M.H., Hultcrantz, M., Lind, K., Ljunggren, H.-G., Malmberg, K.-J & Tullberg, M (2008) IFN-gamma production dominates the early human natural killer

Flodström-cell response to Coxsackievirus infection Cellular microbiology, 10, 426–436

Ishihara, K & Hirano, T (2002) IL-6 in autoimmune disease and chronic inflammatory

proliferative disease Cytokine & growth factor reviews, 13, 357–368

Iwai, M., Masaki, A., Hasegawa, S., Obara, M., Horimoto, E., Nakamura, K., Tanaka, Y., Endo, K., Tanaka, K., Ueda, J., Shiraki, K., Kurata, T & Takizawa, T (2009) Genetic changes of coxsackievirus A16 and enterovirus 71 isolated from hand, foot, and mouth

disease patients in Toyama, Japan between 1981 and 2007 Japanese journal of

infectious diseases, 62, 254–259

Jackson, W.T., Giddings, T.H., Taylor, M.P., Mulinyawe, S., Rabinovitch, M., Kopito, R.R

& Kirkegaard, K (2005) Subversion of cellular autophagosomal machinery by RNA

viruses PLoS biology, 3, e156

Jaks, E., Gavutis, M., Uzé, G., Martal, J & Piehler, J (2007) Differential receptor subunit

affinities of type I interferons govern differential signal activation Journal of molecular

biology, 366, 525–539

Jee, Y.M., Cheon, D.-S., Kim, K., Cho, J.H., Chung, Y.-S., Lee, J., Lee, S.H., Park, K.S., Lee, J.-H., Kim, E.-C., Chung, H.J., Kim, D.-S., Yoon, J.-D & Cho, H.-W (2003) Genetic analysis of the VP1 region of human enterovirus 71 strains isolated in Korea

during 2000 Archives of virology, 148, 1735–1746

Kanda, T., McManus, J.E., Nagai, R., Imai, S., Suzuki, T., Yang, D., McManus, B.M & Kobayashi, I (1996) Modification of viral myocarditis in mice by interleukin-6

Circulation research, 78, 848–856

Kandolf, R., Canu, A & Hofschneider, P.H (1985) Coxsackie B3 virus can replicate in

cultured human foetal heart cells and is inhibited by interferon Journal of molecular and

cellular cardiology, 17, 167–181

Kao, S.J., Yang, F.L., Hsu, Y.H & Chen, H.I (2004) Mechanism of fulminant pulmonary

edema caused by enterovirus 71 Clinical Infectious Diseases, 12, 1784-1788

Kellermann, S., Hudak, S & Oldham, E (1999) The CC Chemokine Receptor-7 Ligands 6Ckine and Macrophage Inflammatory Protein-3ß Are Potent Chemoattractants for In

Vitro- and In Vivo-Derived Dendritic Cells Journal of Immunology, 162,

Khong, W.X., Foo, D.G.W., Trasti, S.L., Tan, E.L & Alonso, S (2011) Sustained high levels

of interleukin-6 contribute to the pathogenesis of enterovirus 71 in a neonate mouse

model Journal of virology, 85, 3067–3076

Kishimoto, T (2006) Interleukin-6: discovery of a pleiotropic cytokine Arthritis Research

and therapy, 8, 2

Kjerrulf, M., Grdic, D., Ekman, L., Schön, K., Vajdy, M & Lycke, N.Y (1997) gamma receptor-deficient mice exhibit impaired gut mucosal immune responses but

Interferon-intact oral tolerance Immunology, 92, 60–68

Kok, C.C., Phuektes, P., Bek, E & McMinn, P.C (2012) Modification of the untranslated

regions of human enterovirus 71 impairs growth in a cell-specific manner J Virol, 86,

542–552

Komatsu, H., Shimizu, Y., Takeuchi, Y., Ishiko, H & Takada, H (1999) Outbreak of severe

neurologic involvement associated with Enterovirus 71 infection Pediatric neurology,

20, 17–23

Kopf, M., Baumann, H., Freer, G., Freudenberg, M., Lamers, M., Kishimoto, T., Zinkernagel, R., Bluethmann, H & Köhler, G (1994) Impaired immune and acute-phase

responses in interleukin-6-deficient mice Nature, 368, 339–342

Kosai, K., Seki, M., Yanagihara, K., Nakamura, S., Kurihara, S., Izumikawa, K., Kakeya, H., Yamamoto, Y., Tashiro, T & Kohno, S (2008) Gabexate mesilate suppresses influenza

pneumonia in mice through inhibition of cytokines The Journal of international medical

research, 36, 322–328

Kramer, M., Schulte, B.M., Toonen, L.W.J., de Bruijni, M.A.M., Galama, J.M.D., Adema, G.J & van Kuppeveld, F.J.M (2007) Echovirus infection causes rapid loss-of-function