s2fieldsofstudy: Biology Medicine

text:
Identification of PTCSC3 as a Novel Locus for Large‐Vessel Ischemic Stroke: A Genome‐Wide Association Study

Background Ischemic stroke is a major cause of death and disability in the world. A major ischemic stroke subtype, large‐vessel ischemic stroke (large artery atherosclerosis; LAA), has been shown to have some genetic components in individuals of European ancestry. However, it is not clear whether the genetic predisposition to LAA stroke varies among ethnicities. We sought to identify genetic factors that contribute to LAA stroke in 2 independent samples of Han Chinese individuals. Methods and Results Novel genetic variants that predispose individuals to LAA stroke were identified using a genome‐wide association study (GWAS) of 444 individuals with LAA stroke and 1727 controls in a Han Chinese population residing in Taiwan. The study was replicated in an independent Han Chinese population comprising an additional 319 cases and 1802 controls. We identified 5 single‐nucleotide polymorphisms, including rs2415317 (P=3.10×10−8), rs934075 (P=4.00×10−9), rs944289 (P=3.57×10−8), rs2787417 (P=1.76×10−8), and rs1952706 (P=2.92×10−8), at one novel locus on chromosome 14q13.3 within PTCSC3 (encoding papillary thyroid carcinoma susceptibility candidate 3) that were associated with LAA stroke at genome‐wide significance (P<5×10−8). Conclusions Our data provide strong support for future studies on the role of PTCSC3 in the pathogenesis of LAA stroke and the association between LAA stroke development and thyroid function. In addition, these findings provide insights into the genetic basis of LAA stroke and identify a novel pathway that might be applicable for future therapeutic intervention.

S troke is a major cause of acquired disability and the second-leading cause of death in the world. 1,2 The primary causes of stroke are unclear 3 ; however, family studies indicate that genetic factors may be involved. 4,5 A previous study demonstrated that 70% to 85% of strokes are ischemic, with the percentage varying by ethnicity. 6 Based on a nationwide surveillance program in Taiwan, including clinical data from 30 599 stroke admissions, 7 ischemic stroke is most frequent (74%), followed by intracerebral hemorrhage (16.1%), transient ischemic attack (6.7%), subarachnoid hemorrhage (2.8%), and cerebral venous thrombosis (0.2%).
Ischemic stroke includes 3 major subtypes according to the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) criteria: large-artery atherosclerosis (LAA); cardioembolism; and small-vessel occlusion. These subtypes may have different etiologies, resulting in disease subtype-specific processes.
The LAA subtype, for example, has been shown to be more strongly correlated with family history than other subtypes. 8 Recent genome-wide association studies (GWASs) of LAA stroke subtypes conducted in Australian populations have reported 1 susceptibility loci in 6p21. 1. 9 In addition, the International Stroke Genetics Consortium and the Wellcome Trust Case Control Consortium 2 have also identified a replicated genetic association with ischemic stroke by conducting a GWAS examining the major specific stroke subtypes. 10 Notably, based on 844 LAA stroke cases in a discovery population from 4 centers in Europe, a genetic variant at chromosome 7p21.1 (ie, HDAC9, which encodes the histone deacetylase [HDAC] 9 protein) is specifically associated with the LAA stroke subtype. 10 This association has been replicated in an independent Caucasian group. 11 However, although existing genetic data in Caucasian have demonstrated that unique genetic variants may predispose patients to a specific subtype of ischemic stroke, 9,10 it remains unclear whether other genetic variants are associated with LAA stroke. Furthermore, it is also important to identify genetic factors by conducting a GWAS in other ethnics. Therefore, in this study, we aimed to clarify the contributions of complex genetic effects to the pathogenesis of LAA stroke by identification of novel susceptibility loci and to validate the associations between previously reported loci in different ethnic groups.

Study Design and Patients
This study was approved by the Institutional Review Board and the Ethics Committee of the Institutional Review Board of Chang Gung Memorial Hospital and Academia Sinica, Taiwan. Written informed consent was obtained from the patients or their family members, in accord with institutional requirements and the principles of the Declaration of Helsinki. Individuals with LAA stroke (n=763; including the 444 patients with LAA stroke in the GWAS and the 319 patients with LAA stroke in the replication study) were consecutively recruited at different geographic medical centers, including Chang Gung Memorial Hospital Taipei Branch, Chang Gung Memorial Hospital Linkou Medical Center, Chang Gung Memorial Hospital Chiayi Branch, and Chang Gung Memorial Hospital Kaohsiung Medical Center (from the northern to southern regions of Taiwan). Patient enrollment and data management were performed in collaboration with the Translational Resource Center for Genomic Medicine of Taiwan. All of the cases were diagnosed according to TOAST criteria. 12 In addition, the cases received carotid ultrasound and transcranial color-coded Doppler to screen for large vessel disease. In those cases with suspected large vessel disease, digital subtraction, magnetic resonance, or computed tomographic angiography was performed for further confirmation. The clinical and imaging data were centralized and the classification of intracranial and extracranial LAA was identified and confirmed by Dr Tsong-Hai Lee. In the present study, we have included both intracranial atherosclerosis and extracranial atherosclerosis. Because identification of effects specific to more-refined phenotypes is critical for genetic studies of stroke, 9,10 we focused our GWAS on patients with carotid artery stenosis attributed to atherosclerotic mechanisms. 13 The controls used for our discovery and replication studies were independent groups for comparison in the GWAS study. The 1727 controls in the GWAS were randomly selected from the Taiwan Han Chinese Cell and Genome Bank in Taiwan, as reported previously. 14 The single-nucleotide polymorphism (SNP) genotyping results of another independent group of 1802 controls in the replication study were randomly selected from the publically available summary frequency of the Taiwan Biobank Website (https://taiwanview.twbiobank.org.tw/taiwanview/twbchipinfo.do).

Genotyping and Quality Control
Genomic DNA was extracted from blood using a Puregene DNA Isolation Kit (Gentra Systems, Inc., Minneapolis, MN). Each individual was genotyped using the Axiom Genome-Wide CHB (with 642 832 SNPs), which is the most comprehensive commercially available genome-wide coverage of the Han Chinese population, according to the manufacturer's protocols, at the National Center for Genome Medicine, Academia Sinica. All of the sample call rates were greater than 99%, and the mean individual sample call rate was 99.7AE0.18%. Firstdegree relatives (parent-offspring and full sibling pairs) of patients with LAA stroke disease and controls were identified by kinship analysis and were excluded from further analysis. Genotyping quality control for each SNP was further evaluated by determining the total call rate (successful call rate) and minor allele frequency (MAF) in cases and controls. SNPs were excluded from further analysis if only 1 allele was successfully genotyped in cases and controls to avoid experimental errors. The total call rate was less than 0.95, or the total MAF was less than 0.05 and the total call rate was less than 0.99. In addition, SNPs that departed significantly from Hardy-Weinberg equilibrium were excluded (P<1910 À4 ).

Statistical Analysis
We estimated the variance inflation factor for genomic control. Genome-wide association analysis was carried out to compare allele and genotype frequencies between cases and controls, using the Cochran-Armitage trend test implemented in PLINK 1.07. Heterogeneity tests (I 2 and P values of the Q statistics) between GWASs and replication groups were performed using the described methods. 15

Validation and Replication
The top 58 SNPs (P<5910 À5 ) from the genome-wide association analysis of the 444 patients with LAA stroke and 1727 controls were further validated in 444 patients with LAA stroke, using either matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MassARRAY; Sequenom, Inc., San Diego, CA) or direct sequencing. Fortyone SNP genotypes with a successful rate of over 99% and over 99% concordance between 2 platforms were then genotyped in an additional 319 patients with LAA stroke for replication.

Results
We performed a case-control GWAS to identify loci associated with increased risk of LAA stroke in the Han Chinese population by using an Affymetrix Axiom CHB array containing 642 832 SNP probes (Affymetrix, Inc., San Diego, CA). We initially enrolled 444 patients with LAA stroke and 1727 controls in a Han Chinese population residing in Taiwan. After kinship analysis and strict quality control filtering, we analyzed 570 275 SNPs (representing 89% of array SNPs) in the discovery stage. Multidimensional scaling analysis ( Figure 1A and 1B) and results of permutation tests for identity-by-state revealed no evidence for strong population stratification between LAA and control groups. Quantilequantile (Q-Q) plots were used to examine P-value distributions ( Figure 1C), and the lambda value is 1.07. In total, we found 58 SNPs associated with LAA (P<5910 À5 ). Forty-one SNPs were validated using either Sequenom MassARRAY or direct sequencing ( Figure 2 and Table 1) and subsequently replicated in an independent cohort of 319 patients with LAA and 1802 controls ( Table 2). In a combined analysis of the discovery and replication cohorts, the P values of 8 of the identified SNPs were lower than 10 À6 (Table 3), and 5 exceeded the threshold for genome-wide significance in the joint analysis (P<5910 À8 ; Table 3). We observed no strong evidence of heterogeneity between samples from the discovery and the replication study for these 5 SNPs (I 2 =0).

Discussion
In this study, we sought to identify novel genetic variations that predisposed individuals to LAA stroke in a Han Chinese population residing in Taiwan. From 2 independent groups (Table 4), we found 5 new SNPs within the PTCSC3 gene for LAA stroke that reached genome-wide statistical significance. These findings provide insights into the genetic basis of LAA stroke and identify a novel pathway that may be applicable for future therapeutic interventions.
In the discovery stage, we imputed 134 SNPs with P value <1910 À3 . Notably, the rs934075 and rs944258 SNPs with high P value (<1910 À5 ) were directly genotyped in our discovery sample, whereas rs934075 could be imputed with an equally high statistical value (<1910 À5 ). In addition, the SNP rs944289, located in chromosome 14q13.3, is associated with an increased risk of papillary thyroid cancer (PTC) based on a GWAS including patients with thyroid cancer in Iceland, Spain, and the United States. 16 This SNP is located in the region upstream of a long intergenic noncoding RNA (lincRNA), named PTCSC3, and functional studies have indicated that this lincRNA is involved in PTC susceptibility. 17 PTCSC3 is highly expressed in the thyroid and weakly expressed in the kidney, 17 and the downregulation of PTCSC3 in thyroid tumors of PTC patients is significantly associated with the risk allele (T) in rs944289. 17 In addition, the risk allele (T) for PTC in the SNP rs944289 has also been shown to be associated with a low concentration of thyroid-stimulating hormone in the general population, 16 and a recent report also indicated that hyperthyroidism may be associated with an increased risk for ischemia stroke in young adults. 18 It raises a likelihood that PTCSC3 may affect thyroid function, and the disruption of thyroid function may, in turn, be associated with a worse cardiovascular risk factor profile, potentially leading to progression of atherosclerosis. 19 Therefore, further studies are warranted to investigate the mechanism through which PTCSC3 is involved in the susceptibility for LAA.
In a previous GWAS conducted in Caucasians, the SNP rs11984041, which had the strongest significance (ie, the lowest P values) for LAA stroke, was shown to be located in the final intron of the HDAC9 gene. 10 Because this SNP (rs11984041) is not polymorphic in the Han Chinese population, an additional study investigated other potential SNPs associated with HDAC9 in Han Chinese individuals. 20 This previous report showed that there are 2 SNPs, located in the different intron of HDAC9 (ie, not in LD with rs11984041), which may be associated with LAA in Han Chinese individuals. 20 However, the P values of these SNPs (rs2389995 and rs2240419) did not reach the first criterion (P<5910 À5 ) in the discovery stage of our GWAS. In the present study, 2 novel suggestive SNPs (rs2074633 and rs28688791) represent a different, more significant locus in the HDAC9 gene (ie, in LD with rs11984041) in Han Chinese individuals. The SNPs (rs28688791 and rs2074633) at chromosome 7p21 were associated with LAA in the discovery phase (P<5910 À5 ; Figure 2), and their P values in the joint analysis were close to reaching GWAS significance (rs28688791, P=3.66910 À7 ,  Table 3). The SNP rs2074633 was located in the 3 0 -untranslated region (UTR) exon of HDAC9, and the SNP rs28688791 was located downstream of the HDAC9 locus and in the 3 0 UTR of TWIST1 (encoding twist family bHLH transcription factor 1; Figure 3B) locus. Interestingly, a difference in the locations of the SNPs (rs28688791 and rs2074633) identified in Han Chinese and the SNP (rs11984041) identified in Caucasians implies multiple regulation pathways in expression of HDAC9 gene in LAA stroke.

Study Limitations
This was a case-control GWAS conducted in a Han Chinese population residing in Taiwan for LAA stroke, a subphenotype of ischemia stroke differentiated based on TOAST    criteria. Although the findings identified a novel locus, PTCSC3, associated with LAA stroke and provide insight into the pathogenic mechanism for LAA stroke, the interpretation of the results of this study require caution owing to the limited number of ethnic groups considered. Future investigations of LAA stroke in other populations from Asia will be critical to clarify whether these newly identified genetic variants for LAA stroke are shared in other populations. Although the SNPs with genome-wide significance could be identified based on the combined analysis, there was still an incomplete validation of these SNPs in an independent cohort, which may be one of the study limitation in the present study. Therefore, further validation studies are needed to warrant our findings. Each SNP is plotted with respect to its chromosomal location (x axis) and its Àlog 10 P values (left y axis) for the trend test from the primary GWAS scan and joint analysis at that region of the chromosome. The results from the discovery analysis and joint analysis for key SNPs are indicated using their rs numbers. GWAS indicates genome-wide association study; SNPs, single-nucleotide polymorphisms.  Figure 4. LD structure and logistic regression analyses in PTCSC3 region. A, shows Àlog10 (P values) of SNPs for the best test from the primary scan as a function of genomic positions for PTCSC3 region (B) LD patterns, D' value, and r 2 among the disease-associated SNPs in PTCSC3 region. LD indicates linkage disequilibrium; SNPs, single-nucleotide polymorphisms.

Conclusions
In the present study, we identified novel associations between LAA stroke and polymorphisms in the PTCSC3 gene based on 5 SNPs with GWAS-significant P values. Because the PTCSC3 gene also contains a risk locus correlated with PTC, our data provide strong support for future studies of the association between LAA and PTC. In addition, compared to previous SNP studies in Han Chinese individuals, here, we observed 2 SNPs with more-significant P values located in the HDAC9 locus, which was in close proximity to the most significant SNP identified by GWAS in Caucasian individuals. These results support the notion that genetic predisposition to LAA may vary by ethnicity. In conclusion, our study revealed that the PTCSC3 signaling pathway may be involved in the pathogenesis of LAA stroke and that PTCSC3 may potentially serve as a therapeutic target for stroke prevention.

Sources of Funding
This study was supported by the Academia Sinica Genomic Medicine Multicenter Study, Taiwan (40-05-GMM) and Chang Gung Memorial Hospital (BMRP 274, CMRPG35072, CMRPG 35073, and CMRPG 39082). The funders had no role in study design, data collection or analysis, the decision to publish, or preparation of the manuscript. We gratefully acknowledge the members of the Translational Resource Center (NSC102-2325-B-001-023) of National Research Program for Biopharmaceuticals and the National Center for Genome Medicine (NSC102-2319-B-001-001) of National Core Facility Program for Biotechnology, National Science Council, at Academia Sinica for their support in subject recruitment, genotyping, and statistical analysis.  HDL-C indicates high-density lipoprotein; IQR, interquartile range; LDL-C, low-density lipoprotein; VLDL, very-low-density lipoprotein.


extfieldsofstudy: Biology Medicine

text:
Risk stratification and immunogenetic risk for infections following stem cell transplantation

ABSTRACT Patients undergoing haematopoietic stem cell transplantation (HSCT) are highly exposed to infectious agents. However, it is not known why certain HSCT recipients rapidly develop severe infections while other, despite similar immunosuppressive conditions, do not. Increasing evidence suggests that such differences may be due, in part, to polymorphisms in immune genes. Thus, the identification of genetic factors influencing susceptibility to infections in HSCT recipients may lead to the development of individualized management strategies. However, studies are challenged by several issues, including the relative small size of existing cohorts, the frequent use of prophylactic or preemptive antimicrobial agents, and the fact that genes responsible for immune functions can be inherited either from the donor or the host. Consequently, the major challenge for today's researchers is to overcome these limitations and find associations that are robust enough to be translated into reliable risk stratification strategies for infectious diseases.

KEYWORDS genetic susceptibility; haematopoietic stem cell transplantation; infection; risk stratification; single nucleotide polymorphism

Infectious complications and risk assessment of HSCT recipients
Opportunistic infections caused by bacteria, fungi and viruses represent a major challenge in patients undergoing haematopoietic stem cell transplantation (HSCT). HSCT is characterized by different phases, each associated with a different pattern of immunosuppression. The risk of infection is particularly important for allogeneic HSCT recipients, in which 3 distinct periods are typically described (Table 1). 1,2 The pre-engraftment period starts with the beginning of the conditioning regimen and extends up to 1 month after transplantation. This phase is characterized by profound neutropenia, which together with the alteration of natural host defense barriers (chemotherapy-induced mucositis, presence of venous catheters) make patients at risk to develop severe bacterial infections (i.e. bacteremia due to Enterobacteria, viridans Streptococci, Enterococci or coagulase-negative Staphylococci). The same factors combined with the extensive use of antibacterial agents subsequently expose patients to the risk of developing invasive fungal infections, in particular candidemia, hepatosplenic candidiasis and early onset invasive aspergillosis (IA). 3,4 The preengraftment phase is also characterized by T-cell depletion and can be associated with viral infections, such as Herpes-simplex virus (HSV) reactivation. 1,2 The post-engraftment period starts with neutrophils counts recovery and extends to »100 d after transplantation, at the time when T-cell function starts to recuperate. This phase is characterized by the occurrence of acute graftversus-host disease (mainly in allogeneic transplant recipients), which requires the administration of immunosuppressive drugs that further contribute to the impairment of T cell functions. This is also the time at risk for Cytomegalovirus (CMV) reactivation, which itself exerts some level of immunosuppression. 5,6 During this phase patients are still at risk for invasive fungal infections, i.e., late-onset aspergillosis 3,4 and Pneumocystis jirovecii pneumonia. 1,2 The late risk period is mainly characterized by the occurrence of chronic graft-vs.-host disease, which further extends the period at risk for infection due to CMV, 5,6 Pneumocystis and Aspergillus, and can also be characterized by infection due to Varicella-zoster virus (VZV) and pyogenic bacteria. 1,2 While these phases are well characterized and the occurrence of corresponding infections quite reproducible at the population level, the pattern of infection at the individual level is difficult to predict. Despite similar underlying conditions, similar age group and similar conditioning regimen, some patients rapidly or repeatedly develop severe infections with one or several pathogens, while other do not. An increasing number of studies have shown that individual susceptibility to infections in immunocompetent patients is influenced by genetic polymorphisms. 7-10 By analogy, it has been hypothesized that susceptibility to infections in immunocompromised patients may also result, in part, from polymorphisms in immune genes. However, such investigations in strongly immunocompromised patients raise very specific questions.
First, HSCT represents an iatrogenic situation in which the infectious pattern is different from that observed in the general population, as infections are more frequent, more severe, and often due to different (so-called opportunistic) pathogens. On the one hand, the large number of infections within a limited number of patients may contribute to increase the statistical power to detect genetic associations. On other hand, HSCT patients represent a limited population, making it difficult to gather very large cohorts of patients as it can be done in a much easier way for common diseases. However, the iatrogenic condition may reveal genetic factors influencing hosts and pathogens interactions that have not been encountered during their long co-evolution. Second, because the phases of allogeneic HSCT are typically associated with specific risks, different prophylactic strategies have been developed to prevent the occurrence of frequent infections and/or to limit their consequences, which often differ depending on the center and local epidemiology. The extended use of anti-microbial drugs during these periods (either as a prophylaxis or for the treatment of suspected or proven infections) can dramatically influence the type of infections and pathogens' resistance patterns, thereby inducing major biases and limiting the ability to compare results from studies. Finally, allogeneic transplant recipients represent chimerical individuals in which a part of immunity is inherited from the donor (e.g. white blood cells), while another part comes from the recipient (e.g., epithelial cells, proteins produced in the liver), so that the polymorphisms responsible for susceptibility to infections may originate either from the donor or from the recipient, or both of them.
The correlation between single nucleotide polymorphisms (SNPs) and disease phenotypes can be assessed by performing association studies. Such studies can be conducted in a candidate genes approach, in which polymorphisms are selected based on the hypothesis that they can influence the function of a specific gene or group of genes and subsequent immune response to the pathogen (hypothesis driven approach). They can also be conducted in the genome-wide approach (genomewide association studies, GWAS, or whole genome/ exome sequencing) in which millions of polymorphisms throughout the entire genome are interrogated (hypothesis free approach). 11,12 Up to now, to our knowledge, no genetic study exploring the association of polymorphisms within the whole genome with infectious phenotypes has been published in HSCT recipients. This may be due to the complexity and heterogeneity of the HSCT procedures, as well as the relative small number of patients presenting the phenotypes of interest. Even for candidate genes, the majority of association studies published so far are challenged by a small sample size, heterogeneous definition of cases and controls, differences in patients' managements between different centers. Also, studies were restricted by the lack of replication and functional evaluation of associated polymorphisms, a restricted number of studied genes and SNPs, the lack of corrections for covariates and multiple tests. Despite Anti-viral prophylaxis or empirical treatment Anti-fungal prophylaxis these limitations, some associations seem to be relevant, because they have been replicated by one or several investigators, or are supported by strong functional evidence.
Because the innate immune system is at the interface of host and pathogen, most candidate gene studies focus on innate immune genes. The innate immune system is composed of physical barriers (skin and mucous membranes), cellular elements (monocytes, macrophages, neutrophils, dendritic cells, mast cells, natural killer [NK] cells) as well as soluble factors (cytokines, chemokines and other), which all contribute to contain the spread of infection. At the molecular level, pathogenassociated molecular patterns (PAMPs) are detected by specific receptors (PRRs), including toll-like receptors (TLRs), NOD-like receptors (NODs), c-type lectin receptors (CLRs) and RIG-I-like receptors (RLRs). Microbes can be neutralized by proteins of the complement system, thereby preventing their entrance into the cells, increasing the ability of immune cells to detect the pathogens (opsonization). Effective innate responses are very important for establishing adaptive immune mechanisms, including cytotoxic T-cell responses and B-cell differentiation which are responsible for microbial clearance and maintenance.
Bacterial infections represent an important problem in HSCT recipients, mainly during the pre-engraftment period. Neutropenic patients who develop fever require the immediate administration of broad-spectrum antibiotics, which are usually administered for long periods of time, even when the episode is neither clinically nor microbiologically documented. Thus, the development of bacterial infection is largely determined by environmental factors (e.g. previous administration of antibiotics for the prophylaxis or treatment of neutropenic fever), which may render the role of genetic factors more difficult to uncover. This may explain why only a limited number of studies identified polymorphisms associated with bacterial infections. To our knowledge, polymorphisms from 7 genes have been associated with bacterial infections in HSCT recipients ( Table 2). Among them, 3 associations are relatively robust.
One of the most relevant associations was that of the promoter SNP rs2232582 in the gene encoding lipopolysaccharide-binding protein (LBP) with susceptibility to gram-negative bacterial infections in HSCT recipients. 13 The polymorphic, risk allele was present in the recipient. It was discovered in a group of »1200 patients and subsequently replicated in a smaller group of 230 patients. LBP binds to lipopolysaccharide (LPS), an essential component of gram-negative bacteria cell walls, and interacts with the surface pattern recognition receptor TLR4 (together with other molecules such as CD14 and MD2) to induce the NF-kB signaling pathways and subsequent production of pro-inflammatory molecules by innate immune cells. [14][15][16] This association was further supported by the fact that patients carrying one or 2 copies of rare allele for rs2232571 SNP (a SNP that is in strong linkage disequilibrium with rs2232582) had higher circulating blood LBP levels before transplantation. 17 A haplotypic combination of promoter variants in LBP (containing rs2232571) was associated with severe sepsis in immunocompetent individuals. 18 The second relevant association was observed between the 3020insC frameshift mutation (known as SNP13 or L1007finsC) within nucleotide-binding oligomerization domain containing 2/caspase recruitment domain-containing protein 15 (NOD2/CARD15) and the development of sepsis in HSCT recipients. The risk allele was observed in both the donor and the recipient in an initial study of 430 patients, 19 and in the donor (but not the recipient) in a subsequent, smaller study of 160 patients. 20 NOD2 is a PRR that belongs to NOD-like receptor family. It is the detector of muramyl dipeptide, a component of the cell wall of both gram-positive and gram-negative bacteria. The presence of 3020insC in NOD2/CARD15 is predicted to result in production of a truncated NOD2 protein. In vitro HEK293T cells transfected with plasmids containing 3020insC NOD2/ CARD15 mutant showed diminished relative NF-kB activity in response to LPS when compared with wildtype NOD2. 21 Another relevant association was found for haplotypes expressing low amounts of mannose-binding lectin (MBL2) and different types of bacterial infections in HSCT patients. [22][23][24][25] MBL2 is a PRR involved in complement activation and subsequently opsonization and phagocytosis of invading pathogens as well as apoptosis. MBL2 polymorphisms are frequent in the human population and classically classified into groups of haplotypes/ diplotypes that are strongly correlated with the serum levels (stratified into high, intermediate and low 26 ). Although these studies were limited by a relative small number of patients (e.g., < 150) and analyzed different phenotypes (bacterial infections due to gram-positive bacteria only, 22 bacterial infections due to either gramnegative and gram-positive bacteria 24,25 or bacterial sepsis 23 ) in different populations (autologous 22-24 versus allogeneic HSCT 25 ), they provided relatively consistent results. Overall, low MBL2 expressing haplotypes in autologous and allogeneic HSCT recipient were [23][24][25] or tended to be 22 associated with an increased risk of infection.
Fungal infections are still associated with an important mortality and morbidity in HSCT recipients. The epidemiology of invasive fungal infections is influenced by the use of antifungal prophylaxis (e.g. fluconazole, posaconazole, co-trimoxazole), which often differ according to the center, and influence the ability of investigators to detect genetic associations. To our knowledge, polymorphisms from 18 genes were significantly associated with invasive fungal infections among HSCT recipients, essentially with IA (Table 2). Among them, 3 associations are particularly robust, as they were performed in relatively large cohorts of patients, replicated by different groups and/or supported by in vitro experiments [27][28][29] (Fig. 1 and Table 2).
Two nonsynonymous SNPs in TLR4 (D299G and T399I) were associated with the risk of developing IA in HSCT recipients. 27 The polymorphism at risk was identified in HSCT donors. The association was replicated in the initial study, and subsequently validated by others. 30,31 TLR4 is a PRR essential for recognition of LPS from gram-negative bacteria as well as Olinked mannan from fungi. Functionally those 2 TLR4 polymorphisms were shown to affect lipopolysaccharide recognition by TLR4, 32 however their impact on detection of the fungi has been not clarified yet. From these studies and other emerged the concept by which the risk of developing invasive aspergillosis could be predicted before transplantation, by the identification of specific genetic polymorphisms together with other pre-transplant risk factors, such as CMV serostatus, both in HSCT donors and/ or the recipients. 27 A stop-codon polymorphism (Y238X) in CLEC7A, the gene encoding Dectin-1, was subsequently associated with IA in a cohort of »200 HSCT recipients. 28 A similar trend was observed in a cohort of »180 HSCT recipients. 33 In both studies, the risk was conferred by presence of the risk allele in either HSCT donors or recipients. Dectin-1 is a key PRR for b-glucan, an important component of fungal cell walls, and triggers inflammatory responses toward fungal pathogen, as well as their phagocytosis and killing. [34][35][36] Different lines of experiments suggested an effective role for Dectin-1 in cells from both HSCT donors and recipients. 28,33 SiRNA inhibition of CLEC7A in lung epithelial cells was associated with a diminished production of inflammatory cytokines after A. fumigatus stimulation, thereby suggesting an antifungal role for Dectin-1 in lung cells from the recipient. 28 Human PBMCs and monocytes carrying the Y238X SNP produced lower amounts of inflammatory cytokines upon Aspergillus stimulation compared to WT cells, thereby suggesting that susceptibility to this infection may also depend from impaired Dectin-1 function in donor cells. 28 A HSCT model of IA using different combinations of donor and recipient mice (WT and deficient in Dectin-1) further suggested a role for Dectin-1 in both donor and recipient derived cells in the immune response to Aspergillus. 28 The most relevant association between IA and genetic variant comes from the study showing the influence of donor haplotype in PTX3 (cluster of 281G and 734A SNPs) with IA in HSCT recipient. 29 This association was discovered in a study of 268 HSCT recipients and replicated in another study of 330 recipients. 29 The same haplotype was associated with IA in 2 cohorts of solid organ transplant recipients. 37,38 PTX3 encodes long pentraxin 3, a soluble PRR that detects galactomannan from the fungi. PTX3 induces many immune processes such as pathogen opsonization, phagocytosis, complement activation, as well as clearance of apoptotic cells. [39][40][41] Functional studies revealed the important role of the donor haplotype at risk in immunity to Aspergillus. 29,[42][43][44] The haplotype was associated with reduced PTX3 expression or production as well as reduced anti-Aspergillus phagocytic and killing activity of neutrophils. 29 Different mouse models of IA (immunocompetent, neutropenic and HSCT) using PTX3-deficient vs. WT mice 42,44 or wild type mice 42,43 with/without complementation with soluble PTX3, respectively, further supported the role of long PTX3 in protection from IA. The successful use of soluble PTX3 in animal models suggest that in the future, soluble molecules may be used in the clinical practice to complement defective immune functions in selected patients. 38,43,45 Viral infections represent an important problem during HSCT, mainly during the pre-and post-engraftment period. They result from the reactivation of a pre-existing virus in the recipient or from the transmission of a new virus from the donor. As it is the case for bacterial and fungal infections, prophylactic and preemptive strategies have been developed to reduce the burden of viral infections in these high-risk periods, thereby complicating the interpretation of genetic association studies. To our knowledge, polymorphisms in 13 genes showed significant association with the presentation of viral infections in HSCT recipients, essentially with CMV infection (Table 2). Yet, only few of them were consistently replicated and/or supported by some level of functional evidence.
Several investigators reported associations between the expression pattern of killer immunoglobulin-like receptors (KIR) genes in allogeneic HSCT donors and the risk of CMV infection in the recipient. KIR are encoded by 15 distinct loci within a »200kB region in the leukocyte receptor complex (chromosome 19q13.4) and interact with ligands from HLA class I molecules to regulate the immune function of NK cells. 46 Some KIR exert an activating action on NK cells, while other exerts an inhibitory action. Individuals selectively express    Notes. Ã D refers to the donor and R refers to the recipient.

ÃÃ
The strongest associations with replication or functional assessment are presented in italic.
different combinations of activating and/or inhibitory KIR genes (>50 haplotypes have been described so far), that have been shown to influence susceptibility to infectious diseases. [47][48][49] Four small studies ( 230 subjects) were relatively consistent in showing that recipients of donor haplotypes expressing a high number of activating KIRs had a lower risk of CMV infection compared to recipients of those expressing a lower number; however, these studies used different cut-offs (> 1 activating KIR gene versus < 1, 50,51 or >5 vs. < 5 52,53 ). Two studies showed that HSCT recipients of donor haplotypes containing the activating KIR2DS2 and KIR2DS4 genes had a reduced risk of CMV infection compared to those receiving other donor haplotypes. 53,54 The role of polymorphisms from 7 PRR genes on susceptibility to CMV infections has been investigated among HSCT recipients. Significant associations were observed for polymorphisms in 2 of them, TLR9 and dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN). TLR9 is a PRR recognizing unmethylated CpG motifs within viral DNA. Two TLR9 SNPs in HSCT donors (1174A/G and P545P SNPs, both in strong linkage disequilibrium) were associated with an increased risk of CMV infection in the recipients in 2 independent groups of HSCT patients (138 in the discovery study and 102 in the replication study). 55 Furthermore the -1237C/T promoter polymorphism in TLR9 (that is not in linkage disequilibrium with the 1174A/G and the P545P SNPs) was associated with an increased risk of CMV infection in Caucasian HSCT recipients in a single study of »220 subjects. 56 DC-SIGN is a macrophage and dendritic cell specific PRR recognizing viral glycoproteins. The presence of 2 promoter polymorphisms in DC-SIGN (-139C/T and -939G/A) was associated with and increased risk of CMV infection and/or disease in another study of HSCT recipients (»130 subjects). 57 Immature DCs from individuals carrying 2 copies of -139T and -939G alleles showed decreased expression of DC-SIGN as compared to DCs from individuals carrying 2 copies of -139C and -939A of those polymorphisms. 57 Yet, so far, this association was not investigated by others.
A study of 72 HSCT recipients reported associations between polymorphisms in 2 gene influencing T cells functions (CD28 and cytotoxic T-lymphocyte antigen-4, CTLA4) and CMV infection in HSCT recipients. 58 CD28 is a co-stimulatory molecules constitutively expressed by naive T cells. CD28 can bind CD80 and CD86 molecules on antigen presenting cells (APC) to initiate signaling pathway important in T-cell activation. 59 CTLA4 is a coinhibitory molecule that is expressed by T-cells upon stimulation and compete with CD28 to bind to CD80 and CD86, thereby playing a key role in the regulation of adaptive immune responses by T lymphocytes. 59 Both molecules are recognized as important factors determining the outcome of HSCT.

Conclusions/future standpoints
Increasing evidence from candidate gene studies suggest that polymorphisms in the donor and/or the recipient can influence susceptibility to bacterial, fungal and viral infections after HSCT. The identification of such markers may be useful in the clinical practice, as they may lead to the development of tailored strategies for the management of such patients. For instance, antimicrobial prophylaxes may be administered to patients undergoing a high risk to develop infections, thereby preventing the use of costly and sometimes toxic agents in patients who are at lower risk. However, many centers already use indiscriminate prophylaxis and/or preemptive treatment strategies that have proven to significantly reduce the infectious burden in this population. Alternatively, the use of immunosuppressive drugs after HSCT may be tailored to the individual risk, and/or novel immunomodulators (such as soluble pattern recognition receptors) may be developed to compensate for immune functions that are relatively deficient in selected individuals. However, studies have not translated into personalized management strategies so far. The major limitations include the fact that most studies identified genetic associations of a single polymorphism with a single pathogen. In the clinical practice, patients develop several infections either at the same time or sequentially, and each is influenced by a complex combination of demographic, clinical and genetic factors. The implementation of personalized approaches in the HSCT population would require the integration of these multiple factors within a global risk scoring system. Such approaches are currently limited by the relative small size of existing studies, the small number of available phenotypes as well as the limited number of genes and polymorphisms that have been tested. This goal could probably only be achieved with the implementation of very large cohorts of patients in several centers, with extensive clinical data and samples collection.


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MicroRNA and Pathogenesis of Enterovirus Infection

There are no currently available specific antiviral therapies for non-polio Enterovirus infections. Although several vaccines have entered clinical trials, the efficacy requires further evaluation, particularly for cross-strain protective activity. Curing patients with viral infections is a public health problem due to antigen alterations and drug resistance caused by the high genomic mutation rate. To conquer these limits in the development of anti-Enterovirus treatments, a comprehensive understanding of the interactions between Enterovirus and host cells is urgently needed. MicroRNA (miRNA) constitutes the biggest family of gene regulators in mammalian cells and regulates almost a half of all human genes. The roles of miRNAs in Enterovirus pathogenesis have recently begun to be noted. In this review, we shed light on recent advances in the understanding of Enterovirus infection-modulated miRNAs. The impacts of altered host miRNAs on cellular processes, including immune escape, apoptosis, signal transduction, shutdown of host protein synthesis and viral replication, are discussed. Finally, miRNA-based medication provides a promising strategy for the development of antiviral therapy.

MicroRNAs (miRNAs) are a newly discovered class of small non-protein-coding RNAs that act via endogenous RNA interference [27][28][29][30][31][32]. To date, more than 1000 cellular miRNAs have been identified that mediate the post-transcriptional regulation of more than 30% of animal genes [28,33]. MiRNA biogenesis is initiated by RNA polymerase II or III, and the newly synthesized primary miRNA transcripts (pri-miRNAs) are cleaved by RNase III and Drosha into precursor miRNAs (pre-miRNAs). Precursor miRNAs are transported into the cytoplasm and further processed by Dicer to form miRNA-miRNA duplexes [34][35][36][37][38]. The miRNA-miRNA duplexes harbor a guide strand that generally functions as the mature miRNA. Mature miRNAs associated with an Ago protein form an RNA-induced silencing complex (RISC) by which miRNAs suppress specific mRNAs by targeting complementary sites of specific mRNAs mainly located in mRNA 3 1 untranslated regions (3 1 UTRs) [39][40][41]. Occasionally, 5 1 UTRs and protein-coding regions can act as the potential binding sites for miRNAs [42][43][44][45]. By post-transcriptional regulation, miRNAs govern a wide range of biological functions, including cell proliferation, differentiation, apoptosis and host-pathogen interactions [46][47][48]. Certain DNA viruses encode viral miRNAs that are able to regulate viral or cellular gene expressions and to contribute to viral pathogenesis [49][50][51]. In contrast, some cellular miRNAs play a role in the virus life cycle, including viral genome replication and virus propagation [52,53].
In general, viruses are predisposed to evolve more genetic diversity to overcome environmental stresses and increase competitive advantages. However, this unique virus tendency generates various antigenic variations and potentiates drug resistance that impedes the development of effective antiviral therapies. Moreover, some viruses produce viral regulatory molecules, such as miRNAs and long non-coding RNAs (lncRNAs), to play a part in viral pathogenesis and life cycle [54][55][56][57]. On the other hand, viruses utilize cellular factors to hijack host bio-energy, evade immune attacks and enforce viral replication [58,59]. In this review, we discuss the emerging roles of cellular and virus-encoded miRNAs in host-pathogen interactions and provide potential strategies for antiviral therapies by manipulating such regulatory molecules.

Host miRNAs Participate in Antiviral Responses and Immune Escape in Enterovirus Infections
Generally, certain microbe-unique molecules, such as double-stranded RNA and cytidine-phosphate-guanosine DNA (CpG DNA), are recognized by host pattern-recognition receptors and activate endosomal toll-like receptor (TLR) signaling to produce type I interferons (IFNs), the first-line immune responses against viral infection [60][61][62][63]. The resulting IFNs can establish antiviral machinery in virus-infected subjects by inducing downstream interferon-stimulated genes, promoting T cell proliferation, stimulating IFNγ production and activating dendritic cells or natural killer cells [64][65][66][67]. Intriguingly, EV71 infection fails to elicit type I IFN production efficiently [68,69]. Ho and his colleagues found that miR-146a is induced in EV71 infection and further suppressed two critical components in interferon production, Interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6). Knocking out miR-146a or neutralizing virus-induced miR-146a restores IRAK1 and TRAF6 expression, augments IFNβ production, inhibits viral propagation and finally improves the survival of virus-infected neonatal mice [58]. In this case, the authors provided a clue to help develop preventive and therapeutic strategies against Enterovirus infections by manipulating miRNA expressions. Interferon regulatory factors can mediate the upregulation of miR-526a, which further targets Cylindromatosis (turban tumor syndrome), also known as CYLD, a well-known deubiquitinase, to enforce K63-linked RIG-I ubiquitination and then triggers IRF3 and NF-κB signaling. EV71 3C protease cleaves host Interferon regulatory factor 7 (IRF7) to block IRF-mediated miR-526a upregulation and then suppresses RIG-I-dependent IFNs production [70]. In addition to EV71, Coxsackie virus B3 (CVB3) infection also influences the host miRNA expression profile [71]. Zhang and his coworkers identified five differentially expressed miRNAs in CVB3-infected mice hearts using a miRNA microarray. As determined by bioinformatics analysis, these differentially expressed miRNAs might be involved in certain important immune and antiviral pathways, such as the Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction, Mitogen-activated protein kinase (MAPK) signaling pathway, Janus kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway, and natural killer cell-mediated cytotoxicity. In contrast, the host also manipulates miRNA expression to establish immune attacks or eliminate viral pathogenesis [72,73]. CVB3-infected subjects activate immune responses to attack the virus, while elevated immune responses might result in cardiac myocyte destruction, reparative fibrosis, myocarditis and even heart failure [72,74]. Both miR-155 and miR-148a were upregulated in cardiac biopsies from viral myocarditis patients caused by CVB3 infection. RelA, an important molecule in NF-κB signaling, was demonstrated in vitro as the direct target of these two upregulated miRNAs [72]. Subsequently, the regulatory role of miR-155 was demonstrated in mouse cardiac myocytes and in a mouse infection model due to the sequence conservation of miR-155 between human and mouse. MiR-155 targets RelA and functions as a negative regulator in the host immune system, by which cardiac myoblast cytokine expression is reduced and the infected subjects are protected from an overdriven immune response; thus, the survival is improved in CVB3 infection [72]. In addition, miRNA-548 mimics present an inhibitory effect on IFNλ1 by targeting its 3 1 UTR to regulate the IFNλ1-mediated antiviral response [73]. Li et al. [73] highlighted miR-548 as downregulated in both Vesicular stomatitis virus and EV71 infections, whereas hosts might establish an antiviral response. In these cases, hosts and viruses regulate cellular miRNAs expression to establish immune attacks against the virus infections or evolve immune escape machineries to create a beneficial situation for viral replication, respectively.

Host miRNAs Are Involved in Enterovirus Infection-Induced Apoptosis
Apoptosis is an important cellular defense mechanism, particularly in the early phase of pathogen infection, by which pathogen-infected cells can be eliminated. Although viral spread can be partly restricted, apoptosis may cause tissue damage in the late stages of infection. Several studies have indicated that apoptosis is the major pathogenic feature of Enterovirus infection, resulting in host cell death and tissue damage [75][76][77]. Hence, the roles of miRNAs in Enterovirus infection-induced apoptosis were investigated [55,78,79]. The researchers found that miR-21 expression was suppressed in CVB3-induced myocarditis, while ectopic miR-21 expression significantly alleviated CVB3-induced myocarditis, including myocardial injury recovery, myocarditis score reduction and increased survival rate [78]. Programmed Cell Death 4 (PDCD4) was validated as the other important miR-21 target involved in apoptosis in several cell types [80][81][82]. The authors illustrated the underlying molecular mechanism in which miR-21 serves as an anti-apoptotic molecule in viral myocarditis via targeting PDCD4 [78]. On the other hand, EV71 infection induced let-7b expression in SH-SY5Y cells, which directly targets cyclin D1 (CCND1), a key element in cell cycle and apoptosis processes [79]. In contrast, the inhibition of let-7b by 2 1 -O-Methyl-RNA restored CCND1 expression, reduced the G2/M phase and restored SH-SY5Y proliferation. This evidence suggests that the proliferation retardation and apoptosis of EV71-infected cells are at least partly attributed to miRNA-mediated mechanisms [79]. More recently, Chang and her colleagues found that miR-146a is upregulated, while miR-370 is downregulated in EV71 infection [55]. Son Of Sevenless Homolog 1 (SOS1) and Growth arrest and DNA-damage-inducible β (GADD45β) are the targets of miR-146a and miR-370, respectively. Further functional studies indicated that the silencing of miR-146a restores SOS1 expression and partially attenuates EV71-induced apoptosis, while the ectopic expression of miR-370 decreases EV71-induced GADD45β expression and diminishes apoptosis. Moreover, the co-expression of antagomiR-146a and miR-370 showed an additive effect on attenuating EV71-induced apoptosis [55]. These studies clearly demonstrate that host miRNAs play a critical role in Enterovirus infection-induced apoptosis by regulating the key elements that are involved in cellular apoptosis and imply that miRNAs might act as potential therapeutic candidates by attenuating Enterovirus infection-induced apoptosis [55,78,79]. The miRNAs that are involved in the regulation of Enterovirus-induced apoptosis are elucidated in Figure 1 and summarized in Table 1. EV71-induced GADD45β expression and diminishes apoptosis. Moreover, the co-expression of antagomiR-146a and miR-370 showed an additive effect on attenuating EV71-induced apoptosis [55]. These studies clearly demonstrate that host miRNAs play a critical role in Enterovirus infection-induced apoptosis by regulating the key elements that are involved in cellular apoptosis and imply that miRNAs might act as potential therapeutic candidates by attenuating Enterovirus infection-induced apoptosis [55,78,79]. The miRNAs that are involved in the regulation of Enterovirus-induced apoptosis are elucidated in Figure 1 and summarized in Table 1. Apoptosis is the major pathogenic feature of Enterovirus infections. MiR-21, which targets Programmed Cell Death 4 (PDCD4), is suppressed in CVB3-induced myocarditis and serves as an anti-apoptotic molecule. EV71-induced let-7b directly targets cyclin D1 (CCND1) and contributes to the retardation of host cell proliferation and the trigger of apoptosis. The other two altered miRNAs that were identified in EV71 infection, miR-146a and miR-370, target SOS1 and GADD45β, respectively. The suppression of SOS1 by miR-146a and the relief of GADD45β by miR-370 induce the apoptosis of EV71-infected host cells. These cellular miRNAs play critical roles in Enterovirus infection-induced apoptosis by regulating the key elements in the cellular apoptosis process.

Enterovirus Regulates Host Signaling Modulators Resulting in Pathogenesis
Many studies have clearly indicated that Enteroviral proteases cleave host factors and then lead to cellular or tissue malfunctions, such as apoptosis, translation shutdown and cytokine suppression, and finally cause pathogenesis [83][84][85][86][87][88][89][90]. For example, EV68 3C protease cleaved host cell TIR-domain-containing adapter-inducing interferon-β (TRIF), a key molecule downstream of TLR3, to abolish NF-κB signaling and IFN-β production and enabled viruses to escape the host innate immune response [83]. In addition to viral proteases, miRNAs have also been characterized as a critical players in host signaling transduction [54,55,58,78,79,[91][92][93][94][95][96][97]. In the investigation of Coxsackie virus-induced cardiovascular pathogenesis, the destruction of cell-cell interactions is one of its key mechanisms. Several components of membrane structure are the targets of Coxsackie virus-regulated miRNAs. CBV3-induced miR-21 enhances desmin degradation by targeting a deubiquitinating enzyme, YOD1 [54], and Gap junction protein alpha 1 (GPJ1) is the target of CBV3-induced miR-1 [93]. MiR-21 also activated mitogen-activated protein kinase (MAPK) signaling cascade by targeting sprouty homolog 1 (SPRY1), a native inhibitor of fibroblast growth factor (FGF) pathways [94]. Moreover, CBV3 infection triggered ERK1/2 activation, leading to miR-126 upregulation. The miR-126 played as a pivot to link the ERK1/2 and WNT/β-catenin signaling pathways and promoted CBV3 propagation [96]. Apoptosis is the major pathogenic feature of Enterovirus infections. MiR-21, which targets Programmed Cell Death 4 (PDCD4), is suppressed in CVB3-induced myocarditis and serves as an anti-apoptotic molecule. EV71-induced let-7b directly targets cyclin D1 (CCND1) and contributes to the retardation of host cell proliferation and the trigger of apoptosis. The other two altered miRNAs that were identified in EV71 infection, miR-146a and miR-370, target SOS1 and GADD45β, respectively. The suppression of SOS1 by miR-146a and the relief of GADD45β by miR-370 induce the apoptosis of EV71-infected host cells. These cellular miRNAs play critical roles in Enterovirus infection-induced apoptosis by regulating the key elements in the cellular apoptosis process.

Enterovirus Regulates Host Signaling Modulators Resulting in Pathogenesis
Many studies have clearly indicated that Enteroviral proteases cleave host factors and then lead to cellular or tissue malfunctions, such as apoptosis, translation shutdown and cytokine suppression, and finally cause pathogenesis [83][84][85][86][87][88][89][90]. For example, EV68 3C protease cleaved host cell TIR-domain-containing adapter-inducing interferon-β (TRIF), a key molecule downstream of TLR3, to abolish NF-κB signaling and IFN-β production and enabled viruses to escape the host innate immune response [83]. In addition to viral proteases, miRNAs have also been characterized as a critical players in host signaling transduction [54,55,58,78,79,[91][92][93][94][95][96][97]. In the investigation of Coxsackie virus-induced cardiovascular pathogenesis, the destruction of cell-cell interactions is one of its key mechanisms. Several components of membrane structure are the targets of Coxsackie virus-regulated miRNAs. CBV3-induced miR-21 enhances desmin degradation by targeting a deubiquitinating enzyme, YOD1 [54], and Gap junction protein alpha 1 (GPJ1) is the target of CBV3-induced miR-1 [93]. MiR-21 also activated mitogen-activated protein kinase (MAPK) signaling cascade by targeting sprouty homolog 1 (SPRY1), a native inhibitor of fibroblast growth factor (FGF) pathways [94]. Moreover, CBV3 infection triggered ERK1/2 activation, leading to miR-126 upregulation. The miR-126 played as a pivot to link the ERK1/2 and WNT/β-catenin signaling pathways and promoted CBV3 propagation [96]. Next, we focused on the role of miRNAs in EV71-induced neurological pathogenesis due to the severe neuronal complications in EV71-infected patients [21,24]. The EV71-induced miR-1246 directly repressed the expression of disc-large homolog 3 (DLG3), which is a member of the membrane-associated guanylate kinase protein family and is associated with mental disorders [95]. MiR-1246 might contribute to EV71-associated neurological pathogenesis by targeting DLG3. In addition to the direct destruction of neuronal components, the inflammatory reaction is one of main causes contributing to tissue damage, such as meningitis and encephalitis [101,102]. The increased Cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) induced by Enterovirus infection accelerated EV71 replication and reactive oxygen species (ROS) generation, and the upstream regulation mechanisms have been intensively elucidated [103][104][105]. Among these regulatory mediators, EGFR signaling plays a crucial role in EV71 replication in the human neuroblastoma cell line, SK-N-SH cells [103,104]. Zhang and his colleagues found that the expression of miR-27a is downregulated during EV71 infection and that miR-27a could target EGFR [97]. Further studies have shown that the ectopic expression of miR-27a suppresses EGFR expression and reduces Akt and ERK phosphorylation. Finally, the blockage of the EGFR signaling cascade attenuated EV71 replication [97]. The miRNAs that are involved in the regulation of host signaling pathways are summarized in Table 1.

Host miRNAs Are Involved in Enterovirus Infection-Induced Protein Synthesis Shutdown
It has long been believed that the viral protease-mediated cleavage of host factors that are involved in the host cap-dependent translation process might almost or even thoroughly account for the shutdown of host protein synthesis caused by Picornavirus infection [85,86,90,106,107]. Enterovirus 2A and 3C proteases digested the host eukaryotic initiation factor 4G (eIF4G) and Poly(A)-binding protein (PABP), and these cleavages led to the shutdown of host cell protein synthesis and further promoted apoptosis, along with nuclear condensation and DNA fragmentation [85,86,90]. In addition to viral proteases, miRNAs have also been characterized as critical players in host protein synthesis shutdown and signaling regulation [54,55,59,78,79,[91][92][93][94][95][96][97]108]. The role of miRNAs in the virus-induced translational switch has recently begun to be investigated. Ho and his colleagues found that cellular miR-141 is induced by EV71 infection, which targets eIF4E, the cap-dependent translation initiation factor, and results in the shutdown of host protein synthesis, while the silencing of virus infection-induced miR-141 almost recovers host protein synthesis and blocks viral propagation up to 1000-fold [59]. Hence, virus infection-induced miR-141 expedites the translational switch from cap-dependent translation to cap-independent translation. This study largely increased the understanding of how miRNA facilitates Enterovirus-causing host protein synthesis in addition to the classical concept [59,109].

Cellular miRNAs Target Vial Genome to Suppress Viral Replication
The host and virus influence cellular miRNA expressions during virus infection, and the altered miRNAs target cellular molecules to enforce viral pathogenesis or establish host defense machineries. Recently, several studies have suggested that host miRNAs could target viral genomes and result in the suppression of virus replication [56,57,[98][99][100]. The induction of miRNA-296-5p was found in EV71-infected human rhabdomyosarcoma (RD) and SK-N-SH cells, and the silicon analysis results predicted that miRNA-296-5p might target both the VP1 and VP3 regions of the viral genome; this speculation was confirmed by molecular biological assays (Figure 2) [56]. Meanwhile, the suppression of endogenous miR-296-5p promoted EV71 replication, and the introduction of mutations into binding sites on the viral genome led EV71 to escape the inhibitory effects of miR-296-5p [56]. Wang et al. [99] and Wen et al. [98] and also found similar phenomena in CVB3 and EV71 infections, respectively [98,99]. The expression of miR-23b was downregulated in EV71 infection, while the restoration of miR-23b by specific mimic inhibited EV71 production ( Figure 2). A further study has evidenced that miR-23b suppressed EV71 replication by targeting the EV71 VPl protein [98]. Wang and the coworkers discovered that miR-342-5p could significantly inhibit CVB3 replication by directly targeting the 2C-coding region of the viral genome ( Figure 2) [99]. Both miR-373 and miR-542-5p target the 5 1 untranslated region (5 1 UTR) of EV71 vRNA and thus attenuate viral propagation as assayed in RD cells (Figure 2) [100]. protein [98]. Wang and the coworkers discovered that miR-342-5p could significantly inhibit CVB3 replication by directly targeting the 2C-coding region of the viral genome ( Figure 2) [99]. Both miR-373 and miR-542-5p target the 5′ untranslated region (5′ UTR) of EV71 vRNA and thus attenuate viral propagation as assayed in RD cells (Figure 2) [100]. Cellular miRNAs play a regulatory role in the viral life cycle to suppress or promote virus replication by targeting the viral genome. MiR-296-5p is upregulated, while miR-23b is downregulated in EV71 infection. EV71 infection-induced miR-296-5p could target both viral VP1 and VP3 regions and result in the inhibition of virus replication. The downregulation of miR-23b in EV71 infection relieves EV71 viral RNAs (vRNAs) from miRNA-mediated inhibition. In addition to endogenous miRNAs, the ectopic introduction of miRNAs also affects virus replication. The ectopic expression of miR-342-5p, miR-373 or miR-542-5p suppresses Enterovirus replication, but miR-10* facilitates CVB3 replication. MiR-342-5p targets the CVB3 2C-coding region and thus inhibits CVB3 replication. In contrast, miR-10a* acts as a functional RNA molecule to facilitate CVB3 replication by targeting the viral 3D-coding region. Similarly, miR-373 and miR-542-5p attenuate EV71 propagation by targeting the 5′ UTR of EV71 vRNAs.

Cellular miRNAs Target the Viral Genome to Promote Viral Propagation
Intriguingly, miR-10a*, the passenger strand of the miRNA duplex, is a functional RNA molecule in CVB3 infection, in which miR-10a* directly targets the nt6818-nt6941 sequence of the CVB3 3D-coding region, facilitating CVB3 replication (Figure 2) [57]. Furthermore, miR-10a* was abundant in the cardiac tissues of suckling Balb/c mice, indicating that miR-10a* might be involved in CVB3-induced myocarditis [57]. Despite a large number of reports evidently showing that miRNAs suppress target gene expression via the RISC complex, a few cases have shown that miRNAs are capable of positively regulating target gene expressions [53,110,111]. As mentioned in 2005, a host miRNA, miR-122, targeted Hepatitis C virus (HCV) 5′ noncoding region, facilitating viral RNA replication by promoting RNA folding or sequestration in the replication complexes [53]. This evidence implied that miRNAs might serve as potential therapeutic agents for Enterovirus infection treatment. The miRNAs that are involved in the manipulation of viral genomes are provided in Figure 2 and summarized in Table 1. Cellular miRNAs play a regulatory role in the viral life cycle to suppress or promote virus replication by targeting the viral genome. MiR-296-5p is upregulated, while miR-23b is downregulated in EV71 infection. EV71 infection-induced miR-296-5p could target both viral VP1 and VP3 regions and result in the inhibition of virus replication. The downregulation of miR-23b in EV71 infection relieves EV71 viral RNAs (vRNAs) from miRNA-mediated inhibition. In addition to endogenous miRNAs, the ectopic introduction of miRNAs also affects virus replication. The ectopic expression of miR-342-5p, miR-373 or miR-542-5p suppresses Enterovirus replication, but miR-10* facilitates CVB3 replication. MiR-342-5p targets the CVB3 2C-coding region and thus inhibits CVB3 replication. In contrast, miR-10a* acts as a functional RNA molecule to facilitate CVB3 replication by targeting the viral 3D-coding region. Similarly, miR-373 and miR-542-5p attenuate EV71 propagation by targeting the 5 1 UTR of EV71 vRNAs.

Cellular miRNAs Target the Viral Genome to Promote Viral Propagation
Intriguingly, miR-10a*, the passenger strand of the miRNA duplex, is a functional RNA molecule in CVB3 infection, in which miR-10a* directly targets the nt6818-nt6941 sequence of the CVB3 3D-coding region, facilitating CVB3 replication (Figure 2) [57]. Furthermore, miR-10a* was abundant in the cardiac tissues of suckling Balb/c mice, indicating that miR-10a* might be involved in CVB3-induced myocarditis [57]. Despite a large number of reports evidently showing that miRNAs suppress target gene expression via the RISC complex, a few cases have shown that miRNAs are capable of positively regulating target gene expressions [53,110,111]. As mentioned in 2005, a host miRNA, miR-122, targeted Hepatitis C virus (HCV) 5 1 noncoding region, facilitating viral RNA replication by promoting RNA folding or sequestration in the replication complexes [53]. This evidence implied that miRNAs might serve as potential therapeutic agents for Enterovirus infection treatment. The miRNAs that are involved in the manipulation of viral genomes are provided in Figure 2 and summarized in Table 1.

Conclusions and Perspective
A complete molecular understanding of virus pathogenesis is necessary to develop more efficient strategies against virus infection. Since the discovery of miRNAs in 1993, miRNA is becoming the largest family of gene regulators, accounting for the regulation of one-third of human genes. In the case of Enterovirus infection, virus-induced cellular miRNAs modulate the cellular and infection processes and contribute to pathogenesis by targeting either host mRNAs or virus RNAs. This growing evidence demonstrates the important role of miRNAs in Enterovirus infection-related pathogenesis and accelerates the progress of anti-virus strategies. There are no effective antiviral treatments, approved medications or vaccines for Enterovirus infections except for Poliovirus [16,112,113]. Routine treatment is supportive for reducing clinical symptoms. The efficacy of passive immunoglobulin treatments is not often effective or satisfying for infected subjects. Unfortunately, quarantine might be the best solution to prevent Enterovirus outbreaks. Although anti-EV vaccines are undergoing clinical trials and may soon be approved, the cross-protection of these vaccines requires further evaluation. Herein, miRNAs provide an opportunity to develop alternative miRNA-based therapeutic and preventive strategies for non-polio Enterovirus infections. In fact, the phase I clinical trial of MRX34, a miR-34 mimic, in solid tumors and hematological malignancies is expected to be completed by the end of 2015 [114]. A phase IIa clinical trial was conducted to assess the safety and efficacy of Miravirsen, a 15-nucleotide locked nucleic acid-modified antisense oligonucleotide against mature miR-122 in HCV carriers, and the preliminary results showed exciting therapeutic potential with acceptable adverse drug effects (ADE) [115]. Furthermore, the phase II clinical trial of RG101, another anti-miR-122 antagonist, combined with an anti-HCV agent in HCV patients has been underway since August 2015. For EV infection control, miR-146a-based medication provides new insight into the potential anti-EV71 therapy by showing that the neutralization of EV71-induced miR-146a prevents death, as demonstrated in an EV71 infection mouse model via the restoration of type I interferon production [58]. The potential long-term cytotoxicity and genotoxicity might not be a critical consideration in EV infections compared to chronic infections or cancers due to the short therapeutic period. Moreover, targeting host components is a new strategy to overcome the drug resistance and antigenic variation of viruses, particularly RNA viruses. Since the mystery of miRNAs in Enterovirus infection is generally understood, the preventive and therapeutic activities of miRNAs might be conspicuous in the foreseablefuture.