Pathogenicity and landscape of differential gene expression in mice orally infected with clinical coxsackievirus A6 (CA6).

Hand, foot, and mouth disease (HFMD) is caused by more than 20 pathogenic enteroviruses belonging to the Picornaviridae family and Enterovirus genus. Since the introduction of the enterovirus-71 (EV71) vaccine in 2016, the number of HFMD cases caused by EV71 has decreased. However, cases of infections caused by other enteroviruses, such as coxsackievirus A6 (CA6) and coxsackievirus A10, have been increasing accordingly. In this study, we used a clinical isolate of CA6 to establish an intragastric infection mouse model using 7-day-old mice to mimic the natural transmission route, by which we investigated the differential gene expression profiles associated with virus infection and pathogenicity. After intragastric infection, mice exhibited hind limb paralysis symptoms and weight loss, similar to those reported for EV71 infection in mice. The skeletal muscle was identified as the main site of virus replication, with a peak viral load reaching 2.31 × 107 copies/mg at 5 dpi and increased infiltration of inflammatory cells. RNA sequencing analysis identified differentially expressed genes (DEGs) after CA6 infection. DEGs in the blood, muscle, brain, spleen, and thymus were predominantly enriched in immune system responses, including pathways such as Toll-like receptor signaling and PI3K-Akt signaling. Our study has unveiled the genes involved in the host immune response during CA6 infection, thereby enhancing our comprehension of the pathological mechanism of HFMD.IMPORTANCEThis study holds great significance for the field of hand, foot, and mouth disease (HFMD). It not only delves into the disease's etiology, transmission pathways, and severe complications but also establishes a novel mouse model that mimics the natural coxsackievirus A6 infection process, providing a pivotal platform to delve deeper into virus replication and pathogenic mechanisms. Additionally, utilizing RNA-seq technology, it unveils the dynamic gene expression changes during infection, offering valuable leads for identifying novel therapeutic drug targets. This research has the potential to enhance our understanding of HFMD, offering fresh perspectives for disease prevention and treatment and positively impacting children's health worldwide.

Molecular characterization of a novel clade echovirus 3 isolated from patients with hand-foot-and-mouth disease in southwest China.

Echovirus 3 (E3) belongs to the species Enterovirus B. Currently, three nearly whole-genome sequences of E3 are available in GenBank in China. In this study, we determined the whole genomic sequences of six E3 strains isolated from the stools of patients with hand-foot-and-mouth disease in Southwest China in 2022. Their nucleotide and amino acid sequences shared 82.1%-86.4% and 96.6%-97.2% identity with the prototype Morrisey strain, respectively, and showed 87.1% and 97.2% mutual identity. The six E3 strains are not clustered with other Chinese strains and formed a novel subgenotype (C6) with the recent American and British strains. Recombination analyses revealed that intertype recombination had occurred in the 2 C and 3D regions of the six E3 strains with coxsackieviruses B5 and B4, respectively. This study augments the nearly whole-genome sequences of E3 in the GenBank database and extends the molecular characterization of this virus in China.

Mechanism of RNA m6 A methylation modification regulating NLRP3 inflammasome activation for hand, foot, and mouth disease progression.

Some children infected with hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) progressed to severe disease with various neurological complications in the short term, with a poor prognosis and high mortality. Studies had revealed that RNA N6 -methyladenosine (m6 A) modification had a significant impact on EV71 replication, but it was unknown how m6 A modification regulated the host cell's innate immune response brought on by EV71 infection. We used MeRIP-seq (methylation RNA immunoprecipitation sequencing), RNA-seq (RNA sequencing), cell transfection, and other techniques. MeRIP-seq and RNA-seq results showed the m6 A methylation modification map of control and EV71-infected groups of RD cells. And multilevel validation indicated that decreased expression of demethylase FTO (fat mass and obesity-associated protein) was responsible for the elevated total m6 A modification levels in EV71-infected RD cells and that thioredoxin interacting protein (TXNIP) may be a target gene for demethylase FTO action. Further functional experiments showed that demethylase knockdown of FTO promoted TXNIP expression, activation of NLRP3 inflammasome and promoted the release of proinflammatory factors in vitro, and the opposite result occurred with demethylase FTO overexpression. And further tested in an animal model of EV71 infection in vitro, with results consistent with in vitro. Our findings elucidated that depletion of the demethylase FTO during EV71 infection increased the m6 A modification level of TXNIP mRNA 3' untranslated region (UTR), enhancing mRNA stability, and promoting TXNIP expression. Consequently, the NLRP3 inflammasome was stimulated, leading to the release of proinflammatory factors and facilitating HFMD progression.

Gut Microbiota in Children with Hand Foot and Mouth Disease on 16S rRNA Gene Sequencing.

Hand foot and mouth disease (HFMD) is a contagious and seasonal viral disease in children. The gut microbiota of HFMD children is not clear now. The study aimed to explore the gut microbiota of HFMD children. The 16S rRNA gene of the gut microbiota of ten HFMD patients and ten healthy children were sequenced on the NovaSeq and PacBio platforms respectively. There were significant differences in gut microbiota between the patients and healthy children. The diversity and abundance of gut microbiota in HFMD patients were lower than that in healthy children. The species Roseburia inulinivorans and Romboutsia timonensis were more abundant in healthy children than those in HFMD patients, which suggests that the two species may be used as probiotics for adjusting the gut microbiota of HFMD patients. Meanwhile, the results of 16S rRNA gene sequences from the two platforms were different. The NovaSeq platform identified more microbiota and has the characteristics of high throughput, short time and low price. However, the NovaSeq platform has low resolution at the species level. The PacBio platform has high resolution based on its long reads length, which is more suitable for species-level analysis. But, the shortcomings of the high price and low throughput of PacBio still need to be overcome. With the development of sequencing technology, the reduction in sequencing price and the increase in throughput will promote the third-generation sequencing technology used in the study of gut microbes.

A large scale 16S ribosomal RNA gene amplicon dataset of hand, foot and mouth patients and healthy individuals.

There is evidence linking hand, foot and mouth disease (HFMD) to gut microbiota dysbiosis, and this relationship was corroborated in a large HFMD patient population in our previous study. Here, we present a bacterial 16S rRNA gene dataset from faecal samples of 713 individuals (254 HFMD patients, 459 healthy controls) aged 2 to 7 years residing in Heyuan and Jiangmen counties, Guangdong Province, southern China. Microbiome analysis indicated a significant increase in genus Prevotella, Cetobacterium, and Megamonas was observed in patients with HFMD, whereas a large increase in genus Bacteroides, Ruminococcus, and Faecalibacterium were seen in the control group. We also share the bioinformatic analytical pipeline for this analysis, from data preprocessing to data filtering and amplicon sequence variant (ASV) table generation. We expect that the dataset will be reprocessed, evaluated and fully analysed with various analysis methods to further elucidate the role of the gut microbiota in HFMD development.

Association of TLR3 gene 1377C/T (rs3775290) and TLR7 gene C/G (rs3853839) polymorphism with hand, foot, and mouth disease caused by human enterovirus 71 infection susceptibility and severity in the Chinese Han population: A meta-analysis of case-control studies.

BACKGROUND: Several case-control studies have been conducted on the relationship between rs3775290 C/T and rs3853839 C/G single nucleotide polymorphisms of the Toll-like receptor (TLR) gene and hand, foot, and mouth disease (HFMD) susceptibility and severity. This meta-analysis aimed to offer a systemic review of HFMD susceptibility and severity among the Chinese Han population associated with the C/T (rs3775290) polymorphism of the TLR3 gene or C/G (rs3853839) polymorphism of the TLR7 gene. METHODS: A computer search was conducted using PubMed, Web of Science, Embase, CNKI, CBM, VIP, and WanFang databases. The time ranges were from database establishment to 30/7/2021. Articles selected according to the inclusion and exclusion criteria underwent data extraction and methodological quality evaluation. RevMan 5.4 and Stata 16.0 were adopted for meta-analysis, and the incorporated odds ratio (OR) values and 95% confidence intervals (CIs) were calculated. Sensitivity and publication bias assessments were performed. RESULTS: 8 articles with 9 studies were selected. Among them, there were 858 cases and 577 controls in TLR3 rs3775290 studies as well as 2151 cases and 1554 controls in TLR7 rs3853839 studies. Regarding rs3775290 of TLR3, susceptibilities of the severe type of T-possessing individuals were larger than those of C-possessing individuals [OR = 1.34, 95%CI (1.10, 1.64), P = .004]. The susceptibility of individuals with the severe TT genotype was 1.61 times that of individuals with the CC genotype [95%CI (1.07, 2.43), P=0.02], while susceptibility to HFMD was not influenced by the genotype. In terms of the rs3853839 of the TLR7 gene, C allele carriers have a higher risk of developing HFMD than G allele carriers. The susceptibility to HFMD in CC+CG individuals was 1.24 times than that in GG individuals [95%CI (1.07, 1.43), P = .004]. However, no relationship was found between this polymorphism and severity of the severe type. No significant publication bias was observed in this study. CONCLUSIONS: rs3775290 (C/T) of TLR3 is associated with susceptibility to the severe type, whereas rs3853839 (C/G) of TLR7 is associated with susceptibility to HFMD. However, owing to the limited quantity and quality of the research, the aforementioned conclusions are yet to be justified by more high-quality research.

Roles of Non-Coding RNAs in Virus-Host Interaction About Pathogenesis of Hand-Foot-Mouth Disease.

Noncoding RNAs (ncRNAs) represent the largest and main transcriptome products and play various roles in the biological activity of cells and pathological processes. Accumulating evidence shows that microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA) are important ncRNAs that play vital regulatory roles during viral infection. Hand-foot-mouth disease (HFMD) virus causes hand-foot-mouth disease, and is also associated with various serious complications and high mortality. However, there is currently no effective treatment. In this review, we focus on advances in the understanding of the modulatory role of ncRNAs during HFMD virus infection. Specifically, we discuss the generation, classification, and regulatory mechanisms of miRNA, lncRNA, and circRNA in the interaction between virus and host, with a particular focus on their influence with viral replication and infection. Analysis of these underlying mechanisms can help provide a foundation for the development of ncRNA-based antiviral therapies.

Genomic analysis of a recombinant coxsackievirus A19 identified in Xinxiang, China, in 2019.

Coxsackievirus A19 (CV-A19) is an enterovirus belonging to the species Enterovirus C, and the prototype strain 8663 was isolated from a patient with Guillain-Barré syndrome in Japan. In this study, we determined the complete genome sequence of a CV-A19 isolate identified in a stool sample from a child with hand, foot, and mouth disease in Xinxiang, Henan, China, in 2019 and named it CV-A19 strain 2019103106/XX/CHN/2019 - 2019103106 for short. The genome of this virus consists of 7409 nucleotides, including a 6624-nucleotide open reading frame encoding a potential polyprotein precursor of 2207 amino acids. Compared with strain 8663, strain 2019103106 showed 85.1% nucleotide sequence identity in the complete genome and 85.6% identity in the VP1 coding region, reflecting their genetic divergence. Phylogenetic analysis of strain 2019103106 and other representative EV-C strains with sequences available in the GenBank database showed that CV-A19 strains could be grouped into two clusters based on the complete or 214-nucleotide partial VP1 coding regions, and 2019103106 belonged to cluster 1, with the closest relationship to CV-A19 strain SWG82 from Shandong, China. Phylogenetic trees based on the P2 and P3 coding regions highlighted the divergence between strains 2019103106 and 8663, implying that strain 2019103106 had undergone recombination. Further recombination analysis suggested that strains V18A-like CV-A1 and BBD26-like CV-A19 probably recombined to yield strain 2019103106. The present study points out the genetic diversity of CV-A19. It expands our understanding of the evolution of the CV-A19 genome, but more genome sequences of epidemic strains are needed to explain the phylogeny and evolutionary history of CV-A19 comprehensively.

Isolation and characterization of a novel clade of coxsackievirus B2 associated with hand, foot, and mouth disease in Southwest China.

Coxsackievirus B2 (CVB2) is an enterovirus B (EV-B) species and can cause aseptic meningitis, myocarditis and hand, foot, and mouth disease (HFMD). We characterized a novel CVB2 (YN31V3) associated with HFMD in Yunnan, Southwest China, in 2019. Although YN31V3 and other Mainland China epidemic strains mainly belonged to genotype C, YN31V3 formed an independent branch. The genome sequence of the strain YN31V3 from this study showed a 12.91% nucleotide difference to its closest strain RW41-2/YN/CHN/2012. Recombination analyses showed that the newly isolated YN31V3 was probably a recombinant, which was closely related to CVB2 strains in the genomic P1 region and other EV-B strains in the P2 and P3 regions, respectively. YN31V3 strain had a temperature-sensitive phenotype. The challenge of suckling BALB/c mice with YN31V3 could cause symptoms of disease and severe pathological lesions.

Enterovirus 71 Activates GADD34 via Precursor 3CD to Promote IRES-Mediated Viral Translation.

Enterovirus 71 (EV71) is the major pathogen of hand, foot, and mouth disease. In severe cases, it can cause life-threatening neurological complications, such as aseptic meningitis and polio-like paralysis. There are no specific antiviral treatments for EV71 infections. In a previous study, the host protein growth arrest and DNA damage-inducible protein 34 (GADD34) expression was upregulated during EV71 infection determined by ribosome profiling and RNA-sequencing. Here, we investigated the interactions of host protein GADD34 and EV71 during infections. Rhabdomyosarcoma (RD) cells were infected with EV71 resulting in a significant increase in expression of GADD34 mRNA and protein. Through screening of EV71 protein we determined that the non-structural precursor protein 3CD is responsible for upregulating GADD34. EV71 3CD increased the RNA and protein levels of GADD34, while the 3CD mutant Y441S could not. 3CD upregulated GADD34 translation via the upstream open reading frame (uORF) of GADD34 5'untranslated regions (UTR). EV71 replication was attenuated by the knockdown of GADD34. The function of GADD34 to dephosphorylate eIF2α was unrelated to the upregulation of EV71 replication, but the PEST 1, 2, and 3 regions of GADD34 were required. GADD34 promoted the EV71 internal ribosome entry site (IRES) activity through the PEST repeats and affected several other viruses. Finally, GADD34 amino acids 563 to 565 interacted with 3CD, assisting GADD34 to target the EV71 IRES. Our research reveals a new mechanism by which GADD34 promotes viral IRES and how the EV71 non-structural precursor protein 3CD regulates host protein expression to support viral replication. IMPORTANCE Identification of host factors involved in viral replication is an important approach in discovering viral pathogenic mechanisms and identifying potential therapeutic targets. Previously, we screened host proteins that were upregulated by EV71 infection. Here, we report the interaction between the upregulated host protein GADD34 and EV71. EV71 non-structural precursor protein 3CD activates the RNA and protein expression of GADD34. Our study reveals that 3CD regulates the uORF of the 5'-UTR to increase GADD34 translation, providing a new explanation for how viral proteins regulate host protein expression. GADD34 is important for EV71 replication, and the key functional domains of GADD34 that promote EV71 are PEST 1, 2, and 3 regions. We report that GADD34 promotes viral IRES for the first time and this process is independent of its eIF2α phosphatase activity.

The Disruption of the Endothelial Barrier Contributes to Acute Lung Injury Induced by Coxsackievirus A2 Infection in Mice.

Sporadic occurrences and outbreaks of hand, foot, and mouth disease (HFMD) caused by Coxsackievirus A2 (CVA2) have frequently reported worldwide recently, which pose a great challenge to public health. Epidemiological studies have suggested that the main cause of death in critical patients is pulmonary edema. However, the pathogenesis of this underlying comorbidity remains unclear. In this study, we utilized the 5-day-old BALB/c mouse model of lethal CVA2 infection to evaluate lung damage. We found that the permeability of lung microvascular was significantly increased after CVA2 infection. We also observed the direct infection and apoptosis of lung endothelial cells as well as the destruction of tight junctions between endothelial cells. CVA2 infection led to the degradation of tight junction proteins (e.g., ZO-1, claudin-5, and occludin). The gene transcription levels of von Willebrand factor (vWF), endothelin (ET), thrombomodulin (THBD), granular membrane protein 140 (GMP140), and intercellular cell adhesion molecule-1 (ICAM-1) related to endothelial dysfunction were all significantly increased. Additionally, CVA2 infection induced the increased expression of inflammatory cytokines (IL-6, IL-1ß, and MCP-1) and the activation of p38 mitogen-activated protein kinase (MAPK). In conclusion, the disruption of the endothelial barrier contributes to acute lung injury induced by CVA2 infection; targeting p38-MAPK signaling may provide a therapeutic approach for pulmonary edema in critical infections of HFMD.

Relationship between polymorphism of receptor SCARB2 gene and clinical severity of enterovirus-71 associated hand-foot-mouth disease.

BACKGROUND: To investigate the relationship between polymorphism of scavenger receptor class B member 2 (SCARB2) gene and clinical severity of enterovirus (EV)-71 associated hand-foot-mouth disease (HFMD). METHODS: Among the 100 recruited cases, 56 were in the severe HFMD group (case group) and 44 were in the general HFMD group (control group). By screening functional single nucleotide polymorphisms (SNPs) and hot SNPs, and performing SNP site optimization, some SNP sites of SCARB2 gene were selected for analysis. Genotyping was performed using a MassArray platform. PLINK software was used for statistical processing and analysis of the correlation differences between the mutant genotypes in the severe and general HFMD groups. The relationship between the SNPs and clinical severity of enterovirus (EV)-71 associated HFMD was assessed. RESULTS: 28 SNPs in SCARB2 were selected by site optimization. Then three loci were not in agreement with the minor allele frequency (MAF) in the 1000 Han Chinese in Beijing (CHB) dataset. Another three loci could not be detected. Nine loci were not suitable for further analysis (MAF < 0.01 and Hardy-Weinberg [HWE] P < 0.001). A total of 13 sites were subsequently analyzed. Through Fisher analysis, the frequency of the rs6812193 T allele was 0.134 and 0.034 in the severe and general HFMD groups, respectively (P 0.023 < 0.05, odds ratio [OR] 4.381 > 1). Logistic regression analysis of rs6812193 T alleles between the severe and general HFMD groups, respectively (P 0.023 < 0.05, OR 4.412 > 1, L95 1.210 > 1). Genotype logistic regression analysis of the rs6812193 alleles CT + TT versus CC gave an OR of 4.56 (95% confidence interval [95% CI] 1.22-17.04, P = 0.012). CONCLUSION: The rs6812193 T allele was a susceptibility SNP for SHFMD, and the rs6812193 polymorphism might be significantly associated with the susceptibility to EV-71 infection.

TLR3 polymorphisms are associated with the severity of hand, foot, and mouth disease caused by enterovirus A71 in a Chinese children population.

Hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) is a contagious viral disease, and toll-like receptors (TLRs) play essential roles in resisting the pathogen. The aim of this study was to assess the potential relationship between several TLRs polymorphisms and the HFMD severity in a Chinese children population. A total of 328 Chinese children with HFMD were included in the present study. The polymorphisms of TLR3 (rs3775290, rs3775291, rs3775296, rs1879026, rs5743312, rs5743313, rs5743303, rs13126816, and rs3775292), TLR4 (rs4986790, rs4986791, rs2149356, rs11536889, and rs41426344), TLR7 (rs179009, rs179010, rs179016, rs3853839, rs2302267, rs1634323, and rs5741880), and TLR8 (rs3764880, rs2159377, rs2407992, rs5744080, rs3747414, rs3764879, and rs5744069) genes were selected. The study indicated that individuals with the GG genotype of TLR3 single-nucleotide polymorphism rs1879026 had a higher risk of developing severe cases (GG vs. GT: OR = 1.875; 95% CI, 1.183-2.971; p = .007). Meanwhile, TLR3 rs3775290 CC genotype and C allele were associated with lower disease severity in females (CC vs. CT: OR = 0.350; 95% CI, 0.163-0.751; p = .006; C vs. T: OR = 0.566; 95% CI, 0.332-0.965; p = .036). TLR3 rs3775291 CC genotype showed 2.537 folds higher risk of developing severe cases in females (CC vs. CT: OR = 2.537; 95% CI, 1.108-5.806; p = .026). Moreover, TLR3 rs1879026 GG genotype was found to be related to increased risk of severe cases in males (GG vs. GT: OR = 2.076; 95% CI, 1.144-3.768; p = .016). The current findings show that the genetic variants of TLR3 rs1879026, rs3775290, and rs3775291 are associated with the severity of EV-A71-associated HFMD in a Chinese children population.

Efficacy of a coxsackievirus A6 vaccine candidate in an actively immunized mouse model.

Coxsackievirus A6 (CV-A6) has been emerging as a major pathogen of hand, foot and mouth disease (HFMD). Study on the pathogenesis of CV-A6 infection and development of vaccines is hindered by a lack of appropriate animal models. Here, we report an actively immunized-challenged mouse model to evaluate the efficacy of a Vero-cell-based, inactivated CV-A6 vaccine candidate. The neonatal Kunming mice were inoculated with a purified, formaldehyde-inactivated CV-A6 vaccine on days 3 and 9, followed by challenging on day 14 with a naturally selected virulent strain at a lethal dose. Within 14 days postchallenge, all mice in the immunized groups survived, while 100% of the Alum-only inoculated mice died. Neutralizing antibodies (NtAbs) were detected in the serum of immunized suckling mice, and the NtAb levels correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak in the immunized mice compared with those in Alum-only inoculated control mice. Elevated levels of interleukin-4, 6, interferon γ and tumour necrosis factor α were also observed in Alum-only control mice compared with immunized mice. Importantly, the virulent CV-A6 challenge strain was selected quickly and conveniently from a RD cell virus stock characterized with the natural multi-genotypes. The virulent determinants were mapped to V124M and I242 V at VP1. Together, our results indicated that this actively immunized mouse model is invaluable for future studies to develop multivalent vaccines containing the major component of CV-A6 against HFMD.

Polymorphisms in the DC-SIGN gene and their association with the severity of hand, foot, and mouth disease caused by enterovirus 71.

Severe hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) infection is associated with high mortality and disability. DC-SIGN, a receptor for EV71, is widely distributed in dendritic cells and may influence the severity of HFMD caused by EV71 infection. This observational study attempts to explore whether single-nucleotide polymorphisms (SNPs) in DC-SIGN are related to the severity of EV71-associated HFMD. Based on linkage disequilibrium and functional predictions, two DC-SIGN SNPs were selected and tested to explore their potential association with the severity of HFMD caused by EV71 infection. Two hundred sixteen Han Chinese children with HFMD caused by EV71 were enrolled to obtain clinical data, including the severity of HFMD, serum DC-SIGN levels, and DC-SIGN SNPs. We found a significant association between the rs7248637 polymorphism (A vs. G: OR = 0.644, 95% CI = 0.515-0.806) and the severity of HFMD caused by EV71 infection, as well as the rs4804800 polymorphism (A vs. G: OR = 1.539, 95% CI =1.229-1.928). These two DC-SIGN SNPs may have an effect on the severity of HFMD caused by EV71 infection.

DNA methylation and SNP in IFITM3 are correlated with hand, foot and mouth disease caused by enterovirus 71.

OBJECTIVES: To explore the mechanisms of interferon-induced transmembrane protein 3 (IFITM3) in response to enterovirus-71-associated hand, foot and mouth disease (EV71-HFMD), in terms of DNA methylation, single-nucleotide polymorphism (SNP) genotype and gene expression. METHODS: In total, 120 patients with EV71-HFMD (60 with mild EV71-HFMD and 60 with severe EV71-HFMD) and 60 healthy controls were enrolled in this study. SNP genotype, IFITM3 promoter methylation and mRNA expression of peripheral blood mononuclear cells were examined using the improved multi-temperature ligase detection reaction, quantitative reverse transcriptase polymerase chain reaction and MiSeq, respectively. RESULTS: The distribution of methylation in patients with EV71-HFMD was significantly lower compared with healthy controls, and the severe EV71-HFMD group showed the lowest frequency of IFITM3 promoter methylation. The average level of IFITM3 promoter CpG methylation was negatively correlated with IFITM3 mRNA expression, and hypermethylation of several specific CpG units contributed to IFITM3 downregulation. IFITM3 expression and promoter methylation correlated with EV71 infection progression, especially in the severe EV71-HFMD group. Compared with mild cases, genotype GG and the G allele of rs12252 were over-represented in patients with severe EV71-HFMD. CONCLUSIONS: IFITM3 methylation status and SNP genotyping may help clinicians to choose the correct treatment strategy for patients with EV71-HFMD.

Antiviral peptides against Enterovirus A71 causing hand, foot and mouth disease.

The emergence of new and resistant viruses is a serious global burden. Conventional antiviral therapy with small molecules has led to the development of resistant mutants. In the case of hand, foot and mouth disease (HFMD), the absence of a US-FDA approved vaccine calls for urgent need to develop an antiviral that could serve as a safe, potent and robust therapy against the neurovirulent Enterovirus A71 (EV-A71). Natural peptides such as lactoferrin, melittin and synthetic peptides such as SP40, RGDS and LVLQTM have been studied against EV-A71 and have shown promising results as potent antivirals in pre-clinical studies. Peptides are considered safe, efficacious and pose fewer chances of resistance. Poor pharmacokinetic features of peptides can be overcome by the use of chemical modifications to improve in vivo delivery particularly by oral route. The use of nanotechnology can remarkably assist in the oral delivery of peptides and enhance stability in vivo. This can greatly increase patient compliance and make it more attractive as antiviral therapy.

Emergence of genotype C1 Enterovirus A71 and its link with antigenic variation of virus in Taiwan.

An outbreak of the hand-foot-mouth disease with severe neurological cases, mainly caused by the genotype C1 enterovirus A71 (EV-A71), occurred in Taiwan between 2018 and early 2019. In the recent decade, the most dominant EV-A71 genotypes in Taiwan were B5 and C4 but changed to C1 in 2018. Antibody-mediated immunity plays a key role in limiting the EV-A71 illness in humans. However, the level of neutralizing activities against genotype C1 virus by human polyclonal and monoclonal antibodies (MAbs) remains largely unclear. In the study, we demonstrated that that 39% (9 in 23) of post-infection sera from the genotype B5- or C4-infected patients in 2014-2017 exhibit reduced titers with the 2018-2019 genotype C1 viruses than with the earlier B5 and C4 viruses tested. This finding with polyclonal sera is confirmed with human MAbs derived from genotype B5 virus-infected individuals. The 2018-2019 genotype C1 virus is resistant to the majority of canyon-targeting human MAbs, which may be associated with the residue change near or at the bottom of the canyon region on the viral capsid. The remaining three antibodies (16-2-11B, 16-3-4D, and 17-1-12A), which target VP1 S241 on the 5-fold vertex, VP3 E81 on the 3-fold plateau and VP2 D84 on the 2-fold plateau of genotype C1 viral capsid, respectively, retained neutralizing activities with variable potencies. These neutralizing antibodies were also found to be protective against a lethal challenge of the 2018-2019 genotype C1 virus in an hSCARB2-transgenic mice model. These results indicate that the EV-A71-specific antibody response may consist of a fraction of poorly neutralizing antibodies against 2018-2019 genotype C1 viruses among a subset of previously infected individuals. Epitope mapping of protective antibodies that recognize the emerging genotype C1 virus has implications for anti-EV-A71 MAbs and the vaccine field.

Genome-wide association study identifies TPH2 variant as a novel locus for severe CV-A6-associated hand, foot, and mouth disease in Han Chinese.

BACKGROUND: Little is known about the association between genetic susceptibility and the severity of hand, foot, and mouth disease (HFMD) infected with coxsackievirus A6 (CV-A6). METHODS: Three hundred and sixty-four CV-A6 HFMD patients were enrolled, including 115 severe and 249 mild patients. A genome-wide association study (GWAS) was performed involving eight DNA pools of 115 severe and 115 mild CV-A6 HFMD patients pair-matched by age and gender. Differences in relative allele signal scores of SNPs in Illumina Human OmniZhongHua-8 BeadChips were compared between the two groups. The tag SNPS for potentially functional SNPs or their high linked SNPs were selected for individual genotyping in all 364 patients and assessed for their associations with severe CV-A6 HFMD using multivariable logistic regression analyses. RESULTS: The top 30 significant SNPs obtained from pooled DNA GWAS analysis were checked for biological functions and their high linkage disequilibrium (LD) SNPs. Four tag SNPs (rs1558206, rs6927647, rs9375728 and rs10879355) were selected for further individual genotyping in 364 CV-A6 patients. Only SNP rs10879355 was associated with severe CV-A6 HFMD, with CC genotype having a greater risk of severe illness than TT+TC genotypes (OR=2.48, 95%CI: 1.34, 4.56). SNP rs4290270 is in complete LD with rs10879355 in Chinese Han children. CONCLUSIONS: This is the first report that one potentially functional SNP rs4290270 in the TPH2 gene may be associated with the risk of severe CV-A6 HFMD.

Aquaporin­4 deletion ameliorates enterovirus 71 infection in mice.

Aquaporin-4 (AQP4) is a major water channel of the central nervous system. The present study was designed to determine whether AQP4 deletion could ameliorate enterovirus (EV) 71 infection­induced hand, foot and mouth disease (HFMD) by inhibiting inflammation and apoptosis in mice. EV 71 strains were injected into neonatal BALB/c mice to induce HFMD. Western blotting and ELISA were used to measure the protein expression and cytokine levels. The levels of AQP4 mRNA and protein in the brain were increased in EV 71­infected mice, while the survival rate and health score were improved in AQP4­knockout (KO) mice with EV 71 infection. The EV 71 infection­induced increases of tumor necrosis factor­α, interleukin (IL)­1ß, IL­6, monocyte chemotactic protein­1, interferon (IFN)­α and IFN­Î³ in plasma and brain were inhibited in AQP4­KO mice. AQP4 deletion reversed the decreased levels of Bcl2 and Bcl2/Bax, and the increased levels of Bax induced by EV 71 infection in the brain. These results demonstrated that AQP4 deletion ameliorated EV 71 induced­HFMD via inhibiting inflammation and apoptosis in mice.