Enterovirus 71 VP1 Research Articles

Immunogenicity and immunodominant linear B-cell epitopes of a new DNA-based tetravalent vaccine against four major enteroviruses causing hand, foot, and mouth disease

Hand, foot, and mouth disease (HFMD) poses a significant public health threat primarily caused by four major enteroviruses: enterovirus 71 (EV71), coxsackieviruses A16, A10, and A6. Broadly protective immune responses are essential for complete protection against these major enteroviruses. In this study, we designed a new tetravalent immunogen for HFMD, validated it in silico, in vivo evaluated the immunogenicity of the DNA-based tetravalent vaccine in mice, and identified immunogenic B-cell and T-cell epitopes. A new tetravalent immunogen, VP1me, was designed based on the chimeric protein and epitope-based vaccine principles. It contains a complete EV71 VP1 protein and six reported neutralizing B-cell epitopes derived from the four major enteroviruses causing HFMD. In silico validation using multiple immunoinformatic tools indicated good attributes of the VP1me immunogen suitable for vaccine development. The VP1me-based DNA vaccine efficiently induced both humoral and cellular immune responses in BALB/cAJcl mice. A combination of in silico prediction and immunoassays enabled the identification of immunogenic linear B-cell and CD8 T-cell epitopes within the VP1me immunogen. Immunodominant linear B-cell epitopes were identified in six regions of VP1me, with one epitope located at the N-terminus of the VP1 protein (aa 9–23) regarded as a novel epitope. Interestingly, some B-cell epitopes could also induce the CD8 T-cell response, suggesting their dual functions in immune stimulation. These results lay the groundwork for further development of VP1me as a new vaccine candidate.

Clinical and morphological myocardial changes in patients with post-COVID-19 syndrome

Aim. To characterize the clinical manifestations and myocardial morphological changes in patients with cardiovascular complications of post-Covid syndrome.Material and methods. A total of 38 patients (Group I) with cardiac symptoms in the post-COVID-19 period underwent endomyocardial biopsy (EMB). Patients from group I were divided into 2 subgroups depending on the presence of morphologically verified myocarditis. For morphological comparison, group II was formed (41 patients without COVID-19 and with EMB performed before 2019). Group II was also divided into two subgroups with and without morphologically verified myocarditis. In addition to the analysis of clinical and paraclinical data, a histological and immunohistochemical study of myocardial tissue was carried out with anti-CD3, CD68, CD16, HLA-DR, MHC1, C1q, enterovirus VP1, SARS-CoV-2 spike protein, ACE2, Ang1, von Willebrand factor, VEGF antibodies, as well as a PCR of myocardial tissue after reverse transcription. Additionally, an immunofluorescent study of myocardial tissue was performed using antibody cocktails to SARS-CoV-2/ CD16 spike protein, SARS-CoV-2/CD68 spike protein, CD80/CD163. Statistical processing was performed using the NumPy, SciPy, Pandas and Matplotlib libraries in the Python programming language.Results. Among patients in group I, 65% had morphologically verified lymphocytic myocarditis (subgroup 1, n=29). Other 35% of patients had no signs of myocardial inflammation (subgroup 2, n=9). The comparison group (group II) included 33 patients with myocarditis (80%) and 8 patients (20%) without it. There was a pronounced expression of CD68+ macrophages in the myocardial tissue in patients with postCOVID-19 syndrome in comparison with group II, both with morphological criteria for myocarditis (p=0,014) and without it (p=0,007), mainly due to M2 macrophages. More pronounced expression of CD68+ macrophages was observed when EMB was performed at earlier stages. If SARS-CoV-2 spike protein was detected in group I on the endothelium and inflammatory infiltrate by the immunohistochemistry, PCR of myocardial tissue for SARS-CoV-2 (n=5) was performed, which did not reveal SARS-CoV-2 particles in any of the analyzed samples.Conclusion. Cardiac symptoms of post-COVID-19 syndrome are caused by myocardial remodeling, including in patients without morphologically verified myocarditis. The direct involvement of SARS-CoV-2 in the development of myocarditis in the post-acute period of COVID-19 has not been proven.

Comparative Study of the Myocardium of Patients from Four COVID-19 Waves.

Few studies have compared COVID-19 patients from different waves. This study aims to conduct a clinical and morphological analysis of patients who died from COVID-19 during four waves. The study involved 276 patients who died from COVID-19 during four waves, including 77 patients in the first wave, 119 patients in the second wave, and 78 patients in the third wave. We performed a histological examination of myocardium samples from autopsies and additionally analyzed the samples by PCR. We conducted immunohistochemistry of the myocardium for 21 samples using antibodies against CD3, CD45, CD8, CD68, CD34, Ang1, VWF, VEGF, HLA-DR, MHC1, C1q, enteroviral VP1, and SARS-CoV-2 spike protein. We also did immunofluorescent staining of three myocardial specimens using VP1/SARS-CoV-2 antibody cocktails. Further, we ran RT-ddPCR analysis for 14 RNA samples extracted from paraffin-embedded myocardium. Electron microscopic studies of the myocardium were also performed for two samples from the fourth wave. Among the 276 cases, active myocarditis was diagnosed in 5% (15/276). Of these cases, 86% of samples expressed VP1, and individual cells contained SARS-CoV-2 spike protein in 22%. Immunofluorescence confirmed the co-localization of VP1 and SARS-CoV-2 spike proteins. ddPCR did not confidently detect SARS-CoV-2 RNA in the myocardium in any myocarditis cases. However, the myocardium sample from wave IV detected a sub-threshold signal of SARS-CoV-2 by qPCR, but myocarditis in this patient was not confirmed. Electron microscopy showed several single particles similar to SARS-CoV-2 virions on the surface of the endothelium of myocardial vessels. A comparison of the cardiovascular complication incidence between three waves revealed that the incidence of hemorrhage (48 vs. 24 vs. 17%), myocardial necrosis (18 vs. 11 vs. 4%), blood clots in the intramural arteries (12 vs. 7 vs. 0%), and myocarditis (19 vs. 1 vs. 6%) decreased over time, and CD8-T-killers appeared. Immunohistochemistry confirmed the presence of endotheliitis in all 21 studied cases. This study compared myocardial damage in patients who died during three COVID-19 waves and showed a decrease in the incidence of endotheliitis complications (thrombosis, hemorrhage, necrosis) and myocarditis over time. However, the connection between myocarditis and SARS-CoV-2 infection remains unproven.

The role of conserved arginine and proline residues in enterovirus VP1 protein.

High diversity of VP1 protein among enteroviruses has been a barrier in developing universally effective antiviral drugs. To maintain structure stability during evolution, several residues of VP1 protein of enteroviruses are conserved. Therefore, investigation of highly conserved residues in VP1 protein may provide information for antiviral drug candidates against enteroviruses. To identify highly conserved amino acid sequences of the VP1 in enterovirus genus, the Consurf and CABS-flex 2.0 web software were applied. Through the combination with secondary structure information, we focused on conserved amino acids of VP1 property analysis. Most conserved residues of VP1 were in the interior and interacted with VP2, VP3 and VP4 capsid proteins. Structure of EV-A71 (PDB code 4AED) showed conserved residues were at hydrophobic pocket and close to the junction between the loop and β-barrel. Interestingly, arginine was the most common conserved residue of VP1. Proline was the second most common conserved residue and was found in the loop and β-barrel intersection areas. VP1 protein flexibility was associated with the secondary structure. Conserved residues of VP1 in β-barrel showed significantly low flexibility. Through large scale sequence analysis, we identified the amino acid distribution and location of conserved residues in VP1. This knowledge can be extrapolated for the Enterovirus genus and may contribute to developing the potential compound as an anti-enteroviral agent.

Detection of enterovirus RNA in peripheral blood mononuclear cells correlates with the presence of the predisposing allele of the type 1 diabetes risk gene IFIH1 and with disease stage

Aims/hypothesisEnteroviral infection has been implicated consistently as a key environmental factor correlating with the appearance of autoimmunity and/or the presence of overt type 1 diabetes, in which pancreatic insulin-producing beta cells are destroyed by an autoimmune response. Genetic predisposition through variation in the type 1 diabetes risk gene IFIH1 (interferon induced with helicase C domain 1), which encodes the viral pattern-recognition receptor melanoma differentiation-associated protein 5 (MDA5), supports a potential link between enterovirus infection and type 1 diabetes.MethodsWe used molecular techniques to detect enterovirus RNA in peripheral blood samples (in separated cellular compartments or plasma) from two cohorts comprising 79 children or 72 adults that include individuals with and without type 1 diabetes who had multiple autoantibodies. We also used immunohistochemistry to detect the enteroviral protein VP1 in the pancreatic islets of post-mortem donors (n=43) with type 1 diabetes.ResultsWe observed enhanced detection sensitivity when sampling the cellular compartment compared with the non-cellular compartment of peripheral blood (OR 21.69; 95% CI 3.64, 229.20; p<0.0001). In addition, we show that children with autoimmunity are more likely to test positive for enterovirus RNA than those without autoimmunity (OR 11.60; 95% CI 1.89, 126.90; p=0.0065). Furthermore, we found that individuals carrying the predisposing allele (946Thr) of the common variant in IFIH1 (rs1990760, Thr946Ala) are more likely to test positive for enterovirus in peripheral blood (OR 3.07; 95% CI 1.02, 8.58; p=0.045). In contrast, using immunohistochemistry, there was no correlation between the common variant in IFIH1 and detection of enteroviral VP1 protein in the pancreatic islets of donors with type 1 diabetes.Conclusions/interpretationOur data indicate that, in peripheral blood, antigen-presenting cells are the predominant source of enterovirus infection, and that infection is correlated with disease stage and genetic predisposition, thereby supporting a role for enterovirus infection prior to disease onset.Graphical abstract

Pancreas Whole Tissue Transcriptomics Highlights the Role of the Exocrine Pancreas in Patients With Recently Diagnosed Type 1 Diabetes.

Although type 1 diabetes (T1D) is primarily a disease of the pancreatic beta-cells, understanding of the disease-associated alterations in the whole pancreas could be important for the improved treatment or the prevention of the disease. We have characterized the whole-pancreas gene expression of patients with recently diagnosed T1D from the Diabetes Virus Detection (DiViD) study and non-diabetic controls. Furthermore, another parallel dataset of the whole pancreas and an additional dataset from the laser-captured pancreatic islets of the DiViD patients and non-diabetic organ donors were analyzed together with the original dataset to confirm the results and to get further insights into the potential disease-associated differences between the exocrine and the endocrine pancreas. First, higher expression of the core acinar cell genes, encoding for digestive enzymes, was detected in the whole pancreas of the DiViD patients when compared to non-diabetic controls. Second, In the pancreatic islets, upregulation of immune and inflammation related genes was observed in the DiViD patients when compared to non-diabetic controls, in line with earlier publications, while an opposite trend was observed for several immune and inflammation related genes at the whole pancreas tissue level. Third, strong downregulation of the regenerating gene family (REG) genes, linked to pancreatic islet growth and regeneration, was observed in the exocrine acinar cell dominated whole-pancreas data of the DiViD patients when compared with the non-diabetic controls. Fourth, analysis of unique features in the transcriptomes of each DiViD patient compared with the other DiViD patients, revealed elevated expression of central antiviral immune response genes in the whole-pancreas samples, but not in the pancreatic islets, of one DiViD patient. This difference in the extent of antiviral gene expression suggests different statuses of infection in the pancreas at the time of sampling between the DiViD patients, who were all enterovirus VP1+ in the islets by immunohistochemistry based on earlier studies. The observed features, indicating differences in the function, status and interplay between the exocrine and the endocrine pancreas of recent onset T1D patients, highlight the importance of studying both compartments for better understanding of the molecular mechanisms of T1D.

Conserved Residues Adjacent to ß-Barrel and Loop Intersection among Enterovirus VP1 Affect Viral Replication: Potential Target for Anti-Enteroviral Development.

Enterovirus genus has over one hundred genotypes and could cause several kinds of severe animal and human diseases. Understanding the role of conserved residues in the VP1 capsid protein among the enterovirus genus may lead to anti-enteroviral drug development. The highly conserved residues were found to be located at the loop and ß-barrel intersections. To elucidate the role of these VP1 residues among the enterovirus genus, alanine substitution reverse genetics (rg) variants were generated, and virus properties were investigated for their impact. Six highly conserved residues were identified as located near the inside of the canyon, and four of them were close to the ß-barrel and loop intersection. The variants rgVP1-R86A, rgVP1-P193A, rgVP1-G231A, and rgVP1-K256A were unable to be obtained, which may be due to disruption in the virus replication process. In contrast, rgVP1-E134A and rgVP1-P157A replicated well and rgVP1-P157A showed smaller plaque size, lower viral growth kinetics, and thermal instability at 39.5°C when compared to the rg wild type virus. These findings showed that the conserved residues located at the ß-barrel and loop junction play roles in modulating viral replication, which may provide a pivotal role for pan-enteroviral inhibitor candidate.

A microfluidic platform for the ultrasensitive detection of human enterovirus 71

A microfluidic platform integrated with two generated monoclonal antibodies (mAbs) was innovatively fabricated for ultrasensitive detection of enterovirus 71 (EV71). To date, this is the first report on EV71 detection using a microfluidic chip-based immunoassay. The mAbs 1F4 and 2H2 against the major capsid protein VP1 of EV71 were conjugated with carboxyl-functionalized magnetic beads (MBs) and carboxyl-functionalized quantum dots (QDs), respectively. MB-conjugated mAbs separated VP1 from samples, while QD-conjugated mAbs provided fluorescence detection signals and formed a sandwich structure. The monolayer of MBs and QDs captured the VP1 sandwich structure showed stronger fluorescence signal in the microfluidic channel, which could be directly evaluated by fluorescence intensity in response to 365 nm ultraviolet (UV) excitation. The limit of detection (LOD) of the microfluidic platform for VP1 was 10 pg mL−1, which was a 31-fold increase compared to sandwich enzyme-linked immunosorbent assay (ELISA, 310 pg mL−1) using identical antibodies. The microfluidic platform performed well with clinical samples, especially for fecal samples with complex compositions and impurities. The assay is quick to perform (within 30 min), and apparently requires just one syringe without other complex liquid flow system, making it is easy to operate and more suitable for on-site detection and future commercial application.

MiR-103/miR-107 inhibits enterovirus 71 replication and facilitates type I interferon response by regulating SOCS3/STAT3 pathway.

Enterovirus71 (EV71), the major cause of hand, foot, and-mouth disease (HFMD), has increasingly become a public health challenge. Type I interferons (IFNs) can regulate innate and adaptive immune responses to pathogens. MicroRNAs (miRNAs) play regulatory roles in host innate immune responses to viral infections. However, the roles of miR-103 and miR-107 in EV71 infection remain unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expression of miR-103, miR-107, suppressor of cytokine signaling 3 (SOCS3), VP1, IFN-α, and IFN-β. Virus titers were measured by 50% tissue culture infectious dose (TCID50) assay. Western blot assay was conducted to detect the protein levels of VP1, IFN-α, IFN-β, SOCS3, signal transducer and activator of transcription 3 (STAT3), and phospho-STAT3 (p-STAT3). Immunofluorescence assay was used to detect the protein level of VP1. The concentrations of IFN-α and IFN-β were examined by Enzyme-linked immunosorbent assay (ELISA). The interaction between SOCS3 and miR-103/miR-107 was predicted by starBase and verified by dual-luciferase reporter assay and RNA pull-down assay. MiR-103 and miR-107 were downregulated and SOCS3 was upregulated in serum from patients with EV71 and EV71-infected cells. Overexpression of miR-103 and miR-107 repressed EV71 replication by inhibiting EV71 titers and VP1 expression. Moreover, upregulation of miR-103 and miR-107 enhanced EV71-triggered the production of type I IFNs. In addition, miR-103 and miR-107 directly targeted SOCS3, and SOCS3 upregulation reversed the effects of miR-103 and miR-107 on EV71 replication and type I IFN response. Importantly, miR-103 and miR-107 increased STAT3 phosphorylation by targeting SOCS3 after EV71 infection. MiR-103 and miR-107 suppressed EV71 replication and increased the production of type I IFNs by regulating SOCS3/STAT3 pathway, which might provide a novel strategy for developing effective antiviral therapy.

Molecular epidemiological analysis of a HFMD epidemic caused by CV-A6

Objective To identify the pathogen that caused the cluster of HFMD epidemic in a middle school of Fuxin City in September 2019, and to find out the homogeneity of the pathogen. Methods The samples were preliminary identified by real-time PCR. Partial VP1 sequences were amplified by nested PCR. The virus type was identified by sequencing and blast comparison. and the homology and phylogenetic analyes of VP1 region were performed by bioinformatics software with CV-A6 reference sequences. Results Samples of 5 cases were tested by real-time fluorescent PCR, and the results showed that the enterovirus universal nucleic acid was positive, and both CV-A16 and EV71 nucleic acids were negative. They were judged to be non-CV-A16 and non-EV71 enterovirus infection. Online BLAST comparison of partial nucleotide sequences of the enterovirus VP1 region showed that samples of 5 cases were all positive for CV-A6 enterovirus, The homology of VP1 nucleotide sequence among 5 samples were 100%. Phylogenetic analysis showed that 5 samples in this study belonged to the CV-A6 D3 subtype which was widely epidemic in China, and they were clustered with the dominant strains in recent years in Liaoning province. Conclusion This cluster of HFMD epidemic is induced by CV-A6. It is closely related to the prevalent CV-A6 strains in Liaoning province in recent years, and no significant variation is detected. 摘要：目的 对2019年9月辽宁省阜新市某中学一起手足口病聚集性疫情的未分型肠道病毒进行病原学鉴定, 分 析其种系发生情况。 方法 使用实时荧光PCR对标本进行初步病原学鉴定, 使用巢式PCR对未分型肠道病毒VP1区 部分核苷酸序列进行扩增和测序, BLAST比对确定肠道病毒型别, 利用生物信息学软件将VP1区全长序列与柯萨奇病 毒A6型(CV-A6)各亚型参考序列进行同源性和系统进化分析。 结果 5份标本经实时荧光PCR检测, 结果 显示肠道 病毒通用型核酸阳性 , CV-A16型和EV71型核酸均为阴性 , 判断为非CV-A16非EV71型肠道病毒感染。对肠道病毒 VP1区部分核苷酸序列进行在线BLAST比对 , 结果 显示5例患者标本均为CV-A6型肠道病毒阳性, 其VP1区核苷酸序 列同源性为100%。系统进化分析显示, 5例标本属于我国广泛流行的CV-A6 D3亚型 , 且与辽宁省近几年的优势流行 株在同一个进化树分支上。 结论 引起此次聚集性手足口病疫情的病原体为CV-A6型肠道病毒, 与近年来辽宁省流 行的CV-A6毒株亲缘关系密切, 未发生明显变异。

The Upregulation of a Novel Long Noncoding RNA AK097647 Promotes Enterovirus 71 Replication and Decreases IFN-λ1 Secretion

Background: Enterovirus 71 (EV71) infects millions of children every year in China and has become a challenge to public health. However, there is no effective treatment for EV71 infection. Long noncoding RNAs (lncRNAs) have been found to play various roles in virus replication and infection. Objective: We aimed to explore the role of a novel long noncoding RNA AK097647 (lncRNA-AK097647) during EV71 infection. Methods: To assess the role of lncRNA-AK097647 during EV71 infection, siRNAs were used to silence lncRNA-K097647 expression. RT-qPCR assay and Western blotting were applied to measure the mRNA and protein levels of EV71 VP1 and the phosphorylation of NF-κB. ELISA was used to detect the level of IFN-λ1 expression. Results: The novel lncRNA-AK097647 was upregulated in human rhabdomyosarcoma cells and the blood of hand, foot, and mouth disease patients infected with EV71, as demonstrated by RT-qPCR. Interestingly, RNAi-mediated knockdown of lncRNA-AK097647 dramatically increased the level of IFN-λ1 expression, resulting in the suppression of EV71 replication. In contrast, overexpression of lncRNA-AK097647 decreased the level of IFN-λ1 expression and resulted in increased EV71 replication. In addition, we found that lncRNA-AK097647 could inhibit the phosphorylation of NF-κB. Conclusion: These results suggest a novel mechanism by which EV71 evades the IFN-mediated host antiviral response by increasing lncRNA-AK097647 expression.

Recombinant Enterovirus 71 Viral Protein 1 Fused to a Truncated Newcastle Disease Virus NP (NPt) Carrier Protein.

Enterovirus 71 (EV71) is the major causative agent in hand, foot, and mouth disease (HFMD), and it mainly infects children worldwide. Despite the risk, there is no effective vaccine available for this disease. Hence, a recombinant protein construct of truncated nucleocapsid protein viral protein 1 (NPt-VP1198–297), which is capable of inducing neutralizing antibody against EV71, was evaluated in a mouse model. Truncated nucleocapsid protein Newcastle disease virus that was used as immunological carrier fused to VP1 of EV71 as antigen. The recombinant plasmid carrying corresponding genes was constructed by recombinant DNA technology and the corresponding protein was produced in Escherichia coli expression system. The recombinant NPt-VP1198–297 protein had elicited neutralizing antibodies against EV71 with the titer of 1:16, and this result is higher than the titer that is elicited by VP1 protein alone (1:8). It was shown that NPt containing immunogenic epitope(s) of VP1 was capable of inducing a greater functional immune response when compared to full-length VP1 protein alone. It was capable to carry larger polypeptide compared to full-length NP protein. The current study also proved that NPt-VP1198–297 protein can be abundantly produced in recombinant protein form by E. coli expression system. The findings from this study support the importance of neutralizing antibodies in EV71 infection and highlight the potential of the recombinant NPt-VP1198–297 protein as EV71 vaccine.

Molecular strategy for the direct detection and identification of human enteroviruses in clinical specimens associated with hand, foot and mouth disease.

BackgroundDiseases caused by human enteroviruses (EVs) are a major global public health problem. Thus, the effective diagnosis of all human EVs infections and the monitoring of epidemiological and ecological dynamic changes are urgently needed.MethodsBased on two comprehensive virological surveillance systems of hand, foot and mouth disease (HFMD), real-time PCR and nested RT-PCR (RT-snPCR) methods based on the enteroviral VP1, VP4-VP2 and VP4 regions were designed to directly detect all human EVs serotypes in clinical specimens.ResultsThe results showed that the proposed serotyping strategy exhibit very high diagnostic efficiency (Study 1: 99.9%; Study 2: 89.5%), and the variance between the study was due to inclusion of the specific Coxsackie virus A6 (CVA6) real-time RT-PCR and VP4 RT-snPCR in Study 1 but not Study 2. Furthermore, only throat swabs were collected and analyzed in Study 2, whereas in Study 1, if a specific EV serotype was not identified in the primary stool sample, other sample types (rectal swab and throat swab) were further tested where available. During the study period from 2013 to 2018, CVA6 became one of the main HFMD causative agents, whereas the level of enterovirus A71 (EV-A71) declined in 2017.ConclusionThe findings of this study demonstrate the appropriate application of PCR methods and the combination of biological sample types that are useful for etiological studies and propose a molecular strategy for the direct detection of human EVs in clinical specimens associated with HFMD.

Clinical efficacy of atrial fibrillation ablation depending on histological changes in the myocardium

There is a group of patients with so-called idiopathic atrial fibrillation (AF) without the causes of arrhythmia established by a standard examination. Aim. To study the effect of histological changes in the myocardium in patients with idiopathic AF on the effectiveness of ablation. Material and methods. The study included 101 patients with idiopathic AF All patients underwent ablation, during which a myocardial biopsy was performed. Depending on the results of intervention, 3 groups were formed: 1 — no AF within 12 months, 2 — recurrent arrhythmia within first 3 months of follow-up, 3 — recurrent arrhythmia after first 3 months of follow-up. To assess inflammatory changes and the severity of fibrosis, histological and immunohistochemical tests of myocardial biopsies were performed. Results . Histological criteria for lymphocytic myocarditis in group 1 observed in 47,5% of patients, in groups 2 and 3 in 27,3% and 25%, respectively. Infiltration of less than 7 cells per 1 mm 2 by CD3+ lymphocytes prevailed in group 3. The activity of inflammation in the studied groups did not significantly differ. The minimum severity of fibrosis was significantly less frequently recorded in group 1 than in group 2 and 3. Nonexpression of viral antigens in the first group was significantly less common than in the second and third groups. Moreover, the combination of expression of enterovirus VP1 and human herpesvirus 6 antigens was significantly more often recorded in the first group. Positive correlation was found between the expression of viral antigens and markers of endo- and myocardial fibrosis. Conclusion. The primary risk factor for recurrent arrhythmia was the initial stage of fibrosis, while inflammatory changes and viral infection were not risk factors. The presence of viral antigens in the myocardium had an indirect effect on the clinical outcome.

Inhibition of Enterovirus 71 by Selenium Nanoparticles Loaded with siRNA through Bax Signaling Pathways.

Enterovirus 71 (EV71) is the principal pathogen leading to severe cases of hand, foot, and mouth disease (HFMD). Specific drugs for EV71 are not discovered currently. Small interfering RNA (siRNA) provides a promising antiviral treatment pathway, but it is difficult to cross cell membranes and is easy to degrade. Nanoparticles are promising for their carrying capacity currently. In this study, the siRNA targeting EV71 VP1 gene was loaded with selenium nanoparticles (SeNPs) and surface decorated with polyethylenimine (PEI) (Se@PEI@siRNA). Se@PEI@siRNA showed a remarkable interference efficiency in the nerve cell line SK-N-SH and prevented the cells to be infected. The mechanism study revealed that Se@PEI@siRNA could lighten the extent of SK-N-SH cells for staying in the sub-G1 phase. Activation of Bax apoptosis signaling was restrained either. Taken together, this study demonstrated that Se@PEI@siRNA is a promising drug against EV71 virus.

Mutated Human P-Selectin Glycoprotein Ligand-1 and Viral Protein-1 of Enterovirus 71 Interactions on Au Nanoplasmonic Substrate for Specific Recognition by Surface-Enhanced Raman Spectroscopy

Protein tyrosine sulfation is a common post-translational modification that stimulates intercellular or extracellular protein-protein interactions and is responsible for various important biological processes, including coagulation, inflammation, and virus infections. Recently, human P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to serve as a functional receptor for enterovirus 71 (EV71). It has been proposed that the capsid viral protein VP1 of EV71 is directly involved in this specific interaction with sulfated or mutated PSGL-1. Surface-enhanced Raman spectroscopy (SERS) is used to distinguish PSGL-1 and VP1 interactions on an Au nanoporous substrate and identify specific VP1 interaction positions of tyrosine residue sites (46, 48, and 51). The three tyrosine sites in PSGL-1 were replaced by phenylalanine (F), as determined using SERS. A strong phenylalanine SERS signal was obtained in three regions of the mutated protein on the nanoporous substrate. The mutated protein positions at (51F) and (48F, 51F) produced a strong SERS peak at 1599–1666 cm−1, which could be related to a binding with the mutated protein and anti-sulfotyrosine interactions on the nanoporous substrate. A strong SERS effect of the mutated protein and VP1 interactions appeared at (48F), (51F), and (46F, 48F). In these positions, there was less interaction with VP1, as indicated by a strong phenylalanine signal from the mutated protein.

Genotyping of enteroviruses and human parechoviruses highlights their diversity in Northwest Ethiopia.

Enteroviruses (EVs) and human parechoviruses (HPeVs) infections are associated with various forms of disease, including gastroenteritis. As information on the molecular epidemiology of these viruses is limited in Ethiopia, the genetic diversity of EV and HPeV was investigated in the Northwestern part of the country. Of the total 450 stool samples obtained from infants and young children with diarrhea, 157 (34.9%) were positive for EV and 49 (10.9%) for HPeV RNA when tested by real-time reverse transcription polymerase chain reaction. Genotyping was performed by sequencing of the EV VP1 gene and the HPeV VP3/VP1 gene, respectively. Genotyping of EV was successful in 118 samples. Thereof, 82 (69.5%) belonged to non-polio EVs as a broad range of genotypes within species C, B, and A. Sabin polioviruses were found in 36 cases. HPeV sequences were also heterogeneous with a relative dominance of genotype 3. In conclusion, diverse EV and HPeV genotypes were found cocirculating in Northwest Ethiopia. The findings highlight the importance of continuous surveillance of these viruses in Ethiopia.

Sequence analysis of VP1 and VP4 genes of enterovirus 71 strains isolated from children with severe and mild hand-foot and mouth disease

Objective To analyze and compare VP1 and VP4 genetic characteristics of human enterovirus 71(EV71) isolated from children with severe and mild hand-foot and mouth disease(HFMD). Methods EV71 strains isolated from severe HFMD patients (6 cases) and mild HFMD patients (6 cases) in Taizhou district, Zhejiang province were included during 2016.Total virus RNA was extracted by Viral RNA Mini Extraction Kit, and reverse transcription polymerase chain reaction(RT-PCR) was used to amplify the sequence of the VP1 and VP4 genes of EV71.And then the sequencing results were compared with those of A, B, C genotype reference EV71 strains from GenBank by nucleotide alignment and amino acid alignment analysis. Results There were no statistically significant differences in sex and age between the two groups(χ2=14.51, t=2.82, all P<0.05). The homogeneity between EV71 strains from severe patients and mild patients was 95.8%~99.6% and 99.1%~100.0% for VP1 nucleotide sequences and amino acid sequences, respectively, while 95.0%~99.9% and 99.0%~100.0% for VP4 nucleotide sequences and amino acid sequences, respectively.The twelve EV71 strains isolated from HFMD patients in Taizhou shared the highest identity with EV71-genotype C, especially genotype C4a.In addition, compared with the mild patients, three respective strains from severe HFMD patients showed mutations at the residue 170 in the VP1 protein(V→L), residue 293 in the VP1 protein(A→S) and residue 7 in the VP4 protein(T→A). Conclusion All EV71 strains isolated from severe and mild HFMD patients in Taizhou district share high homology of nucleotide and amino sequence, and all of them belong to subgenogroup C4a.The mutations in the VP1 and VP4 of EV71 might be related to HFMD disease severity. Key words: Hand-foot and mouth disease; Enterovirus 71; Genotype; Disease severity; Mutation

Enterovirus 71 Infection Shapes Host T Cell Receptor Repertoire and Presumably Expands VP1-Specific TCRβ CDR3 Cluster.

Enterovirus 71 (EV71) has become an important public health problem in the Asia-Pacific region in the past decades. EV71 infection might cause neurological and psychiatric complications and even death. Although an EV71 vaccine has been currently approved, there is no effective therapy for treating EV71-infected patients. Virus infections have been reported to shape host T cell receptor (TCR) repertoire. Therefore, understanding of host TCR repertoire in EV71 infection could better the knowledge in viral pathogenesis and further benefit the anti-viral therapy development. In this study, we used a mouse-adapted EV71 (mEV71) model to observe changes of host TCR repertoire in an EV71-infected central nervous system. Neonate mice were infected with mEV71 and mouse brainstem TCRβ repertoires were explored. Here, we reported that mEV71 infection impacted host brainstem TCRβ repertoire, where mEV71 infection skewed TCRβ diversity, changed VJ combination usages, and further expanded specific TCRβ CDR3 clones. Using bioinformatics analysis and ligand-binding prediction, we speculated the expanded TCRβ CDR3 clone harboring CASSLGANSDYTF sequence was capable of binding cleaved EV71 VP1 peptides in concert with major histocompatibility complex (MHC) molecules. We observed that mEV71 infection shaped host TCRβ repertoire and presumably expanded VP1-specific TCRβ CDR3 in mEV71-infected mouse brainstem that integrated EV71 pathogenesis in central nervous system.

Downregulation of miR-155-5p facilitates enterovirus 71 replication through suppression of type I IFN response by targeting FOXO3/IRF7 pathway

ABSTRACT Enterovirus 71 (EV71), the major cause of hand-foot-and-mouth disease (HFMD), has evolved diverse strategies to counter the type I interferon (IFN-I) response during infection. Recently, microRNAs have regulatory roles in host innate immune responses to viral infections; however, whether EV71 escapes the IFN-I antiviral response through regulation of miRNAs remains unclear. Using a microarray assay, microRNA-155-5p (miR-155-5p) was found to be significantly up-regulated in serum from patients with EV71 infection and the increased expression of miR-155-5p was further confirmed in vivo and in vitro in response to EV71 infection. miR-155-5p overexpression suppressed EV71 titers and VP1 protein level, while miR-155-5p inhibition had an opposite result. Moreover, we found that miR-155-5p overexpression enhanced EV71 triggered IFN I production and the expressions of IFN-stimulated genes (ISGs), while inhibition of miR-155-5p suppressed these processes. Furthermore, bioinformatics analysis and luciferase reporter assay demonstrated that miR-155-5p directly targeted forkhead box protein O3 (FOXO3) and negatively regulated FOXO3/IRF7 axis, an important regulatory pathway for type I IFN production during EV71 infection. Inhibition of FOXO3 reversed the effects of miR-155-5p inhibitor on EV71 replication and the type I IFN production. Importantly, in EV71 infection mice, agomir-155-5p injection resulted in a significant reduction of viral VP1 protein expressions in brain and lung tissues, increased IFN-α/β production and increased mice survival rate. In contrast, antagomir-155-5p enhanced EV71 induced these effects. Collectively, our study indicates that weaken miR-155-5p facilitates EV71 replication through suppression of type I IFN response by FOXO3/IRF7 pathway, thereby suggesting a novel strategy for developing effective antiviral therapy.