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@#Objective To explore the absorption characteristics of aluminum hydroxide adjuvant with inactivated enterovirus 71(EV71).Methods The morphology,purity and particle size distribution of inactivated EV71 particles were analyzed by transmission electron microscope,size exclusion chromatography HPLC(SEC-HPLC) and dynamic light scatter(DLS),and the morphology of aluminum hydroxide adjuvant nanoparticles was observed by transmission electron microscope.Using inactivated EV71 antigen content(4 000,6 000,8 000,10 000,11 000,12 000,13 000,14 000,20 000,30 000 U/mL),aluminum hydroxide adjuvant concentration [0.35,0.25,0.17,0.085 mg/mL(aluminum content)],adsorption time(0.30 and 120 min),ionic strength(sodium chloride concentration of 0.15,0.75 and 1.25 mol/L)and phosphorus-aluminum molar ratio(P/Al,0.15,0.64,2.08 and 7.87) as variables,the adsorption characteristics of aluminum hydroxide adjuvant with inactivated EV71 antigen were investigated.Results Inactivated EV71 particles mainly existed in the form of intact virus particles with a diameter of about 30 nm;Aluminum hydroxide adjuvant showed the characteristics of nanocrystallization,and the particle size was distributed within 200～700 nm.The inactivated EV71 antigen at the concentration of no more than 11 000 U/mL was completely absorbed by aluminum hydroxide adjuvant of 0.35 mg/mL(aluminum content),and no free antigen was detected in the supernatant;while from 12 000 U/mL,the content of free antigen in the supernatant increased with the increase of antigen content;the inactivated EV71 antigen of 250 U/mL was completely absorbed by various concentrations of aluminum hydroxide adjuvant,and no free antigen was detected in the supernatant;the adsorption effect of aluminum hydroxide adjuvant consistent after incubation for different time,and no free antigen was detected in the supernatant.Under the conditions of single dose of vaccine with aluminum hydroxide content(0.35 mg/mL) and inactivated EV71 antigen content(250 U/mL),sodium chloride ion strength had no effect on the adsorption of inactivated EV71 virus,while phosphate ion concentration significantly effected the adsorption.Conclusion Aluminum hydroxide adjuvant in a single dose of vaccine completely absorbed inactivated EV71 antigen,and group replacement played an important role.
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@#Objective To investigate the effect of astragalus membranaceus（AM）injection on apoptosis and autophagy of human gastric epithelial cell line（GES⁃1）induced by enterovirus 71（EV71）. Methods GES⁃1 cells were cultured in vitro and divided into infected group（EV71 infected at a MOI of 3 and control group（no virus infected）. The morpho⁃logical changes of EV71 infected cells were observed by inverted microscope. The level of VP1 in GES⁃1 cells infected with EV71 was detected by Western blot；CCK⁃8 assay was used to detect the viability of GES⁃1 cells infected with EV71；Nuclear staining with DAPI was used to observe the morphological changes of nuclear apoptosis infected with EV71. GES⁃1 cells were divided into control group（without virus infection），infection group and AM intervention group with final concentration of 1，2. 5，5 and 10 μg／mL，respectively. Western blot was used to detect the effect of AM intervention on the expression of apoptosis⁃related proteins Caspase⁃3，PARP and autophagy⁃related proteins LC3 and P62 in GES⁃1 cells infected withEV71. CCK⁃8 method was used to detect the effect of AM intervention on the viability of GES⁃1 cells infected with EV71. Results GES⁃1 cells were round，shrunken with nuclear pyknosis and uneven size；VP1 level increased（t = 41. 56，P < 0. 01），cell viability decreased（t = 19. 07，P < 0. 01），Caspase⁃3 and PARP proteins were cut off（t = 35. 29 and 3. 648， P < 0. 01 and 0. 021 8，respectively），LC3Ⅱ／LC3Ⅰ ratio increased（t = 10. 16，P = 0. 000 5）and P62 protein was degraded（t = 68. 68，P < 0. 01）；AM inhibited the degradation of Caspase⁃3，PARP and P62 proteins induced by EV71 （t = 52. 66，59. 60 and 40. 22，respectively，each P < 0. 01）and increased the ratio of LC3Ⅱ／LC3Ⅰ（t = 5. 521，P = 0. 005 3），andreducedtheinhibitoryeffectofEV71ontheviabilityofGES⁃1cells（t =4. 420，P =0. 0115）. Conclusion EV71 infection induced apoptosis of GES⁃ 1 cells and AM intervention inhibited EV71 induced apoptosis by inhibiting EV71 induced autophagy.
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Objective To investigate the vaccine efficacy (VE) of two doses of inactivated enterovirus 71 (EV71) vaccine on severe hand, foot, and mouth disease (HFMD) in eligible children. Methods A retrospective case-control study was conducted in this project. A total of 109 patients with EV71 severe HFMD aged between 6 months to 5 years old who were admitted to the Pediatric Intensive Care Unit (PICU) of Guangxi Maternity and Child Health Hospital from September 2016 to September 2020, and reported to the Chinese Disease Prevention and Control Information System were selected as the case group. According to 1:1 matching,109 healthy children aged between 6 months to 5 years old were selected as the control group. The vaccine efficacy of EV71 inactivated vaccine was calculated. Results The vaccination rate of the two doses of inactivated EV71 vaccine in the case group was significantly lower than that in the control group（9.17% to 54.13%，χ2=50.911, P<0.05；OR=0.086(95%CI:0.040-0.182)). The vaccine efficacy of two doses of inactivated EV71 vaccine was 91.44% (95% CI:81.8%-96%). Conclusion The inactivated EV71 vaccine has a good protective effect on hand, foot, and mouth disease caused by EV71. The vaccination of EV71 inactivated vaccine should be continuously promoted.
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Objective To determine the epidemiological and etiological characteristics of hand，foot，and mouth disease （HFMD） in Pujiang County from 2008 through 2020， so as to provide scientific evidence for prevention and control measures. Methods Descriptive epidemiological analysis was used to analyze HFMD cases retrieved in the Chinese Information System for Disease Control and Prevention in Pujiang County during 2008‒2020. Results From 2008 through 2020， a total of 9 393 cases were documented in Pujiang County， with an annual incidence of 182.3 per 100 000， showing a trend of alternating high-incidence and low-incidence years. The seasonal distribution was bimodal， and the incidence peaked in May-July and November-December. The incidence of HFMD in urban areas was significantly higher than that in rural areas， and the incidence was positively correlated with population density. Majority of cases （94.9%） were children under 5 years old， of which boys had higher incidence than girls. The number of documented cases was the highest among preschool children living in families and kindergartens， accounting for 97.3%. Totally， 558 cases were laboratory confirmed in Pujiang County in 2008-2020， of which severe cases were all caused by EV71 infection. Conclusion Enterovirus serotypes in HFMD have continually changed from 2008 through 2020 in Pujiang County. However， severe HFMD remains principally attributable to EV71 infection， suggesting that it is necessary to strengthen the surveillance on the etiology of HFMD. In addition， it warrants further promotion of EV71 vaccination.
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Objective To determine the epidemiological and etiological characteristics of hand，foot，and mouth disease （HFMD） in Pujiang County from 2008 through 2020， so as to provide scientific evidence for prevention and control measures. Methods Descriptive epidemiological analysis was used to analyze HFMD cases retrieved in the Chinese Information System for Disease Control and Prevention in Pujiang County during 2008‒2020. Results From 2008 through 2020， a total of 9 393 cases were documented in Pujiang County， with an annual incidence of 182.3 per 100 000， showing a trend of alternating high-incidence and low-incidence years. The seasonal distribution was bimodal， and the incidence peaked in May-July and November-December. The incidence of HFMD in urban areas was significantly higher than that in rural areas， and the incidence was positively correlated with population density. Majority of cases （94.9%） were children under 5 years old， of which boys had higher incidence than girls. The number of documented cases was the highest among preschool children living in families and kindergartens， accounting for 97.3%. Totally， 558 cases were laboratory confirmed in Pujiang County in 2008-2020， of which severe cases were all caused by EV71 infection. Conclusion Enterovirus serotypes in HFMD have continually changed from 2008 through 2020 in Pujiang County. However， severe HFMD remains principally attributable to EV71 infection， suggesting that it is necessary to strengthen the surveillance on the etiology of HFMD. In addition， it warrants further promotion of EV71 vaccination.
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Objective:To investigate the prevention and control effect of hand， foot and mouth disease （HFMD） after entervirus （EV） 71 vaccine immunization， evaluate economic benefit of EV71 vaccine immunization，and provide evidence for developing HFMD vaccine immunization strategies． Methods:Descriptive analysis was performed on comparing epidemiological characteristics of HFMD before and after EV71 vaccine immunization. Cost-benefit analysis was conducted， in which disease burden of HFMD， coverage of EV71 vaccine immunization， and costs of EV71 vaccine immunization were collected to evaluate the benefit-cost ratio （BCR）. Results:The average incidence of HFMD was 202.17/10 million from 2017 to 2018， which decreased by 43.8% compared with the average of the previous six years（χ2 = 395.49，P<0.05）； the average proportion of severe case decreased by 88.7%（χ2=40.84，P<0.05）. The prevalence of EV71 in the outpatients from 2017 to 2018 were 2.56%（18/704）， which decreased by 88.7% compared with the previous six years（χ2=124.74，P<0.05）. The severe and fatal cases were mainly caused by EV71 （84.5% -100.0%）. The average cost of EV 1 vaccine immunization was RMB 526， which was approximately 30.2% of the average cost of HFMD diagnosis and treatment. Coverage of EV71 vaccine was 40.12% from 2017 to 2018 in Minhang. The total costs of EV71 vaccination in Minghang from 2016 to 2018 was 174.89 million RMB. It was estimated that EV71 vaccination strategies had prevented 2 491 outpatients， 232 ordinary inpatients and 54 severe cases. The BCR was determined to be 0.35∶1. Conclusion:EV71 vaccine may reduce the incidence of HFMD， the incidence of severe cases， and the disease burden. It is highly recommended to reduce the immunization costs to increase the immunization coverage.
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Enterovirus 71 (EV71) infection is more likely to cause hand, foot and mouth disease (HFMD) in children, which can lead to neurogenic complications and higher mortality. As a commonly used clinical medicine, Reduning injection (RDN) helps to shorten the symptoms of patients with HFMD and facilitate the early recovery of children. However, the regulatory mechanism of RDN on the HFMD immune system disorder caused by EV71 remains to be discussed. This study collected detailed treatment data of 56 children with HFMD who entered the affiliated Children's Hospital of Nanjing Medical University during 2019. Retrospective analysis of clinical data showed that the symptoms of the RDN treatment group were improved compared with the untreated group. To explore its mechanism, the relevant detection indicators were detected by flow cytometry, enzyme-linked immunosorbent assay and real-time quantitative PCR. It was found that the number and function of innate immune (ILCs) and adaptive immunity (Th1, Th2 and secreted cytokines) were reduced, suggesting that RDN plays a role by regulating cellular immunity. The in vitro differentiation inhibition test further confirmed that RDN affected Th1 differentiation by inhibiting the expression of transcription factors on the basis of Th1 cell differentiation in vitro.
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Objective To analyze the epidemic characteristics of the hand-foot-mouth disease (HFMD) in four years before and after EV71 vaccine inoculation, and to provide a basis for better EV71 vaccination and prevention and control of HFMD. Methods The descriptive epidemiology method was used to analyze the monitoring data of HFMD from 2013 to 2020 and EV71 vaccination from 2017 to 2020 in Xinwu District of Wuxi City. Results A total of 9 589 HFMD cases including 104 severe cases (accounting for 1.08%) were reported in four years before EV71 vaccination, and the reported average annual incidence rate was 431.15/100 000. A total of 7 396 HFMD cases including 21 severe cases (accounting for 0.28%) were reported in four years after vaccine inoculation, and the reported average annual incidence rate was 325.28/100,000. Annual incidence rate and severe illness rate decreased significantly before and after vaccine inoculation. The HFMD incidences displayed two epidemic peaks, from May to June and from October to November. The three streets with the highest average annual incidence were Jiangxi, Meicun, and Shuofang. The HFMD cases were mainly children under 5 years old, and there were more men than women. A total of 394 samples were sent for examination from 2013-2020, and 231 were positive, with a total positive rate of 58.63%. There were significant changes in the etiological composition before and after vaccine inoculation. The composition of EV71 virus decreased significantly (χ2=69.70, P2=22.35, P<0. 05). From 2017 to 2020, a total of 12 472 people were inoculated with EV71 vaccine in Xinwu District of Wuxi City, with estimated annual vaccination rates of 3.78%, 10.96%, 8.40% and 7.63%, respectively. Conclusion There is no significant change in time, region and population distribution of HFMD before and after EV71 vaccination in Xinwu District of Wuxi City, but the annual incidence rate and severe illness rate show a decreasing trend, and the dominant intestinal pathogens have changed. It is suggested to strengthen the use of EV71 vaccine and the development of multivalent HFMD-related vaccine.
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Objective:To analyze differentially expressed microRNAs (miRNAs) and target genes in human respiratory epithelial cells (16HBE) after enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) infection using high-throughput sequencing.Methods:TargetScan and miRDB databases were used to predict the target genes of miRNAs that were both up-regulated or down-regulated after EV71 and CVA16 infection. The genes that were both up-regulated and down-regulated were screened out. GO and pathway analysis of the target genes were conducted to screen immune-related target genes and their corresponding miRNAs. The target genes and their corresponding miRNAs that were up-regulated or down-regulated in both immune-related GO and pathway were further screened. Some miRNAs and their target genes were selected for qRT-PCR verification.Results:There were 598 target genes of up-regulated miRNAs and 1 311 target genes of down-regulated miRNAs and 62 target genes that might be up-regulated or down-regulated simultaneously were screened out. The number of up-regulated target genes involved in immune-related GO and pathway were 17 and 13, respectively, and the number of corresponding miRNAs were 15 and 17, respectively. There were 58 and 47 down-regulated target genes involved in immune-related GO and pathway, respectively, and the number of corresponding miRNAs were 30 and 42, respectively. Three up-regulated target genes were involved in both immune-related GO and pathway and regulated by four miRNAs. Nine down-regulated target genes were involved in both immune-related GO and pathway and regulated by 13 miRNAs.Conclusions:This study was conducive to elucidate the host-pathogen interaction after EV71 and CVA16 infection, and provided reference for studying the pathogenesis of hand, foot and mouth disease.
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Enterovirus 71 (EV71) is one of the main pathogens causing hand, foot and mouth disease (HFMD), which seriously threatens the health of infants and young children and causes major infectious diseases in the Asia-Pacific region. The host′s innate immune response is the most effective way to fight against viral infections. However, EV71 has evolved a series of strategies, escaping from innate immune surveillance and destroying its antiviral function in the process of gaming with the host. This article mainly focuses on the research progress of EV71′s innate immune escape mechanism, and provides help for the development of anti-EV71 infection drugs.
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Aim To investigate the antiviral activity and mechanism of myricetin against enterovirus 71 (E V 7 1) infection. Methods The cytopathic effect (CPE) and plaque assay were used to observe the antiviral effect of myricetin against EV71 in Vero cell. The cells were treated with myricetin at different concentrations combined with crystal violet staining to detectthe cytotoxicity of myricetin. The effect of myricetin on VP1 protein expression was detected by Western blot. The effect of myricetin on VP1 gene expressionwas evaluated byRT-PCR. Results Myricetin pretreatment at 2. 5-20 fimol L-1' significantly inhibitedcell death induced by EV71 infection in a dose-dependent manner with the IC50 value of 5. 6 jxmol • L-1. Compared to virus control group, myricetin could significantly reduce the viral titer at the concentration of 2. 5 ~ 20 u,mol • L-1. The results of Western blot and RT-PCR showed that myricetin could markedlyreduce the gene and protein expression levels of viral capsid protein VP1. Conclusion Myricetin has significant antiEV71 activity in vitro.
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Objective@#To investigate the influence of dendritic cell specific intercellular adhesion molecule-3 grabbing non integrin (DC-SIGN) expression on the infection of enterovirus type 71 (EV71) for dendritic cells (DCs) and 293T cells. @*Methods@#Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood by gradient density centrifugation and induced to DCs. The DC-SIGN gene was amplified by polymerase chain reaction (PCR) for construction of transient expression vector pLB-DC-SIGN which was then transfected into 293T cells using Lipofectamine TM 2000. After 293T cell and 293T-pLB-DC-SIGN was infected with EV71, EV71 mRNA was identified by fluorescence quantitative PCR. EV71/VP1 was identified by western blot. After the expression of DC-SIGN on DCs was blocked by yeast mannan, EV71 mRNA was identified by fluorescence quantitative PCR and EV71/VP1 was identified by western blot. @*Results@#EV71 viral load and EV71/VP1 expression level in 293T cells overexpressed DC-SIGN was significantly higher than that of control 293T, while the viral load and EV71/VP1 expression level in DCs blocked with DC-SIGN inhibitor was lower than that of control DCs (P＜0.05). @*Conclusion@#DC-SIGN may be one of the receptors of EV71 who promote EV71 infection in DCs and 293T cells in vitro.
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Objective To investigate the effect of Reduning Injection on Vero cells and suckling mice infected by EV71 virus and its mechanism in vitro and in vivo. Methods The anti-virus ability was exhibited in Vero cells which were infected by 33 TCID50 and 25 TCID50 titers of EV71 in vitro. Meanwhile, the antiviral activity in vivo was evaluated on suckling mice model, in which suckling mice were intraperitoneal injected with EV71 at 100 μL. After 2 h infection, Reduning Injection 2.60, 1.30, and 0.65 g/kg and ribavirin 10 mg/kg were followed for treatment. To further understand the antiviral effects of Reduning Injection, weigh of suckling mice, clinical symptoms, expression of IL-1β, IL-6, MCP-1, and TNF-α in skeletal muscle were recorded. Moreover, histological examination of skeletal muscle was examined. Results The results demonstrated that Reduning Injection 5.0 mg/mL could obviously inhibit Vero cells lesion induced by EV71 in vitro. Compared with the model group, suckling mouse model results showed the high and middle dose of Reduning Injection could reduce clinical manifestations scores, increase the weight and improve living conditions of model mice, decrease the expression of IL-1β, IL-6, TNF-α, and MCP-1 in skeletal muscle of suckling mice (P<0.05, 0.01). Histological damages in skeletal muscle were also alleviated with Reduning Injection treatment. Conclusion In a suckling mouse model, Reduning Injection can reduce mortality and obviously prolong the survival time of suckling mice caused by EV71 infection. It can alleviate clinical symptoms and growth inhibition caused by the EV71 virus. Inhibition of IL-1β, IL-6, TNF-α, and MCP-1 levels may be the important mechanism of Reduning Injection in the treatment of hand-foot-and-mouth disease.
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Objective To establish an enterovirus 71(EV71) infection model of tree shrew primary renal cells.Methods Tree shrew primary renal cells were obtained by trypsin digestion.After subculture and purification,EV71 virus was used to infect these primary cells.The culture supernatant of these EV71-infected cells was collected for virus titer detection at 1,2,4,6 and 8 days post-infection.The cells were collected for detection of EV71 VP1 protein by Western blot assay.Furthermore,the expression and location of VP1 protein in the infected cells were detected by indirect immunofluorescence assay.Vero cells were taken as positive control to evaluate the infectivity of EV71 virus to tree shrew primary renal cells.Results Morphologically,the cultured cells were proved to be majorly consisted of the primary renal cells after subculture and purification.The obtained primary cells were infected by EV71 virus.The virus titer was up to 1.3×106 TCID 50/mL during 48-96 h post-infection,proving that EV71 virus infected and proliferated in the tree shrew primary renal cells.Western blot showed that the viral VP1 protein was detected from infected primary cells at 2 to 8 d post infection.VP1 protein was also observed in the cytoplasm at 2 to 6 d post infection by indirect immunofluorescence.Compared with Vero cells,the infectivity of EV71 virus to tree shrew primary renal cells and its proliferation were confirmed.Conclusions Based on the successful establishment of cell culture of tree shrew primary renal cells,the infectivity to the obtained cells and proliferation of EV71 virus in the cells are confirmed.The model of EV71 virus-infected tree shrew primary renal cells is initially established.
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Objective To observe the effects of Ganlu Xiaodu Dan and its incomplete prescription on expressions of MiR-146a and TLR4 mRNA in RD cells infected by EV71. Methods With technique of cell culturing, Ganlu Xiaodu Dan therapy group, incomplete Qing prescription therapy group, incomplete Li prescription therapy group, normal cells control group, model control group and ribavirin control group were set, and tests for virus toxicity and medicine toxicity in cells were taken, then expressions of miRNA-146a and TLR4 mRNA in these RD cells 24 hours after intervention with medicine were detected. Results Compared with normal cells control group, miR-146a in mRNA model control group decreased and TLR4 mRNA increased. Compared with model control group, miR-146a mRNA in Ganlu Xiaodu Dan therapy group, incomplete Qing prescription therapy group and incomplete Li prescription therapy group all increased while TLR4 mRNA decreased, and differences between ribavirin control group and model control group were not significant. Compared with Ganlu Xiaodu Dan therapy group, both expressions of miR-146a and TLR4 mRNA in incomplete Qing prescription therapy group were lower; miR-146a increased and TLR4 mRNA decreased in incomplete Li prescription therapy group. Compared with incomplete Qing prescription therapy group, miR-146a mRNA in incomplete Li prescription therapy group increased, but expression of TLR4mRNA between them was not significant. Conclusion Ganlu Xiaodu Dan can regulate the immune reactions caused by infection of EV71 by increasing expression of miR-146a mRNA and reducing expression of TLR4 mRNA. There may be antagonism effect between incomplete Qing prescription and incomplete Li prescription.
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Objective To identify the role of RNA-editing enzyme ADAR1 (adenosine deaminase acting on RNA) in EV71 infection and virus mutation.Methods RNAi technology was applied to establish ADAR1 knock-down stable cell lines.Then the cells were served to evaluate the role of ADAR1 in EV71 infection by MTT assay for detecting virus-induced cell viability,virus plaque assay for quantification of the virus titer and the cellular susceptibility to the virus,and Western blot for virus protein expressions.ADAR1-mediated RNA editing can result in the genetic A-G and T-C mutations.To further determine whether the effects of ADAR1 on EV71 infection were correlated with ADAR1-mediated EV71 RNA editing and therefore increased the viral mutations during the infection,the characteristics of EV71 mutation were analyzed based on the different full-length viral genomes from epidemic regions.The viral genome was also sequenced from the infected ADAR1 knock-down cells.Results After ADAR1 knock-down,the cell viability decreased quickly after the virus infection,and formed much more and larger sizes of plaques than the control cells.The virus capsid protein VP1 expressions and virus titer in the cells culture media were both increased in ADAR1 knockdown cells.Statistic analysis showed that A-G and T-C mutations were the major mutations of EV71,which were believed to be the hot sites for RNA-editing.However,the results of viral RNA genomic sequencing data indicated that ADAR1 did not edit EV71 genome directly.Conclusions ADAR1 was a restriction factor for controlling EV71.However,ADAR1 does not directly edit EV71 genome.
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Objective To study the epidemiological characteristics of Hand-Foot-Mouth Disease of chil-dren in Zhongshancity from 2011 to 2015 and provide etiological evident for the prevention and control of Hand -Foot -Mouth Disease of children in Zhongshan city. Methods We analyzed the data of children with Hand-Foot-Mouth Disease in Zhongshan Boai 's Hospital from 2011 to 2015. The enterovirus nucleic acid of Hand-Foot-Mouth Diseaseof children were detected. Results Atotal of 31164 cases of Hand-Foot-Mouth Disease were diagnosed in Zhongshan Boai′s Hospital during 2011-2015,the ratio of male to female was 1.62:1. Every year,the peak inci-dence of the disease is not the same. During 2011-2015,there were 10147 cases of Hand-Foot-Mouth Diseaseof children,among which,through viral nucleic acid EV71and CoxA16testing,2062 cases were positive and the positve rate was 20.32%. Pathogen of Hand-Foot-Mouth Diseasewas mainly based on EV71 in 2011,2012, 2013and2015,accounting for the total positive rate of 79.04%,72.46%,81.60%,73.73%respectively;Pathogen of Hand -Foot -Mouth Diseasewas mainly based on Cox16 in 2014,accounted for 54.47% of the total positive. In different years EV71 and CoxA16 positive rate was significant different.There were 1982 cases repeated Hand-Foot-MouthDiseaseinfectioins .Conclusion The incidence of Hand-Foot-Mouth Disease of children is related to age , sex and season in Zhongshan city. It is mainly based on clinical diagnosis ,the preventive measures and the clinical recognition in time to provide help for the treatment and control of Hand-Foot-Mouth Disease of children.
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Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.
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Animals , Humans , Mice , Amino Acid Sequence , Antibodies, Monoclonal , Chemistry , Genetics , Metabolism , Binding Sites , Cell Line , Crystallography, X-Ray , Enterovirus A, Human , Genetics , Allergy and Immunology , Fibroblasts , Virology , Gene Expression , HEK293 Cells , Immunoglobulin Fab Fragments , Chemistry , Genetics , Metabolism , Lysosomal Membrane Proteins , Chemistry , Genetics , Allergy and Immunology , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, Scavenger , Chemistry , Genetics , Allergy and Immunology , Receptors, Virus , Chemistry , Genetics , Allergy and Immunology , Recombinant Fusion Proteins , Chemistry , Genetics , Allergy and Immunology , Sequence Alignment , Sequence Homology, Amino Acid , Sf9 Cells , Spodoptera , ThermodynamicsABSTRACT
Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD), particularly in infants and children below 4 years of age. Shikonin is a bioactive compound with anti-inflammatory, antiviral, and antibacterial activities derived from the roots of the Chinese medicinal herb Lithospermum erythrorhizon. This study aimed to examine the antiviral activity of PMM-034, a shikonin ester derivative, against EV71 in rhabdomyosarcoma (RD) cells. Cytotoxicity of PMM-034 on RD cells was determined using WST-1 assay. Dose- and time-dependent effects of PMM-034 on EV71 replication in RD cells were determined using plaque reduction assay. mRNA expression levels of EV71/VP1 and pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) were determined by real-time RT-PCR, and EV71/VP1 and phospho-p65 protein expressions were determined by western blot analysis. PMM-034 exhibited only weak cytotoxicity against RD cells. However, PMM-034 exhibited significant antiviral activity against EV71 in RD cells with 50% inhibitory concentration of 2.31 μg/mL. The VP1 mRNA and protein levels were significantly reduced in cells treated with PMM-034. Furthermore, relative mRNA expression levels of IL-1β, IL-6, IL-8, and TNF-α significantly decreased in the cells treated with PMM-034, while the phospho-p65 protein expression was also significantly lower in the treated cells. These results indicated that PMM-034 suppressed the expressions of pro-inflammatory cytokines in RD cells, exhibiting antiviral activity against EV71, as evidenced by the reduced VP1 mRNA and protein levels in PMM-034-treated cells. Thus, PMM-034 is a promising candidate for further development as an EV71 inhibitor.
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Humans , Antiviral Agents/pharmacology , Enterovirus A, Human/drug effects , Naphthoquinones/pharmacology , Rhabdomyosarcoma/virology , Blotting, Western , Cell Line, Tumor , Cytokines/analysis , Dose-Response Relationship, Drug , Real-Time Polymerase Chain Reaction , Toxicity Tests , Viral Plaque Assay , Virus Replication/drug effectsABSTRACT
Enterovirus 71 (EV71) is the main pathogen of severe hand-foot-mouth disease. As the characteristics and the pathological mechanism of EV71 gradually become clear, the therapeutic strategies of EV71 infection are clarified. Acting on specific target can directly interfere the process of EV71 infection, while regulating the host immune can clean the virus, ease inflammation and protect uninfected cells. In addition, some cytokines like interferon with both broad-spectrum antiviral function and immune regulation are used in clinical. In addition, drug combination could enhance the antiviral effect and reduce toxicity and drug resistance. At present, most of the therapeutics are still in pre-clinical phase, while only a few are in clinical application. This article reviews the different types of EV71 infection treatment strategies and methods recently reported, in order to provide help for further research on anti-EV71 infection.