Enterovirus Research Articles

Rational design of a DNA-launched live attenuated vaccine against human enterovirus 71

Human Enterovirus 71 (EV71) has emerged as one of the predominant causative agents of hand, foot and mouth disease (HFMD) with global impact. Despite the inactivated vaccine being licensed, other vaccine candidates based on advanced technology platforms are under development. In this report, we rationally designed and constructed two DNA-launched live attenuated vaccine candidates (pDL-EV71) under the control of specific promoters. In vitro and in vivo transfection with pDL-EV71 driven by the CMV promoter successfully yielded fully infectious EV71. More importantly, the administration of pDL-EV71 did not cause clinical symptoms following intracranial or intramuscular inoculation in neonatal and IFNα/βR−/− mice, demonstrating its safety profile. Moreover, a single-dose or two-dose immunization with pDL-EV71 elicited robust neutralizing antibodies against EV71 as well as an antigen-specific cellular response in mice. A single-dose immunization with 10 ​μg of pDL-EV71 conferred complete protection against lethal EV71 infection in neonates born to immunized maternal mice. Overall, our present results demonstrate that pDL-EV71 is a safe and effective vaccine candidate against EV71 for further development.

Tanomastat exerts multi-targeted inhibitory effects on viral capsid dissociation and RNA replication in human enteroviruses

Global cyclical outbreaks of human enterovirus infections has positioned human enterovirus A71 (EV-A71) as a neurotropic virus of clinical importance. However, there remains a scarcity of internationally approved antivirals and vaccines. In pursuit of repurposing drugs for combating human enteroviruses, we employed a comprehensive pharmacophore- and molecular docking-based virtual screen targeting EV-A71 capsid protein VP1-4, 3C protease, and 3D polymerase proteins. Among 15 shortlisted ligand candidates, we dissected the inhibitory mechanism of Tanomastat in cell-based studies and evaluated its invivo efficacy in an EV-A71-infected murine model. We demonstrated that Tanomastat exerts dose-dependent inhibition on EV-A71 replication, with comparable efficacy profiles in enterovirus species A, B, C, and D invitro. Time-course studies suggested that Tanomastat predominantly disrupts early process(es) of the EV-A71 replication cycle. Mechanistically, live virus particle tracking and docking predictions revealed that Tanomastat specifically impedes viral capsid dissociation, potentially via VP1 hydrophobic pocket binding. Bypassing its inhibition on entry stages, we utilized EV-A71 replication-competent, 3Dpol replication-defective, and bicistronic IRES reporter replicons to show that Tanomastat also inhibits viral RNA replication, but not viral IRES translation. We further showed that orally administered Tanomastat achieved 85% protective therapeutic effect and alleviated clinical symptoms in EV-A71-infected neonatal mice. Our study establishes Tanomastat as a broad-spectrum anti-enterovirus candidate with promising pre-clinical efficacy, warranting further testing for potential therapeutic application. MOE Tier 2 grants (MOE-T2EP30221-0005, R571-000-068-592, R571-000-076-515, R571-000-074-733) and A∗STARBiomedical Research Council (BMRC).

Characterization of cross-reactivity of coxsackievirus A2 VP1-specific polyclonal antibodies with enterovirus A71, coxsackievirus A16, and coxsackievirus A6

Coxsackievirus A2 (CVA2) is associated with multiple diseases in children. Currently, there is limited research on immunological detection methods for CVA2. Herein, the VP1 gene of CVA2 strain 201711, belonging to cluster 2 within genotype D, was analyzed. The structures of VP1 from CVA2 strains 201711, 7-1 and 12-1, enterovirus A71 (EV-A71) strain 201713, coxsackievirus A16 (CVA16) strain 201717, and coxsackievirus A6 (CVA6) strain JLS10 were compared. The Escherichia coli BL21(DE3)/pET vector system was employed to express the recombinant protein containing the entire VP1 of CVA2 strain 201711. Mice were immunized with the purified protein, and the sera were collected and used to specifically identify the VP1 in CVA2-infected RD cells by Western blot and immunofluorescence assay. There was no evident cross-reactivity of the sera with the VP1 of EV-A71, CVA16, and CVA6 strains mentioned above. Therefore, this study provided mouse-specific anti-CVA2 VP1 polyclonal antibodies for CVA2 detection.

Biomarkers and genotypes in patients with Central nervous system infection caused by enterovirus

Purpose Enteroviruses (EV) comprises many different types and are the most common cause of aseptic meningitis. How the virus affects the brain including potential differences between types are largely unknown. Measuring biomarkers in CSF is a tool to estimate brain damage caused by CNS infections. Methods A retrospective study was performed in samples from 38 patients with acute neurological manifestations and positive CSF-EV RNA (n = 37) or serum-IgM (n = 1). The EV in 17 samples were typed by sequencing. The biomarkers neurofilament light (NFL), glial fibrillary acidic protein (GFAP), S-100B protein, amyloid-β (Aβ) 40 and Aβ42, total-tau (T-tau) and phosphorylated tau (P-tau) were measured and compared with data derived from a control group (n = 19). Results There were no increased levels of GFAP (p ≤ 0.1) nor NFL (p ≤ 0.1) in the CSF of patients with EV meningitis (n = 38) compared with controls. However, we found decreased levels of Aβ42 (p < 0.001), Aβ40 (p < 0.001), T-tau (p ≥ 0.01), P-tau (p ≤ 0.001) and S-100B (p ≤ 0.001). E30 (n = 9) and CVB5 (n = 6) were the most frequent EV-types identified, but no differences in biomarker levels or other clinical parameters were found between the infecting virus type. Seven patients who were followed for longer than one month reported remaining cognitive impairment, although no correlations with biomarker concentrations were observed. Conclusion There are no indication of neuronal or astrocyte damage in patients with EV meningitis. Yet, decreased concentrations of Aβ40, Aβ42, P-tau and T-tau were shown, a finding of unknown importance. Cognitive impairment after acute disease occurs, but with only a limited number of patients analysed, no conclusion can be drawn concerning any association with biomarker levels or EV types.

Clinical application of phage immunoprecipitation sequencing to diagnose enterovirus D68 as the underlying etiology in a case of Gullain-Barré syndrome.

Gullain-Barré syndrome (GBS) is an acute peripheral neuropathy often preceded by respiratory or gastrointestinal infections, though molecular testing of cerebrospinal fluid (CSF) is often inconclusive. In a recent case of severe pediatric GBS in British Columbia, Canada, we detected CSF antibodies against enterovirus D (EV-D) to link GBS with prior EV-D68 respiratory infection.

Detection of hotspots and coldspots of the incidence of hand, foot and mouth diseases: A case study in Ho Chi Minh city, Vietnam

Background: Hand, foot, and mouth disease (HFMD) is a common pediatric illness mostly caused by Coxsackievirus A16 (CV-A16) and Human Enterovirus 71 (EV-A71). HFMD has grown significantly over the last 20 years, and it has drawn a lot of attention. To better understand of the epidemiology of HFMD, this study aims to investigate the use of Getis-Ord’s 𝐺𝑖 ∗ statistic in the identification of hotspots and coldspots of HFMD in Ho Chi Minh city, Vietnam. Methods: Getis-Ord’s 𝐺𝑖 ∗ statistic was first applied to identify hotspots and coldspots of HFMD incidence including highhigh, low-low spatial clusters. HFMD cases and infection rates collected in Ho Chi Minh were then used to explore the spatial clusters of HFMD Finally, the main findings will be discussed and summarised. Results: It was found that the large number of hand, foot and mouth disease cases were mainly concentrated in the western area of Ho Chi Minh City. A total of 04 hotspot clusters and 05 coldspots of HFMD infection rate were detected in the 29th week of 2024 in the west, center and north of Ho Chi Minh City, respectively. Whereas, 06 hotspots and 04 coldspots of HFMD infection rate were detected in the 30th week of 2024 in the west, south and center of Ho Chi Minh City, respectively. Conclusions: The results of this investigation confirm the value of Getis-Ord's 𝐺𝑖 ∗ statistic in the detection of hotspots and coldspots associated with confirmed cases of HFMD.

Long-Term Immunogenicity Study of an Aluminum Phosphate-Adjuvanted Inactivated Enterovirus A71 Vaccine in Children: An Extension to a Phase 2 Study.

Enterovirus A71 (EV-A71) causes hand, foot, and mouth disease in infants and children with potential for fatal complications such as encephalitis and acute flaccid myelitis. This study examined the long-term immunity conferred by EV71vac, an inactivated EV-A71 vaccine adjuvanted with aluminum phosphate, in children from the age of 2 months to <6 years, for up to 5 years after the first immunization. A total of 227 participants between 2 months and <6 years of age who had previously received either EV71vac or placebo in the phase two clinical study were enrolled. Subjects were divided into age groups: 2 years to <6 years (Group 2b), 6 months to <2 years (Group 2c), and 2 months to <6 months (Group 2d). At Year 5, the neutralizing antibody titers against the B4 subgenotype remained high at 621.38 to 978.20, 841.40 to 1159.93, and 477.71 to 745.07 for Groups 2b, 2c, and 2d, respectively. Cross-neutralizing titers at Year 5 remained high against B5 and C4a subgenotypes, respectively. No long-term safety issues were reported. Our study provides novel insights into the long-term immunity conferred by EV71vac in children aged from two months to six years, particularly in those who received EV71vac between two and six months of age.

Metabolic immaturity and breastmilk bile acid metabolites are central determinants of heightened newborn vulnerability to norovirus diarrhea

The pathogenic outcome of enteric virus infections is governed by a complex interplay between the virus, intestinal microbiota, and host immune factors, with metabolites serving as a key mediator. Noroviruses bind bile acid metabolites, which are produced by the host and then modified by commensal bacteria. Paradoxically, bile acids can have both proviral and antiviral roles during norovirus infections. Working in an infant mouse model of norovirus infection, we demonstrate that microbiota and their bile acid metabolites protect from norovirus diarrhea, whereas host bile acids promote disease. We also find that maternal bile acid metabolism determines the susceptibility of newborn mice to norovirus diarrhea during breastfeeding. Finally, targeting maternal and neonatal bile acid metabolism can protect newborn mice from norovirus disease. In summary, neonatal metabolic immaturity and breastmilk bile acids are central determinants of heightened newborn vulnerability to norovirus disease.

Comparison of four concentration methods of adenovirus, norovirus and rotavirus in tap water

Human enteric viruses, as adenovirus (HAdV), norovirus (HuNoV) and rotavirus (RVA) are significant causes of gastroenteritis associated with consumption of contaminated water worldwide. Various methods have been described for their detection and monitoring in water. The aim of this study was to compare the performance of four conditions for concentrating HAdV, HuNoV and RVA from water matrices, in order to develop a single protocol that could simultaneously concentrate all target viruses from tap water. The tested conditions were based on the adsorption-elution using electronegative filters, in which we evaluated cation-coated filtration by MgCl2 with or without acid rinse by H2SO4 and two elution buffers, namely NaOH and tris-glycine-beef extract. Genomic material was extracted and amplified by real-time PCR and real-time RT-PCR using commercial kits. Based on the statistical analysis of amplification results (cycles of quantification), the condition involving cation-coated filtration by MgCl2 using electronegative filters with acid rinse by H2SO4 combined with NaOH elution allowed efficient recovery of both HAdV, HuNoV and RVA from tap water compared to the other conditions. These findings confirm the effectiveness of the approach used to monitor three major enteric viruses in tap water.

Epidemiology of childhood enterovirus infections in Hangzhou, China, 2019–2023

Human enteroviruses are highly prevalent world-wide. Up to more than 100 subtypes of enteroviruses can cause several diseases, including encephalitis, meningitis, myocarditis, hand-foot-mouth disease, conjunctivitis, respiratory diseases, and gastrointestinal diseases, thus posing a great threat to human health. This study aimed to investigate the epidemiological characteristics of enterovirus in children in Hangzhou, China before and after the COVID-19 outbreak. Systematic monitoring of enterovirus infections was performed by collecting samples from the children admitted to the inpatient wards and outpatient departments in the Children’s Hospital, Zhejiang University School of Medicine, between January 2019 and May 2023. A commercial real-time RT PCR kit was utilized to detect enteroviruses. Among the 34,152 samples collected, 1162 samples, accounting for 3.4% of the samples, were tested positive for enteroviruses. The annual positive rates of the enteroviruses were 5.46%, 1.15%, 4.43%, 1.62%, and 1.96% in 2019, 2020, 2021, 2022, and May 2023, respectively. The positivity rate of the enteroviruses was highest among children aged 3–5 years and 5–7 years. Moreover, the monthly positivity rate of enterovirus infection ranged from 0.32% to 10.38%, with a peak in June and July. Serotypes, especially EV71 and CA16, causing severe symptoms such as HFMD, were decreasing, while the proportion of unidentified serotypes was on the rise. The incidence of enteroviruses in Hangzhou was higher in children aged 1–3 years and 7–18 years.

A Useful Method to Provide Infectious and Cultivable In Vitro Naked Viral Particles of Hepatitis A Virus.

Hepatitis A virus (HAV) is an enteric virus mainly transmitted by the faecal-oral route. Belonging to the Picornaviridae family, HAV was first described as small naked particles, like all viruses of this family. However, for about a decade, it was demonstrated that HAV particles can exist surrounded by a lipid bilayer. This type of particle, called enveloped HAV (eHAV), acquires its lipid bilayer by hijacking a part of cell membranes during the virion egress in the last steps of the viral cycle. In vitro culture systems produce mainly eHAV, and so, to date, most of the studies on HAV have been carried out using this type of viral particle. In this study, a method based on lipid bilayer removal by chemical delipidation is proposed for the production of naked HAV particles. The resulting naked HAV particles conserve their infectivity and are therefore fully cultivable in vitro. By using this method, naked HAV particles can easily be produced in vitro and can be useful to perform further studies such as inactivation processes for the food industry, as HAV is a main concern for food safety.

Enteric viruses and SARS-CoV-2 in environmental water in rural communities of Osun State, Nigeria

Enteric viruses and SARS-CoV-2 in environmental water in rural communities of Osun State, Nigeria

Monitoring the VDPV2 outbreak in Egypt during 2020–2021 highlights the crucial role of environmental surveillance and boosting immunization in combating Poliovirus

BackgroundPoliovirus is a highly infectious enterovirus (EV) that primarily affects children and can lead to lifelong paralysis or even death. Vaccine-derived polioviruses (VDPVs) are a great threat since they are derived from the attenuated virus in the Oral Poliovirus Vaccine (OPV) and can mutate to a more virulent form. The purpose of this study was to identify VDPV serotype 2 through the year 2020–2021 via surveillance of sewage samples collected from different localities and governorates in Egypt and stool specimens from Acute Flaccid Paralysis (AFP) cases. Both were collected through the national poliovirus surveillance system and according to the guidelines recommended by the WHO.MethodsA total of 1266 sewage samples and 3241 stool samples from January 2020 to December 2021 were investigated in the lab according to World Health Organization (WHO) protocol for the presence of Polioviruses by cell culture, molecular identification of positive isolates on L20B cell line was carried out using real-time polymerase chain reactions (RT-PCR). Any positive isolates for Poliovirus type 2 and isolates suspected of Vaccine Derived Poliovirus Type 1 and type 3 screened by (VDPV1) or Vaccine Poliovirus Type 3 (VDPV3) assay in RT-PCR were referred for VP1 genetic sequencing.ResultsThe outbreak was caused by circulating VDPV2 (cVDPV2) strains started in January 2021. By the end of February 2021, a total of 11 cVDPV2s were detected in sewage samples from six governorates confirming the outbreak situation. One additional cVDPV2 was detected later in the sewage sample from Qena (June 2021). The first and only re-emergence of VDPV2 in stool samples during the outbreak was in contact with Luxor in June 2021. By November 2021, a total of 80 VDPVs were detected. The Egyptian Ministry of Health and Population (MOHP), in collaboration with the WHO, responded quickly by launching two massive vaccination campaigns targeting children under the age of five. Additionally, surveillance systems were strengthened to detect new cases and prevent further spread of the virus.ConclusionThe continued threat of poliovirus and VDPVs requires ongoing efforts to prevent their emergence and spread. Strategies such as improving immunization coverage, using genetically stable vaccines, and establishing surveillance systems are critical to achieving global eradication of poliovirus and efficient monitoring of VDPVs outbreaks.

Copper I-II-containing composites and coatings with high and broad-spectrum antimicrobial activity

Recently, antimicrobial surface coatings have emerged as an effective strategy to mitigate microbial contamination and disease transmission. The application of cuprous (I) oxide (Cu2O) and copper (II) pyrithione (CPT) in antimicrobial surfaces faces several challenges such as deep color, weak stability, limited functionality, and poor dispersion in coatings. These challenges complicate the design of novel copper-based antimicrobial agents. In this study, high-purity CPT was synthesized innovatively using Cu2O as a copper source, differing from traditional methods. Following this approach, diatomaceous earth/cuprous oxide/copper pyrithione (DME/Cu2O/CPT) composites with enhanced antimicrobial activity and a lighter color than Cu2O and CPT were prepared by adjusting the SPT dosage. Notably, in DME/Cu2O/CPT composites, Cu2O existed as nanoscale fragments adhering to the surfaces of CPT. Furthermore, coatings made from composites and latex paint exhibited a lighter color difference, higher stability, and exceptional antimicrobial activity against pathogens such as S. aureus, E. coli, P. aeruginosa, C. albicans, and human enterovirus 71. The DME/Cu2O/CPT composites displayed high, stable, and broad-spectrum antimicrobial efficacy, attributing to the synergistic enhancement effects of released Cu+ and Cu2+. Consequently, this study introduced a simple and scalable method for preparing DME/Cu2O/CPT composites, serving as a coating additive with broad-spectrum antimicrobial activity and long-term stability for various solid surfaces.

Antiviral Activity of Rhein against Enterovirus 71 through Inhibiting Viral Replication and Stability

Background: Enterovirus 71 (EV71) is one of the most prevalent pathogens responsible for hand, foot, and mouth disease in the Asia-Pacific region. Severe EV71 infections can be fatal in children under the age of 5. As of now, there are no proven anti-EV71 drugs available. Rhein is an anthraquinone derivative, mainly derived from rhubarb plants. Several beneficial pharmacological properties of rhein have been linked to anti-inflammatory, antioxidant, anticancer, and antiviral effects. Aim: This research aims to evaluate rhein’s antiviral activity against Enterovirus EV71 in vitro. Methods: The cytotoxicity of rhein was assessed using a Cell Counting Kit-8 kit. The antiviral activities of rhein were characterized by viral RNA level, protein expression level, and infectious ability using quantitative reverse transcription polymerase chain reaction, Western blot assay, and immunofluorescence assay, respectively. The mechanism by which rhein suppresses virus life cycles was examined utilizing a time-of-addition assay. An inactivation assay was performed to evaluate whether rhein directly impaired the virion stability. Results: The findings indicated that rhein exhibited anti-EV71 activity by reducing viral RNA synthesis, protein expression, and infectivity. Rhein demonstrates potent antiviral effects against EV71 at the late-life stage and inhibition of virion stability. Conclusion: Our findings strongly support further research into rhein as a potential treatment for EV71.

Anti-enterovirus 71 activity of native fucosylated chondroitin sulfates and their derivatives

Enterovirus 71 (EV71) is recognized as a major causative agent of hand, foot, and mouth disease (HFMD), posing a significant global public health concern due to its widespread impact and resulting in a major public health issue worldwide. Despite its prevalence, current clinical therapy lacks effective antiviral agents. Fucosylated chondroitin sulfates (FCS) derived from sea cucumber exhibits a range of biological activities including potent antiviral effects. This study provides compelling evidence of the potent antiviral efficacy of FCS against EV71. To further elucidate the impact of structural variations on the anti-EV71 activity, native FCSs with diverse sulfation patterns and a varity of FCS derivatives were prepared and analyzed. Notably, this study presents the detailed structural characterization of FCSs from the sea cucumbers Holothuria scabra Jaege and Holothuria fuscopunctata. Analysis of the structure-activity relationships revealed that molecular weight, sulfated fucose branches, and sulfation pattern were all crucial factors contributing to the potent inhibitory effects of FCS against EV71. Interestingly, molecular weight emerged as the most significant structural determinant of the antiviral potency. These findings suggest the promising potential of utilizing FCS as an innovative EV71 entry inhibitor for the treatment of HFMD.

TUDCA inhibits EV71 replication by regulating ER stress signaling pathway and suppressing autophagy

Tauroursodeoxycholic acid (TUDCA) is a naturally occurring hydrophilic bile acid that alleviates endoplasmic reticulum (ER) stress and inhibits apoptosis, thereby protecting cells. Previous studies have shown that enterovirus 71 (EV71) infection modulates ER stress and induces autophagy to assist viral replication. This study observed the effects of TUDCA pretreatment on HeLa and Vero cells infected with EV71, finding that TUDCA inhibits EV71 replication in TUDCA pretreated HeLa and Vero cells in a dose-dependent manner. We found that TUDCA pretreatment inhibited EV71 replication by regulating three branches of UPR, that is up-regulated ATF6, down-regulated both PERK and IRE1. The results also indicated that autophagy which is downstream of UPR, was inhibited either. The results indicate that TUDCA inhibits EV71 replication by regulating UPR sensor proteins and autophagy following ER stress.

Exploring waterborne viruses in groundwater: Quantification and Virome characterization via passive sampling and targeted enrichment sequencing

Aquifers, which provide drinking water for nearly half the world's population, face significant challenges from microbial contamination, particularly from waterborne viruses such as human adenovirus (HAdV), norovirus (NoV) and enterovirus (EV). This study, conducted as part of the UPWATER project, investigates the sources of urban groundwater contamination using viral passive sampling (VPS) and target enrichment sequencing (TES). We assessed the abundance of eight viral pathogens (HAdV, EV, NoV genogroup I and II, rotavirus, influenza A virus, hepatitis E virus and SARS-CoV-2) and investigated the virome diversity of groundwater in the aquifer of the Besòs River Delta in Catalonia. Over a period of 7 months, we collected 114 samples from the aquifer using nylon and nitrocellulose membranes to adsorb viruses over a 10-day period. Human faecal contamination was detected in nearly 50 % of the groundwater samples, with mean HAdV total counts ranging from 1.23E+02 to 3.66E+03 GC, and occasional detections of EV and NoV GI and GII. In addition, deep sequencing revealed a diverse virome in the aquifer, with detection of human pathogens, including adenovirus, astrovirus, calicivirus, enterovirus, herpesvirus, papillomavirus and rotavirus. Time-integrated sampling using VPS increases the likelihood of virus detection and, when combined with TES, can provide a deeper understanding of virus prevalence in this important water compartment. This approach is expected to streamline long-term monitoring efforts and enable small communities or water managers with limited resources to effectively manage their groundwater reservoirs.

Evaluation of inhibitory activity of essential oils and natural extracts on foodborne viruses.

Enteric viruses are recognized as a major concern in health care and in the food sector in Canada. Novel clean-label strategies for controlling enteric viruses are sought in the food industry. In this study, we examined the antiviral potential of plant extracts and essential oils on murine norovirus 1 (MNV-1), hepatitis A virus (HAV), and herpes simplex virus 1 (HSV-1). Inactivation of the viruses by grape seed, blueberry, green tea, and cranberry extracts and by rosemary and thyme essential oils was measured using plaque formation assay. Concentrations ranging from 50 to 200 000ppm with a contact time of 90min were tested. Grape seed extract at 10 000ppm was the most effective (P<0.05) at reducing MNV-1 and HAV infectious titers, respectively, by 2.85±0.44 log10 and 1.94±0.17 log10. HSV-1 titer was reduced by 3.81±0.40 log10 at 1000ppm grape seed extract. Among the plant products tested, grape seed extract was found the most effective at reducing the infectious titers of MNV-1, HAV, and HSV.

Correlations of PSGL-1 VNTR polymorphism with the susceptibility to severe HFMD associated with EV-71 and the immune status after infection

Enterovirus 71 (EV-71) has strong neurotropism, and it is the main pathogen causing severe hand, foot, and mouth disease (HFMD). In clinical observations, significant differences were observed in the severity and prognosis of HFMD among children who were also infected with EV-71. Genetic differences among individuals could be one of the important causes of differences in susceptibility to EV-71–induced HFMD. As P-selectin glycoprotein ligand-1 (PSGL-1) is an important receptor of EV-71, the correlation between single-nucleotide polymorphisms (SNPs) in PSGL-1 and the susceptibility to severe HFMD following EV-71 infection is worth studying. Given the role of PSGL-1 in immunity, the correlations between PSGL-1 SNPs and the immune status after EV-71 infection are also worth studying. Meanwhile, PSGL-1 variable number of tandem repeats (VNTR) represents a research hotspot in cardiovascular and cerebrovascular diseases, but PSGL-1 VNTR polymorphism has not been investigated in HFMD caused by EV-71 infection. In this study, specific gene fragments were amplified by polymerase chain reaction, and PSGL-1 VNTR sequences were genotyped using an automatic nucleic acid analyzer. The correlations of PSGL-1 VNTR polymorphism with the susceptibility to EV-71–associated severe HFMD and the post-infection immune status were analyzed. The PSGL-1 VNTR A allele was identified as a susceptible SNP for severe HFMD. The risk of severe HFMD was higher for AA + AB genotype carriers than for BB genotype carriers. The counts of peripheral blood lymphocyte subsets were lower in AA + AB genotype carries than in BB genotype carries. In conclusion, PSGL-1 VNTR polymorphism is associated with the susceptibility to EV-71–induced severe HFMD and the immune status after infection. PSGL-1 VNTR might play a certain role in the pathogenesis of severe cases.