Enterovirus Genotypes Research Articles

Adaptation of Enterovirus 71 to Adult Interferon Deficient Mice

Non-polio enteroviruses, including enterovirus 71 (EV71), have caused severe and fatal cases of hand, foot and mouth disease (HFMD) in the Asia-Pacific region. The development of a vaccine or antiviral against these pathogens has been hampered by the lack of a reliable small animal model. In this study, a mouse adapted EV71 strain was produced by conducting serial passages through A129 (α/β interferon (IFN) receptor deficient) and AG129 (α/β, γ IFN receptor deficient) mice. A B2 sub genotype of EV71 was inoculated intraperitoneally (i.p.) into neonatal AG129 mice and brain-harvested virus was subsequently passaged through 12 and 15 day-old A129 mice. When tested in 10 week-old AG129 mice, this adapted strain produced 100% lethality with clinical signs including limb paralysis, eye irritation, loss of balance, and death. This virus caused only 17% mortality in same age A129 mice, confirming that in the absence of a functional IFN response, adult AG129 mice are susceptible to infection by adapted EV71 isolates. Subsequent studies in adult AG129 and young A129 mice with the adapted EV71 virus examined the efficacy of an inactivated EV71 candidate vaccine and determined the role of humoral immunity in protection. Passive transfer of rabbit immune sera raised against the EV71 vaccine provided protection in a dose dependent manner in 15 day-old A129 mice. Intramuscular injections (i.m.) in five week-old AG129 mice with the alum adjuvanted vaccine also provided protection against the mouse adapted homologous strain. No clinical signs of disease or mortality were observed in vaccinated animals, which received a prime-and-boost, whereas 71% of control animals were euthanized after exhibiting systemic clinical signs (P<0.05). The development of this animal model will facilitate studies on EV71 pathogenesis, antiviral testing, the evaluation of immunogenicity and efficacy of vaccine candidates, and has the potential to establish correlates of protection studies.

Neutralizing Antibodies Induced by Recombinant Virus-Like Particles of Enterovirus 71 Genotype C4 Inhibit Infection at Pre- and Post-attachment Steps

BackgroundEnterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia–Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear.Methods/FindingsIn the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs). Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC) located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment.ConclusionsCollectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development.

Cross-reactive Neutralizing Antibody Responses to Enterovirus 71 Infections in Young Children: Implications for Vaccine Development

BackgroundRecently, enterovirus 71 (EV71) has caused life-threatening outbreaks involving neurological and cardiopulmonary complications in Asian children with unknown mechanism. EV71 has one single serotype but can be phylogenetically classified into 3 main genogroups (A, B and C) and 11 genotypes (A, B1∼B5 and C1∼C5). In Taiwan, nationwide EV71 epidemics with different predominant genotypes occurred in 1998 (C2), 2000–2001 (B4), 2004–2005 (C4), and 2008 (B5). In this study, sera were collected to measure cross-reactive neutralizing antibody titers against different genotypes.MethodsWe collected historical sera from children who developed an EV71 infection in 1998, 2000, 2005, 2008, or 2010 and measured cross-reactive neutralizing antibody titers against all 11 EV71 genotypes. In addition, we aligned and compared the amino acid sequences of P1 proteins of the tested viruses.ResultsSerology data showed that children infected with genogroups B and C consistently have lower neutralizing antibody titers against genogroup A (>4-fold difference). The sequence comparisons revealed that five amino acid signatures (N143D in VP2; K18R, H116Y, D167E, and S275A in VP1) are specific for genogroup A and may be related to the observed antigenic variations.ConclusionsThis study documented antigenic variations among different EV71 genogroups and identified potential immunodominant amino acid positions. Enterovirus surveillance and vaccine development should monitor these positions.

Phylogenetic Analysis of Enterovirus 71 Circulating in Beijing, China from 2007 to 2009

The major pathogens of hand, foot and mouth disease (HFMD) in Beijing, China from 2007 to 2009 were identified in this study. A total of 186 HFMD cases were included, and 136 cases (73%) were positive for enterovirus (EV). In 2007, 75% (27/36) were Coxsackievirus A16 (CA16) positive and 19% (7/36) were Enterovirus 71 (EV71) positive cases. However, EV71 was the predominant virus in 2008, when 56% (31/55) of the cases were positive for EV71 and 22% (12/55) were positive for CA16. In 2009, EV71 and CA16, with positive rates of 36% (16/45) and 29% (13/45), respectively, were still the major pathogens of HFMD. Phylogenetic analysis revealed that the dominant genotype of EV71 was C4, with co-circulation of genotype A in 2009. The prevalent cluster of the EV71 subgenotype C4 changed over time. A proposed new sublineage of EV71, C4a-2, was the predominant virus associated with the Beijing and nationwide HFMD outbreaks since 2008 and amino acid substitution, which possibly link to the central nervous system tropism of EV71, was found in genotype A viruses. Persistent surveillance of HFMD-associated pathogens is required for predicting potential emerging viruses and related disease outbreaks.

Development of A Sensitive and Specific Epitope-Blocking ELISA for Universal Detection of Antibodies to Human Enterovirus 71 Strains

BackgroundHuman Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease (HFMD) in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections.Methodology/Principal FindingIn the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6) that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization.ConclusionThe epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

Epidemiology and Clinical Characteristics of Enterovirus Infections in Children: A Single Center Analysis from 2006 to 2010

† , Jecheon Myongji Hospital, Jecheon-si, Chungbuk, Korea Purpose:This study was performed to investigate the epidemiology of enterovirus (EV) infections in children at a secondary hospital during recent 5 years. Methods:We collected the cerebrospinal fluid, stool and throat swab samples from the pediatric patients with suspected EVinfections in KEPCO Medical Center, Seoul, Korea from July 2006 to September 2010. EVdetection and genotype identi- fication were performed by RT-PCR at Korea Centers for Disease Control and Prevention. Results:A total of 386 samples were collected from 277 patients during study period. Ninety-eight patients (35.4%) were diagnosed with EV infections. The RT-PCR positive rate was the highest in throat swab samples (48.3%). The median age of patient was 4.7 years (range, 0.1-12.5 years). Aseptic meningitis (50, 51.0%) was the most common clinical manifestation; herpangina (22, 22.4%) and hand-foot-mouth disease (18, 18.4%). One hundred EVs were isolated from 98 patients and 20 genotypes of EV were identified; Echovirus 30 (28 cases, 28%), Enterovirus 71 (12 cases, 12%), Echovirus 25 (10 cases, 10%), Echovirus 9 (9 cases, 9%) and Coxsackievirus A6 (8 cases, 8%). Aseptic meningitis caused by Echovirus 30 was the most common manifestation in 2008. There was no complicated case caused by Enterovirus 71. Conclusion:This study showed the epidemiology of confirmed EV infection in children from 2006 to 2010. There is a need for continuous surveillance of EV infections and its clinical manifestations. (Korean J Pediatr Infect Dis 2013;20:81-88)

Circulation of Coxsackievirus A10 and A6 in Hand-Foot-Mouth Disease in China, 2009–2011

Coxsackieviruses A10 (CV-A10) and A6 (CV-A6) have been associated with increasingly occurred sporadic hand-foot-mouth disease (HFMD) cases and outbreak events globally. However, our understanding of epidemiological and genetic characteristics of these new agents remains far from complete. This study was to explore the circulation of CV-A10 and CV-A6 in HFMD and their genetic characteristics in China. A hospital based surveillance was performed in three heavily inflicted regions with HFMD from March 2009 to August 2011. Feces samples were collected from children with clinical diagnosis of HFMD. The detection and genotyping of enteroviruses was performed by real-time PCR and sequencing of 5′UTR/VP1 regions. Phylogenetic analysis and selection pressure were performed based on the VP1 sequences. Logistic regression model was used to identify the effect of predominant enterovirus serotypes in causing severe HFMD. The results showed 92.0% of 1748 feces samples were detected positive for enterovirus, with the most frequently presented serotypes as EV-71 (944, 54.0%) and CV-A16 (451, 25.8%). CV-A10 and CV-A6 were detected as a sole pathogen in 82 (4.7%) and 44 (2.5%) cases, respectively. Infection with CV-A10 and EV-71 were independently associated with high risk of severe HFMD (OR = 2.66, 95% CI: 1.40–5.06; OR = 4.81, 95% CI: 3.07–7.53), when adjusted for age and sex. Phylogenetic analysis revealed that distinct geographic and temporal origins correlated with the gene clusters based on VP1 sequences. An overall ω value of the VP1 was 0.046 for CV-A10 and 0.047 for CV-A6, and no positively selected site was detected in VP1 of both CV-A10 and CV-A6, indicating that purifying selection shaped the evolution of CV-A10 and CV-A6. Our study demonstrates variety of enterovirus genotypes as viral pathogens in causing HFMD in China. CV-A10 and CV-A6 were co-circulating together with EV-71 and CV-A16 in recent years. CV-A10 infection might also be independently associated with severe HFMD.

Detection of human enterovirus 71 and Coxsackievirus A16 in an outbreak of hand, foot, and mouth disease in Henan Province, China in 2009

During 2009, an outbreak of hand, foot, and mouth disease (HFMD) enrolled 490 people in Henan Province, causing the death of two children. In order to investigate the pathogens responsible for this outbreak and characterize their genetic characteristics, a total of 508 clinical specimens (stool, throat swab, and vesicle fluid) were collected from the Center for Disease Control and Prevention of Henan Province. Virological investigations (virus isolation, conventional reverse transcription PCR, and real-time reverse transcription PCR) and phylogenetic analysis were performed. It was found that human enterovirus 71 (EV71) was the main pathogen causing this outbreak, while Coxsackievirus A16 (CoxA16) played only a subsidiary role. Phylogenetic analysis of 24 EV71 isolates collected during the period from March 11 to July 24, 2009 showed that they belonged to subgenotypes C4 and C5. Our study for the first time characterizes the epidemiology of HFMD and EV71 infection in Henan Province in 2009 and provides the first direct evidence of the genotype of EV71 circulating in Henan Province at that time. Our study should facilitate the development of public health measures for the control and prevention of HFMD and EV71 infection in at-risk individuals in China.

Infections nosocomiales à entérovirus en néonatologie : du diagnostic à la preuve, à propos d’une observation d’infection neuroméningée

Although enteroviruses generally cause asymptomatic or mild disease, neonates are at higher risk for severe illnesses, among which systemic disease characterized by multiorgan involvement is a potentially fatal condition. Enterovirus neonatal infections may be the source of nosocomial infections in neonatology or in pediatric intensive care units. We report central nervous system infections due to Echovirus 11 in two neonates and the molecular evidence of nosocomial transmission of this strain in a neonatal unit by enterovirus genotyping and phylogenetic analysis. This report illustrates the importance of including enterovirus genome detection in the sepsis screening concomitantly with bacteriological investigations performed at admission of a neonate. Rapid diagnosis and subsequent genotyping could have a beneficial impact on clinical practices at the individual level (reducing the length of antibiotic therapy) and public health policy at the collective level by reinforcing hygiene measures to prevent nosocomial infections, with nurseries and neonatal units being at greater risks.

Nearly Constant Shedding of Diverse Enteric Viruses by Two Healthy Infants

Stool samples from two healthy infant siblings collected at about weekly intervals during their first year of life were analyzed by PCR for 15 different enteric viral genera. Adenovirus, Aichi virus, Anellovirus, Astrovirus, Bocavirus, Enterovirus, Parechovirus, Picobirnavirus, and Rotavirus were detected. Not detected were Coronavirus, Cardiovirus, Cosavirus, Salivirus, Sapovirus, and Norovirus. Long-term virus shedding, lasting from one to 12 months, was observed for adenoviruses, anelloviruses, bocaviruses, enteroviruses, parechoviruses, and picobirnaviruses. Repeated administration of oral poliovirus vaccine resulted in progressively shorter periods of poliovirus detection. Four nonpolio enterovirus genotypes were also detected. An average of 1.8 distinct human viruses were found per time point. Ninety-two percent (66/72) of the fecal samples tested contained one to five different human viruses. Two British siblings in the mid-1980s showed nearly constant fecal viral shedding. Our results demonstrate that frequent enteric infections with diverse viruses occur during early childhood in the absence of severe clinical symptoms.

Seroepidemiology of Enterovirus 71 infection prior to the 2011 season in children in Shanghai

BackgroundIn 2010, China experienced the largest outbreak on record of Enterovirus 71 (EV71)-associated Hand Foot and Mouth Disease (HFMD) with more than 1.7 million cases, 27,000 patients with severe neurological complications and 905 deaths. Understanding of the seroprevalence of neutralizing antibodies (NAb) against EV71 and their protective role against HFMD in children is crucial for the implementation of future therapeutic and prophylactic intervention. ObjectivesTo correlate the prevalence of NAb against EV71 genotype C4a in children prior to the 2011 epidemic season with severe EV71-associated HFMD disease during the subsequent 2011 epidemic season. Study design614 sera samples were collected from children without HFMD. EV71 NAb were tested by a quantitative PCR assay. Samples with NAb ≥1:8 were scored as positive. Results122 (19.9%) of 614 sera were EV71-seropositive. The NAb seroprevalence was highest in infants 0–5 months of age (28.6%) and lowest in children 1–1.9 years of age (13.4%). 64.1% of severe EV71-associated HFMD occurred in children 1–2.9 years. ConclusionsDespite the large 2010 outbreak, the overall seroprevalence of EV71 in children is relatively low. The seropositive rate of EV71 NAb prior to the 2011 season was inversely correlated with the number of EV71-infected severe cases in 2011. Loss of maternal antibodies in infants and lack of acquired anti-EV71 immunity are responsible for increased proportion of severe HFMD in the 1–2 years age group. Our data suggest that future vaccination campaigns should be initiated as early as 6 months.

Detection of human enterovirus 71 and coxsackievirus A16 in children with hand, foot and mouth disease in China

The aims of the present study were to investigate the genetic characteristics of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) strains in China and to evaluate the relationship between the genotypes of CVA16 and EV71 and their geographical distribution. A total of 399 stool specimens were collected from children with symptoms of hand, foot and mouth disease (HFMD) in Zhejiang Province. The presence of enteroviruses was determined using reverse transcription-semi-nested PCR targeted to the VP1 gene of all human enteroviruses and DNA sequencing. EV71 and CVA16, the major etiological agents of HFMD, were detected in 38.4% (38/99) and 35.4% (35/99) of HEV-A species-positive cases, respectively. Based on the phylogenetic analysis of the VP1 gene, EV71 strains identified in this study belong to subgenotype C4, and CVA16 strains herein were classified into clusters B2a and B2b within the genotype B2. Taking into consideration other published data, we conclude that the genetic characteristics of enteroviruses in China reflect the pattern of the endemic circulation of the subgenotype C4 to EV71 and clusters B2a and B2b within genotype B2 to CVA16, which have been continuously circulating in China since 1997. This observation indicates that the genetic characteristics of enteroviruses in China seem to depend on their special geographical and climatical features allowing them to be sustained with little external effect.

Immunological Evaluation and Comparison of Different EV71 Vaccine Candidates

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are major causative agents of hand, foot, and mouth diseases (HFMDs), and EV71 is now recognized as an emerging neurotropic virus in Asia. Effective medications and/or prophylactic vaccines against HFMD are not available. The current results from mouse immunogenicity studies using in-house standardized RD cell virus neutralization assays indicate that (1) VP1 peptide (residues 211–225) formulated with Freund's adjuvant (CFA/IFA) elicited low virus neutralizing antibody response (1/32 titer); (2) recombinant virus-like particles produced from baculovirus formulated with CFA/IFA could elicit good virus neutralization titer (1/160); (3) individual recombinant EV71 antigens (VP1, VP2, and VP3) formulated with CFA/IFA, only VP1 elicited antibody response with 1/128 virus neutralization titer; and (4) the formalin-inactivated EV71 formulated in alum elicited antibodies that cross-neutralized different EV71 genotypes (1/640), but failed to neutralize CVA16. In contrast, rabbits antisera could cross-neutralize strongly against different genotypes of EV71 but weakly against CVA16, with average titers 1/6400 and 1/32, respectively. The VP1 amino acid sequence dissimilarity between CVA16 and EV71 could partially explain why mouse antibodies failed to cross-neutralize CVA16. Therefore, the best formulation for producing cost-effective HFMD vaccine is a combination of formalin-inactivated EV71 and CAV16 virions.

The clinical features of 15 cases with severe enterovirus 71 ( EV71 ) infection and the genotype of EV71

Objective To explore the clinical feature of severe enterovirus 71 (EV71) associated hand foot and mouth disease (HFMD),and genotype of EV71.Methods Fluorescent quantitation PCR was done for detecting EV71.RT-PCR was performed to amplify VP1 for sequencing and identifying genotype.A retrospective analysis was performed based on the clinical data of 15 cases with severe EV71 infection.Results EV71 nucleotide was positive in all 15 cases.The genotype of EV71 was C4.All cases had abnormal temperature and followed with nervous symptoms in the early stage.Average time was 1.26 days from onset to severe complications appearance.Eleven cases progressed to neurogenic pulmonary edema.Four cases accepted nasal continuous positive airway pressure.Eleven cases accepted oral trachea cannula mechanical ventilation.Except for 3 cases died,one case abandoned,others 11 cases were cured.Conclusion The isolated strains of EV71 in this study are all C4 genotype.All cases with severe EV71 infection were followed with nervous symptoms in the early stage,most of whom would progress to neurogenic pulmonary edema.The mortality would be cut down by using mechanical ventilation in early stage. Key words: Hand, foot and mouth disease; Critical illness; Enterovirus 71 ; Genotype

Characterization of an Outbreak of Hand, Foot, and Mouth Disease in Nanchang, China in 2010

Recent outbreaks of human enterovirus 71 (EV71) infection and EV71-associated hand, foot, and mouth disease (HFMD) in China have affected millions and potentially lead to life-threatening complications in newborns. Furthermore, these outbreaks represent a significant global public health issue in the world. Understanding the epidemiology of HFMD and EV71 infection and their transmission patterns in China is essential for controlling outbreaks. However, no studies on the outbreaks of HFMD and EV71 infection in China during 2010 have been reported. In this report, we carried out an epidemiological analysis to study an outbreak of HFMD and EV71 infection in 2010 in the city of Nanchang in the Jiangxi province of People's Republic of China. From April 7 to May 11, 2010, a total of 109 HFMD cases were reported, and in this report the HFMD cases were studied by both epidemiological and laboratory analyses. The epidemiological study indicates that children aged younger than 8 years old represented more than 90% of the reported cases, with the age group of 1–3 years containing the highest number of cases. Laboratory studies detected a high prevalence of EV71 amongst the cases in our study, suggesting EV71 as a common enterovirus found in HFMD cases in Nanchang. Phylogenetic analysis of the sequence of the VP1 region of four EV71 isolates indicated that the Nanchang strains belong to the C4 subgenotype commonly found in China during outbreaks in 2008 but contain distinct variations from these strains. Our study for the first time characterizes the epidemiology of HFMD and EV71 infection in China in 2010 and furthermore, provides the first direct evidence of the genotype of EV71 circulating in Nanchang, China. Our study should facilitate the development of public health measures for the control and prevention of HFMD and EV71 infection in at-risk individuals in China.

The etiological identification of an aseptic encephalitis outbreak (ten cases) in a hospital of Xiamen city, 2011

To identify the etiology of an aseptic encephalitis outbreak (ten cases) in a hospital of Xiamen city from 11 to 17 May, 2011. A total of ten patients' throat swabs, anal swabs and cerebrospinal fluid were collected and detected by RT-PCR for pan-enterovirus. The samples containing detectable pan-enterovirus were tested by PCR with genotype-specific general primers located in VP1 region of enterovirus genotype A, B and C (HEV-A, B and C). The PCR products of VP1 segment were purified and sequenced, and phylogenetic analysis was performed. Meanwhile, the pathogens in those samples were isolated in Vero cell culture. Homologous analysis of VP1 sequences were carried out for the cultured virus samples and the original clinical samples to identify the outbreak etiology. Among the ten cases, seven cases were positive for pan-enterovirus nucleic acid. When tested by genotype-specific PCR, the throat and anal swab samples from those 7 patients were positive with HEV-B VP1 primers. Meanwhile, the HEV-B VP1 segments were sequenced and phylogenetic analyzed, which indicated the seven cases were all infected by enterovirus Echo 30. The sequences from those samples had homology of 95.3% - 97.1% with the epidemic strains in Zhejiang, 2004. Out of the seven cases, the sequences of XM2, XM3, XM4, XM8 throat swab samples and XM3, XM6 throat samples showed 99.4% - 100.0% homology which were different from the sequence of XM1, and the homology was 92.8% - 93.4%. Furthermore, the viruses were isolated using Vero cells from XM1, XM2, XM3, XM4 and XM8 throat swab samples, and the VP1 sequence showed more than 99.9% homology with the original specimens. The local outbreak of aseptic encephalitis was caused by Echo 30 of enterovirus genotype B, and the epidemic strains may have different genetic background.

Identification and characterization of a cross-neutralization epitope of Enterovirus 71

Enterovirus 71 (EV71) infections in children manifest as exanthema and are most commonly known as hand-foot-and-mouth disease (HFMD). Because it can cause severe neurological complications like poliomyelitis, EV71 has now emerged as an important neurotropic virus in Asia. EV71 virus has been shown to consist of 3 (A, B and C) genotypes and many subgenotypes. Although EV71 vaccine development has recently yielded promising preclinical results, yet the correlation between the content of antigen(s) in vaccine candidates and the level of protective antibody responses is not established. The neutralization epitope(s) of EV71 antigens could be used as the surrogate biomarker of vaccine potency. Using peptide ELISA, antisera generated from animals immunized with formalin-inactivated EV71 virion vaccine formulated in alum, EV71-specific neutralizing monoclonal antibody (nMAb) and a panel of 153 overlapping synthetic peptides covering the entire sequences of VP1, VP2 and VP3 of EV71, we screened for immunodominant linear neutralization epitope(s). Synthetic peptide VP2-28, corresponding to residues 136–150 of VP2, was found to bind to and inhibit the binding to EV71 of nMAb MAB979 that was found to have cross-neutralizing activity against different genotypes of EV71 virus. In addition, VP2-28 was found to be recognized only by neutralizing antisera generated from rabbits immunized with the formalin-inactivated whole EV71 virion vaccine but not by antisera from immunized mice and rats. During the epitope mapping, a murine EV71 genotype- and strain-specific linear neutralization epitope VP1–43 was identified within residues 211–220 of VP1. Furthermore, based on sequence alignment and structure prediction analysis using poliovirus as the template for molecular modeling, the VP1-43 and VP2-28 epitopes were shown to run in parallel within 0.1nm and form a rim of the canyon at the junction site of VP1 and VP2 in the viral capsid. In mouse, rat and rabbit immunogenicity studies, a dose-dependent relationship between the number of VP2-28 epitope units measured by a quantitative assay in vaccine preparations and the magnitude of neutralizing titers was demonstrated. VP2-28 has amino acid sequences that are highly conserved among EV71 genotypes, is not affected by formalin-treatment and long-term storage. Thus, VP2-28 could be used as the surrogate biomarker in the potency testing of candidate EV71 vaccines.

Molecular epidemiology of human enterovirus 71 strains in Qingdao region, Shandong province, 2007 - 2009

To study the molecular epidemiology of human enterovirus 71 (EV71) isolated from patients with hand-foot-mouth disease (HFMD) in Qingdao region between 2007 and 2009. Throat swabs of HFMD were detected for total enterovirus (EV), EV71 and CA16 by fluorescence reverse transcription-polymerase chain reaction (RT-PCR). The VP1 region was amplified from positive EV71 specimens and 51 nucleotide sequences of VP1 gene were successfully sequenced. EV71 genotypes were characterized by phylogenetic analysis. 51 Qingdao EV71 strains identified between 2007 and 2009 belonged to C4a cluster of subgenotype C4 and 5.3% nucleotide divergence and 1.7% amino acid divergence were found among them. The strains in 2008 and 2009 were respectively divided into several lineages on phylogenetic trees but there was a main lineage (namely prominent strain) every year. The Qingdao prominent strains identified in 2008 had high identities with the Linyi prominent strains in 2007. The Qingdao prominent strains in 2009 and the secondary prominent strains in 2008 had high identities with the Fuyang prominent strains in 2008. Two Qingdao strains in 2007 had high identities with the Linyi popular strains in 2007. Data showed that several EV71 transmission chains were co-circulating in Qingdao between 2008 and 2009. The main transmission chain in 2008 became the minor one but the minor transmission chain in 2008 was transferred into the main transmission chain in 2009.

Chimeric virus-like particles presenting common neutralizing epitopes of enterovirus 71

To develop an effective vaccine against multiple genotypes of enterovirus 71 (EV71), we generated chimeric virus-like particles (VLPs) repetitively displaying the common neutralizing epitopes of EV71 and evaluated their immunogenicities in mice. The two conserved epitopes, encompassing amino acids 163-177 and 208-222 of VP1 of EV71, were fused to hepatitis B core antigen (HBcAg) and expressed in E.coli. The resulting fusion proteins were found to assemble into chimeric VLPs. Both unmodified HBcAg and chimeric VLPs induced HBcAg-specific antibody responses in mice, however, only chimeric VLP-immunized sera possessed EV71 epitope-specific IgG antibodies and efficiently neutralized different EV71 strains. Collectively, our results indicate that the chimeric VLP is capable of eliciting broadly neutralizing antibody responses and is therefore a promising EV71 vaccine candidate.

Enterovirus 71 Infection with Central Nervous System Involvement, South Korea

We assessed neurologic sequelae associated with an enterovirus 71 (EV71) outbreak in South Korea during 2009. Four of 94 patients had high signal intensities at brainstem or cerebellum on magnetic resonance imaging. Two patients died of cardiopulmonary collapse; 2 had severe neurologic sequelae. Severity and case-fatality rates may differ by EV71 genotype or subgenotype.