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Abstract Background Enterovirus 71(EV71)-associated hand, foot and mouth disease (HFMD) decreased dramatically in Beijing from 2009 to 2019. This study was to investigate the epidemiological characteristics, evolutionary dynamics, geographic diffusion pathway, and other features of EV71 in Beijing, China. Methods We conducted a retrospective study of EV71-associated HFMD and its causative agent in Beijing, China, from 2009 to 2019. Phylogenetic and phylogeographic methods based on the EV71 genome were used to determine the evolution features, origin, and spatiotemporal dynamics. Positive selection sites in the VP1 gene were identified and exhibited in the tertiary structure. Bayesian birth-death skyline model was used to estimate the effective reproductive number (Re). Results EV71-associated HFMD decreased greatly in Beijing. From 2009 to 2019, EV71 strains prevalent in Beijing shared high homology in each gene segment and evolved with a rate of 4.99*10− 3 substitutions per site per year. The genetic diversity of EV71 first increased and peaked in 2012 and then decreased with fluctuations. The time to the most recent common ancestor (TMRCA) of EV71 in Beijing was estimated around 2003 when the EV71 strains were transmitted to Beijing from east China. Beijing played a crucial role in seeding EV71 to central China as well. Two residues (E145Q/G, A293S) under positive selection were detected from both the VP1 dataset and the P1 dataset. They were embedded within the loop of the VP1 capsid and were exposed externally. Mean Re estimate of EV71 in Beijing was about 1.007. Conclusion In recent years, EV71 was not the primary causative agent of HFMD in Beijing. The low Re estimate of EV71 in Beijing implied that strategies for preventing and controlling HFMD were performed effectively. Beijing and east China played a crucial role in disseminating EV71 to other regions in China.

Background: Enterovirus 71(EV71)-associated hand, foot and mouth disease (HFMD) decreased dramatically in Beijing from 2009 to 2019. This study was to investigate the epidemiological characteristics, evolutionary dynamics, geographic diffusion pathway, and other features of EV71 in Beijing, China. Methods: We conducted a retrospective study of EV71-associated HFMD and its causative agent in Beijing, China, from 2009 to 2019. Phylogenetic and phylogeographic methods based on the EV71 genome were used to determine the evolution features, origin, and spatiotemporal dynamics. Positive selection sites in the VP1 gene were identified and exhibited in the tertiary structure. Bayesian birth-death skyline model was used to estimate the effective reproductive number (Re). Results: EV71-associated HFMD decreased greatly in Beijing. From 2009 to 2019, EV71 strains prevalent in Beijing shared high homology in each gene segment and evolved with a rate of 4.99*10− 3 substitutions per site per year. The genetic diversity of EV71 first increased and peaked in 2012 and then decreased with fluctuations. The time to the most recent common ancestor (TMRCA) of EV71 in Beijing was estimated around 2003 when the EV71 strains were transmitted to Beijing from east China. Beijing played a crucial role in seeding EV71 to central China as well. Two residues (E145Q/G, A293S) under positive selection were detected from both the VP1 dataset and the P1 dataset. They were embedded within the loop of the VP1 capsid and were exposed externally. Mean Re estimate of EV71 in Beijing was about 1.007. Conclusion: In recent years, EV71 was not the primary causative agent of HFMD in Beijing. The low Re estimate of EV71 in Beijing implied that strategies for preventing and controlling HFMD were performed effectively. Beijing and east China played a crucial role in disseminating EV71 to other regions in China. [ABSTRACT FROM AUTHOR]

As a global public health problem, hand, foot and mouth disease（HFMD） is extremely harmful and can cause neurological diseases in children.It has the characteristics of high infectivity, diverse modes of transmission and complex and diverse pathogens.The pathogens causing HFMD in children are mainly Coxsackie virus A and human enterovirus 71（EV71）.As one of the main pathogens of HFMD,EV71 is highly infectious.At present, the effective drugs against EV71 have been developed, which is a key way to prevent HFMD.This paper summarized the research progress of EV71 and anti-EV71 drugs, so as to provide valuable reference for the research and development of new drugs in the future. [ABSTRACT FROM AUTHOR]

Background: There have been sporadic and periodic large‑scale epidemics of hand, foot, and mouth disease (HFMD) with cases at risk for significant morbidity and mortality particularly in Southeast Asia since 1997 and in India since early 2003. Method: We retrospectively studied 403 cases recorded from 2009 to 2019 and reviewed relevant Indian literature published between 2004 and 2019 to understand clinical, epidemiological, and virological attributes of this long‑lasting Indian epidemic. Result: There were 96.8% children and adolescents (M:F 1.6:1) aged 2 months to 18 years and 84% were aged <5 years. Adult family contacts comprised 3.2%. Only 12 sporadic cases occurred during 2009‑2011 followed by increased number from 2012 to 2015 peaking with 30.8% cases in 2013 and declining slowly until the year 2019 with small resurge in 2018. The major peaks occurred during summers with small peaks in autumns. Literature review showed 3332 cases presenting between 2004 and 2019 across Indian states with similar epidemiological trends whereas serotyping identified Coxsackievirus A16 (CV A16) in 83%, Coxsackievirus A6 (CV A6) in 17%, Enterovirus 71 in 4.1%, and multiple strains in 11.7% samples, respectively. Conclusion: The overall features of this long‑lasting HFMD epidemic; affecting children aged <5 years more often than adults, none or minimum neurological or pulmonary complications in few patients, peaks occurring during summer and autumn months, and identity of the pathogenic virus coincide with global trends. However, the continuous spread of the disease across the country appears in sync with pre‑epidemic periods of China and Taiwan. It calls for a continuous surveillance and making HFMD a notifiable disease in India. [ABSTRACT FROM AUTHOR]

Abstract Background Prophylactic vaccines are critical in preventing hand, foot, and mouth disease (HFMD) primarily caused by human enterovirus 71 (EV71) infection. Children aged less than 5 years are especially susceptible to EV71 infections. In addition to the development of vaccines containing the inactivated virus, those containing virus-like particles (VLPs) with repeated antigens also constitute an effective preventive strategy for EV71 infections, with safety and productivity advantages. We previously developed a fusion protein composed with truncated peptides of the EV71 capsid protein, which assembled into spherical particles. This study aimed to assess the immunoprotective effects of this fusion protein as a vaccine candidate in a mouse model of EV71 infection. Methods To evaluate the protective effect of fusion protein vaccine candidate, neonatal mice born by immunized female mice, as well as normal neonatal mice immunized twice were infected with EV71 virus. Whereafter, the survival rates, clinical scores and viral loads were measured. Results The high dosage and booster immunization helped induce specific serum antibodies with high neutralization titers, which were transferred to neonatal mice, thereby facilitating effective resistance towards EV71 infection. An active immune response was also observed in neonatal mice which generated following immunization. Conclusions The present results suggest that this fusion protein is a suitable vaccine candidate in treating EV71 infections.

Background and Objectives: Human Enterovirus 71 (EV-A71) is the causative agent for many dermal to neurological diseases especially polio-like paralysis outbreaks around the world. This study, the first of this kind in Iran, aimed to find neutralizing antibodies against EV-A71 in serum of healthy individuals in different age groups based on neutralization test (NT). Materials and Methods: In this cross-sectional study, 547 serum samples were collected from healthy individuals who were referring for routine checkup tests (aged from under 6 months to over 31 years old) to Imam-Khomeini Hospital in Tehran during January-December 2015. Serum samples were examined by NT in cell culture to detect neutralizing antibodies against EV-A71. In the next step, some of the positive samples were subjected to complete titration to determine the exact titer of anti-EV-A71 antibodies. Results: Of 547 samples, 310 (56.7%) were positive for EV-A71 neutralizing antibody. The presence of the antibody increased with age (p

Background and Objectives: Human Enterovirus 71 (EV-A71) is the causative agent for many dermal to neurological diseases especially polio-like paralysis outbreaks around the world. This study, the first of this kind in Iran, aimed to find neutralizing antibodies against EV-A71 in serum of healthy individuals in different age groups based on neutralization test (NT). Materials and Methods: In this cross-sectional study, 547 serum samples were collected from healthy individuals who were referring for routine checkup tests (aged from under 6 months to over 31 years old) to Imam-Khomeini Hospital in Tehran during January-December 2015. Serum samples were examined by NT in cell culture to detect neutralizing antibodies against EV-A71. In the next step, some of the positive samples were subjected to complete titration to determine the exact titer of anti-EV-A71 antibodies. Results: Of 547 samples, 310 (56.7%) were positive for EV-A71 neutralizing antibody. The presence of the antibody increased with age (p<0.001), and there was a significant statistical relationship between sex and the presence of antibody (p=0.009). Conclusion: Our results demonstrated an apparent but limited circulation of EV-A71 in our society. After the worldwide eradication of poliovirus, EV-A71 which can cause polio-likes syndrome, might be the new challenge for our health care system as regard more in depth research is however needed. [ABSTRACT FROM AUTHOR]

Therapeutic strategies for severe hand, foot, and mouth disease (HFMD) are currently either inconsequent or deficient in evidence. We retrospectively surveyed HFMD outbreaks in Xiangyang from June 2008 to December 2013. HFMD is staged from I to V according to clinical severity. Severe HFMD is defined as a case involving the central nervous system (CNS). We analyzed risk factors for fatality of severe cases and compared the efficiency and outcome of some therapies by binary logistic regression. The overall HFMD cases included 637 (1.26%) severe cases and 38 fatalities (0.075%). Analyses indicate that age (40 mg/L), and cardiac troponin I (>0.04 ng/ml) are risk factors for fatality (all P < 0.05). Intravenous immunoglobulin (IVIG) and mechanical ventilation applied only in early stage IV significantly improved HFMD progression (both P < 0.05) with odds ratios of 0.24 (95% CI: 0.10–0.57) and 0.01 (95% CI: 0.00–0.10), respectively. Neither methylprednisolone nor milrinone administered in any stage made any significant difference on mortality (all P > 0.05). Precise recognition of the severe HFMD cases in early stage IV and prompt IVIG and mechanical ventilation application may reduce mortality. Mechanical ventilation training programs and dispatch of specialists to hospitals where there is no chance of transferring critical cases to the severe HFMD designated hospitals are two key measures to reduce fatality.

Background: Prophylactic vaccines are critical in preventing hand, foot, and mouth disease (HFMD) primarily caused by human enterovirus 71 (EV71) infection. Children aged less than 5 years are especially susceptible to EV71 infections. In addition to the development of vaccines containing the inactivated virus, those containing virus-like particles (VLPs) with repeated antigens also constitute an effective preventive strategy for EV71 infections, with safety and productivity advantages. We previously developed a fusion protein composed with truncated peptides of the EV71 capsid protein, which assembled into spherical particles. This study aimed to assess the immunoprotective effects of this fusion protein as a vaccine candidate in a mouse model of EV71 infection. Methods: To evaluate the protective effect of fusion protein vaccine candidate, neonatal mice born by immunized female mice, as well as normal neonatal mice immunized twice were infected with EV71 virus. Whereafter, the survival rates, clinical scores and viral loads were measured. Results: The high dosage and booster immunization helped induce specific serum antibodies with high neutralization titers, which were transferred to neonatal mice, thereby facilitating effective resistance towards EV71 infection. An active immune response was also observed in neonatal mice which generated following immunization. Conclusions: The present results suggest that this fusion protein is a suitable vaccine candidate in treating EV71 infections. [ABSTRACT FROM AUTHOR]

Phyllanthus is a flowering plant used in Ayurvedic Healing for treating various harmful diseases. It is an Hepatoprotective agent and is used to treat various diseases of the Urogenital system and it also possesses Antiseptic properties. Pharmacological uses of Phyllanthus are Intestinal infections, Diabetes, Hepatitis, Dysentery, Skin infections, Gonorrhea, Heavy menstrual bleeding, Expectorant, etc. Phyllanthus extract contains Corilagin that can possibly act against Human Enterovirus 71 and Coxsackie Virus. Phyllanthus urinaria loses detectable Hepatitis B e antigen from serum. The Phyllanthus amarus plant suppresses HBV mRNA transcription in vitro and exhibits therapeutic potential in chronic HBV carriers. Phyllanthus urinaria attacks the Hbe Antigen in serum. The aqueous extract of Phyllanthus urinaria inhibited the viral entry, thereby down regulating the viral gB, gD and VP16 which are essential for viral attachment and entry, It inhibits the manipulation of the hostactin-cytoskeleton dynamics by the virus and it also prevents the manipulation of the host proteins by the virus. Phyllanthus amarus derived preparations block interaction HIV-1 gp120 and the Polyphenol Extract and Gallic Acid of Lipid Lowering activity of Phyllanthus niruri is mediated through inhibition of hepatic cholesterol biosynthesis, increased faecal bile acids excretion and enhanced plasma lecithin: cholesterol acyltransferase activity. The standardized methanol extracts of P. amarus and P. urinaria and their biomarkers phyllanthin and hypophyllanthin were able to modulate the innate immune response of phagocytes especially on the chemotactic migration of phagocytes, and on the release of ROS. Among the extracts, P. amarus consistently showed strong inhibition at different steps of the phagocytosis, emphasizing its potential as an immunomodulating product. Hence this is about the conventional way of treating various viral diseases with Phyllanthus and its species. [ABSTRACT FROM AUTHOR]

Since the outbreak of the enterovirus 71 (EV71) infection in Malaysia in 1997, large epidemics of EV71 have occurred in the Asia-Pacific region. Many children and infants have died from serious neurological complications during these epidemics, and EV71 infection has become a serious public health problem in these areas. EV71 infection causes hand, foot and mouth disease (HFMD) in children, and usually resolves spontaneously. However, EV71 occasionally involves the central nervous system (CNS), and induces diverse neurological complications such as brainstem encephalitis, aseptic meningitis, and acute flaccid paralysis. Among those complications, brainstem encephalitis is the most critical neurological manifestation because it can cause neurogenic pulmonary hemorrhage/edema leading to death. The characteristic clinical symptoms such as myoclonus and ataxia, cerebrospinal fluid (CSF) pleocytosis, and brainstem lesions on magnetic resonance imaging, in conjunction with the skin rash of HFMD and the isolation of EV71 from a stool, throat-swab, or CSF sample are typical findings indicating CNS involvement of EV71 infection. Treatment with intravenous immunoglobulin and milrinone are recommended in cases with severe neurological complications from EV71 infection, such as brainstem encephalitis. Despite the recent discovery of receptors for EV71 in human cells, such as the scavenger receptor B2 and P-selection glycoprotein ligand 1, it is not known why EV71 infection predominantly involves the brainstem. Recently, 3 companies in China have completed phase III clinical trials of EV71 vaccines. However, the promotion and approval of these vaccines in various countries are problems yet to be resolved.

Outbreaks of human enterovirus 71 (EV-71) in Asia are related to high illness and death rates among children. To gain insight into the potential threat for the population of Europe, we determined the neutralizing activity in intravenous immunoglobulin (IVIg) batches and individual serum samples from donors in the Netherlands against EV-71 strains isolated in Europe and in Asia. All IVIg batches and 41%, 79%, and 65% of serum samples from children ≤5 years of age, women of childbearing age, and HIV-positive men, respectively, showed high neutralizing activity against a Dutch C1 strain, confirming widespread circulation of EV-71 in the Netherlands. Asian B3–4 and C4 strains were efficiently cross-neutralized, predicting possible protection against extensive circulation and associated outbreaks of those types in Europe. However, C2 and C5 strains that had few mutations in the capsid region consistently escaped neutralization, emphasizing the importance of monitoring antigenic diversity among circulating EV-71 strains.

Background: Subgenotype C4 of enterovirus 71 (EV71) is the predominant agent of Hand Foot and Mouth disease (HFMD) circulating in the mainland of China. For the first time, a subgenotype C2 of EV71 named SY30-2 was isolated from a HFMD case in Beijing, China. Since it is uncertain whether antibodies raised against subgenotype C4 of EV71 can protect C2 EV71, it is important to monitor and check the presence of cross-reactive antibodies against new EV71 subgenotypes. To find out the causes for the different NtAb, this study is to investigate the relationships between amino acid residue variations and cross-reactive antibodies against EV71 subgenotypes C2 and C4.Methods: Nucleotide and amino acid sequences from full-length genome sequence of SY30-2 were compared to EV71 reference strains. A microneutralization test was used to detect neutralizing antibody (NTAb) in the sera of subgenotype C4 of EV71 infected cases against SY30-2 and FY17 (a C4 isolate). The 3D structure of the viral capsid protein of SY30-2 was constructed.Results: Genome sequence and similarity plot analyses showed that SY30-2 shared the highest identity with subgenotype C2 of EV71 strains in every fragment of the genome. While the microneutralization test result showed that children infected with subgenotype C4 of EV71 had higher NTAb titers against FY17 than SY30-2 (p < 0.001). The amino acid sequence comparison revealed that four amino acid residues VP1-22, VP1-31, VP1-249 and VP3-93 were highly conserved in subgenotype C4 of EV71 compared with the corresponding amino acid residues on subgenotype C2 of EV71 (p < 0.05). Furthermore, the 3D-structure of viral capsid protein showed that VP1-22, VP1-31 and VP3-93 were located on the surface of virion.Conclusion: This is the first report of an EV71 subgenotype C2 isolated from HFMD in Beijing, China. Only a few antigenic variations on subgenotype C2 of EV71 could have led to a great decrease in NTAb titer. Thus, imported new genotypes and subgenotypes of EV71 should be closely monitored. The efficacy of available vaccines against new viruses should be evaluated as well.

Human enterovirus 71 (EV-A71) causes hand, foot and mouth disease (HFMD). EV-A71 circulates in many countries and has caused large epidemics, especially in the Asia-Pacific region, since 1997. In April 2012, an undiagnosed fatal disease with neurological involvement and respiratory distress occurred in young children admitted to the Kantha Bopha Children’s Hospital in Phnom Penh, Cambodia. Most died within a day of hospital admission, causing public panic and international concern. In this study, we describe the enterovirus (EV) genotypes that were isolated during the outbreak in 2012 and the following year. From June 2012 to November 2013, 312 specimens were collected from hospitalized and ambulatory patients and tested by generic EV and specific EV-A71 reverse transcription PCR. EV-A71 was detected in 208 clinical specimens while other EVs were found in 32 patients. The VP1 gene and/or the complete genome were generated. Our phylogenetic sequencing analysis demonstrated that 80 EV-A71 strains belonged to the C4a subgenotype and 3 EV-A71 strains belonged to the B5 genotype. Furthermore, some lineages of EV-A71 were found to have appeared in Cambodia following separate introductions from neighboring countries. Nineteen EV A (CV-A6 and CV-A16), 9 EV B (EV-B83, CV-B3, CV-B2, CV-A9, E-31, E-2 and EV-B80) and 4 EV C (EV-C116, EV-C96, CV-A20 and Vaccine-related PV-3) strains were also detected. We found no molecular markers of disease severity. We report here that EV-A71 genotype C4 was the main etiological agent of a large outbreak of HFMD and particularly of severe forms associated with central nervous system infections. The role played by other EVs in the epidemic could not be clearly established.

Background: Subgenotype C4 of enterovirus 71 (EV71) is the predominant agent of Hand Foot and Mouth disease (HFMD) circulating in the mainland of China. For the first time, a subgenotype C2 of EV71 named SY30-2 was isolated from a HFMD case in Beijing, China. Since it is uncertain whether antibodies raised against subgenotype C4 of EV71 can protect C2 EV71, it is important to monitor and check the presence of cross-reactive antibodies against new EV71 subgenotypes. To find out the causes for the different NtAb, this study is to investigate the relationships between amino acid residue variations and cross-reactive antibodies against EV71 subgenotypes C2 and C4. Methods: Nucleotide and amino acid sequences from full-length genome sequence of SY30-2 were compared to EV71 reference strains. A microneutralization test was used to detect neutralizing antibody (NTAb) in the sera of subgenotype C4 of EV71 infected cases against SY30-2 and FY17 (a C4 isolate). The 3D structure of the viral capsid protein of SY30-2 was constructed. Results: Genome sequence and similarity plot analyses showed that SY30-2 shared the highest identity with subgenotype C2 of EV71 strains in every fragment of the genome. While the microneutralization test result showed that children infected with subgenotype C4 of EV71 had higher NTAb titers against FY17 than SY30-2 (p < 0.001). The amino acid sequence comparison revealed that four amino acid residues VP1-22, VP1-31, VP1-249 and VP3-93 were highly conserved in subgenotype C4 of EV71 compared with the corresponding amino acid residues on subgenotype C2 of EV71 (p < 0.05). Furthermore, the 3D-structure of viral capsid protein showed that VP1-22, VP1-31 and VP3-93 were located on the surface of virion. Conclusion: This is the first report of an EV71 subgenotype C2 isolated from HFMD in Beijing, China. Only a few antigenic variations on subgenotype C2 of EV71 could have led to a great decrease in NTAb titer. Thus, imported new genotypes and subgenotypes of EV71 should be closely monitored. The efficacy of available vaccines against new viruses should be evaluated as well. [ABSTRACT FROM AUTHOR]

Vaccine development and pathogenesis studies for human enterovirus 71 are limited by a lack of suitable animal models. Here, we report the development of a novel neonatal gnotobiotic pig model using the non-pig-adapted neurovirulent human enterovirus 71 strain BJ110, which has a C4 genotype. Porcine small intestinal epithelial cells, peripheral blood mononuclear cells and neural cells were infected in vitro. Oral and combined oral–nasal infection of 5-day-old neonatal gnotobiotic pigs with 5×108 fluorescence forming units (FFU) resulted in shedding up to 18 days post-infection, with viral titers in rectal swab samples peaking at 2.22×108 viral RNA copies/mL. Viral capsid proteins were detected in enterocytes within the small intestines on post-infection days (PIDs) 7 and 14. Additionally, viral RNA was detected in intestinal and extra-intestinal tissues, including the central nervous system, the lung and cardiac muscle. The infected neonatal gnotobiotic pigs developed fever, forelimb weakness, rapid breathing and some hand, foot and mouth disease symptoms. Flow cytometry analysis revealed increased frequencies of both CD4+ and CD8+ IFN-γ-producing T cells in the brain and the blood on PID 14, but reduced frequencies were observed in the lung. Furthermore, high titers of serum virus-neutralizing antibodies were generated in both orally and combined oral–nasally infected pigs on PIDs 7, 14, 21 and 28. Together, these results demonstrate that neonatal gnotobiotic pigs represent a novel animal model for evaluating vaccines for human enterovirus 71 and for understanding the pathogenesis of this virus and the associated immune responses.

Human enterovirus 71 (HEV71) has been considered as an essential human pathogen, which causes hand, foot and mouth disease in young children. Several HEV71 outbreaks have been observed in many Asia-Pacific countries for the past two decades with significant fatalities. However, there are no competent vaccines or antivirals against HEV71 infection to date. Thus, it is of critical priority to delve into the search for anti-HEV71 agents. Prior to this, there is a need to gain knowledge about the distinct targets of HEV71 that are available and that have been exploited for antiviral therapy. This review aims to provide a better understanding of HEV71 virology and feature potential antivirals for progressive clinical development with respect to their elucidated mechanistic actions. [ABSTRACT FROM AUTHOR]

PurposeHand-foot-mouth disease (HFMD) is a common viral illness in children, which is usually mild and self-limiting. However, in recent epidemics of HFMD in Asia, enterovirus 71 (EV71) has been recognized as a causative agent with severe neurological symptoms with or without cardiopulmonary involvement. HFMD was epidemic in Korea in the spring of 2009. Severe cases with complications including death have been reported. The clinical characteristics in children with neurologic manifestations of EV71 were studied in Ewha Womans University Mokdong Hospital.MethodsExaminations for EV71 were performed from the stools, respiratory secretion or CSF of children who presented neurologic symptoms associated with HFMD by realtime PCR. Clinical and radiologic data of the patients were collected and analyzed.ResultsEV71 was isolated from the stool of 16 patients but not from respiratory secretion or CSF. Among the 16 patients, meningitis (n=10) was the most common manifestation, followed by Guillain-Barré syndrome (n=3), meningoencephalitis (n=2), poliomyelitis-like paralytic disease (n=1), and myoclonus (n=1). Gene analysis showed that most of them were caused by EV71 subgenotype C4a, which was prevalent in China in 2008.ConclusionBecause EV71 causes severe complications and death in children, a surveillance system to predict upcoming outbreaks should be established and maintained and adequate public health measures are needed to control disease.

Enterovirus 71 (EV71) outbreaks occur periodically in the Asia-Pacific region. In 2006, Brunei reported its first major outbreak of EV71 infections, associated with fatalities from neurologic complications. Isolated EV71 strains formed a distinct lineage with low diversity within subgenogroup B5, suggesting recent introduction and rapid spread within Brunei.

Background: Enterovirus (EV) 71 is the main pathogen associated with hand, foot and mouth disease (HFMD) or herpangina. Outbreaks of HFMD caused by EV71 infection are associated severe neurological disease and high mortality rates in children. Several sporadic cases of EV71 infection occurred in the Republic of Korea (ROK) in 2000, and EV71 infections were not reported thereafter until 2006. In this prospective study, we report the epidemic and virologic characteristics of the EV71 endemic from 2007 to 2012 in the Republic of Korea.Methods: We analyzed characteristics of the EV71 infection-associated epidemic from collected specimens and clinical information from 9987 patients with suspected EV infection from the National EV Surveillance System in ROK. To identify the EV71 subgenotype, the homology of viral protein 1 sequences obtained using reverse transcription polymerase chain reaction was compared with the sequences on other countries available from GenBank database.Results: EV71 was detected in 585 (16.7 %) specimens (cerebrospinal fluid, stool or rectal swabs, throat swabs and blood) during study period and was most frequently observed during epidemic seasons in 2009-2012. Major manifestations due to EV71 infection were HFMD (62.2 %) and HFMD with severe neurological complications (28.4 %). Five deaths (0.9 %) due to EV71 infection occurred, with an increased mortality rate during the period after 2009. Most patients (476; 81.4 %) were less than 5 years of age. Analysis of the monthly distribution showed that there was an obvious seasonal pattern to the epidemics, with infections appearing from June to August. The major subgenotype of EV71 isolates circulating in ROK was the C4a strain, which has also appeared in China, Japan and Vietnam.Conclusions: This surveillance provided valuable data on the epidemic characteristics of EV71 infections in ROK during a 6-year period. Our findings provide data to assist during future outbreaks of EV71 and associated acute neurologic disease. [ABSTRACT FROM AUTHOR]

Hand, foot, and mouth disease (HFMD) has caused public health concerns worldwide. We aimed to investigate the effect of meteorological factors on the HFMD epidemic in Qingdao, a port city in China. A total of 78641 cases were reported in Qingdao between January 2007 and December 2014. Of those, 71084 (90·39%) occurred in children aged 0-5 years, with an incidence of 1691·2/100000. The incidence increased from early spring, peaked between spring and summer, and decreased in late summer. Aetiological agents in all severe cases and selected mild cases were characterized by examining throat swabs. Except for enterovirus 71 (EV71) and coxsackievirus A16 (CA16), other EVs caused >50% of the HFMD cases between 2011 and 2014. EV71 was more frequent in the off-peak months than in the peak months and prone to causing more severe cases compared to CA16 (χ 2 = 46·3, P < 0·001). CA10 caused more severe HFMD than did CA6 (χ 2 = 20·49, P < 0·001) and all non-CA10 EVs (χ 2 = 41·01, P < 0·001). Community-derived HFMD cases accounted for 65·11%. Spearman rank correlation analysis showed that HFMD incidence in children aged 0-5 years was positively correlated with atmospheric temperature (r s = 0·77, P < 0·001), relative humidity (r s = 0·507, P < 0·001), and precipitation (r s = 0·328, P < 0·001). Climate changes and CA10 surveillance in communities should be integrated into the current prophylactic programme. [ABSTRACT FROM AUTHOR]

This study provides a comprehensive overview of the molecular epidemiology of human enterovirus 71 (HEV71) in the Asia-Pacific region from 1997 through 2002. Phylogenetic analysis of the VP4 and VP1 genes of recent HEV71 strains indicates that several genogroups of the virus have been circulating in the Asia-Pacific region since 1997. The first of these recent outbreaks, described in Sarawak (Malaysian Borneo) in 1997, was caused by genogroup B3. This outbreak was followed by large outbreaks in Taiwan in 1998, caused by genogroup C2, and in Perth (Western Australia) in 1999, where viruses belonging to genogroups B3 and C2 cocirculated. Singapore, Taiwan, and Sarawak had HEV71 epidemics in 2000, caused predominantly by viruses belonging to genogroup B4; however, large numbers of fatalities were observed only in Taiwan. HEV71 was identified during an epidemic of hand, foot and mouth disease in Korea; that epidemic was found to be due to viruses constituting a new genogroup, C3.

Human enterovirus 71 (EV71) infection has emerged as a major threat to children; however, no effective antiviral treatment or vaccine is currently available. Antibody-based treatment shows promises to control this growing public health problem of EV71 infection, and a few potent monoclonal antibodies (mAbs) targeting viral capsid protein have been well described. Here, we generated an EV71-specific mouse mAb 2G8 that conferred full protection against lethal EV71 challenge in a suckling mouse model. 2G8 belonged to IgM isotype and neutralized EV71 at the attachment stage. Biochemical assays mapped the binding epitope of 2G8 to the SP70 peptide, which spanning amino acid residues 208-222 on the VP1 protein. Alanine scanning mutagenesis defined the essential roles of multiple residues, including Y208, T210, G212, K215, K218, L220, E221, and Y222, for 2G8 binding. Then, a panel of single mutation was individually introduced into the EV71 infectious clone by reverse genetics, and three mutant viruses, K215A, K218A, and L220A, were successfully recovered and characterized. Biochemical and neutralization assays revealed that K218A mutant partially escaped 2G8 neutralization, while L220A completely abolished 2G8 binding and neutralization. In particular, neutralization assays with human sera demonstrated that K218A and L220A substitutions are also critical for antibody neutralization in natural infection population. These findings not only generate a protective mAb candidate with therapeutic potential but also provide insights into antibody-mediated EV71 neutralization mechanism. [ABSTRACT FROM AUTHOR]

Human enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are major causative agents of hand, foot, and mouth disease (HFMD) especially in infants and children under 5 years of age. Despite recent outbreaks of HFMD, there are no approved therapeutics against EV71 and CA16 infection. Moreover, in a small percentage of cases, the disease progression can lead to serious complications of the central nervous system. In this study, we investigated the antiviral effect of corilagin and Phyllanthus urinaria extract, which contains corilagin as a major component, on EV71 and CA16 infection in vitro. Our results indicate that corilagin reduces the cytotoxicity induced by EV71 or CA16 on Vero cells with and IC value of 5.6 and 32.33 μg/mL, respectively. We confirmed the presence of corilagin in EtOAc and BuOH fractions from P. urinaria extract and this correlated with antiviral activity of the fractions against EV71 or CA16. Future studies will be required to confirm the antiviral activity of corilagin and P. urinaria extract in vivo. Challenging a model with a lethal dose of viral infection will be required to test this. Collectively, our work provides potential candidates for the development of novel drugs to treat HFMD. [ABSTRACT FROM AUTHOR]

Purpose: Since 1980s, human enterovirus-71 virus (HEV-71) is one of the common infectious disease in Asian Pacific region since late 1970s without effective commercial antiviral or pro- tective vaccine is unavailable yet. The work examines the role of vaccine adjuvant particle size and the route of administration on postvaccination antibody response towards HEV-71 vaccine adsorbed to calcium phosphate (CaP) adjuvant. Materials and Methods: First, CaP nano-particles were compared to a commercial micro-size and vaccine alone. Secondly, intradermal reduced dosage was compared to the conventional intramuscular immunization. Killed HEV-71 vaccines adsorbed to CaP nano-size (73 nm) and commercial one of micro-size (1.7 µm) were administered through intradermal, intramuscular, rabbits received vaccine alone and unvaccinated animals. Results: CaP nano-particles adsorbed HEV-71 vaccine displayed higher antibody than the micro-size or unadsorbed vaccine alone, through both parenteral immunization routes. Moreover, the intradermal route (0.5 µg/mL) of 0.1-mL volume per vaccine dose induced equal IgG antibody level to 1.0-mL intramuscular route (0.5 µg/mL). Conclusion: The intradermal vaccine adsorbed CaP nano-adjuvant showed safer and significant antibody response after one-tenth reduced dose quantity (0.5 µg/mL) of only 0.1-mL volume as the most suitable protective, cost effective and affordable formulation not only for HEV-71; but also for developing further effective vaccines toward other human pathogens. [ABSTRACT FROM AUTHOR]

Cytokine profiles may impact the pathogenicity and severity of hand, foot, and mouth disease caused by human enterovirus (HEV) 71. In 91 severe or mild HEV 71-associated hand, foot, and mouth disease children, serum was collected between days 2 and 10 or day >10. Serum cytokines including Type 1 T helper (Th1) cytokines: interleukin (IL)-2, interferon-gamma (IFN-γ), IL-12, and IL-18, Type 1 T helper (Th2) cytokines: IL-4, IL-10, IL-13, proinflammatory cytokines: IL-1α, IL-1β, IL-6, IL-8, IL-17, and tumor necrosis factor alpha (TNF-α), were assessed during the early stage and recovery. In the patients with mild illness, the peaks of IL-8 and IL-10 were observed on day 6 and that of IL-18 was on day 4. In the patients with severe illness, all cytokines spiked on day 3 and peaked on day 11. All cytokines except IL-6, IL-8, IL-18, and TNF-α were significantly correlated with immunoglobulin M levels by the end of the disease course. Cytokine profile variations between the patients with mild and severe illness may indicate prognosis and strain virulence, useful in clinical treatment of patients. J. Med. Virol. 86:1377-1385, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background Human enterovirus 71 (EV71) is an important pathogen caused large outbreaks in Asian- Pacific region with severe neurological complications and may lead to death in young children. Understanding of the etiological spectrum and epidemic changes of enterovirus and population's immunity against EV71 are crucial for the implementation of future therapeutic and prophylactic intervention. Results A total of 1,182 patients who presented with the symptoms of hand foot and mouth disease (67.3%) or herpangina (HA) (16.7%) and admitted to the hospitals during 2008-2013 were tested for enterovirus using pan-enterovirus PCR targeting 5'-untranslated region and specific PCR for viral capsid protein 1 gene. Overall, 59.7% were pan-enterovirus positive comprising 9.1% EV71 and 9.1% coxsackievirus species A (CV-A) including 70.5% CV-A6, 27.6% CV-A16, 1.1% CV-A10, and 0.8% CV-A5. HFMD and HA occurred endemically during 2008-2011. The number of cases increased dramatically in June 2012 with the percentage of the recently emerged CV-A6 significantly rose to 28.4%. Co-circulation between different EV71 genotypes was observed during the outbreak. Total of 161 sera obtained from healthy individuals were tested for neutralizing antibodies (NAb) against EV71 subgenotype B5 (EV71-B5) using microneutralization assay. The seropositive rate of EV71-B5 was 65.8%. The age-adjusted seroprevalence for individuals was found to be lowest in children aged >6 months to 2 years (42.5%). The seropositive rate remained relatively low in preschool children aged > 2 years to 6 years (48.3%) and thereafter increased sharply to more than 80% in individuals aged > 6 years. Conclusions This study describes longitudinal data reflecting changing patterns of enterovirus prevalence over 6 years and demonstrates high seroprevalences of EV71-B5 NAb among Thai individuals. The rate of EV71 seropositive increased with age but without gender-specific significant difference. We identified that relative lower EV71 seropositive rate in early 2012 may demonstrate widely presented of EV71-B5 in the population before account for a large outbreak scale epidemic occurred in 2012 with due to a relatively high susceptibility of the younger population. [ABSTRACT FROM AUTHOR]

Abstract: Enterovirus 71 infection causes hand, foot and mouth disease in children, and can produce diverse neurologic complications. Epidemics occurring in Korea between 2009 and 2012 resulted in the death of some patients. The present study aimed to clarify the correlation between clinical features and MRI findings in patients presenting with acute neurologic manifestations related to enterovirus 71 infection. Based on their clinical features, the patients were classified into four clinical groups: (1) brainstem encephalitis (n=17), characterized by myoclonus, tremor, ataxia, and autonomic dysregulation such as pulmonary hemorrhage; (2) aseptic meningitis (n=2); (3) encephalitis (n=2), characterized by decreased consciousness, seizure, and fever without myoclonus, tremor, ataxia, and autonomic dysregulation; and (4) acute flaccid paralysis (n=1). Thirteen of the 17 patients with brainstem encephalitis showed characteristic lesions in the dorsal brainstem and bilateral cerebellar dentate nuclei on brain MRI, whereas three had no abnormality. One of the two patients with meningitis had a small lesion in the left dorsal pons. Two patients with encephalitis had no apparent MRI abnormality. One patient with acute flaccid paralysis of the right leg had contrast-enhancement of the bilateral ventral nerve roots at the lumbar spine level on MRI. Five of 13 patients with lesions in the bilateral dentate nuclei of the cerebellum exhibited no cerebellar symptoms, while two with no cerebellar lesions developed ataxia. Although most patients presenting with neurologic manifestations of enterovirus 71 infection had characteristic clinical features together with typical MRI findings, the clinical features were not necessarily consistent with MRI findings. [Copyright &y& Elsevier]

Abstract: The detection of human enterovirus 71 (EV 71), an important etiological agent of foot, hand and mouth disease, by a hybrid voltammetric immunosensor was proposed in this paper for the first time. Poly(o-phenylenediamine) (PoPD) film was formed on a glassy carbon electrode (GCE) by utilizing electropolymerization, which yielded an interface with amino groups for the subsequent assembly of gold nanoparticles (AuNPs). The electrochemical characteristics of the immunosensor were investigated by cyclic voltammetry (CV). The parameters influencing the performance of the resulting immunosensor were studied in detail. The current response of the proposed immunosensor decreased linearly in the EV 71 concentration range from 0.1 to 80ngmL−1 with a detection limit of 0.04ngmL−1 (signal-to-noise ratio of 3). Due to its simple preparation, good reproducibility and long-time stability, the proposed voltammetric immunosensor will be promising in the point-of-care diagnostics application of clinical screening of multiple diseases. [Copyright &y& Elsevier]

To study epidemiological features and etiological characteristics of hand, foot and mouth disease (HFMD), 42,012 cases were investigated from April 2008 to December 2011 in Huizhou, China. The average incidence rate was 21.16 per 10,000. The highest peak of HFMD incidence was detected between April and July each year, accounting for 55.93 % of all reported cases; another peak occurred in October through December, accounting for 24.17 % of the cases. Of the reported cases, 89.75 % were in children less than 4 years old, and there was a slight predominance of HFMD in boys and children living in rural and suburban areas. The major pathogens causing HFMD were enterovirus 71 (EV71) and coxsackievirus A16 (CA16). C4a was the most prominent EV71 subgenotype circulating in Huizhou. This founding provides insight for developing public-health interventions for the control and prevention of HFMD, especially for reducing the risk of HFMD in high-risk individuals by taking precautions against enterovirus infections. [ABSTRACT FROM AUTHOR]

Abstract: Background: Human enterovirus 71 (HEV71) is a common cause of severe outbreaks of hand-foot- and mouth disease, aseptic meningitis and encephalitis in Asian populations but has not caused such epidemics in all populations. Objectives: To analyze the frequency of HEV71 in the background childhood population in Finland by screening in stool and serum samples and by measuring neutralizing antibodies against HEV71 in serum and to compare the genetic relationship of virus strains detected in asymptomatic children and those causing severe illness in Finland to the strains found in other countries. Study design: 4185 stool samples and 5686 serum samples were collected and clinical symptoms recorded from children who were observed from birth. Additional stool samples were available from four children hospitalized due to EV71 infection. Samples were screened for the presence of RNA of human enteroviruses using RT-PCR and HEV71 amplicons were identified by sequencing. Phylogenetic analyses were carried out to study genetic relationships between different virus strains. Neutralizing antibodies against HEV71 were screened from 522 children. Results: A total of 0.3% of stool samples and two serum samples from healthy children were positive for HEV71 genome. 1.6% of the children had neutralizing antibodies against HEV71. Most infections were asymptomatic or mild in contrast to the clear symptoms in the children hospitalized due to HEV71. All viruses were C strains. Conclusions: HEV71 is circulating in Finland but it is rare. No clear difference was seen between strains circulating in the Finnish background population and those found in hospitalized patients or those causing severe outbreaks worldwide. [Copyright &y& Elsevier]

Hand, foot, and mouth disease (HFMD), first reported in New Zealand in 1957 is caused by Coxsackievirus A16 (CVA16) and human enterovirus 71 (HEV71) and occasionally by Coxsackievirus A4-A7, A9, A10, B1-B3, and B5. This is characterized by erythematous papulo vesicular eruptions over hand, feet, perioral area, knees, buttocks and also intraorally mostly in the children. HFMD has been known for its self limiting course. Only small scale outbreaks have been reported from United States, Europe, Australia, Japan and Brazil for the first few decades. However, since 1997 the disease has conspicuously changed its behavior as noted in different Southeast Asian countries. There was sharp rise in incidence, severity, complications and even fatal outcomes that were almost unseen before that period. Following the near complete eradication of poliovirus, HEV71, the non-polio enterovirus, may become the greatest threat to cause significant neurological complications. This adds to the fact that effective therapy or vaccine is still a far reaching goal. There are reports of disease activity in different corners of India since 2004. Although of milder degree, continuous progress to affect larger parts of the country may indicate vulnerability of India from possible future fatal outbreaks. Low level of awareness among the health care providers may prove critical. [ABSTRACT FROM AUTHOR]

Most human enterovirus 71 (EV71) infections present with mild conditions such as hand-foot-mouth disease or herpangina. However, EV71 can invade the central nervous system, causing neurologic diseases such as brainstem encephalitis, meningitis and acute flaccid paralysis despite the near complete eradication of polioviruses. Neurological complications from EV71 infection occur within a few days and can lead to death even though rare. There is no report for EV71 infection with severe neurological manifestation in Jeju Island so far. We therefore report a case of EV 71 infection with bilateral acute flaccid paralysis for the first time in Jeju Island with review of related articles. [ABSTRACT FROM AUTHOR]

Abstract: The replication of human enterovirus 71 (HEV71) in cell culture is inhibited by concentrations of guanidine that do not have an observable adverse effect on host cell metabolism. Although the HEV71 non-structural protein 2C is known to play an important role in viral RNA replication, its precise biochemical activities and structure have not been fully determined. Here we describe amino acid substitutions in HEV71 protein 2C that confer resistance to guanidine. Three guanidine-resistant virus populations were independently isolated and found to contain five mutations in protein 2C, one of which, A4657T (2C-M193L), was present in two of the independently selected populations. This mutation was introduced into a HEV71 infectious cDNA clone and was sufficient to confer complete resistance to growth inhibition in the presence of 4mM guanidine. In the first guanidine-resistant population selected, the 2C-M193L mutation occurred in association with an additional mutation, A4459G (2C-I127V), located in the putative cis-acting replication element (cre) of coding region 2C. This mutation conferred only partial guanidine resistance when introduced into the HEV71-26M infectious clone. When the 2C-I127V and 2C-M193L mutations were introduced into HEV71-26M together, the 2C-I127V mutation did not increase the level of guanidine resistance due to the 2C-M193L mutation alone. This study confirms that guanidine resistance can be readily selected in HEV71 and is attributable to mutations within protein 2C. [Copyright &y& Elsevier]

Abstract: Human enterovirus 71 (EV71) has become a major public health threat across Asia Pacific. The virus causes hand, foot, and mouth disease which can lead to neurological complications in young children. There are no specific antivirals or vaccines against EV71 infection. The major neutralizing epitope of EV71 is located in the carboxy-terminal half of the VP1 protein at amino acid positions 215–219 (). To study the immunogenicity of VP1 we have developed a baculovirus vector which displays VP1 as a type II transmembrane protein, providing an accessible C-terminus. Immunization of mice with this recombinant baculovirus elicited neutralizing antibodies against heterologous EV71 in an in vitro microneutralization assay. Passive protection of neonatal mice confirmed the prophylactic efficacy of the antisera. Additionally, EV71 specific T cell responses were stimulated. Taken together, our results demonstrate that the display of VP1 as a type II transmembrane protein efficiently stimulated both humoral and cellular immunities. [Copyright &y& Elsevier]

Abstract: Most human enterovirus 71 (HEV71) strains infect only primates and are unable to cause clinically apparent infection in mice. Here we describe a mouse-adapted HEV71 strain that belongs to sub-genogroup B5 with increased virulence in newborn BALB/c mice. The mouse-virulent strain was initially selected by serial passage of a HEV71 clinical isolate (HEV71-B5) in Chinese hamster ovary (CHO) cells (CHO-B5), followed by serial passage in newborn mice. Virus from the fifth mouse passage was cultured twice on Vero cells and designated as MP-B5. MP-B5 induces severe disease of high mortality in newborn mice in a dose-dependent manner. Skeletal muscle is the primary site of virus replication and results in severe myositis. CHO-B5 harbours a single amino acid substitution (K149 →I) in the VP2 capsid protein. Five additional nucleotide sequence changes were identified in MP-B5, two of which are located in the 5′ UTR and the three within the open reading frame (ORF). Two of the ORF mutations resulted in deduced amino acid changes in the capsid protein VP1: S241 →L and K244 →E; the third ORF mutation was a synonymous C→T change at nucleotide position 6072 within the 3D polymerase gene. Infectious cDNA clone-derived mutant virus populations of HEV71 belonging to sub-genogroup B3 (CHO-26M) that contain the VP1 mutations identified in MP-B5 were generated in order to determine the mutation(s) responsible for mouse virulence. Only viruses expressing the VP1 (K244 →E) mutation were virulent in 5-day-old BALB/c mice, indicating that the VP1 (K244 →E) change is the critical genetic determinant of mouse adaptation and virulence in this model. [Copyright &y& Elsevier]

Abstract: We selected Chinese hamster ovary (CHO) cell-adapted strains of human enterovirus 71 (HEV71) belonging to sub-genogroups B5 (HEV71-B5) and C2 (HEV71-C2) by serial passage in CHO cells at a high multiplicity of infection. During the course of CHO cell passage, virus growth improved significantly, with increasing virus titres and the presence of cytopathic effect observed. A study of virus growth kinetics revealed that the CHO cell-adapted strains of HEV71-B5 (CHO-B5) and HEV71-C2 (CHO-C2) grew efficiently in CHO cells with maximum titres >100-fold higher than unadapted parental virus. Both CHO-B5 and CHO-C2 harboured single amino acid mutations within the VP2 capsid protein gene. CHO-B5 has an amino acid substitution of K149 →I in VP2 and CHO-C2 has an amino acid substitution of K149 →M in VP2. An isolate of sub-genogroup C4 (HEV71-C4) failed to adapt to CHO cells during serial passage. Infectious cDNA clone-derived populations of HEV71-C4 containing the mutations K149 →I or K149 →M in VP2 were generated by site-directed mutagenesis. Both mutations resulted in the ability of the virus to replicate efficiently in CHO cells, indicating that amino acid position 149 in VP2 is critical for the adaptation of HEV71 to growth in CHO cells. [Copyright &y& Elsevier]

Abstract: A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of human enterovirus 71 subgenotype C4 (EV71-C4) and Coxsackievirus A16 (CVA16) infection, respectively. The reaction was performed in one step in a single tube at 65°C for 60min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limits of the RT-LAMP assay were 0.33 and 1.58 of a 50% tissue culture infective dose (TCID50) per reaction based on 10-fold dilutions of a titrated EV71 or CVA16 strain, respectively. No cross-reaction was observed with Coxsackievirus A (CVA) viruses (CVA2, 4, 5, 7, 9, 10, 14, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3, 6, 11, and 19). The assay was further evaluated with 47 clinical stool specimens diagnosed previously with EV71, CVA16 or other human enterovirus infections. Virus isolates from stool samples were confirmed by virus neutralization testing and sequencing. RT-LAMP with HNB dye was demonstrated to be a sensitive and cost-effective assay for rapid visual detection of human EV71-C4 and CVA16. [Copyright &y& Elsevier]

Abstract: Evolutionary history of human enterovirus 71 (EV71) in the Netherlands shows displacement of virus subgenogroups, that only partly can be explained by antigenic changes. Additionally, occasional epidemics have occurred that remain to be explained. Previous studies have shown subgenogroup specific recombination events in the genome of Asian EV71 strains. To find clues on the role of genome recombination in evolution of the EV71 subgenogroups found in Europe and in the evolution of strains capable of causing outbreaks, we analyzed the genomes of 19 strains representing the genetic diversity of EV71 in the Netherlands between 1963 and 2010. We selected viruses from EV71 endemic and epidemic years (1986 and 2007). Subgenogroup specific genome recombination events were detected for subgenogroup B0, B1 and B2 viruses, in line with observed genome recombination events in Asian subgenogroup B3 and B4 viruses. Considering recombination events distinguishing strains from epidemic years from those of non-epidemic years, breakpoints for recombination were detected in the 5′UTR of B2 viruses from the outbreak in 1986, with highest similarity of the 5′UTR to B4 and B3 strains isolated during outbreaks in the Asian Pacific region. No indications for recombination were found in genogroup C isolates. Except for the ‘86 B2 isolates’ Dutch isolates phylogenetically interspersed with international reference strains of the same subgenogroup, indicating a global dissemination of (recombinant) EV71 viruses. The difference observed in the 5′UTR of EV71 strains isolated in endemic versus epidemic years suggests that changes in the 5′UTR cause evolution of strains capable of causing outbreaks. [Copyright &y& Elsevier]

Abstract: Human enterovirus 71 (HEV71) has emerged as a major cause of epidemics of hand, foot and mouth disease associated with severe neurological sequelae in the Asia-Pacific region. In this study, a passive protection mouse model was used to evaluate the protective efficacy of formalin-inactivated HEV71 vaccines derived from a Chinese C4 genotype strain. Pregnant mice were immunised using a prime/boost strategy and ≥50U of vaccine protected five-day-old pups from lethal challenge with a mouse-adapted (B3 genotype) strain of HEV71. Immunised mice developed a neutralising antibody response to both the immunising C4 strain and to the mouse-adapted strain. Mice born to immunised dams showed significantly less myositis and reduced viral loads in tissues. [Copyright &y& Elsevier]

Abstract: Background: An outbreak of hand, foot, and mouth disease (HFMD) included 1149 people in Linyi City, Shandong Province, China, in 2007: three children died. Objectives: To characterize the pathogens responsible for this outbreak and to analyze their genetic features. Study design: A total of 233 clinical specimens were collected from 105 hospitalized patients, including 11 patients with severe HFMD. Virological investigations (direct RT-PCR, viral isolation and molecular identification) and phylogenetic analysis were performed. Results: Human enterovirus 71 (HEV71) was the main pathogen that caused this outbreak, based on clinical manifestations, epidemiological data, and laboratory results. Phylogenetic analysis indicated that the Shandong HEV71 isolates belonged to 3 lineages in subgenotype C4. Subgenotype C4 could be further divided into two clusters (C4a and C4b), which corresponded to two time periods. Cluster C4a HEV71 has been the predominant virus circulating in mainland China in the past 5 years. Conclusions: The 2007 HFMD outbreak was mainly caused by HEV71 subgenotype C4 with 3 transmission chains. This virus has been continuously circulating in China since 1998. The Shandong strains co-evolved with isolates from other provinces in mainland China and neighboring countries. [Copyright &y& Elsevier]

Abstract: Although several cell-based reporter assays have been developed for screening of viral protease inhibitors, most of these assays have a significant limitation in that numerous false positives can be generated for the compounds that are interfering with reporter gene detection due to the cellular viability. To improve, we developed a mammalian cell-based assay based on the reverse two-hybrid system to monitor the proteolytic activity of human enterovirus 71 (EV71) 3C protease and to validate the cytotoxicity of compounds at the same time. In this system, the GAL4 DNA binding domain (M3) and transactivation domain (VP16) were fused, in-frame, with 3C or 3Cmut. The 3Cmut was an inactivated protease with mutations at the predicted catalytic triad. The reporter plasmid contains a secreted alkaline phosphatase (SEAP) gene under the control of GAL4 activating sequences. We demonstrated that M3-3C-VP16 failed to turn on the expression of SEAP due to the separation of M3 and the VP16 domains by self-cleavage of 3C. In contrast, SEAP expression was induced by the M3-3Cmut-VP16 fusion protein or the M3-3C-VP16 in cells treated with AG7088, a potent inhibitor of human rhinoviruses (HRVs) 3C protease. Potentially, this protease detection system should greatly facilitate anti-EV71 drug discovery through a high-throughput screening. [Copyright &y& Elsevier]

Abstract Background In China, a rapid expansion of Hand, foot, and mouth disease (HFMD) outbreaks has occurred since 2004 and HFMD has become an important issue for China. However, people are still only concerned with human enterovirus 71(HEV-71) and coxsackie virus A16 (CV-A16). Much of what is known about the other enterovirus infections relies on fractional evidence and old epidemic data, with little knowledge concerning their distribution. To alert potential threatens of the other enteroviruses, our study genetically characterized specimens from different regions of China and yielded novel information concerning the circulating and phylogenetic characteristics of enteroviral strains from HFMD cases. Findings A total of 301 clinical throat swabs were randomly obtained from patients suffering from HFMD from the southern, northern and central regions of China during outbreaks in 2009. 266 of 301 (88.4%) HFMD cases were found positive for HEV and seven genotypes, HEV-71, CV-A16, -B5, -A4, -A6, -A10, and -A12, were detected. Conclusions The HFMD pathogen compositions in the different regions of China were significantly different. HFMD epidemics might persist for a long time in China due to the multiple pathogen compositions, the enteroviral characteristic of recombination and co-infection, the ever-increasing travel and migration and the deficiency of effective vaccine. Our study deserves the attention on HFMD control and vaccine development.

Abstract Human enterovirus 71 (EV71) infection causes hand, foot and mouth disease in children under 6 years old and this infection occasionally induces severe neurological complications. No vaccines or drugs are clinical available to control EV71 epidemics. In present study, we show that treatment with lycorine reduced the viral cytopathic effect (CPE) on rhabdomyosarcoma (RD) cells by inhibiting virus replication. Analysis of this inhibitory effect of lycorine on viral proteins synthesis suggests that lycorine blocks the elongation of the viral polyprotein during translation. Lycorine treatment of mice challenged with a lethal dose of EV71 resulted in reduction of mortality, clinical scores and pathological changes in the muscles of mice, which were achieved through inhibition of viral replication. When mice were infected with a moderate dose of EV71, lycorine treatment was able to protect them from paralysis. Lycorine may be a potential drug candidate for the clinical treatment of EV71-infected patients.