Objective: To estimate the serotype and age-specific hospitalization burden associated with hand, foot and mouth disease (HFMD) in Anhua county of Hunan province, between October 2013 and September 2016. Methods: We collected hospitalization records of HFMD patients from 6 virological surveillance hospitals, and reimbursement records through new rural cooperative medical system from 23 township health centers to estimate the age-specific hospitalization burden of HFMD in Anhua. Combined with the results of virological surveillance, the serotype-specific hospitalization burden of HFMD in Anhua, was estimated. Results: During the three years, it was estimated that 3 541 clinical diagnosed HFMD cases, including 3 146 laboratory-confirmed HFMD cases, were hospitalized in Anhua, but only one was diaguosed as being severe. The estimated average hospitalization rate was 723/100 000(95%CI: 699/100 000-747/100 000) for clinical diagnosed HFMD and 642/100 000 (95%CI: 620/100 000-665/100 000) for laboratory-confirmed HFMD between October 2013 and September 2016. The cases caused by Cox A16 (208/100 000) and Cox A6 (202/100 000) had higher hospitalization rates compared with the cases caused by EV71 (130/100 000), Cox A10 (38/100 000) and other enterovirus (64/100 000), and the difference was statistically significant (P<0.001). HFMD-associated hospitalization rates peaked in children aged 1 year (3 845/100 000), and then decreased with age. Compared with the hospitalized HFMD caused by EV71 and Cox A16, Cox A6-associated hospitalizations mainly occurred in younger age groups (P<0.001). Conclusion: Our study revealed a substantial hospitalization burden associated with mild HFMD caused by EV71, Cox A16, Cox A6 and Cox A10, especially in young children, in Anhua.
Child ; China/epidemiology* ; Enterovirus ; Enterovirus A, Human/isolation & purification* ; Enterovirus Infections/virology* ; Hand, Foot and Mouth Disease/virology* ; Hospitalization/statistics & numerical data* ; Hospitals/statistics & numerical data* ; Humans ; Infant ; Serogroup

OBJECTIVETo eliminate the side effects of aluminum adjuvant and His-tag, we constructed chimeric VLPs displaying the epitope of EV71 (SP70) without His-tagged. Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.

METHODSThe fusion protein was constructed by inserting SP70 into the MIR of truncated HBcAg sequence, expressed in E. Coli, and purified through ion exchange chromatography and density gradient centrifugation. Mice were immunized with the VLPs and sera were collected afterwards. The specific antibody titers, IgG subtypes and neutralizing efficacy were detected by ELISA, neutralization assay, and EV71 lethal challenge. IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.

RESULTSHBc-SP70 proteins can self-assemble into empty VLPs. After immunization with HBc-SP70 VLPs, the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge. There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not. The specific IgG subtypes were mainly IgG1 and IgG2b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.

CONCLUSIONThe fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation. In the absence of adjuvant, they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant. Furthermore, the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.

Adjuvants, Immunologic ; Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; blood ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; immunology ; virology ; Epitopes ; immunology ; metabolism ; Escherichia coli ; metabolism ; Female ; Immunity, Cellular ; Immunity, Humoral ; Mice ; Recombinant Fusion Proteins ; immunology

OBJECTIVE: Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses. METHODS: A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA. RESULTS: Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system. CONCLUSION Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.
Collagen ; Drug Combinations ; Enterovirus ; growth & development ; isolation & purification ; Enterovirus Infections ; virology ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; virology ; Human bocavirus ; growth & development ; isolation & purification ; Humans ; Laminin ; Parvoviridae Infections ; virology ; Primary Cell Culture ; methods ; Proteoglycans ; Real-Time Polymerase Chain Reaction ; Respiratory Mucosa ; virology ; Virus Cultivation

Enterovirus 71 is a neuroinvasive virus that is associated with severe neurological complications. We had earlier suggested that the replication capacity of a severe strain was higher than that of a mild strain. The recombinant 3CRV and 3CDRV virus strains were successfully rescued in our previous study. In the present study, we found no difference in virulence between 3CRV and severe strains. However, the capacity of replication and to cause cell injury of 3CDRV strain decreased in vitro, especially at 39.5 °C. Replacement of 3CD region in the severe strain led to milder symptoms, less body weight loss, and lower viral load in ICR mice. Histopathological findings indicated less severe injury in mice infected with 3CDRV strain. This study suggests that the 3CD region contributes to the attenuation of the severe strain, including its replication capacity and temperature sensitivity.
Animals ; Cytopathogenic Effect, Viral ; Enterovirus A, Human ; genetics ; pathogenicity ; Enterovirus Infections ; pathology ; virology ; Gene Expression Regulation, Viral ; Mice ; Mice, Inbred ICR ; Mutation ; Viral Load ; Viral Proteins ; genetics ; metabolism ; Virulence ; Virus Replication

OBJECTIVEKnowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes.

METHODSIn this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing.

RESULTSWithin the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run.

CONCLUSIONMinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.

Child, Preschool ; Enterovirus ; genetics ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; virology ; Feces ; Genome, Viral ; Hand, Foot and Mouth Disease ; virology ; Humans ; Nucleic Acid Amplification Techniques ; instrumentation ; methods

We analyzed the genetic characteristics of coxsackievirus A4 (CV-A4) based on the entire VP1 coding region. Samples were isolated from patients with acute flaccid paralysis (AFP) in Shaanxi, China from 2006 to 2010. We wished to ascertain the predominant genotype and the relationship between CV-A4 infection and AFP. Sixty-eight non-polio enteroviruses were inoculated onto RD cells (to increase the virus titer) and molecular typing was undertaken. The entire VP1 coding region was amplified. Percentage of CV-A4 was 10.3% (7/68). Analyses of genetic identify and creation of phylogenetic trees revealed that CV-A4 could be classified into A, B and C genotypes. Seven CV-A4 strains from Shaanxi and other CV-A4 strains from China formed an independent evolution lineage located in group 4 and belonged to the C2 sub-genotype. These data suggested that CV-A4 strains of sub-genotype C2 were the predominant genotypes in China. These strains co-evolved and co-circulated with those from other provinces in China, so continued monitoring of CV-A4 (by clinical and genetic surveillance) should be enhanced.
China ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; virology ; Genotype ; Humans ; Paralysis ; virology ; Phylogeny ; Viral Proteins ; genetics

In the present study, the complete genomes of four common (4/EV71/Wenzhou/CHN/2014, 15/ EV71/Wenzhou/CHN/2014, 116/EV71/Wenzhou/ CHN/2014, and 120/EV71/Wenzhou/CHN/2014) and two virulent (11/EV71/Wenzhou/CHN/2014 and 109/EV71/Wenzhou/CHN/2014) enterovirus 71 (EV71) isolates were sequenced and described. They are 7405 bp in length and belong to EV71 sub-genotype C4 (C4a cluster). Nucleotide sequence alignment revealed six nucleotide variations (GP151→TP151, GP199→AP199, GP261→TP261, AP328→CP328, GP422→AP422, and GP437→TP437) in the two virulent isolates within the 5'UTR of the IRES element. RNA secondary structure predictions of IRES and FCE indicated that the common isolates shared similar structures, which were different from those of the virulent isolates. Moreover, the GP114→CP114 and GP151→TP151 mutations in the virulent isolates contributed to the formation of the unique RNA secondary structures in SL II. Furthermore, nucleotide/amino acid sequence alignments of 82 EV71 isolates indicated that six sites (TP488 and CP577 in the 5'UTR; AsnP57 in 2A; IleP56 in 3C; CP10 and AP47 in the 3'UTR) are potentially associated with the neurovirulence of EV71. Finally, the 3D structures of 2A were analogous, whereas the structures of VP1 and 3C were variable.
Base Sequence ; Central Nervous System ; virology ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; pathogenicity ; Enterovirus Infections ; virology ; Genome, Viral ; Genomics ; Genotype ; Humans ; Molecular Sequence Data ; Nucleic Acid Conformation ; Phylogeny ; RNA, Viral ; chemistry ; genetics ; Virulence

A retrospective surveillance study on enterovirus D68 was performed in Beijing, China, following the largest and most widespread EV-D68 infection, which occurred in the USA. From January 2011 to July 2015, EV-D68 was identified in 12 individuals with respiratory infections in Beijing, China. The phylogenetic relationships based on the genomic sequence alignment showed that there were two lineages circulating in Beijing from 2011 to 2015. Eight EV-D68 strains belonged to group 1 and four belonged to group 3. All EV-D68 strains from Beijing in 2014 were separately clustered into subgroup II of group 1. Based on these results, we concluded that the Beijing EV-D68 strains had little association with the EV-D68 strains circulating in the 2014 USA outbreak.
Adolescent ; Aged ; Beijing ; epidemiology ; Child ; Child, Preschool ; Enterovirus D, Human ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; epidemiology ; virology ; Female ; Genome, Viral ; Humans ; Male ; Phylogeny ; Retrospective Studies

Enterovirus 71 frequently involves the central nervous system and may present with a variety of neurologic manifestations. Here, we aimed to describe the clinical features, magnetic resonance imaging (MRI) findings, and cerebrospinal fluid (CSF) profiles of patients presenting with neurologic complications of enterovirus 71 infection. We retrospectively reviewed the records of 31 pediatric patients hospitalized with acute neurologic manifestations accompanied by confirmed enterovirus 71 infection at Ulsan University Hospital between 2010 and 2014. The patients' mean age was 2.9 ± 5.5 years (range, 18 days to 12 years), and 80.6% of patients were less than 4 years old. Based on their clinical features, the patients were classified into 4 clinical groups: brainstem encephalitis (n = 21), meningitis (n = 7), encephalitis (n = 2), and acute flaccid paralysis (n = 1). The common neurologic symptoms included myoclonus (58.1%), lethargy (54.8%), irritability (54.8%), vomiting (48.4%), ataxia (38.7%), and tremor (35.5%). Twenty-five patients underwent an MRI scan; of these, 14 (56.0%) revealed the characteristic increased T2 signal intensity in the posterior region of the brainstem and bilateral cerebellar dentate nuclei. Twenty-six of 30 patients (86.7%) showed CSF pleocytosis. Thirty patients (96.8%) recovered completely without any neurologic deficits; one patient (3.2%) died due to pulmonary hemorrhage and shock. In the present study, brainstem encephalitis was the most common neurologic manifestation of enterovirus 71 infection. The characteristic clinical symptoms such as myoclonus, ataxia, and tremor in conjunction with CSF pleocytosis and brainstem lesions on MR images are pathognomonic for diagnosis of neurologic involvement by enterovirus 71 infection.
Acute Disease ; Brain/diagnostic imaging ; Central Nervous System Diseases/etiology/*pathology ; Child ; Child, Preschool ; Encephalitis/pathology ; Enterovirus A, Human/genetics/*isolation & purification ; Enterovirus Infections/drug therapy/*pathology/virology ; Feces/virology ; Female ; Humans ; Immunoglobulins/administration & dosage ; Infant ; Injections, Intravenous ; Leukocytes/cytology ; Leukocytosis/cerebrospinal fluid/pathology ; Magnetic Resonance Imaging ; Male ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Republic of Korea ; Retrospective Studies ; Seasons

OBJECTIVETo investigate the etiology of severe hand-foot-mouth disease (HFMD) in children in Henan province.

METHODSA total of 244 HFMD cases admitted to a hospital in Zhengzhou from April to June of 2014 were recruited for research sampling, Real-time RT-PCR, virus isolation, VP1 sequencing and alignment methods were used to test the enterovirus-related etiology. SPSS 17.0 was used in performing statistical analysis.

RESULTSThere were 109 severe and 135 mild cases among all the 244 HFMD cases. The number of enterovirus positive stool samples was 229, with positive rate as 93.85%. EV71, Cox A16 and Cox A10 made up 83.84%, 5.68% and 8.30% of the enterovirus etiologicy, strains, respectively. EV71 infection caused 8 HFMD cases with heart-lung failure and 2 death, Cox A10 infection led to 1 HFMD case with heart-lung failure and death. There were statistically differences seen regarding the enterovirus infection rates between severe and the mild HFMD cases (χ(2)=5.312,P=0.021). Statistically significant difference was seen in the constituent ratio of EV71, Cox A16 and the others by Fisher' s exact test (P=0.048). There was statistically significant difference seen between the cardiorespiratory failure rate and the fatality rate by EV71 and Cox A10 infection (χ(2)=0.051,P=0.821; χ(2)=2.198,P=0.138). Cox A10 strains idenfied in Henan in 2014 belonged to genotype 6. The rates on homology of nucleotide and amino acid among the Cox A10 strains in Henan in 2014 were 94.3%-99.7% and 96.3%-100.0% respectively.

CONCLUSIONSEV71 still remained the most common pathogen that causing severe HFMD in children, with the increasing Cox A10 percentage in the pathogens spectrum of HFMD infection. Cox A10 strains in Henan in 2014 belonged to genotype 6. Genotype 6 Cox A10 had appeared and widely distributed in Henan for long time, but not yet variated or reconstructed. Cox A10 infection could lead to cardio-respiratory failure thus called for the monitoring program on non-EV71 and non-Cox A16 enterovirus, especially Cox A10 to be strenthened.

Amino Acids ; genetics ; Biometry ; Child ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; epidemiology ; virology ; Evolution, Molecular ; Genotype ; Hand, Foot and Mouth Disease ; epidemiology ; prevention & control ; virology ; Hospitals ; statistics & numerical data ; Humans ; Real-Time Polymerase Chain Reaction