Electrodes ; Enterovirus A, Human ; isolation & purification ; Gold ; Metal Nanoparticles ; Nanotubes, Carbon

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

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

Objective: To analyze the epidemiological characteristics of hand foot and mouth disease (HFMD) cases caused by Coxsackie virus A16 (Cox A16) in Guangdong province from 2012 to 2016. Methods: The data of mild HFMD cases caused by Cox A16 were collected from 8 sentinel hospitals in 8 prefecture-level cities in Guangdong to estimate Cox A16 infection status and its population and time distribution characteristics. Results: (1) The highest estimated incidence of Cox A16 infection was in 2014 (113.0/100 000), followed by 2016 (86.4/100 000) and 2012 (79.1/100 000), while the estimated incidence was lower in 2015 (29.0/100 000) and 2013 (28.8/100 000). (2) Cox A16 was confirmed to be the predominant pathogen causing HFMD outbreaks (54.6%, 89/163). The number of outbreaks in the year with high incidence (28 outbreaks) was 11.2 times higher than that in the year with low incidence (2.5 outbreaks). (3) Across all age groups, the annual estimated incidence of Cox A16 infection decreased with age (trend χ(2)=853 905.63, P<0.01). The incidence was highest in age group 1 year (1 449.2/100 000), followed by that in age group 3 years (1 097.0/100 000), in age group 2 years (1 083.5/100 000), in age group 4 years (687.8/100 000) and in age group 0 year (604.9/100 000). Among the age groups <12 months, the estimated incidence increased with age (trend χ(2)=5 541.77, P<0.01), which was highest in age group 11-months (2 105.1/100 000), followed by that in age groups 10-months (1 448.6/100 000), 9-months (938.3/100 000), 8-months (703.3/100 000) and 6-months (664.6/100 000). (4) The annual incidence peak was during May (143.9/100 000)-June (131.5/100 000). Conclusion: The prevalence of Cox A16 infection differed with year in Guangdong during 2012-2016. When the incidence of Cox A16 infection was high, more outbreaks occurred. The prevalence occurred mainly in nurseries and kindergartens from May to June each year. Children aged 0-4 years were the high risk group for Cox A16 infection, children aged 6-11 months were at high risk for Cox A16 infection.
Animals ; Child ; Child, Preschool ; China/epidemiology* ; Cities ; Coxsackievirus Infections/epidemiology* ; Disease Outbreaks ; Enterovirus A, Human/isolation & purification* ; Hand, Foot and Mouth Disease/epidemiology* ; Hospitals ; Humans ; Incidence ; Infant ; Schools

OBJECTIVETo provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples.

METHODSViruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline.

RESULTSNGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events.

CONCLUSIONThe improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.

Clinical Laboratory Techniques ; DNA Barcoding, Taxonomic ; DNA Primers ; Enterovirus ; classification ; genetics ; isolation & purification ; Herpesvirus 4, Human ; genetics ; isolation & purification ; Humans ; Influenza B virus ; genetics ; isolation & purification ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA ; methods ; Sequence Analysis, RNA ; methods

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

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

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