The study aims to investigate the impacts of prolyl oligopeptidase (POP) on the replication of foot-and-mouth disease virus (FMDV) in BHK-21 cells. Firstly, the effects of FMDV replication on POP expression in BHK-21 cells were analyzed by Western blotting and Real-time reverse transcription polymerase chain reaction (RT-qPCR). Secondly, a eukaryotic expression plasmid for POP was constructed, and the effects of POP overexpression on the replication of two different serotypes of FMDV were assessed by Western blotting, RT-qPCR, and virus titer assays. Thirdly, specific small interfering RNAs (siRNAs) targeting POP were synthesized, and their efficiency in interfering with endogenous POP expression was identified by RT-qPCR. The impacts of downregulating endogenous POP expression on FMDV replication were further evaluated by Western blotting, RT-qPCR, and virus titer assays. The results indicated that FMDV infection did not significantly affect POP expression in BHK-21 cells. Overexpression of POP dose-dependently enhanced the replication of both FMDV/O and FMDV/A serotypes. Conversely, siRNA-mediated downregulation of endogenous POP expression markedly suppressed FMDV/O replication. This study is the first to demonstrated that the role of the host POP protein in promoting FMDV replication in BHK-21 cells, thereby providing a critical theoretical foundation and potential molecular targets for developing efficient candidate cell strains for foot-and-mouth disease inactivated vaccines.
Foot-and-Mouth Disease Virus/genetics* ; Virus Replication/genetics* ; Prolyl Oligopeptidases ; Serine Endopeptidases/physiology* ; Animals ; Cell Line ; RNA, Small Interfering/genetics* ; Foot-and-Mouth Disease/virology* ; Cricetinae

BACKGROUND: Severe hand-foot-and-mouth disease (HFMD) is a life-threatening contagious disease among young children and infants. Although enterovirus A71 has been well acknowledged to be the dominant cause of severe HFMD, there still remain other unidentified risk factors for severe HFMD. Previous studies mainly focused on identifying the individual-level risk factors from a clinical perspective, while rare studies aimed to clarify the association between regional-level risk factors and severe HFMD, which may be more important from a public health perspective. METHODS: We retrieved the clinical HFMD counts between 2008 and 2014 from the Chinese Center for Disease Control and Prevention, which were used to calculated the case-severity rate in 143 prefectural-level cities in mainland China. For each of those 143 cities, we further obtained city-specific characteristics from the China City Statistical Yearbook (social and economic variables) and the national meteorological monitoring system (meteorological variables). A Poisson regression model was then used to estimate the associations between city-specific characteristics (reduced by the principal component analysis to avoid multicollinearity) and the case-severity rate of HFMD. The above analysis was further stratified by age and gender to examine potential modifying effects and vulnerable sub-populations. RESULTS: We found that the case-severity rate of HFMD varied dramatically between cities, ranging from 0 to 8.09%. Cities with high case-severity rates were mainly clustered in Central China. By relating the case-severity rate to city-specific characteristics, we found that both the principal component characterized by a high level of social and economic development (RR = 0.823, 95%CI 0.739, 0.916) and another that characterized by warm and humid climate (RR = 0.771, 95%CI 0.619, 0.960) were negatively associated with the case-severity rate of HFMD. These estimations were consistent across age and gender sub-populations. CONCLUSION Except for the type of infected pathogen, the case-severity rate of HFMD was closely related to city development and meteorological factor. These findings suggest that social and environmental factors may also play an important role in the progress of severe HFMD.
Adolescent ; Child ; Child, Preschool ; China/epidemiology* ; Cities/epidemiology* ; Female ; Hand, Foot and Mouth Disease/virology* ; Humans ; Incidence ; Infant ; Infant, Newborn ; Male ; Risk Factors

To improve the specific recognition and presentation of virus-like particle (VLPs), and to develop immune-targeted VLPs vaccine, the gene fragment encoding OVA₂₅₇₋₂₆₄ peptide was inserted into the VP3 gene of foot-and-mouth disease virus (FMDV) between the 171th and 172th amino acids (aa) or 173th and 174th aa by reverse PCR. The recombinant proteins were expressed by using Escherichia coli and assembled into chimeric VLP (VLP(OVA)) in vitro after purification. The VLP(OVA) was measured by dynamic light scattering and transmission electron microscopy. The recombinant protein and the assembled VLPs were evaluated by Western blotting, enzyme-linked immunosorbent assay and laser scanning confocal microscopy to confirm the insertion of OVA₂₅₇₋₂₆₄ peptide into VP3 and its location. The results show that insertion of OVA₂₅₇₋₂₆₄ into the 173th and 174th aa of FMDV VP3 did not affect the assembly of VLPs. The VLP(OVA) in size was larger than VLPs, and the OVA₂₅₇₋₂₆₄ peptide was located on the surface of VLP(OVA).
Animals ; Escherichia coli ; genetics ; Foot-and-Mouth Disease ; virology ; Foot-and-Mouth Disease Virus ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Vaccines, Virus-Like Particle

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

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

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

OBJECTIVETo study the molecular epidemiology of hand-foot-mounth disease (HFMD) associated Coxsackievirus A10 (Cox A10) identified in Fujian province.

METHODSA total of 1 525 specimens from non-EV71 non-Cox A16 HFMD patients were collected during 2011-2014. Isolated virus strains were identified and sub-typed. Full-length coding regions for the VP1 gene of the predominant serotype Cox A10 isolates were amplified and sequenced.

RESULTSAmong the 407 non-EV71 non-Cox A16 HFMD cases confirmed by virus isolation and molecular subtyping, 103 (25.3%) were caused by Cox A10, accounting for 11.0%, 6.0%, 18.4% and 9.2% among the HFMD-associated entero-viruses identified in 2011, 2012, 2013 and 2014, respectively, in Fujian province. Compared to the general features observed in the HFMD epidemics, no differences on the Cox A10-specificity rates were observed among factors as geographical origins, gender or age groups, but all with high rates of severity. Data from the nucleotide sequence analyses on VP1 genes showed low homology levels of 76.0%-77.1% among Cox A10 strains from Fujian province, in contrast to the prototype Cox A10 strain, but with high levels of homology in the amino acid sequences (91.9%-93.6%). RESULTS from the Phylogenetic analysis also indicated that Cox A10 isolates from Fujian province were distinct from the prototype strain or other isolates from other countries but was homologous to domestic strains, but the Fujian isolates clustered into multiple branches.

CONCLUSIONSCox A10 remained one of the predominant serotypes of HFMD in Fujian province. Cox A10 isolates identified in Fujian province were co-circulating and co-evolving with other domestic strains.

Benzeneacetamides ; Child ; Child, Preschool ; China ; epidemiology ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Epidemics ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; genetics ; virology ; Humans ; Infant ; Male ; Molecular Epidemiology ; Molecular Sequence Data ; Open Reading Frames ; Phylogeny ; Piperidones ; Serogroup

Integrin alphavbeta3 plays a major role in various signaling pathways, cell apoptosis, and tumor angiogenesis. To examine the functions and roles of alphavbeta3 integrin, a stable CHO-677 cell line expressing the murine alphavbeta3 heterodimer (designated as "CHO-677-malphavbeta3" cells) was established using a highly efficient lentiviral-mediated gene transfer technique. Integrin subunits alphav and beta3 were detected at the gene and protein levels by polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA), respectively, in the CHO-677-malphavbeta3 cell line at the 20th passage, implying that these genes were successfully introduced into the CHO-677 cells and expressed stably. A plaque-forming assay, 50% tissue culture infective dose (TCID50), real-time quantitative reverse transcription-PCR, and IFA were used to detect the replication levels of Foot-and-mouth disease virus (FMDV) in the CHO-677-malphavbeta3 cell line. After infection with FMDV/O/ZK/93, the cell line showed a significant increase in viral RNA and protein compared with CHO-677 cells. These findings suggest that we successfully established a stable alphavbeta3-receptor-expressing cell line with increased susceptibility to FMDV. This cell line will be very useful for further investigation of alphavbeta3 integrin, and as a cell model for FMDV research.
Animals ; Animals, Suckling ; CHO Cells ; Cloning, Molecular ; Cricetulus ; DNA, Complementary/genetics/metabolism ; Disease Susceptibility/virology ; Foot-and-Mouth Disease/*genetics/virology ; Foot-and-Mouth Disease Virus/*physiology ; Integrin alphaVbeta3/*genetics/metabolism ; Mice

OBJECTIVETo study the genetic characteristics of VP1 region of coxsackievirus A10 (Cox A10) strains isolated from hand foot and mouth disease (HFMD) cases in Ningxia Hui Autonomous Region (Ningxia) in 2013.

METHODSA total of 280 specimens, which were identified as non-enterovirus 71 and non-Cox A16 by real-time PCR, were collected and cultured by using RD cell, and the VP1 genes of isolated strains were amplified by using reverse transcriptase PCR (RT-PCR) with degenerated primers and sequenced. The sequencing results were aligned with the sequences in GenBank with BLAST algorithm to identify the virus genotypes. Homologous comparison and phylogenetic analysis were conducted for all the Cox A10 strains identified.

RESULTSAmong 36 virus strains isolated from 280 clinical specimens, 6 were identified as Cox A10. The homologies of nucleotide and amino acid of the Cox A10 strains isolated in Ningxia were 97.0%-99.8% and 99.0%-99.7% respectively, and the Cox A10 strains isolated in Ningxia shared 76.3%-77.2%, 81.6%-83.1%, 94.4%-98.9% and 80.0%-82.3% nucleotide homologies respectively and shared 92.3%-93.0%, 94.0%-95.3%, 98.0%-99.7% and 90.6%-94.0% amino acid homologies respectively with the representative strains of A, B, C and D genotypes. Phylogenetic tree analysis revealed that Cox A10 strains isolated in Ningxia belonged to genotype C.

CONCLUSIONCox A10 is one of the most common pathogen causing HFMD in Ningxia in 2013. All the Cox A10 stains isolated from HFMD patients in Ningxia belonged to genotype C.

Algorithms ; Amino Acids ; China ; Databases, Nucleic Acid ; Enterovirus A, Human ; genetics ; isolation & purification ; Genotype ; Hand, Foot and Mouth Disease ; virology ; Humans ; Phylogeny ; Real-Time Polymerase Chain Reaction

OBJECTIVETo understand the infection status of enterovirus 71 (EV71) and coxsackievirus A16 (Cox A16) among children receiving health examination for child care setting entrance in Beijing and their related medical care seeking practice and provide evidence for the estimation of disease burden caused by hand foot and mouth disease (HFMD).

METHODSSerological survey was conducted in the local children receiving health examination for child care setting entrance. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect anti-EV71 and anti-Cox A16 IgG and IgM.

RESULTSA total of 813 children were surveyed (mean age: 3.5 ± 1.0 year old). The seropositive rate was 61.9% and 4.4% for anti-Cox A16 IgG and IgM. The seropositive rate was 9.3% and 1.1% for anti-EV71 IgG and IgM. No significant difference was observed in sex specific seropositive rate (P > 0.05). However, significant differences were found in seropositive rate among different age groups (P < 0.05). Among the children who were anti-Cox A16 positive, 7.8% had ever had rashes on their hands and feet, mouth or buttocks (HFMD-like rashes). Among the children who were anti-EV71 positive, 10.7% had ever had HFMD-like rashes. For the children who were anti-Cox A16 or anti-EV71 positive, only 7.1% were brought to see doctors by their parents. However, among the seropositive children with rashes, 80.5% were brought to see doctors.

CONCLUSIONIn the healthy children at the age to go to child care setting in Beijing, most had ever infected with Cox A16. The anti-EV71 positive rate was much lower than the anti-Cox A16 positive rate. It was necessary to strengthen the prevention and control of EV71 infection in child cares settings.

Antibodies, Anti-Idiotypic ; blood ; Beijing ; epidemiology ; Child Health Services ; utilization ; Child, Preschool ; Cost of Illness ; Enterovirus A, Human ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Male ; Parents ; psychology ; Patient Acceptance of Health Care ; statistics & numerical data