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

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

Hand, foot, and mouth disease (HFMD) is a viral infectious disease regarded to be a public-health problem worldwide. Since the 1990s, HFMD began to spread in the Asia-Pacific region (especially in South-East Asia). HFMD outbreaks have occurred in mainland China frequently since 2008, and the morbidity and mortality of HFMD has continued to increase in recent years. In mainland China, enterovirus A serotype enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) have been the major pathogens of HFMD during these years. However, the etiological spectrum of HFMD changes with time. This review focuses mainly on the etiological spectrum of HFMD and changes in epidemic patterns in mainland China.
China ; epidemiology ; Disease Outbreaks ; Enterovirus ; classification ; genetics ; isolation & purification ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Prevalence

OBJECTIVETo investigate the genetic characteristics of coxsackievirus A10(CV-A10) strains isolated from hand, foot and mouth disease (HFMD) cases in Ningxia province.

METHODSBased on the HFMD laboratory network surveillance system, 2 470 patients clinical specimens including 450 faeces and 2 020 throat swaps were collected from various regions people's hospital in Ningxia Hui Autonomous Region during January, 2013 to December, 2014. All specimens were isolated using rhabdomyosarcoma cells. VP1 regional gene of isolated strains was amplified by RT-PCR using degenerate primers and sequenced. Sequences were compared with the database of GenBank by the Blast algorithm to identify the enterovirus genotypes. All the CV-A10 strains were performed the homology and phylogenetic evolution analysis.

RESULTS450 specimens identified as non-EV-A71, non-CV-A16 enterovirus were collected and 36 CV-A10 strains were isolated, 6 strains were isolated in 2013 and 30 strains were isolated in 2014. The homology of nucleotides and amino acids among 36 CV-A10 strains were 90.6%-100.0% , and 90.2%-100.0%, respectively. Compared 36 strains with genotype A, B, C, D representative strains, it has the highest homology with the genotype C, the nucleotide and amino acids homogeneity were 90.2%-98.9% and 95.7%-99.7%. The phylogenetic tree showed 36 strains and genotype C representative strains located in the same evolutionary branch.

CONCLUSIONCV-A10 was one of the most common pathogen of HFMD in Ningxia Hui Autonomous Region. All CV-A10 strains belonged to genotype C and contained wide homology range.

China ; Enterovirus ; genetics ; Genotype ; Hand, Foot and Mouth Disease ; virology ; Humans ; Phylogeny ; Sequence Homology, Amino Acid ; Sequence Homology, Nucleic Acid

Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease (HFMD). belongs to family Picornaviridae, genus Enterovirus, species A. EV71 infection usually affects subjects aged <5 years. HFMD caused by EV71 infection is usually mild in children. However, in some cases EV71 infection can lead to severe neurogenic disease and even death. EV71 infection has caused epidemic worldwide (especially in the Asia Pacific). HFMD caused by EV71 has become a major public-health prol lem across the Asia Pacific. In EV71 infection, the pathogenesis is determined by viral and host factor, Here, we review research on host susceptibility and how EV71 suppresses immune and intracellular ri
Animals ; Enterovirus A, Human ; genetics ; pathogenicity ; physiology ; Hand, Foot and Mouth Disease ; virology ; Humans ; Virulence ; Virus Attachment ; Virus Replication

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

The aim of this study was to understand the enterovirus types and biological features of pediatric cases of HFMD in Sanmenxia City during 2011, and compare the latter to a cohort of healthy children. Stool samples of 55 cases of HFMD and 60 healthy children were collected for the isolation and identification of enteroviruses using RNA extraction and real-time RT-PCR assays. EV71 and CA16 were identified by nucleotide sequencing using virus-specific VP1 primers; for the other enteroviruses, 012/011 and 008/013 primers were used for amplification and sequencing. The results were analysed by sequence alignment with known sequences, and the characteristics of the EV71 VP1 gene were also analyzed. The detection rates for enteroviruses in cases of HFMD and healthy children were 52.73% (29/55) and 18.33% (11/60), respectively. Among these, there were 22 cases of EV71, four cases of CA16 and three cases of other enteroviruses in the cases with HFMD. Eleven healthy children had intestinal viruses, of which nine were Coxsackie B virus strains (81.82%, 9/11). Gene sequencing of the 19 EV71 strains illustrated that they were all subgenotype C4a, but the evolutionary tree showed an obvious clustering between cases from Lingbao City and Lushi County. This study demonstrates that the EV71 subgenotype C4a and CA16 strains were the most common cause of HFMD in Sanmenxia City in 2011, and that Coxsackie B strains were prevalent in healthy children. This finding may indicate that there is a widespread source of recessive infection in the community.
Child ; Child, Preschool ; China ; epidemiology ; Cities ; epidemiology ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Infant ; Male ; Phylogeny ; Viral Proteins ; genetics