Reverse-genetic engineering of foot and mouth disease virus (FMDV) can improve the productivity, antigen matching, antigen stability, immune response ability, and biological safety of vaccines, so vaccine candidates with anticipated biological characteristics can be promptly achieved. Negative influence in taming of virulent strains can also be decreased or avoided. Reverse genetics not only make up for deficiencies like limitation of viral nature, low success rate, and time and energy consuming, but also realize more active designing of vaccines. Therefore, reverse genetics is significant in improving integral quality and efficiency of vaccines. In this review, we use FMDV vaccines as an example to summarize improvement in biological characteristics of virulent strains and provide a reference for related researches.
Animals ; Antibodies, Viral ; immunology ; Foot-and-Mouth Disease ; immunology ; prevention & control ; virology ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; Reverse Genetics ; Viral Vaccines ; genetics ; immunology

After sequencing, we amplified and cloned foot-and-mouth disease virus (FMDV) O/QYYS/s/06 whole genome by three fragments. These three fragments were cloned into vector P43 one by one to construct recombinant plasmid P43C, which carried the full-length cDNA of FMDV O/QYYS/s/06. Then, plasmid P43C and plasmid T7 expressing T7 RNA polymerase were co-transfected into BHK-21 cells. After 48 h, we harvested the culture broth from transfected BHK-21 cells and inoculated into 2-3 day-old sucking mice. After four generation passage, the virus harvested from sucking mice was confirmed to be type O FMDV by the indirect hemagglutination test, sucking mice's neutralization test and sequencing. The results showed that we have successfully constructed the full-length cDNA clone of FMDV O/QYYS/s/06 strain.
Animals ; Animals, Newborn ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Viral ; biosynthesis ; genetics ; Foot-and-Mouth Disease ; virology ; Foot-and-Mouth Disease Virus ; classification ; genetics ; pathogenicity ; Mice ; Transcription, Genetic ; Transfection

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

OBJECTIVETo learn about the pathogen spectrum and genetic characterization of HFMD with encephalitis in Yantai city.

METHODSStool samples and cerebrospinal fluids (CSF) collected from HFMD with encephalitis cases in Yantai. Virus were isolated from stool samples and identified by fluorescence reverse transcription-polymerase chain reaction. The VPl region was amplified and sequenced from positive specimens. Genetic characterization was identified by sequence analysis.

RESULTSGetting 3 virus strains from 10 stool specimens and all of them belong to EV71. The nucleotide and amino acid homogeneity with the representative isolates of C4a were 98%-99% and 98.90%-99.45% respectively.

CONCLUSIONThe pathogen of HFMD with encephalitis in Yantai city were mainly EV71 wich belong to subgenogroup C4 cluster C4a.

Capsid Proteins ; genetics ; Encephalitis, Viral ; virology ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Hand, Foot and Mouth Disease ; complications ; virology ; Humans ; Phylogeny

OBJECTIVETo investigate the characteristics of molecular epidemiology and molecular evolution of 5 EV 71 (enterovirus 71, EV71) strains from 5 Shenzhen patients with hand-food-mouth disease associated with EV 71 infection.

METHODS5 EV 71 strains were isolated, and sequenced to analyzed the full length gene sequences in order to compare nucleotide and amino acid homology with other EV71 strains from other regions and countries as well as previous strains across the world through bioinformatics software.

RESULTS5 strains of EV 71 belonged to sub-genotype C4 by analysis of nucleotide sequences of VP1 and VP4 of EV 71. The differences of nucleotide and amino acid sequences were much small with nucleotide homology of 93% and amino acid homology of 98% among these 5 strains. A phylogenetic tree analysis indicated that 2008 Shenzhen epidemic strains were the most close to 2004 Shenzhen circulating strains, and also much close to 1998 Shenzhen epidemic strains and 2008 Fuyang Anhui strains. The dead strain was very close to 2008 Fuyang Anhui epidemic strains.

CONCLUSIONIt can be speculated that this epidemic strains of EV 71 probably originate from the same ancient strain in the history, may from 1998 Shenzhen strain.

China ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Evolution, Molecular ; Hand, Foot and Mouth Disease ; virology ; Humans ; Phylogeny

China ; epidemiology ; Enterovirus ; genetics ; isolation & purification ; Genes, Viral ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans

Nine primers were designed for the full-length genome of O/CHINA/99 and each sequence fragment was obtained by RT-PCR, and cloned into pOK12 vecter, the full-length genome cDNA clone of O/CHINA/99 was identified by restriction enzymes digestion, PCR, and the whole genome sequencing. The results showed that the O/CHINA/99 whole genome was formed with the length of 8200 nt. The nucleotide sequence of the full-length cDNA shared 99.1% homology with its prototype. RNA synthesized in vitro by means of a bacteriophage T7 promter inserted in front of the cDNA led to the production of infectious particle upon transfection of BHK-21 cell using lipofectamine reagent, as shown by cytopathic effects. The rescued virus had high pathogenicity in mice by endermic infection too. All the results showed that an infectious molecular clone was successfully constructed and rescued virus could be obtained.
Animals ; Animals, Newborn ; Cell Line ; Cloning, Molecular ; Cricetinae ; DNA, Complementary ; genetics ; Foot-and-Mouth Disease ; virology ; Foot-and-Mouth Disease Virus ; genetics ; pathogenicity ; Humans ; Mice ; Models, Genetic ; Polymerase Chain Reaction

In order to study the genotypes and molecular evolution of human enterovirus (HEV) A species in Shandong Province, Stool samples were collected from AFP and HFMD patients in Shandong Province and virus isolation was performed. Reverse Transcription-Polymerase Chain Reactions (RT-PCR) specific for EV71 and CVA16 were performed with the virus isolates from HFMD patients. Positive isolates were selected for entire VP1 coding gene amplification and sequencing. Isolates with negative PCR results and isolates from AFP patients were selected for entire VP1 coding gene amplification and sequencing using primers specific for HEV A species. Phylogenetic tree was constructed among these VP1 nucleotide sequences and of other strains. Altogether 293 strains classified into 8 genotypes were isolated. The homologous comparison and phylogenetic analysis showed Shandong strains were distinct with prototype strains in every genotype. This report presents an overview of HEV-A in Shandong Province.
Cell Line ; China ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; virology ; Feces ; virology ; Genotype ; Hand, Foot and Mouth Disease ; virology ; Humans ; Molecular Sequence Data ; Paraplegia ; virology ; Phylogeny

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