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

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

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

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

An etiology study on HFMD in Qingdao region during 2008-2009 was conducted. The virus RNA were isolated from throat swabs of HFMD,the EV, EV71 and CVA16 were detected by multiplex realtime RT-PCR. For those specimens with EV positive and both EV71 and CVA16 negative,a reverese transcription-seminested polymerase chain reaction (RT-snPCR) was perfomed to amplify part sequence of the VP1 gene for sabsequent analysis to identify the serotype. The results indicated that EV71 and CVA16 were the major pathogens of HFMD in Qingdao during 2008-2009. The proportion of EV71 was greater than CVA16 in either mild or serious HFMD cases. Sequence analysis showed that 5 non-EV71 and non-CVA16 serotypes (8 specimens) were obtained in 2008 including Coxsackievirus A5, A6, A10, A12 and Echovirus 9, which were well distributed. Three serotypes(13 specimens) were obtained in 2009 including Coxsackievirus A9, A12 and B2, of which CVA12 was of a big proportion (11/13). CVA12 became a new relatively major pathogen of HFMD in Qingdao during 2009.
China ; epidemiology ; Enterovirus ; classification ; genetics ; isolation & purification ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Phylogeny ; RNA, Viral ; genetics ; Serotyping

A rapid detection of foot-and-mouth disease virus (FMDV) was established by using reverse transcription loop-mediated isothermal amplification (RT-LAMP) method, meanwhile its specificity and sensitivity were assessed. The results showed that the FMDV RNA could be amplified by incubation at 65degrees C for only 1h using six primers designed based on FMDV polyprotein gene and the amplification products could be detected easily by naked-eye. There is no cross reaction with other virus such as SVDV, SFV and PPV by detecting their RNA samples. The detection limit of this method was found to be 10(-5) dilution of RNA sample which was 100-fold higher than that of PCR and 10-fold higher than real-time PCR.
Animals ; DNA Primers ; Foot-and-Mouth Disease ; prevention & control ; virology ; Foot-and-Mouth Disease Virus ; genetics ; isolation & purification ; Genome, Viral ; genetics ; Nucleic Acid Amplification Techniques ; RNA, Viral ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Swine Vesicular Disease ; virology