It was reported that the sera of convalescent animals contain antibodies to foot and mouth disease (FMD) virus (FMDV) 2C, highly conserved nonstructural protein (NSP), whereas the sera of vaccinated animals do not. But ELISA methods using this protein were not reported and developed until recently. In this study, NSP 2C peptides were synthesized within the amino acid sequence of the conserved 2C nonstructural region of FMDV according to the sequences from Genbank database and used for identifying antigenic determinants. One of the synthesized thirteen peptides gave strong positive reactivity with most of the sera from 13 FMD infected farms, but not with sera from vaccinated and non-infected animals. Moreover, with the sera collected through serial bleedings from four cattle and five goats infected with FMDV O/SKR/2000 experimentally, positive results were obtained in two species after 10 days post infection (DPI). Therefore, we tried to develop and evaluate this ELISA based on 2C peptides. In comparison with the commercial NSP ELISA, the 2C peptide based ELISA method showed good specificity and sensitivity. These results demonstrate that the synthetic 2C peptide ELISA can be a complementary marker to differentiate FMDV-infected from vaccinated on a herd basis.
Animals ; Cattle ; Enzyme-Linked Immunosorbent Assay/veterinary ; Foot-and-Mouth Disease/*diagnosis ; Foot-and-Mouth Disease Virus/*isolation&purification ; Goats ; Sensitivity and Specificity ; Vaccination ; Viral Nonstructural Proteins/*chemical synthesis ; Viral Vaccines

One step TaqMan real-time reverse transcription polymerase chain reaction (R/T RT-PCR)using a set of primers/probes was developed for the detection of foot-and-mouth disease (FMD)virus. The gene-specific probes labeled fluorogen for the internal ribosomal entry site, Leader sequence and 2B regions were used to detect FMD virus (FMDV). This assay specifically detected FMDV both in cell culture preparations and clinical samples, and was capable of distinguishing FMD from other viral diseases similar to clinical signs (swine vesicular disease, vesicular stomatitis and bovine viral diarrhea). This assay was shown to be 1000-fold more sensitive than the conventional RT-PCR method. The detection limits of this assay was 1 TCID 50 /ml of the FMDV RNA concentration. Quantification was obtained by a standard curves plotting threshold cycle values versus known infectivity titer. The assay was sensitive, specific and rapid enough to detect FMDV RNA genome in probang samples. As such, the described method is reliable and provides faster disease diagnostics than the conventional RT-PCR procedure to detect FMDV.
Animals ; Foot-and-Mouth Disease/*diagnosis/virology ; Foot-and-Mouth Disease Virus/*isolation&purification ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; Sensitivity and Specificity ; Taq Polymerase

To establish a sensitive, rapid and simple gold immunochromatography assay (GICA) for detecting Asia1 type of foot-and-mouth disease virus (FMDV) from the field samples. The purified anti-FMDV type Asia1 monoclonal antibody labeled with colloidal gold and the goat anti-Guinea pig IgG were wrapped onto nitrocellulose membrane as the test line (T line) and the control line (C line), respectively. The strip was then further optimized. A total of 87 field samples were detected. The results indicated a correct rate of 98.8% for detecting FMDV Asia1 type. No cross reaction was found with swine vesicular disease (SVD) and FMDV O, A and C type antigen. The sensitivity of the strip can reach to 10(-4) (TCID50 6.25). It had the same results for positive and negative specimens tested in three times. This strip could be stored at 4 degrees C for three months. In this study, the established gold immunochromatographic strip test kit is simple, rapid, sensitive and specific for detecting FMDV type Asial, and is potentially useful for the for pen-side diagnosis.
Animals ; Antibodies, Monoclonal ; Chromatography ; methods ; Foot-and-Mouth Disease ; diagnosis ; Foot-and-Mouth Disease Virus ; classification ; immunology ; isolation & purification ; Gold Colloid ; Immunoassay ; methods ; Immunoglobulin G ; immunology ; Reagent Strips ; Sensitivity and Specificity

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

We compared genetic variations in the VP1 gene of foot-and-mouth disease viruses (FMDVs) isolated since 2000 from various region of the world. We analyzed relative synonymous codon usage (RSCU) and phylogenetic relationship between geographical regions, and calculated the genetic substitution patterns between Korean isolate and those from other countries. We calculated the ratios of synonymously substituted codons (SSC) to all observed substitutions and developed a new analytical parameter, EMC (the ratio of exact matching codons within each synonymous substitution group) to investigate more detailed substitution patterns within each synonymous codon group. We observed that FMDVs showed distinct RSCU patterns according to phylogenetic relationships in the same serotype (serotype O). Moreover, while the SSC and EMC values of FMDVs decreased according to phylogenetic distance, G + C composition at the third codon position was strictly conserved. Although there was little variation among the SSC values of 18 amino acids, more dynamic differences were observed in EMC values. The EMC values of 4- and 6-fold degenerate amino acids showed significantly lower values while most 2-fold degenerate amino acids showed no significant difference. Our findings suggest that different EMC patterns among the 18 amino acids might be an important factor in determining the direction of evolution in FMDV.
Animals ; Capsid Proteins/*genetics ; Cattle ; Codon/*genetics ; Evolution, Molecular ; Foot-and-Mouth Disease/diagnosis/epidemiology/*virology ; Foot-and-Mouth Disease Virus/*genetics/isolation & purification ; Gene Frequency ; Geography ; Korea ; Phylogeography ; Polymorphism, Genetic ; RNA, Viral/*analysis ; Species Specificity

We compared genetic variations in the VP1 gene of foot-and-mouth disease viruses (FMDVs) isolated since 2000 from various region of the world. We analyzed relative synonymous codon usage (RSCU) and phylogenetic relationship between geographical regions, and calculated the genetic substitution patterns between Korean isolate and those from other countries. We calculated the ratios of synonymously substituted codons (SSC) to all observed substitutions and developed a new analytical parameter, EMC (the ratio of exact matching codons within each synonymous substitution group) to investigate more detailed substitution patterns within each synonymous codon group. We observed that FMDVs showed distinct RSCU patterns according to phylogenetic relationships in the same serotype (serotype O). Moreover, while the SSC and EMC values of FMDVs decreased according to phylogenetic distance, G + C composition at the third codon position was strictly conserved. Although there was little variation among the SSC values of 18 amino acids, more dynamic differences were observed in EMC values. The EMC values of 4- and 6-fold degenerate amino acids showed significantly lower values while most 2-fold degenerate amino acids showed no significant difference. Our findings suggest that different EMC patterns among the 18 amino acids might be an important factor in determining the direction of evolution in FMDV.
Animals ; Capsid Proteins/*genetics ; Cattle ; Codon/*genetics ; Evolution, Molecular ; Foot-and-Mouth Disease/diagnosis/epidemiology/*virology ; Foot-and-Mouth Disease Virus/*genetics/isolation & purification ; Gene Frequency ; Geography ; Korea ; Phylogeography ; Polymorphism, Genetic ; RNA, Viral/*analysis ; Species Specificity

The highly-pathogenic EV71 strain is the primary cause of mortality in hand-foot-and-mouth disease (HFMD) associated with neurological symptoms, for which no clinically effective drugs or vaccines exist. This study aimed to construct infectious cDNA clones of the EV71 highly-pathogenic strain and the cell-culture adapted strain using a reverse genetics approach. The genomic RNAs of EV71 parent strains were used as the templates for RT-PCR amplification, and then the PCR products that overlapped the full-length genome were connected into the pBR322 vector to produce infectious clones of pEV71 (HP) and pEV71 (CCA), respectively. The results showed that the HP strain propagated much more quickly than the CCA strain. The rescued viruses derived from the infectious clones not only maintained their consistency with their parent strains in terms of genomic sequences, but also retained their respective biological phenotypes. This research will contribute to our understanding of EV71 pathogenesis and the development of novel vaccines against HFMD.
Animals ; Cercopithecus aethiops ; DNA, Complementary ; Enterovirus A, Human ; genetics ; growth & development ; isolation & purification ; pathogenicity ; Hand, Foot and Mouth Disease ; virology ; Humans ; Phylogeny ; Vero Cells ; Virus Cultivation

To develop a sensitive and specific ELISA for detection of antibodies to the nonstructural protein of FMDV. We cloned and expressed FMDV nonstructural protein 3AB in Escherichia coli expression system. The recombinant protein 3AB was purified with Ni-NTA HisBind Resins and characterized by Western blotting. An indirect ELISA based on purified protein 3AB as a coating antigen was established. The specificity and sensitivity of this assay were evaluated by comparison with a commercial 3ABC-ELISA kit in detecion of serum samples. The results showed that the recombinant protein 3AB was expressed as a formation of inclusion bodies in Escherichia coli. The purified protein could specificially react with FMDV infection antibodies in Western blotting assay, but no reaction with the immune antibodies induced with vaccine. Two assays were no significant differences in specificity and sensitivity for detection of field samples (P>0.05). Therefore, we speculated that the recombinant protein 3AB is a promising molecular marker, which may effectively differentiate FMD-infected from vaccinated animals in a herd.
Animals ; Antibodies, Viral ; analysis ; Antigens, Viral ; biosynthesis ; genetics ; immunology ; Cattle ; Cattle Diseases ; diagnosis ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease ; diagnosis ; immunology ; Foot-and-Mouth Disease Virus ; chemistry ; genetics ; isolation & purification ; Genetic Vectors ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology

The complete gene encoding the structural protein of FMDV(VP1) was subcloned into expression vector pPROex-HT, resulting in the fusion expression plasmid pPROexHT-VP1. After transformed into E. coli BL21(DE3) and induced by IPTG, the fusion protein was expressed in high level. Western blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against FMDV. Based on the fusion protein further purified, a novel indirect ELISA (VP1-ELISA) was developed to detect FMDV antibody in pigs. Comparison between VPl-ELISA and the government standard kit (liquid phase block ELISA) showed the two methods had 96.25 percent agreement by detecting 80 serum samples, indicating that the indirect VP1-ELISA was specific and sensitive.
Animals ; Antibodies, Viral ; blood ; Capsid Proteins ; biosynthesis ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease ; diagnosis ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; isolation & purification ; Genetic Vectors ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Sensitivity and Specificity ; Swine

OBJECTIVETo reveal the etiological agent of hand, foot and mouth disease in children in Beijing.

METHODSThroat swabs were collected from 6 infants and young children with hand, foot and mouth disease who visited the affiliated Children's Hospital from May to June 2007. Aspirated fluid from tracheal intubatton, serum and cerebral spinal fluid (CSF) were collected from a 9 years old girl (No.4243) having central neural system complication of severe hand, foot and mouth disease and admitted to the hospital from the Emergency Department. Throat swab and aspirated fluid were inoculated into the cell lines Hep-2, MDCK and Vero for virus isolation. RNAs were extracted by Trizol from 6 throat swab specimens and aspirated fluid, serum while CSF was from that severe case (No.4243). The gene fragment from 5' UTR of enterovirus was amplified from throat swabs and aspirated fluid by reverse transcription-polymerase chain reaction (RT-PCR) with the primer pairs located at the untranslated region of all enterovirus. EV71 was identified by RT-PCR with the 2 and half primer pairs located at different parts of VP1 gene of EV71. The PCR products for VP1 encoding gene of EV71 from the specimens were sequenced and sequence analysis was performed by comparing those published VP1 genes of EV71. EV71 and CA16 specific primers were used to identify the isolates by RT-PCR and the sequences were directly determined from PCR products.

RESULTSGene fragments with expected molecular weight were amplified from all 6 throat swabs and the aspirate by the primer pairs universal for the 5' UTR of enterovirus, suggesting that these patients with hand, foot and mouth disease were infected by entorovirus. Out of these 7 specimens, 2 throat swabs and the aspirate were also showing positive for the VP1 of the EV71 by different primer sets. Sequence analysis revealed that the sequences for the amplicons from 1 throat swab (No. F4211) and the aspirate shared highest homology with those published EV71, indicating that these specimens were truly positive for EV71. The sequences amplified from these specimens shared 100% and 98.9% homology to each other and were closer to the sequences of EV71 identified from Zhejiang province than those from Taiwan and strain BrCr. Gene fragments for 5' UTR of enterovirus were obtained by RT-PCR after CPE appeared in 6 out of 7 inoculations including that aspirate fluid in Vero cell, indicating that enteroviruses were isolated from these specimens. Virus isolates from one throat swab (No. F4211) and the aspirate (No. 4243) were positive by RT-PCR with the primer pairs for EV71, which was consistent with RT-PCR amplification directly from specimens. Virus isolates from other 4 specimens were CA16 by RT-PCR and sequence analysis.

CONCLUSIONThese data suggested that hand, foot and mouth disease recently appeared in children in Beijing was related with EV71 and CA16. EV71 could cause severe clinical manifestations with central nerve system complications even in the child older than 5 years.

Animals ; Cercopithecus aethiops ; Child ; China ; epidemiology ; Enterovirus A, Human ; genetics ; isolation & purification ; Enterovirus Infections ; complications ; epidemiology ; Female ; Hand, Foot and Mouth Disease ; etiology ; virology ; Humans ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, RNA ; Vero Cells ; Virus Cultivation