Evidence of recombination in a new isolate of foot-and-mouth disease virus serotype Asia 1.

Phylogenetic analysis of the nucleotide sequence of VP1 revealed that a new isolate of foot-and-mouth disease virus (FMDV) serotype Asia 1 identified in Mongolia in 2005 was related to Chinese and Russian strains isolated during the same year. In this study, these strains were defined as East Asian strains having a common geographical origin, and the complete genomic sequence of the Mongolian strain (As1/MOG/05) was determined and compared to other strains of serotype Asia 1. As1/MOG/05 showed 100% identity with an East Asian strain from China (As1/Qinghai/CHA/05) in terms of its VP1 nucleotide sequence. However, the Mongolian strain has a four-amino acid extension in 3D that is missing from all other strains of serotype Asia 1, and which is not due to an insertion. A full genomic scan revealed that the Mongolian strain is closer to the East Asian strain As1/JS/CHA/05 than to all other strains of serotype Asia 1 in nearly all genomic regions. Within the narrow region of low similarity between the two sequences, As1/JS/CHA/05 was found to have a mosaic structure with a partial 2C fragment supposedly transferred from Hong Kong strain As1/HNK/CHA/05. The genomic mosaicism and extension detected in non-structural protein-coding regions in this study may be used to trace the origins and evolution of problematic strains of serotype Asia 1 that have arisen in East Asia since 2005.

Cross-protective efficacy of the O1 Manisa + O 3039 bivalent vaccine and the O 3039 monovalent vaccine against heterologous challenge with FMDV O/Jincheon/SKR/2014 in pig.

After massive foot-and-mouth disease (FMD) outbreaks originated from Jincheon County from Dec. 2014 to Apr. 2015, the effectiveness of the previous FMD vaccine containing only the O1 Manisa as the O antigen, O1 Manisa + A Malaysia 97 + Asia 1 Sharmir trivalent vaccine, was questioned in South Korea, and a change in the O antigen in FMD vaccines was demanded to control the FMD caused by FMDV O/Jincheon/SKR/2014, the O Jincheon strain. Therefore, the efficacies of O1 Manisa + O 3039 bivalent vaccine and O 3039 monovalent vaccine were studied for cross-protection against heterologous challenge with the O Jincheon strain. In this study, the efficacy of the O1 Manisa + O 3039 bivalent vaccine was better than that of the O 3039 monovalent vaccine, even though the serological relationship (r1 value) between O Jincheon and O 3039 was matched according to the OIE Terrestrial Manual. According to serological test results from vaccinated specific pathogen free pigs, virus neutralization test titers against Jincheon were good estimates for predicting protection against challenge. A field trial of the O1 Manisa + O 3039 bivalent vaccine was performed to estimate the possibility of field application in conventional pig farms, especially due to concerns about the effect of maternally derived antibodies (MDA) in field application of the FMD vaccine. According to the result of the field trial, the O1 Manisa + O 3039 bivalent vaccine was considered to overcome MDA. The results of the efficacy and field trials indicated that the O1 Manisa + O3039 vaccine could be suitable to replace previous FMD vaccines to control the FMD field situation caused by O Jincheon FMDV.

Control of type O foot-and-mouth disease by vaccination in Korea, 2014-2015.

On December 3, 2014, a type O foot-and-mouth disease (FMD) outbreak began in Korea. Although vaccinations were administered, FMD cases increased steadily for five months, and reached 185 cases by April 2015. Most of the affected animals were pigs, which are vulnerable to vaccination. The FMD virus belonged to the South-East Asia (SEA) topotype that had been observed three times in Korea between April 2010 and July 2014. However, the FMD virus isolated in December 2014 had a unique feature; that is, partial deletion of the 5´ non-coding region, a deletion not seen in previous SEA topotype isolates identified in Korea. We conclude that this outbreak included the introduction of a new FMD strain to Korea, and that Korea was now affected by genetically similar FMD virus strains that are related to those from neighboring countries.

Development of an epitope-blocking-enzyme-linked immunosorbent assay to differentiate between animals infected with and vaccinated against foot-and-mouth disease virus.

An epitope-blocking ELISA (EB-ELISA) was developed to distinguish animals infected with foot-and-mouth-disease (FMDV) from those immunized with commercial vaccines. The assay used monoclonal antibodies to target the 3B core repeat motif (QKPLK) and purified recombinant 3AB proteins from the major B cell line epitopes of FMDV. Sera from uninfected and regularly vaccinated cattle, pigs, goats, and sheep (raised in FMDV free areas) were screened to evaluate the specificity of the EB-ELISA. The specificity scores of the assays were 99.8-100% and 100%, respectively. Reference sera from cattle, pigs, goats, and sheep experimentally infected with FMDV tested positive, with only a single exception. Antibodies formed in response to FMDV 3B appeared 1 week after infection and persisted at high levels for more than 8 weeks within the sera collected from serial bleeding of animals infected with FMDV O/SKR/2000. The EB-ELISA was used to differentiate between farms vaccinated against and those infected with FMDV (FMDV Asia serotype) during the 2005 epidemic in Mongolia by detecting antibodies against the FMDV Asia serotype in outbreak farms. This EB-ELISA method shows promise as an effective tool for FMDV control and eradication.

Enhanced immune response with foot and mouth disease virus VP1 and interleukin-1 fusion genes.

The capsid of the foot and mouth disease (FMD) virus carries the epitopes that are critical for inducing the immune response. In an attempt to enhance the specific immune response, plasmid DNA was constructed to express VP1/interleukin-1alpha (IL-1alpha) and precursor capsid (P1) in combination with 2A (P1-2A)/IL-1alpha under the control of the human cytomegalovirus (HCMV) immediateearly promoter and intron. After DNA transfection into MA104 (monkey kidney) cells, Western blotting and an immunofluorescence assay were used to confirm the expression of VP1 or P1-2A and IL-1alpha. Mice were inoculated with the encoding plasmids via the intradermal route, and the IgG1 and IgG2a levels were used to determine the immune responses. These results show that although the immunized groups did not carry a high level of neutralizing antibodies, the plasmids encoding the VP1/ IL-1alpha, and P1-2A /IL-1alpha fused genes were effective in inducing an enhanced immune response.

Development of synthetic peptide ELISA based on nonstructural protein 2C of foot and mouth disease virus.

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.

Development of a Lightcycler-based reverse transcription polymerase chain reaction for the detection of foot-and-mouth disease virus.

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.

Identification and antigenic site analysis of foot-and-mouth disease virus from pigs and cattle in Korea.

From May to June 2002, a total of 16 foot-and mouth disease (FMD) outbreaks due to the serotype O virus, Pan Asia strain, were recorded in Korea. The viruses were identified by antigen ELISA, RT-PCR and sequence analysis. The overall nucleotide sequence divergence of the VP1 region among the 4 isolates in 2002 was 0 to 1.4%, but between O/SKR/2002 and O/SKR/2000 isolates was 1.9-4.9%. Phylogenetic analysis with the some known strains from East Asian countries showed that the 4 Korean isolates in 2002 formed one distinct cluster, which different from clusters of Korean isolates in 2000, with in the same lineage of the ME-SA topotype strains. Deduced amino acid sequences around neutralizable antigenic site on VP1 site of O/SKR/2002 isolates were aligned and compared with other strains. At the antigenic site 1, the replacements of the critical amino acid residues at position 144 from V to L and at position 152 from A to T were observed in O/SKR/2002 viruses. For antigenic site 2 and 4, there were not significant variations in general. At the antigenic site 3, the substitutions of amino acid residues were present at positions 54 and 56 in O/SKR/2002 isolates and an alternative residue I at position 54 are observed only at the sequence of O/SKR/AS/2002 (cow) virus. And the substitution (L-->P) of significant residue at position 144 was detected at the amino acid sequence of the O/SKR/2002 (cow) virus.

Simple and rapid lateral-flow assay for the detection of foot-and-mouth disease virus.

A simple lateral-flow assay (LFA) based on a monoclonal antibody (MAb 70-17) was developed for the detection of foot-and-mouth disease virus (FMDV) under nonlaboratory conditions. The LFA was evaluated with epithelial suspensions (n = 704) prepared from current and historical field samples which had been submitted to the Pirbright Laboratory (United Kingdom) and from negative samples (n = 100) collected from naïve animals in Korea. Four FMDV serotypes (type O, A, Asia 1, and C) were detected in the LFA, but not the remaining three FMDV serotypes (SAT 1, SAT 2, and SAT 3). The diagnostic sensitivity of the LFA for FMDV types O, A, C, and Asia 1 was similar, at approximately 87.3%, to that of 87.7% obtained with antigen enzyme-linked immunosorbent assay (Ag-ELISA). The diagnostic specificity of the LFA was 98.8%, compared to 100% for the Ag-ELISA. These results demonstrate that the LFA using the FMDV MAb 70-17 to detect FMDV is a supportive method for taking rapid measurements at the site of a suspected foot-and-mouth disease outbreak in Asia before diagnosing the disease in the laboratory, thereby offering the possibility of implementing control procedures more rapidly.

Characterization of recombinant foot-and-mouth disease virus pentamer-like structures expressed by baculovirus and their use as diagnostic antigens in a blocking ELISA.

Non-infectious recombinant pentamer-like structures of the foot-and-mouth disease virus (FMDV) were expressed by baculovirus, and the antigenicity and immunogenicity of the proteins were analyzed in a blocking ELISA for the detection of FMDV antibodies. The recombinant pentamer-like structures were produced in insect (Sf9) cells that were inoculated with recombinant baculoviruses that expressed, simultaneously, the genes for the P1 and 3C proteins of FMDV from individual promoters. The FMDV pentamer-like structures were processed by viral 3C protease, as shown in Western blots, and were antigenic, as revealed by their reactivities in an indirect ELISA. Analysis by CsCl gradient centrifugation showed that the pentamer-like structures were similar to authentic pentameric subunits from FMDV in terms of sedimentation velocity. Furthermore, the pentamer-like structures induced high levels of FMDV-specific antibodies in mice following immunization. Observations made under the electron microscope revealed that the pentamer-like structures expressed by insect cells self-assembled to form pentameric subunits of 7-8 nm in diameter, which resemble the authentic FMDV (23+/-2 nm in diameter). The results indicate that these pentamer-like structures are as antigenic and immunogenic as authentic FMDV, although the former are smaller in size. Based on these results, a blocking ELISA was developed using the recombinant pentamer-like structure. The ELISA showed specificity of 99.5% and sensitivity of 98.5% when tested with FMDV antibody-negative and -positive sera, respectively. This blocking ELISA is highly specific and offers many advantages over the current ELISAs that use inactivated FMDV antigen. This is the first report of the production and diagnostic application of recombinant pentameric subunits of FMDV.

Comparison and analysis of the complete nucleotide sequence of foot-and-mouth disease viruses from animals in Korea and other PanAsia strains.

During the last 3 years, foot-and-mouth disease virus serotype O, named PanAsia, caused two outbreaks in the Republic of Korea. To determine if there was an obvious genetic relationship between the virus isolated in 2002 (O/SKR/2002) and the O/SKR/2000, and to further analyze the epidemiological relationships between the PanAsia viruses and the viruses identified in Korea, the complete nucleotide sequence of the O/SKR/2002 and the O/SKR/2000 were determined by automatic cycling sequencing and primer walking. The nucleotides and the deduced amino acid (aa) sequences of the strains identified in Korea were compared with each other and also those enrolled in the GenBank database. In comparison and analysis of the viruses identified in Korea, any deletions or insertions in the specific fragment gene of both the O/SKR/2002 and O/SKR/2000 were not identified. However, comparison of the aa sequence of the identified virus in 2002 from pigs with those of other PanAsia strains revealed significant substitutions of 4 aa in the VPI region and 8 aa in the 3A region. In phylogenetic analysis based on the translated region, the identified virus in 2002 appeared to be the divergence of approximately 1% degree with other PanAsia viruses. Also, animal experiments indicated that O/SKR/2000 is not host-restricted and develop the clinical signs in the main susceptible livestock species (cattle and pigs). However, O/SKR/2002 did not develop the clinical signs in cattle and showed severe clinical signs only in pigs. These analytic data suggest that 2002 outbreaks in Korea is not re-occurred but re-introduced from nowhere.