Re-emergence of foot-and-mouth disease in the Republic of Korea caused by the O/ME-SA/Ind-2001e lineage.

The O/ME-SA/Ind-2001e foot-and-mouth disease virus (FMDV) lineage is a pandemic strain that has recently become dominant within East and Southeast Asia. During May 2023, this viral lineage spread to the Republic of Korea, where 11 outbreaks were detected on cattle and goat farms located in Cheongju and Jeungpyeong. Infected animals displayed typical FMD signs including vesicular lesions with drooling and anorexia. Molecular diagnostic testing and genetic analysis (VP1 sequencing) showed that the causative FMDVs belonged to the O/ME-SA/Ind-2001e lineage and shared the closest nucleotide identity (97.95-99.21%) to viruses that have been collected from Mongolia and South-East Asian countries. Phylogenetic analyses showed that these sequences were distinct to those collected from the previous Korean O/ME-SA/Ind-2001e lineage outbreaks in 2019, demonstrating that these cases are due to a new incursion of the virus into the country. Prompt implementation of emergency vaccination using antigenically matched serotype O vaccines (r1 value: 0.74-0.93), together with intensive active surveillance on farms surrounding the infected premises has successfully prevented further spread of FMD. These recent FMD outbreaks reinforce the importance of research to understand the risks associated with transboundary pathways in the region, in order to reduce the possibility of a further reintroduction of FMD into the Republic of Korea.

On-Site Remote Monitoring System with NIR Signal-Based Detection of Infectious Disease Virus in Opaque Salivary Samples.

Infectious disease viruses, such as foot-and-mouth disease virus (FMDV), are highly contagious viruses that cause significant socioeconomic damage upon spreading. Developing an on-site diagnostic tool for early clinical detection and real-time surveillance of FMDV outbreaks is essential to prevent the further spread of the disease. However, early diagnosis of FMDV is still challenging due to the limited sensitivity and time-consuming manual result entry of commercial on-site tests for salivary samples. Here, we report a near-infrared (NIR) signal nanoprobe-based highly accurate detection and remote monitoring system toward FMDVs, which automates the analysis and reporting of diagnosis data. The NIR signal lateral flow immunoassay (LFA) was assembled with a nanoprobe with a stable emission intensity at 800 nm, minimizing the interference signal of opaque salivary samples. We investigated the clinical applicability of the NIR signal LFA at biosafety level 3 (BSL-3) laboratories using 147 opaque salivary samples. The NIR signal LFA achieved a 32-fold lower limit of detection (LOD) than a commercial LFA in detecting live FMDVs, including all isolates occurring in the Republic of Korea during 2010-2017. Our results showed that the NIR signal LFA successfully discriminated the FMDV-positive clinical salivary samples from healthy controls with a sensitivity of 96.9%, specificity of 100.0%, and AUC (area under the receiver operator characteristic curve) value of 0.999. Finally, we substantiated the real-time collection of diagnostic results using a customized portable NIR reader at nine different laboratories of government-certified quarantine institutions for foot-and-mouth disease (FMD).

Enhanced detection and serotyping of foot-and-mouth disease virus serotype O, A, and Asia1 using a novel multiplex real-time RT-PCR.

Rapid and accurate detection and serotyping of foot-and-mouth disease (FMD) virus (FMDV) is essential for implementing control policies against emergent FMD outbreaks. Current serotyping assays, such as VP1 reverse transcription-polymerase chain reaction (RT-PCR)/sequencing (VP1 RT-PCR/sequencing) and antigen detection enzyme-linked immunosorbent assay (ELISA), have problems with increasing serotyping failure of FMDVs from FMD outbreaks. This study was conducted to develop a multiplex real-time RT-PCR for specific detection and differential serotyping of FMDV serotype O, A, and Asia 1 directly from field clinical samples. Primers and probes were designed based on 571 VP1 coding region sequences originated from seven pools. Multiplex real-time RT-PCR using these primers and probes demonstrated serotype-specific detection with enhanced sensitivity compared to VP1 RT-PCR/sequencing for reference FMDV (n = 24). Complete serotyping conformity between the developed multiplex real-time RT-PCR and previous VP1 RT-PCR/sequencing was demonstrated using FMDV field viruses (n = 113) prepared in cell culture. For FMDV field clinical samples (n = 55), the serotyping rates of multiplex real-time RT-PCR and VP1 RT-PCR/sequencing were 92.7% (51/55) and 72.7% (40/55), respectively. Moreover, the developed multiplex real-time RT-PCR demonstrated improved FMDV detection (up to 33.3%) and serotyping (up to 67.7%) capabilities for saliva samples when compared with 3D real-time RT-PCR and VP1 RT-PCR/sequencing, during 10 days of challenge infection with FMDV serotype O, A, and Asia 1. Collectively, this study suggests that the newly developed multiplex real-time RT-PCR assay may be useful for the detection and differential serotyping of FMDV serotype O, A, and Asia 1 in the field.

Identification of the O/ME-SA/Ind-2001e Sublineage of Foot-and-Mouth Disease Virus in Cambodia.

Foot-and-mouth (FMD) is endemic in Cambodia with numerous outbreaks in cattle, pigs and other susceptible animal species reported every year. Historically, these outbreaks were caused by the FMD virus (FMDV) of serotype O PanAsia and Mya-98 lineages and serotype A Sea-97 lineage. However, the trans-pool movement of FMDV between inter-pool regions or countries throughout FMD endemic regions has raised concerns regarding infection with the new genotype or serotype of FMDV in Cambodia. In this study, 19 sequences of VP1 coding region obtained from 33 clinical samples collected from FMDV-affected cattle farms in Cambodia during January to March 2019 were genetically characterized to identify the genotypes/lineages of FMDV. Phylogenetic analysis of VP1 coding sequences revealed that recent field viruses belonged to O/ME-SA/Ind-2001e (15.8%), O/ME-SA/PanAsia (52.7%), and A/ASIA/Sea-97 (31.5%). Besides, the field viruses of O/ME-SA/Ind-2001e in Cambodia showed 93.5-96.8% identity with the VP1 coding sequences of the same sublineage viruses from pool 1 and 2 surrounding Cambodia. This is the first report of O/ME-SA/Ind-2001e infection in Cambodia, suggesting that the trans-pool movement of the new genotype should be closely monitored for efficient control of FMD.

Phage Display Screening of Bovine Antibodies to Foot-and-Mouth Disease Virus and Their Application in a Competitive ELISA for Serodiagnosis.

For serodiagnosis of foot-and-mouth disease virus (FMDV), monoclonal antibody (MAb)-based competitive ELISA (cELISA) is commonly used since it allows simple and reproducible detection of antibody response to FMDV. However, the use of mouse-origin MAb as a detection reagent is questionable, as antibody responses to FMDV in mice may differ in epitope structure and preference from those in natural hosts such as cattle and pigs. To take advantage of natural host-derived antibodies, a phage-displayed scFv library was constructed from FMDV-immune cattle and subjected to two separate pannings against inactivated FMDV type O and A. Subsequent ELISA screening revealed high-affinity scFv antibodies specific to a serotype (O or A) as well as those with pan-serotype specificity. When BvO17, an scFv antibody specific to FMDV type O, was tested as a detection reagent in cELISA, it successfully detected FMDV type O antibodies for both serum samples from vaccinated cattle and virus-challenged pigs with even higher sensitivity than a mouse MAb-based commercial FMDV type O antibody detection kit. These results demonstrate the feasibility of using natural host-derived antibodies such as bovine scFv instead of mouse MAb in cELISA for serological detection of antibody response to FMDV in the susceptible animals.

Probe-based real-time reverse transcription loop-mediated isothermal amplification (RRT-LAMP) assay for rapid and specific detection of foot-and-mouth disease virus.

Rapid and specific detection of foot-and-mouth disease virus (FMDV) is a key factor for promoting prompt control of FMD outbreaks. In this study, a real-time reverse transcription loop-mediated isothermal amplification (RRT-LAMP) assay with high sensitivity, rapidity and reliability was developed using a targeted gene-specific assimilating probe for real-time detection of seven FMDV serotypes. Positive assay signals were generated within 15 min for the lowest concentration of a standard RNA sample at 62°C; this was substantially faster than that achieved by the OIE (World Organisation for Animal Health)-recommended real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. The new assay specifically amplified the 3D gene of all seven FMDV serotypes and did not amplify other viral nucleic acids. The detection limit of the assay was 102  copies/µl which is comparable to that achieved by qRT-PCR. Furthermore, using clinical samples, the results of the RRT-LAMP assay were largely in agreement with those from the qRT-PCR assay with a kappa value (95% confidence interval [CI]) of 0.94 (0.86-1.02). The established RRT-LAMP assay that features assimilating probes is an advanced molecular diagnostic tool that is easily applicable to a wide range of circumstances and has high potential for use as an on-site diagnostic assay for rapid, specific, and reliable detection of FMDVs in clinical samples.

Introduction and Application of the Interferon-γ Assay in the National Bovine Tuberculosis Control Program in South Korea.

Bovine tuberculosis is a chronic disease impacting both public health and the livestock industry. The interferon (IFN)-γ assay has been introduced as an ancillary test for diagnosing bovine tuberculosis to overcome limitations of the skin test. The objective of this study was to assess the IFN-γ assay in terms of diagnostics and as a nationwide surveillance program in South Korea. From 2012 to 2013, cattle (n = 120) with bovine tuberculosis and cattle (n = 426) from bovine tuberculosis free herds were subjected to the IFN-γ assay to evaluate the sensitivity and specificity of the assay, respectively, depending on various cut-offs (0-3.5). When optical density of the cut-off was 0.1, the sensitivity and specificity were found to be 81.7% (74.7-88.6) and 99.5% (98.9-100.0), respectively. After introducing the IFN-γ assay as part of the national control program, the IFN-γ assay and single caudal fold skin test data were collected from 47 regional veterinary services to compare the results of these two tests. Overall, the agreement between the IFN-γ assay and the single caudal fold skin test (n = 492,068) was 98.2%, and Cohen's kappa value for the two methods was 0.47. Serial and parallel use of the IFN-γ assay and skin test for the bovine tuberculosis control program were compared using samples (n = 91) from cattle confirmed as bovine tuberculosis positive in laboratories from 2014 to 2016. Parallel screening for bTB showed much higher sensitivity (86/91, 94.5%) than the following screening approaches: serial (47.2%, 43/91), single screening using CFT (63.7%, 58/91), or the IFN-γ assay (78.0%, 71/91). These results indicate that the IFN-γ assay and single caudal fold skin test are complementary to each other; therefore, parallel use of these two tests is considered a useful approach to reduce the prevalence of bovine tuberculosis in South Korea.

Development of Monoclonal Antibody Specific to Foot-and-Mouth Disease Virus Type A for Serodiagnosis.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease affecting cloven-hoofed livestock worldwide. FMD virus (FMDV) type A is one of the most common causes of FMD outbreaks among the seven FMDV serotypes, and its serological diagnosis is therefore important to confirm FMDV type A infection and to determine FMD vaccine efficacy. Here, we generated monoclonal antibodies (mAbs) specific to FMDV type A via hybridoma systems using an inactivated FMDV type A (A22/Iraq/1964) and found 4 monoclones (#29, #106, #108, and #109) with high binding reactivity to FMDV type A among 594 primary clones. In particular, the #106 mAb had a higher binding reactivity to the inactivated FMDV type A than the other mAbs and a commercial mAb. Moreover, the #106 mAb showed no cross-reactivity to inactivated FMDV type South African territories 1, 2, and 3, and low reactivity to inactivated FMDV type O (O1 Manisa). Importantly, the solid-phase competitive ELISA (SPCE) using horseradish peroxidase (HRP)-conjugated #106 mAb detected FMDV type A-specific Abs in sera from FMD type A-vaccinated cattle more effectively than a commercial SPCE. These results suggest that the newly developed FMDV type A-specific mAb might be useful for diagnostic approaches for detecting Abs against FMDV type A.

Complete Genome Sequence of O/VN1/2014, a Foot-and-Mouth Disease Virus of Serotype O Isolated in Vietnam in 2014.

In this article, we report the complete genome sequence of foot-and-mouth disease virus (FMDV) strain O/VN1/2014 isolated in Vietnam (Lao Cai) in 2014. The virus belongs to serotype O, topotype South East Asia (SEA), and genotype Mya-98 (O/SEA/Mya-98). It is the latest complete genome information for the genotype O/SEA/Mya-98 in Vietnam since 2009.

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.

An improved reverse transcription loop-mediated isothermal amplification assay for sensitive and specific detection of serotype O foot-and-mouth disease virus.

A sensitive and specific swarm primer-based reverse transcription loop-mediated isothermal amplification (sRT-LAMP) assay for the detection of serotype O foot-and-mouth disease virus (FMDV) was developed and evaluated. The assay specifically amplified the VP3 gene of serotype O FMDV, but did not amplify the VP3 gene of other serotype FMDVs or any other viruses. The limit of detection of the assay was 102 TCID50/mL or 103 RNA copies/µL, which is 100 times lower than that of the RT-LAMP assay without swarm primers. The new assay is 10 times more sensitive than reverse transcription-polymerase chain reaction (RT-PCR) and is comparable to the sensitivity of real time RT-PCR (qRT-PCR). Evaluation of the assay using different serotypes of FMDV strains showed 100% agreement with the RT-PCR results. The previously reported serotype O FMDV-specific RT-LAMP assay did not detect 20 out of 22 strains of serotype O FMDVs, probably due to multiple mismatches between the primer and template sequences, showing that it is not suitable for detecting the serotype O FMDVs circulating in Pool 1 region countries, including Korea. In contrast, the developed sRT-LAMP assay with improved primers can rapidly and accurately diagnose serotype O FMDVs circulating in Pool 1 region countries and will be a useful alternative to RT-PCR and qRT-PCR.

A tailored reverse transcription loop-mediated isothermal amplification for sensitive and specific detection of serotype A foot-and-mouth disease virus circulating in pool 1 region countries.

Rapid and accurate diagnosis of foot-and-mouth disease viruses (FMDV) is essential for the prompt control of FMD outbreaks. Reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (qRT-PCR) are used for routine FMDV diagnosis as World Organisation for Animal Health-recommended diagnostic assays. However, these PCR-based assays require sophisticated equipment, specialized labour, and complicated procedures for the detection of amplified products, making them unsuitable for under-equipped laboratories in developing countries. In this study, to overcome these shortcomings, a simple, rapid, and cost-effective reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of serotype A FMDV circulating in the pool 1 region. The amplification could be completed in 40 min at 62°C, and the results could be visually detected by the naked eye without any additional detection systems. The assay specifically amplified the VP1 gene of the Sea-97 genotype of serotype A FMDV, but it did not amplify other viral nucleic acids. The limit of detection of the assay was 102 TCID50 /ml, which is 10 times more sensitive than RT-PCR and is comparable to the sensitivity of qRT-PCR. Evaluation of the assay using different FMDV strain serotypes showed 100% agreement with the results of RT-PCR. Surprisingly, the previously reported RT-LAMP assay did not detect all eight tested strains of serotype A FMDVs, due to multiple mismatches between primer and template sequences, demonstrating that it is not suitable for detecting serotype A FMDVs circulating in pool 1-region countries. Conversely, the newly developed RT-LAMP assay using improved primers can rapidly and accurately diagnose the genotype of Sea-97 strains of serotype A FMDVs from the pool 1 region. The established RT-LAMP assay in this study is a simple, rapid, specific, sensitive, and cost-effective tool for the detection of serotype A FMDV in the resource-limited pool 1-region countries.

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.

Molecular and genomic features of Mycobacterium bovis strain 1595 isolated from Korean cattle.

The aim of this study was to investigate the molecular characteristics and to conduct a comparative genomic analysis of Mycobacterium (M.) bovis strain 1595 isolated from a native Korean cow. Molecular typing showed that M. bovis 1595 has spoligotype SB0140 with mycobacterial interspersed repetitive units-variable number of tandem repeats typing of 4-2-5-3-2-7-5-5-4-3-4-3-4-3, representing the most common type of M. bovis in Korea. The complete genome sequence of strain 1595 was determined by single-molecule real-time technology, which showed a genome of 4351712 bp in size with a 65.64% G + C content and 4358 protein-coding genes. Comparative genomic analysis with the genomes of Mycobacterium tuberculosis complex strains revealed that all genomes are similar in size and G + C content. Phylogenetic analysis revealed all strains were within a 0.1% average nucleotide identity value, and MUMmer analysis illustrated that all genomes showed positive collinearity with strain 1595. A sequence comparison based on BLASTP analysis showed that M. bovis AF2122/97 was the strain with the greatest number of completely matched proteins to M. bovis 1595. This genome sequence analysis will serve as a valuable reference for improving understanding of the virulence and epidemiologic traits among M. bovis isolates in Korea.

Complete Genome Sequence of a Foot-and-Mouth Disease Virus of Serotype O, Isolated from Gochang, Republic of Korea, in 2016.

The complete genome sequence of a foot-and-mouth disease (FMD) serotype O virus isolated from Gochang, Republic of Korea, is reported here.

Complete Genome Sequence of a Foot-and-Mouth Disease Virus of Serotype O Isolated from Gimje, Republic of Korea, in 2016.

The complete genome sequence of a foot-and-mouth disease (FMD) serotype O virus isolated from Gimje, Republic of Korea, is reported here.

Isolation of Mycobacterium bovis from Free-Ranging Wildlife in South Korea.

We demonstrate Mycobacterium bovis infection in wild boar ( Sus scrofa ) in South Korea. During 2012-15, we attempted to isolate M. bovis from 847 wild animals, mainly Korean water deer ( Hydropotes inermis argyropus), raccoon dogs ( Nyctereutes procyonoides ), and wild boar, from 11 regions in South Korea. We isolated M. bovis from three of 118 wild boar (2.5%) captured in Gyeonggi Province, where bovine tuberculosis (bTB) outbreaks have also occurred in livestock. Spoligotypes and mycobacterial interspersed repetitive units-variable number tandem repeats types of these M. bovis isolates (SB0140 and SB1040, 4-2-3-3-7-5-5-4-4-3-4-3 and 5-2-3-3-7-5-5-4-3-10-5-2; MIRU4, MIRU16, MIRU27, MIRU31, ETR-A, ETR-B, ETR-C, QUB11b, QUB26, QUB3336, VNTR2401, and VNTR3171) have also been identified from farmed livestock such as cattle ( Bos taurus coreanae), Formosan sika deer ( Cervus nippon taiouanus), and American elk ( Cervus canadensis ) in the country. In South Korea, bTB appears to be endemic in livestock, and there are numerous opportunities for contact between wild boar and livestock due to high population densities and broad activity ranges. Our results support the hypothesis that M. bovis is transmitted between domestic and wild animals.

Reconstitution of ST2 (IL-1R4) specific for IL-33 activity; no suppression by IL-1Ra though a common chain IL-1R3 (IL-1RAcP) shared with IL-1.

Interleukin-33 (IL-33) receptors are composed of ST2 (also known as IL-1R4), a ligand binding chain, and IL-1 receptor accessory protein (IL-1RAcP, also known as IL-1R3), a signal transducing chain. IL-1R3 is a common receptor for IL-1α, and IL-1ß, IL-33, and three IL-36 isoforms. A549 human lung epithelial cells are highly sensitive to IL-1α and IL-1ß but not respond to IL-33. The lack of responsiveness to IL-33 is due to ST2 expression. ST2 was stably transfected into A549 cells to reconstitute its activity. RT-PCR and FACS analysis confirmed ST2 expression on the cell surface of A549/ST2 cells. Upon IL-33 stimulation, A549/ST2 cells induced IL-8 and IL-6 production in a dose dependent manner while A549/mock cells remained unresponsive. There was no difference in IL-1α and IL-1ß activity in A549/ST2 cells compared to A549/mock cells despite the fact that IL-33 shares IL-1R3 with IL-1α/ß. IL-33 activated inflammatory signaling molecules in a time- and dose-dependent manner. Anti-ST2 antibody and soluble recombinant ST2-Fc abolished IL-33-induced IL-6 and IL-8 production in A549/ST2 cells but the IL-1 receptor antagonist failed to block IL-33-induced cytokines. This result demonstrates for the first time the reconstitution of ST2 in A549 human lung epithelial cell line and verified its function in IL-33-mediated cytokine production and signal transduction.

Complete Genome Sequence of Mycobacterium bovis Clinical Strain 1595, Isolated from the Laryngopharyngeal Lymph Node of South Korean Cattle.

Mycobacterium bovis strain 1595 was isolated from the lymph node of South Korean native cattle. The complete genome sequence of strain 1595 was determined in 2 contigs and was found to be 4,351,712 bp in size, with a 65.64% G+C content and 4,358 predicted protein-coding genes.

Bovine tuberculosis in an Asian small-clawed otter (Aonyx cinerea) in the Republic of Korea.

Bovine tuberculosis caused by Mycobacterium bovis has a wide range of hosts including cattle and humans, but its incidence in otters is very rare. Our report describes a case of bovine tuberculosis in an Asian small-clawed otter (Aonyx cinerea). A deceased female otter ~2-3 years of age that was raised in an aquarium was submitted to the Animal and Plant Quarantine Agency (Anyang, Republic of Korea) for autopsy in June 2013. Following gross pathological examination, many white nodules were observed in the lungs and mesentery. The nodules showed central necrosis infiltrated with lymphocytes and macrophages and surrounded by fibrous tissue. Acid-fast bacteria were detected in the necrotic foci, but no fungi were observed. Molecular analysis led to the detection of M. bovis, which is identified in otters in some European countries such as Spain and France.