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.

Comparison of the protective efficacy between single and combination of recombinant adenoviruses expressing complete and truncated glycoprotein, and nucleoprotein of the pathogenic street rabies virus in mice.

BACKGROUND: Rabies is an important viral zoonosis that causes acute encephalitis and death in mammals. To date, several recombinant vaccines have been developed based on G protein, which is considered to be the main antigen, and these vaccines are used for rabies control in many countries. Most recombinant viruses expressing RABV G protein retain the G gene from attenuated RABV. Not enough is currently known about the protective effect against RABV of a combination of recombinant adenoviruses expressing the G and N proteins of pathogenic street RABV. METHODS: We constructed a recombinant adenovirus (Ad-0910Gsped) expressing the signal peptide and ectodomain (sped) of G protein of the Korean street strain, and evaluated the immunological protection conferred by a single and combination of three kinds of recombinant adenoviruses (Ad-0910Gsped and Ad-0910G with or without Ad-0910 N) in mice. RESULTS: A combination of Ad-0910G and Ad-0910 N conferred improved immunity against intracranial challenge compared to single administration of Ad-0910G. The Ad-0910G virus, expressing the complete G protein, was more immunogenic than Ad-0910Gsped, which expressed a truncated G protein with the transmembrane and cytoplasmic domains removed. Additionally, oral vaccination using a combination of viruses led to complete protection. CONCLUSIONS: Our results suggest that this combination of viruses is a viable new intramuscular and oral vaccine candidate.

A Robust Quadruple Protein-Based Indirect ELISA for Detection of Antibodies to African Swine Fever Virus in Pigs.

African swine fever (ASF) emerged in domestic pigs and wild boars in China in 2018 and rapidly spread to neighboring Asian countries. Currently, no effective vaccine or diagnostic tests are available to prevent its spread. We developed a robust quadruple recombinant-protein-based indirect enzyme-linked immunosorbent assay (QrP-iELISA) using four antigenic proteins (CD2v, CAP80, p54, and p22) to detect ASF virus (ASFV) antibodies and compared it with a commercial kit (IDvet) using ASFV-positive and -negative serum samples. The maximum positive/negative value was 24.033 at a single antigen concentration of 0.25 µg/mL and quadruple ASFV antigen combination of 1 µg/mL at a 1:100 serum dilution. Among 70 ASFV-positive samples, 65, 67, 65, 70, 70, and 14 were positive above the cut-offs of 0.121, 0.121, 0.183, 0.065, 0.201, and 0.122, for CD2v, CAP80, p54, p22-iELISA, QrP-iELISA, and IDvet, respectively, with sensitivities of 92.9%, 95.7%, 92.9%, 100%, 100%, and 20%, respectively, all with 100% specificity. The antibody responses in QrP-iELISA and IDvet were similar in pigs infected with ASFV I. QrP-iELISA was more sensitive than IDvet for early antibody detection in pigs infected with ASFV II. These data provide a foundation for developing advanced ASF antibody detection kits critical for ASF surveillance and control.

Whole Genome Sequencing of SARS-CoV-2 in Cats and Dogs in South Korea in 2021.

SARS-CoV-2 infections have caused unprecedented damage worldwide by affecting humans and various animals. The first reported animal infection was observed in a pet dog in Hong Kong in March 2020. 36 countries reported 692 SARS-CoV-2 infections in 25 different animal species by 31 August 2022. Most outbreaks were caused by contact with SARS-CoV-2 infected humans. In South Korea, the first SARS-CoV-2 infection in an animal was reported in a cat in February 2021. As of 31 December 2021, 74 dogs and 42 cats have been confirmed to have SARS-CoV-2 in South Korea. Here, we identified various SARS-CoV-2 genomic lineages in SARS-CoV-2 confirmed cats and dogs. Among the 40 animal samples sequenced for lineage identification, a total of eight Pango lineages (B.1.1.7 (Alpha variant), B.1.429 (Epsilon variant), B.1.470, B.1.497, B.1.619.1, B.1.620, AY.69 (Delta variant), and AY.122.5 (Delta variant)) were identified. The dominant lineages were AY.69 (Delta variant; 37.5%), B.1.497 (35.0%), and B.1.619.1 (12.5%). This study provides the first reported cases of six lineages (B.1.470, B.1.497, B.1.620, B.1.619.1, AY.69 (Delta variant)), and AY.122.5 (Delta variant) in cats and dogs. Our results emphasize the importance of monitoring SARS-CoV-2 in pets because they are dynamic hosts of variant Pango lineages of SARS-CoV-2.

Development of an indirect ELISA against African swine fever virus using two recombinant antigens, partial p22 and p30.

African swine fever (ASF) is a highly fatal viral disease affecting pigs. It is caused by the ASF virus (ASFV), and causes serious economic losses to the swine industry worldwide, including in Korea. Commercially available enzyme-linked immunosorbent assay (ELISA) kits for detecting anti-ASFV antibodies are used for the diagnosis and surveillance of ASF. In this study, an ELISA was developed to detect anti-ASFV antibodies using two recombinant proteins, p22 and p30, from genotype II ASFV. Recombinant transmembrane domain-deleted p22 (p22∆TM) and p30 were expressed in E.coli vector system pET32a and mixed for use as antigens in indirect ELISA. The p22∆TM/p30-based indirect ELISA was validated using 31 sera from genotype I ASFV-infected pigs and 1133 sera from uninfected pigs. Area under the curve of this test was 0.999 [95 % concentration interval 0.992-1.000], and sensitivity and specificity were 93.5 % and 99.8 %, respectively. The between run coefficient of variation for internal quality control serum was 6.61 %. In the seroconversion analysis, the p22∆TM/p30-based indirect ELISA showed equal or better ability to detect antibodies in pigs experimentally challenged with ASFV p72 genotypes I and II (p < 0.05). In conclusion, the p22∆TM/p30-based indirect ELISA is a reliable diagnostic method for detecting anti-ASFV antibodies.

Surveillance for West Nile virus in dead wild birds, South Korea, 2005-2008.

To investigate the possibility of West Nile virus (WNV) introduction into South Korea, the National Veterinary Research and Quarantine Service has conducted nationwide surveillance of WNV activity in dead wild birds since 2005. Surveillance conducted during 2005-2008 found no evidence of WNV activity.

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.

Isolation and Genetic Characterization of African Swine Fever Virus from Domestic Pig Farms in South Korea, 2019.

On 17 September 2019, the first outbreak of African swine fever in a pig farm was confirmed in South Korea. By 9 October, 14 outbreaks of ASF in domestic pigs had been diagnosed in 4 cities/counties. We isolated viruses from all infected farms and performed genetic characterization. The phylogenetic analysis showed that all of fourteen ASFV isolates in South Korea belong to genotype II and serogroup 8. Additionally, all isolates had an intergenic region (IGR) II variant with additional tandem repeat sequences (TRSs) between the I73R and I329L genes and showed characteristics of central variable region (CVR) 1 of the B602L gene and IGR 1 of MGF 505 9R/10R genes. These are identical to the genetic characteristics of some European isolates and Chinese isolates.

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.

Outbreak of African swine fever in South Korea, 2019.

African swine fever, a fatal haemorrhagic disease of swine, was confirmed in domestic pigs for the first time in South Korea in September 2019. The causative virus belonged to the p72 genotype II and had an additional tandem repeat sequence in the intergenic region (IGR) between the I73R and I329L.

West Nile virus capsid protein induces p53-mediated apoptosis via the sequestration of HDM2 to the nucleolus.

The capsid protein of the West Nile virus (WNV) functions as an apoptotic agonist via the induction of mitochondrial dysfunction and the activation of caspases-9 and -3. Here, we have determined that the WNV capsid (WNVCp) is capable of binding to and sequestering HDM2 into the nucleolus. WNVCp was shown to interfere with the formation of the HDM2 and p53 complex, thereby causing the stabilization of p53 and the subsequent induction of its target apoptotic protein, Bax. Whereas WNVCp was capable of inducing the p53-dependent apoptotic process in wild-type mouse embryonic fibroblasts (MEF) or SH-SY5Y cells, it exerted no significant effects on p53-null MEF or on p53-knockdown SH-SY5Y cells. This suggests that WNVCp-mediated apoptosis requires p53. Furthermore, when WNV was transfected into cells, endogenous Hdm2 and WNVCp were able to interact physically. WNVCp expressed in wild-type MEF proved able to induce the translocation of the endogenous Hdm2 into the nucleolus. Consistently, WNV was highly pathogenic in the presence of p53, and was less so in the absence of p53. The results of these studies suggest that the apoptotic mechanism mediated by WNV might occur in accordance in a fashion similar to that of the tumour-suppressing mechanism mediated by ARF.

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.

Isolation of a recent Korean epizootic strain of Newcastle disease virus from Eurasian Scops Owls affected with severe diarrhea.

Velogenic Newcastle disease virus (NDV) was recovered from two dead Eurasian Scops Owls (Otus scops) from a wildlife rescue center in Korea during 2005. Phylogenetic analysis based on the sequence of the partial fusion (F) protein revealed that the isolates had the highest level of homology to recent Korean NDV strains from poultry.

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.

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.

The DE loop of the domain III of the envelope protein appears to be associated with West Nile virus neutralization.

The envelope (E) protein of WNV plays an important role in the virus neutralization. Using a mAb 5E8, a neutralizing epitope on the domain III of the E of the New York strain of WN virus was characterized. Results from neutralization-escape mutants and site-directed mutagenesis revealed that the 5E8 epitope is a highly conformation dependent epitope consisting of at least residues E330, E332 and E367 on the domain III. Besides known critical neutralizing epitopes E330 and E332, our results indicate that residue E367, a component of DE loop on the domain III, appeared to be associated with neutralization but little with neuroinvasion of the virus, as reported previously (Beasley et al., 2002).

The present and future of veterinary vaccines for Japanese encephalitis in Korea.

Japanese encephalitis (JE) is a mosquito-borne zoonotic disease that affects approximately 50,000 people annually in Asia, causing 10,000 deaths. Considering the role of pigs as the virus-amplifying host and the economic loss in the swine industry, JE is an important disease for both public and animal health. A nationwide JE virus (JEV) vaccination program has been conducted annually for more than 30 years to prevent severe reproductive disorders in the Korean sow population. Remarkable progress in molecular biology has made it possible to analyze the genome of the vaccine strain at the nucleotide and amino acid levels. However, the scientific record of the current JEV veterinary vaccine has not been reported. Therefore, this article outlines the current JEV vaccine strain used in animals and discusses future directions for developing new veterinary JEV vaccines.

Rabbit Hemorrhagic Disease Virus Variant Recombinant VP60 Protein Induces Protective Immunogenicity.

Rabbit hemorrhagic disease virus (RHDV) is highly contagious and often causes fatal disease that affects both wild and domestic rabbits of the species Oryctolagus cuniculus. A highly pathogenic RHDV variant (RHDVa) has been circulation in the Korean rabbit population since 2007 and has a devastating effect on the rabbit industry in Korea. A highly pathogenic RHDVa was isolated from naturally infected rabbits, and the gene encoding the VP60 protein was cloned into a baculovirus transfer vector and expressed in insect cells. The hemagglutination titer of the Sf-9 cell lysate infected with recombinant VP60 baculovirus was 131,072 units/50 µl and of the supernatant 4,096 units/50 µl. Guinea pigs immunized twice intramuscularly with a trial inactivated RHDVa vaccine containing recombinant VP60 contained 2,152 hemagglutination inhibition (HI) geometric mean titers. The 8-week-old white rabbits inoculated with one vaccine dose were challenged with a lethal RHDVa 21 days later and showed 100% survival rates. The recombinant VP60 protein expressed in a baculovirus system induced high HI titers in guinea pigs and rendered complete protection, which led to the development of a novel inactivated RHDVa vaccine.

Monoclonal antibody-based competitive ELISA for simultaneous detection of rinderpest virus and peste des petits ruminants virus antibodies.

An experimental competitive enzyme-linked immunosorbent assay (morbillivirus cELISA) using a recombinant N antigen (rRPV N) expressed in a baculovirus and a ruminant morbillivirus (RPV and PPRV)-specific monoclonal antibody (P-13A9) was developed for simultaneous detection of rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) antibodies and its diagnostic performance was evaluated. A set of known reference antisera against RPV and PPRV belonging to different lineages, experimental sera from cattle vaccinated for a RPV of Asian lineage, and field sera from cattle and sheep/goat populations known to be positive (West Africa) and negative (Korea) for RPV and PPRV were used for the evaluation. Morbillivirus cELISA results on the panel of experimental RPV and PPRV antisera showed high correlation (r=0.97) between the whole virus and the rRPV N antigens, suggesting that the rRPV N contains a ruminant morbillivirus-specific antigenic determinant recognized by the P-13A9 and it may be suitable as an ELISA antigen in place of the whole virus. Morbillivirus cELISA detected anti-RPV and anti-PPRV antibodies in all reference RPV and PPRV antisera containing VN titers >/=1:8, suggesting that the assay can simultaneously detect antibodies against RPV and PPRV. Anti-RPV antibody was detected by morbillivirus cELISA in vaccinated cattle as early as the VNT and continued to be detectable by both the cELISA and the VNT until termination of the study. When applied to field samples from Africa, morbillivirus cELISA showed good agreement with a RP cELISA kit (kappa value of 0.86) in bovine sera and with a peste des petits ruminant cELISA kit (kappa value of 0.81) in caprine/ovine sera. Usefulness of morbillivirus cELISA using the rRPV N protein was discussed.