Genomic Surveillance of SARS-CoV-2: Distribution of Clades in the Republic of Korea in 2020

Since a novel beta-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in December 2019, there has been a rapid global spread of the virus. Genomic surveillance was conducted on samples isolated from infected individuals to monitor the spread of genetic variants of SARS-CoV-2 in Korea. The Korea Disease Control and Prevention Agency performed whole genome sequencing of SARS-CoV-2 in Korea for 1 year (January 2020 to January 2021). A total of 2,488 SARSCoV-2 cases were sequenced (including 648 cases from abroad). Initially, the prevalent clades of SARSCoV-2 were the S and V clades, however, by March 2020, GH clade was the most dominant. Only international travelers were identified as having G or GR clades, and since the first variant 501Y.V1 was identified (from a traveler from the United Kingdom on December 22 nd , 2020), a total of 27 variants of 501Y.V1, 501Y.V2, and 484K.V2 have been classified (as of January 25 th , 2021). The results in this study indicated that quarantining of travelers entering Korea successfully prevented dissemination of the SARS-CoV-2 variants in Korea.

2019 Tabletop Exercise for Laboratory Diagnosis and Analyses of Unknown Disease Outbreaks by the Korea Centers for Disease Control and Prevention

Objectives@#The Korea Centers for Disease Control and Prevention has published “A Guideline for Unknown Disease Outbreaks (UDO).” The aim of this report was to introduce tabletop exercises (TTX) to prepare for UDO in the future. @*Methods@#The UDO Laboratory Analyses Task Force in Korea Centers for Disease Control and Prevention in April 2018, assigned unknown diseases into 5 syndromes, designed an algorithm for diagnosis, and made a panel list for diagnosis by exclusion. Using the guidelines and laboratory analyses for UDO, TTX were introduced. @*Results@#Since September 9th , 2018, the UDO Laboratory Analyses Task Force has been preparing TTX based on a scenario of an outbreak caused by a novel coronavirus. In December 2019, through TTX, individual missions, epidemiological investigations, sample treatments, diagnosis by exclusions, and next generation sequencing analysis were discussed, and a novel coronavirus was identified as the causal pathogen. @*Conclusion@#Guideline and laboratory analyses for UDO successfully applied in TTX. Conclusions drawn from TTX could be applied effectively in the analyses for the initial response to COVID-19, an ongoing epidemic of 2019 - 2020. Therefore, TTX should continuously be conducted for the response and preparation against UDO.

Current status of vaccine development for tularemia preparedness

Tularemia is a high-risk infectious disease caused by Gram-negative bacterium Francisella tularensis. Due to its high fatality at very low colony-forming units (less than 10), F. tularensis is considered as a powerful potential bioterrorism agent. Vaccine could be the most efficient way to prevent the citizen from infection of F. tularensis when the bioterrorism happens, but officially approved vaccine with both efficacy and safety is not developed yet. Research for the development of tularemia vaccine has been focusing on the live attenuated vaccine strain (LVS) for long history, still there are no LVS confirmed for the safety which should be an essential factor for general vaccination program. Furthermore the LVS did not show protection efficacy against high-risk subspecies tularensis (type A) as high as the level against subspecies holarctica (type B) in human. Though the subunit or recombinant vaccine candidates have been considered for better safety, any results did not show better prevention efficacy than the LVS candidate against F. tularensis infection. Currently there are some more trials to develop vaccine using mutant strains or nonpathogenic F. novicida strain, but it did not reveal effective candidates overwhelming the LVS either. Difference in the protection efficacy of LVS against type A strain in human and the low level protection of many subunit or recombinant vaccine candidates lead the scientists to consider the live vaccine development using type A strain could be ultimate answer for the tularemia vaccine development.

GFP-tagged E. coli shows bacterial distribution in mouse organs: pathogen tracking using fluorescence signal

PURPOSE: In vaccine efficacy evaluation, visualization of pathogens in whole organism at each time point would be able to reduce the consuming animals and provide the in vivo information within consistent background with identical organism. MATERIALS AND METHODS: Using IVIS spectrum whole live-animal imaging system, fluorescent intensity was optimized and visualized proportionately by concentrating Escherichia coli MC1061 strain which expresses GFP (E. coli-GFP) in BALB/C mice after injection. RESULTS: Local distribution of disseminated E. coli-GFP was traced in each organ by fluorescence. Detached organ showed more obvious fluorescent signal, and intestine showed strongest fluorescent signal. CONCLUSION: This in vivo imaging method using GFP-tagged pathogen strain suggest quantified infected pathogens by fluorescence intensity in whole animals can provide the information about the localization and distribution after infection.

The Laboratory Diagnosis of Melioidosis in a Korean Patient

Burkholderia pseudomallei is a gram-negative opportunistic intracellular pathogen that causes an acute and fatal septicemic melioidosis in humans. The organism is mainly found in Southeastern Asia and Northern Australia. Recently, we encountered a case of melioidosis in a Korean patient and performed the laboratory diagnosis of melioidosis. As a result, a gram negative bacterium was isolated from a melioidosis patient, and it was identified as B. pseudomallei on DNA sequencing of 16S ribosomal RNA with 99.9% homology and biochemical examination of VITEK gram-negative identification card. Also, DNA from cultured bacteria was tested in multiplex PCR, a 245 bp fragment amplified from the metalloprotease gene and a fragment of variable size ranging from 400~700 bp resulting from amplification of the 10 bp repetitive element for B. pseudomallei were confirmed after electrophoresis. The bacterium was sensitive to ceftazidime, imipenem and meropenem but resistant to ticarcillin. So far, there are no domestic cases of melioidosis in Korea, however, due to the increase in international travelers, the incidence of melioidosis is likely to increase. We report a recent case of melioidosis in a Korean patient.

Detection of Botulinum Neurotoxin Type A by In Vitro Bioassay Based on Endopeptidase Activity

Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that cleaves SNAP-25 (synaptosome-associated protein of 25 kDa), a specific cellular protein essential for neurotransmitter release. As well as mouse bioassay to detect BoNT/A, various assay methods based on its endopeptidase activity have been developed. In this study, we tried to develop a BoNT/A assay system using recombinant SNAP-25 with glutathione S-transferase (GST) tags at both termini as substrate. The recombinant GST-SNAP-25-GST with 70 kDa was expressed and purified in E. coli and synthesized N-terminal 50 kDa and C-terminal 25 kDa fragment after cleavage at the Gln(197)-Arg(198) bond by BoNT/A. To detect both fragments, we obtained rabbit antisera against peptides corresponding to the cleaved ends of each fragment. In the western blotting, the N-terminal fragment was detected by the antibody specifically recognizing the newly exposed C-terminus (corresponding to amino acid residue 191-197). This assay system was able to detect until 3.125 ng of BoNT/A, which corresponded to about 90 fold LD50 in mice. These results suggest that the in vitro endopeptidase assay developed in this study would replace others to detect BoNT/A.

Detection of Botulinum Neurotoxin Type A by In Vitro Bioassay Based on Endopeptidase Activity

Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that cleaves SNAP-25 (synaptosome-associated protein of 25 kDa), a specific cellular protein essential for neurotransmitter release. As well as mouse bioassay to detect BoNT/A, various assay methods based on its endopeptidase activity have been developed. In this study, we tried to develop a BoNT/A assay system using recombinant SNAP-25 with glutathione S-transferase (GST) tags at both termini as substrate. The recombinant GST-SNAP-25-GST with 70 kDa was expressed and purified in E. coli and synthesized N-terminal 50 kDa and C-terminal 25 kDa fragment after cleavage at the Gln(197)-Arg(198) bond by BoNT/A. To detect both fragments, we obtained rabbit antisera against peptides corresponding to the cleaved ends of each fragment. In the western blotting, the N-terminal fragment was detected by the antibody specifically recognizing the newly exposed C-terminus (corresponding to amino acid residue 191-197). This assay system was able to detect until 3.125 ng of BoNT/A, which corresponded to about 90 fold LD50 in mice. These results suggest that the in vitro endopeptidase assay developed in this study would replace others to detect BoNT/A.

Development of Capture ELISA Using a Biotinylated Monoclonal Antibody for Detection of Botulinum Neurotoxin Type A

A capture enzyme-linked immunosorbent assay (capture ELISA) was developed to detect Clostridium botulinum neurotoxin type A (BoNT/A) in assay buffer and human serum. The assay is based upon affinity-purified rabbit polyclonal and biotinylated monoclonal antibodies directed against the BoNT/A complex purified from C. botulinum ATCC19397. For the capture ELISA, the optimized amount (2 microgram/ml) of rabbit polyclonal antibody was immobilized on ELISA plates to detect BoNT/A (ranging from 0 to 500 ng/ml), which was recognized by 2 microgram/ml of the monoclonal antibody. From three independent repeated experiments, standard curves were linear over the range of 0~31.25 ng/ml BoNT/A and the coefficients (r(2)) ranged from 0.9951~0.9999 for all assays. The inter-variations were typically 0.50~6.93% and the specificity was confirmed by showing no cross-reactivity against BoNT/B and /E. The detection limit of capture ELISA was 0.488 ng/ml, which was close to mouse LD(50). In addition, application with BoNT/A-spiking human sera showed a possibility to detect BoNT/A with capture ELISA from the contaminated human sera. Taken together, the newly developed capture ELISA could serve as a rapid and sensitive screening tool for detecting BoNT/A simultaneously from massive specimens.