Genomic Analysis of Monkeypox Virus During the 2023 Epidemic in Korea.

We aimed to characterize the genomes of monkeypox virus isolates from the Far East, providing insights into viral transmission and evolution. Genomic analysis was conducted on 8 isolates obtained from patients with monkeypox virus disease in the Republic of Korea between May 2022 and early 2023. These isolates were classified into Clade IIb. Distinct lineages, including B.1.1, A.2.1, and B.1.3, were observed in 2022 and 2023 isolates, with only the B.1.3 lineage detected in six isolates of 2023. These genetic features were specific to Far East isolates (the Republic of Korea, Japan, and Taiwan), distinguishing them from the diverse lineages found in the Americas, Europe, Africa, and Oceania. In early 2023, the prevalence of the B.1.3 lineage of monkeypox virus identified in six patients with no overseas travel history is considered as an indicator of the potential initiation of local transmission in the Republic of Korea.

Prolonged viral shedding in an immunocompromised Korean patient infected with hMPXV, sub-lineage B.1.3, with acquired drug resistant mutations during tecovirimat treatment.

Following the worldwide surge in mpox (monkeypox) in 2022, cases have persisted in Asia, including South Korea, and sexual contact is presumed as the predominant mode of transmission, with a discernible surge in prevalence among immunocompromised patients. Drugs such as tecovirimat can result in drug-resistant mutations, presenting obstacles to treatment. This study aimed to ascertain the presence of tecovirimat-related resistant mutations through genomic analysis of the monkeypox virus isolated from a reported case involving prolonged viral shedding in South Korea. Here, tecovirimat-resistant mutations, previously identified in the B.1 clade, were observed in the B.1.3 clade, predominant in South Korea. These mutations exhibited diverse patterns across different samples from the same patient and reflected the varied distribution of viral subpopulations in different anatomical regions. The A290V and A288P mutant strains we isolated hold promise for elucidating these mechanisms, enabling a comprehensive analysis of viral pathogenesis, replication strategies, and host interactions. Our findings imply that acquired drug-resistant mutations, may present a challenge to individual patient treatment. Moreover, they have the potential to give rise to transmitted drug-resistant mutations, thereby imposing a burden on the public health system. Consequently, the meticulous genomic surveillance among immunocompromised patients, conducted in this research, assumes paramount importance.

Complete Genome Sequence of Monkeypox Virus Strain MPXV-ROK-P1-2022 Isolated from the First Monkeypox Patient in the Republic of Korea.

We report the complete genome sequence of the monkeypox virus strain MPXV-ROK-P1-2022, isolated from the first patient diagnosed with monkeypox in the Republic of Korea in June 2022. The virus was fully sequenced on the Illumina MiSeq instrument, and the phylogenetic tree showed that the strain belongs to lineage B.1.1.

Isolation and identification of monkeypox virus MPXV-ROK-P1-2022 from the first case in the Republic of Korea.

OBJECTIVES: Monkeypox outbreaks in nonendemic countries have been reported since early May 2022. The first case of monkeypox in the Republic of Korea was confirmed in a patient who traveled to Europe in June 2022, and an attempt was made to isolate and identify the monkeypox virus (MPXV) from the patient's specimens. METHODS: Clinical specimens from the patient were inoculated in Vero E6 cells. The isolated virus was identified as MPXV by the observation of cytopathic effects on Vero E6 cells, transmission electron microscopy, conventional polymerase chain reaction (PCR), and sequencing of PCR products. RESULTS: Cytopathic effects were observed in Vero E6 cells that were inoculated with skin lesion swab eluates. After multiple passages from the primary culture, orthopoxvirus morphology was observed using transmission electron microscopy. In addition, both MPXV-specific (F3L and ATI) and orthopoxvirus-specific genes (A39R, B2R, and HA) were confirmed by conventional PCR and Sanger sequencing. CONCLUSION: These results indicate the successful isolation and identification of MPXV from the first patient in the Republic of Korea. The isolated virus was named MPXV-ROK-P1-2022.

The First Case of Monkeypox in the Republic of Korea.

A rapid outbreak of monkeypox is ongoing in non-endemic countries since May 2022. We report the first case of monkeypox in the Republic of Korea. This occurred in a 34-year-old male patient who traveled to Europe in June 2022. On the day of his return to the Republic of Korea (June 21, 2022), the patient presented with a genital lesion. The results of the monkeypox real-time polymerase chain reaction tests were positive in the penile ulcer, oropharyngeal and nasopharyngeal specimens. The patient subsequently developed fever and skin rash after hospital admission. Careful history taking along physical examination should be conducted in the patients who have epidemiologic risk factors for monkeypox. Moreover, appropriate specimens should be obtained from lesions and tested for the monkeypox virus.

Construction of a bivalent vaccine against anthrax and smallpox using the attenuated vaccinia virus KVAC103.

BACKGROUND: Anthrax and smallpox are high-risk infectious diseases, and considered as potential agents for bioterrorism. To develop an effective countermeasure for these diseases, we constructed a bivalent vaccine against both anthrax and smallpox by integrating a gene encoding protective antigen (PA) of Bacillus anthracis to the genome of the attenuated vaccinia virus strain, KVAC103. RESULTS: Immunization with this bivalent vaccine induced antibodies against both PA and vaccinia virus in a mouse model. We also observed that the efficacy of this vaccine can be enhanced by combined immunization with immunoadjuvant-expressing KVAC103. Mouse groups co-immunized with PA-expressing KVAC103 and either interleukin-15 (IL-15) or cholera toxin subunit A (CTA1)-expressing KVAC103 showed increased anti-PA IgG titer and survival rate against B. anthracis spore challenge compared to the group immunized with PA-expressing KVAC103 alone. CONCLUSIONS: We demonstrated that the attenuated smallpox vaccine KVAC103 is an available platform for a multivalent vaccine and co-immunization of immunoadjuvants can improve vaccine performance.

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.

Draft Genome Sequence of the First South Korean Clinical Isolate of Burkholderia pseudomallei, H0901.

We report here the draft genome sequence of Burkholderia pseudomallei H0901. This strain was isolated in 2003 from the first melioidosis patient in South Korea.

Construction of Luminescence- and Fluorescence-Tagged Burkholderia pseudomallei for Pathogen Tracking in a Mouse Model.

Molecular imaging is a powerful method for tracking various infectious disease-causing pathogens in host organisms. Currently, a dual molecular imaging method that can provide temporal and spatial information on infected hosts at the organism, organ, tissue, and cellular levels simultaneously has not been reported for Burkholderia pseudomallei, a high-risk pathogen that causes melioidosis. In this study, we have established an experimental method that provides spatiotemporal information on infected hosts using luminescent and fluorescent dual-labeled B. pseudomallei. Using this method, we visualized B. pseudomallei infection at the organism, organ, and tissue levels in a BALB/c mouse model by detecting its luminescence and fluorescence. The infection of B. pseudomallei at the cellular level was also visualized by its emitted fluorescence in infected macrophage cells. This method could be an extremely useful and applicable tool to study the pathogenesis of B. pseudomallei-related infectious diseases.

Botulinum neurotoxin type A induces TLR2-mediated inflammatory responses in macrophages.

Botulinum neurotoxin type A (BoNT/A) is the most potent protein toxin and causes fatal flaccid muscle paralysis by blocking neurotransmission. Application of BoNT/A has been extended to the fields of therapeutics and biodefense. Nevertheless, the global response of host immune cells to authentic BoNT/A has not been reported. Employing microarray analysis, we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. We identified 70 genes that were modulated following 1 nM BoNT/A treatment. The altered genes were mainly involved in signal transduction, immunity and defense, protein metabolism and modification, neuronal activities, intracellular protein trafficking, and muscle contraction. Microarray data were validated with real-time RT-PCR for seven selected genes including tlr2, tnf, inos, ccl4, slpi, stx11, and irg1. Proinflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells. Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2) and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK). BoNT/A also suppressed lipopolysaccharide-induced NO and TNFα production from RAW264.7 macrophages at the transcription level by blocking activation of JNK, ERK, and p38 MAPK. As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK.

Clinical features of abdominopelvic actinomycosis: report of twenty cases and literature review.

PURPOSE: Intrabdominal actinomycosis is difficult to diagnose preoperatively. This chronic infection has a propensity to mimic many other diseases and may present with a wide variety of symptoms. The aim of this study was to evaluate the characteristic clinical features with review of the literature. MATERIALS AND METHODS: We retrospectively analyzed 22 patients with intrabdominal actinomycosis between January 2000 and January 2006. RESULTS: There were two men and 20 women with a mean age of 42.8 years (range, 24-69). Twelve patients presented with masses or abdominal pain, whereas 3 patients presented with acute appendicitis. The rate of performing an emergency surgery was 50% due to symptoms of peritonitis. The mean size of tumor was 5.5 cm (range, 2.5-11.0). Sixty percent (n = 12) of female patients had intrauterine device (IUD). The average time to definite diagnosis was 10.6 days. CONCLUSION: Intrabdominal abdominal actinomycosis must first be suspected in any women with a history of current or recent IUD use who presents abdominal pain. If recognized preoperatively, a limited surgical procedure, may spare the patient from an extensive operation.

Inhibitory effects of gallic acid and quercetin on UDP-glucose dehydrogenase activity.

We have examined polyphenols as potential inhibitors of UDP-glucose dehydrogenase (UGDH) activity. Gallic acid and quercetin decreased specific activities of UGDH and inhibited the proliferation of MCF-7 human breast cancer cells. Western blot analysis showed that gallic acid and quercetin did not affect UGDH protein expression, suggesting that UGDH activity is inhibited by polyphenols at the post-translational level. Kinetics studies using human UGDH revealed that gallic acid was a non-competitive inhibitor with respect to UDP-glucose and NAD+. In contrast, quercetin showed a competitive inhibition and a mixed-type inhibition with respect to UDP-glucose and NAD+, respectively. These results indicate that gallic acid and quercetin are effective inhibitors of UGDH that exert strong antiproliferative activity in breast cancer cells.

Small-interfering-RNA-mediated silencing of human glutamate dehydrogenase induces apoptosis in neuroblastoma cells.

In the nervous system, GDH (glutamate dehydrogenase) is enriched in astrocytes and is important for recycling glutamate, a major excitatory neurotransmitter. The function of hGDH (human GDH) may be important in neurodegenerative diseases such as Parkinson's disease. To test the effect of decreased hGDH expression, several vector-based plasmidlinked hGDH siRNAs (small interfering RNAs) were expressed intracellularly in BE(2)C human neuroblastoma cells. Immunoblotting and reverse-transcription-PCR confirmed that expression of hGDH protein and mRNA was knocked down by co-transfection with phGDH-siRNA vectors in BE(2)C human neuroblastoma cells. TUNEL (terminal uridine deoxynucleotidyl transferase dUTP nick-end labelling) and DNA fragmentation assays 48 h after transfection of phGDH-siRNAs revealed that inhibition of hGDH expression induced cellular apoptosis and activated phospho-ERK1/2 (phospho-extracellular-signal-regulated kinase 1/2). These findings show that inhibition of hGDH expression by siRNA is related to apoptosis in neuronal cells.

Identification of amino acid residues responsible for different GTP preferences of human glutamate dehydrogenase isozymes.

Human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) differ markedly in their inhibition by GTP. These regulatory preferences must arise from amino acid residues that are not common between hGDH isozymes. We have constructed chimeric enzymes by reciprocally switching the corresponding amino acid segments 390-465 in hGDH isozymes that are located within or near the C-terminal 48-residue antenna helix, which is thought to be part of the regulatory domain of mammalian GDHs. These resulted in triple mutations in amino acid sequences at 415, 443, and 456 sites that are not common between hGDH1 and hGDH2. The chimeric enzymes did not change their enzyme efficiency (k(cat)/K(m)) and expression level. Functional analyses, however, revealed that the chimeric mutants almost completely acquired the different GTP regulatory preference between hGDH isozymes. These results suggest that the 415, 443, and 456 residues acting in concert are responsible for the GTP inhibitory properties of hGDH isozymes.

Inhibitory properties of nerve-specific human glutamate dehydrogenase isozyme by chloroquine.

Human glutamate dehydrogenase exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. In the nervous system, GDH is enriched in astrocytes and is important for recycling glutamate, a major excitatory neurotransmitter during neurotransmission. Chloroquine has been known to be a potent inhibitor of house-keeping GDH1 in permeabilized liver and kidney-cortex of rabbit. However, the effects of chloroquine on nerve-specific GDH2 have not been reported yet. In the present study, we have investigated the effects of chloroquine on hGDH2 at various conditions and showed that chloroquine could inhibit the activity of hGDH2 at dose-dependent manner. Studies of the chloroquine inhibition on enzyme activity revealed that hGDH2 was relatively less sensitive to chloroquine inhibition than house-keeping hGDH1. Incubation of hGDH2 was uncompetitive with respect of NADH and non-competitive with respect of 2-oxoglutarate. The inhibitory effect of chloroquine on hGDH2 was abolished, although in part, by the presence of ADP and L-leucine, whereas GTP did not change the sensitivity to chloroquine inhibition. Our results show a possibility that chloroquine may be used in regulating GDH activity and subsequently glutamate concentration in the central nervous system.

Alteration of the quaternary structure of human UDP-glucose dehydrogenase by a double mutation.

There are conflicting views for the polymerization process of human UDP-glucose dehydrogenase (UGDH) and no clear evidence has been reported yet. Based on crystal coordinates for Streptococcus pyogenes UGDH, we made double mutant A222Q/S233G. The double mutagenesis had no effects on expression, stability, and secondary structure. Interestingly, A222Q/S233G was a dimeric form and showed an UGDH activity, although it showed increased Km values for substrates. These results suggest that Ala222 and Ser233 play an important role in maintaining the hexameric structure and the reduced binding affinities for substrates are attributable to its altered subunit communication although quaternary structure may not be critical for catalysis.

Protective effect of benzothiazole derivative KHG21834 on amyloid beta-induced neurotoxicity in PC12 cells and cortical and mesencephalic neurons.

We have investigated the effect of KHG21834, a benzothiazole derivative, on the amyloid beta protein (Abeta)-induced cell death in rat pheochromocytoma (PC12) cells and rat cortical and mesencephalic neuron-glia cultures. KHG21834 attenuated the Abeta(25-35)-induced apoptotic death in PC12 cells determined by characteristic morphological alterations and positive in situ terminal end-labeling (TUNEL). In the cortical neuron-glia cultures, KHG21834 reduced the Abeta(25-35)-induced apoptosis determined by TUNEL staining. Immunocytochemical analysis and Western blot analysis of Abeta(25-35)-induced neurotoxicity in mesencephalic neuron-glia cultures with anti-tyrosine hydroxylase (TH) antibody showed that Abeta(25-35) decreased the expression of TH protein by 60% and KHG21834 significantly attenuated the Abeta(25-35)-induced reduction in the expression of TH. Moreover, KHG21834 attenuates Abeta(25-35)-induced toxicity concomitant with the reduction of activation of extracellular signal-regulated kinase (ERK)1/2 to a lesser extent. ERK1 was more sensitively affected than ERK2 in attenuation of Abeta(25-35)-induced phosphorylation by KHG21834. These results demonstrated that KHG21834 was capable of protecting neuronal cells from Abeta(25-35)-induced degeneration.

Amino acid changes within antenna helix are responsible for different regulatory preferences of human glutamate dehydrogenase isozymes.

Human glutamate dehydrogenase (hGDH) exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. Because they differ in only 16 of their 505 amino acids, the regulatory preferences must arise from amino acid residues that are not common between hGDH1 and hGDH2. To our knowledge none of the mutagenesis studies on the hGDH isozymes to date have identified the amino acid residues fully responsible for the different regulatory preferences between hGDH1 and hGDH2. In this study we constructed hGDH1(hGDH2(390-448))hGDH1 (amino acid segment 390-448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1(390-448))hGDH2 (amino acid segment 390-448 of hGDH2 replaced by the corresponding hGDH1 segment) by swapping the corresponding amino acid segments in hGDH1 and hGDH2. The chimeric enzymes by reciprocal swapping resulted in double mutations in amino acid sequences at 415 and 443 residues that are not common between hGDH1 and hGDH2 and are located in the C-terminal 48-residue "antenna" helix, which is thought to be part of the regulatory domain of mammalian GDHs. Functional analyses revealed that the doubly mutated chimeric enzymes almost completely acquired most of the different regulatory preferences between hGDH1 and hGDH2 for electrophoretic mobility, heat-stability, ADP activation, palmitoyl-CoA inhibition, and l-leucine activation, except for GTP inhibition. Our results indicate that substitutions of the residues in the antenna region may be important evolutionary changes that led to the adaptation of hGDH2 to the unique metabolic needs of the nerve tissue.

Regulation of glutamate level in rat brain through activation of glutamate dehydrogenase by Corydalis ternata.

When treated with protopine and alkalized extracts of the tuber of Corydalis ternata for one year, significant decrease in glutamate level and increase in glutamate dehydrogenase (GDH) activity was observed in rat brains. The expression of GDH between the two groups remained unchanged as determined by Western and Northern blot analysis, suggesting a post-translational regulation of GDH activity in alkalized extracts treated rat brains. The stimulatory effects of alkalized extracts and protopine on the GDH activity was further examined in vitro with two types of human GDH isozymes, hGDH1 (house-keeping GDH) and hGDH2 (nerve-specific GDH). Alkalized extracts and protopine activated the human GDH isozymes up to 4.8-fold. hGDH2 (nerve- specific GDH) was more sensitively affected by 1 mM ADP than hGDH1 (house-keeping GDH) on the activation by alkalized extracts. Studies with cassette mutagenesis at ADP-binding site showed that hGDH2 was more sensitively regulated by ADP than hGDH1 on the activation by Corydalis ternata. Our results suggest that prolonged exposure to Corydalis ternata may be one of the ways to regulate glutamate concentration in brain through the activation of GDH.

Inhibition of human UDP-glucose dehydrogenase expression using siRNA expression vector in breast cancer cells.

UDP-glucose dehydrogenase (UGDH) catalyzes two oxidations of UDP-glucose to yield UDP-glucuronic acid. Pathological over-production of extracellular matrix components may be linked to the availability of UDP-glucuronic acid, therefore UGDH is a potential therapeutic target. RNA interference (RNAi) has been adapted to knock down the expression of human UGDH. A UGDH siRNA plasmid was constructed using a pRNA-U6.1/Neo vector and transfected into breast cancer cells, ZR-75-1, with an efficiency of up to 50%. Western blot analysis showed that the UGDH expression was efficiently knocked down at protein levels by RNAi in ZR-75-1 cells.