Etiological agent and clinical characteristics of haemorrhagic fever with renal syndrome in the southern Republic of Korea: a genomic surveillance study.

OBJECTIVES: High incidences of haemorrhagic fever with renal syndrome (HFRS) have been reported in the southern Republic of Korea (ROK). A distinct southern genotype of Orthohantavirus hantanense (HTNV) was identified in Apodemus agrarius chejuensis on Jeju Island. However, its association with HFRS cases in southern ROK remains elusive. We investigated the potential of the southern HTNV genotype as an etiological agent of HFRS. METHODS: Samples from 22 patients with HFRS and 193 small mammals were collected in the southern ROK. The clinical characteristics of patients infected with the southern HTNV genotype were analysed. Amplicon-based MinION sequencing was employed for southern HTNV from patients and rodents, facilitating subsequent analyses involving phylogenetics and genetic reassortment. RESULTS: High-throughput sequencing of HTNV exhibited higher coverage with a cycle of threshold value below 32, acquiring nearly whole-genome sequences from six patients with HFRS and seven A. agrarius samples. The phylogenetic pattern of patient-derived HTNV demonstrated genetic clustering with HTNV from Apodemus species on Jeju Island and the southern Korean peninsula, revealing genetic reassortment in a single clinical sample between the M and S segments. DISCUSSION: These findings imply that the southern HTNV genotype has the potential to induce HFRS in humans. The phylogenetic inference demonstrates the diverse and dynamic characteristics of the southern HTNV tripartite genomes. Therefore, this study highlights the significance of active surveillance and amplicon sequencing for detecting orthohantavirus infections. It also raises awareness and caution for physicians regarding the emergence of a southern HTNV genotype as a cause of HFRS in the ROK.

Seroprevalence of severe fever with thrombocytopenia syndrome using specimens from the Korea National Health & Nutrition Examination Survey.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an acute febrile disease caused by bites from ticks infected with the SFTS virus. In Korea, SFTS patients are observed nationwide, including Jeju Island, but there are currently no data regarding the national prevalence of SFTS, including that of residents of 16 cities and provinces. This study aimed to investigate the seroprevalence of SFTS in Korea. METHODOLOGY/PRINCIPAL FINDINGS: A total of 1500 participants were selected through random sampling from the 2014-2015 Korea National Health and Nutrition Examination Survey (KNHANES). An indirect immunofluorescence antibody assay (IFA) was performed to assess immunoglobulin (Ig) G and IgM antibody titers against SFTS virus. RESULTS: Of the 1500 participants, 55 (3.7%) tested positive for IgG and 1 (0.1%) tested positive for IgM, with antibody titer of ≥ 1:32. Approximately 3.9% and 2.5% of participants in urban and rural areas, respectively, had a positive titer of ≥ 1:32. There was a significant correlation between SFTS incidence per 100,000 population and seroprevalence using an IgG titer ≥ 1:64 as the cut-off value. CONCLUSION: This is the first study to investigate national SFTS seroprevalence in all 16 cities and provinces representing Korea. Our study will also provide useful guidelines for the development of preventive measures against SFTS.

Risk factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA environmental contamination in rooms of patients with coronavirus disease 2019 (COVID-19).

The risk factors of environmental contamination by SARS-CoV-2 are largely unknown. We analyzed 1,320 environmental samples obtained from COVID-19 patients over 1 year. The risk factors for contamination of COVID-19 patients' surrounding environment were higher viral load in the respiratory tract and shorter duration from symptom onset to sample collection.

Whole-genome analysis and mutation pattern of SARS-CoV-2 during first and second wave outbreak in Gwangju, Republic of Korea.

To investigate the specific genomic features and mutation pattern, whole and near-complete SARS-CoV-2 genome sequences were analyzed. Clinical samples were collected from 18 COVID-19-positive patients and subjected to nucleic acid purification. Cell culture was performed to extract various SARS-CoV-2 isolates. Whole-genome analysis was performed using next-generation sequencing, and phylogenetic analyses were conducted to determine genetic diversity of the various SARS-CoV-2 isolates. The next-generation sequencing data identified 8 protein-coding regions with 17 mutated proteins. We identified 51 missense point mutations and deletions in 5' and 3' untranslated regions. The phylogenetic analysis revealed that V and GH are the dominant clades of SARS-CoV-2 circulating in the Gwangju region of South Korea. Moreover, statistical analysis confirmed a significant difference between viral load (P < 0.001) and number of mutations (P < 0.0001) in 2 mutually exclusive SARS-CoV-2 clades which indicates frequent genomic alterations in SARS-CoV-2 in patients with high viral load. Our results provide an in-depth analysis of SARS-COV-2 whole genome which we believe, can shed light in the understanding of SARS-COV-2 pathogenesis and mutation pattern which can aid in the development of prevention methods as well as future research into the pathogenesis of SARS-CoV-2 and therapeutic development.

Accuracy of Real-Time Polymerase Chain Reaction in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is a mild to severe respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The diagnostic accuracy of the Centers for Disease Control and Prevention (CDC)- or World Health Organization (WHO)-recommended real-time PCR (RT-qPCR) primers in clinical practice remains unproven. We conducted a prospective study on the accuracy of RT-qPCR using an in-house-designed primer set (iNP) targeting the nucleocapsid protein as well as various recommended and commercial primers. The accuracy was assessed by culturing or seroconversion. We enrolled 12 confirmed COVID-19 patients with a total of 590 clinical samples. When a cutoff value of the cycle threshold (Ct) was set to 35, RT-qPCRs with WHO RdRp primers and CDC N1, N2, and N3 primers showed sensitivity of 42.1% to 63.2% and specificity of 90.5% to 100% in sputum, and sensitivity of 65.2% to 69.6% and specificity of 65.2% to 69.6% in nasopharyngeal samples. The sensitivity and specificity of iNP RT-qPCR in sputum and nasopharyngeal samples were 94.8%/100% and 69.6%/100%, respectively. Sputum testing had the highest sensitivity, followed by nasopharyngeal testing (P = 0.0193); self-collected saliva samples yielded better characteristics than oropharyngeal samples (P = 0.0032). Our results suggest that iNP RT-qPCR has better sensitivity and specificity than RT-PCR with WHO (P < 0.0001) or CDC (N1: P = 0.0012, N2: P = 0.0013, N3: P = 0.0012) primers. Sputum RT-qPCR analysis has the highest sensitivity, followed by nasopharyngeal, saliva, and oropharyngeal assays. Our study suggests that considerable improvement is needed for the RT-qPCR WHO and CDC primer sets for detecting SARS-CoV-2. IMPORTANCE Numerous research campaigns have addressed the vast majority of clinical and diagnostic specificity and sensitivity of various primer sets of SARS-CoV2 viral detection. Despite the impressive progress made to resolve the pandemic, there is still a need for continuous and active improvement of primers used for diagnosis in clinical practice. Our study significantly exceeds the scale of previously published research on the specificity and sensitivity of different primers comparing with different specimens and is the most comprehensive to date in terms of constant monitoring of primer sets of current usage. Henceforth, our results suggest that sputum samples sensitivity is the highest, followed by nasopharyngeal, saliva, and oropharyngeal samples. The CDC recommends the use of oropharyngeal specimens, leading to certain discrepancy between the guidelines set forth by the CDC and IDSA. We proved that the oropharyngeal samples demonstrated the lowest sensitivity for the detection of SARS-CoV-2.

Viable Severe Acute Respiratory Syndrome Coronavirus 2 Isolates Exhibit Higher Correlation With Rapid Antigen Assays Than Subgenomic RNA or Genomic RNA.

Background: Rapid identification and effective isolation are crucial for curbing the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To meet this requirement, antigen-detection rapid diagnostic tests (Ag-RDTs) are essential. Methods: Between February 2020 and August 2020 we performed a cohort study of patients with confirmed COVID-19. The clinical performance of Ag rapid fluorescence immunoassay (FIA) and Ag Gold was evaluated and compared in parallel with genomic and subgenomic real-time reverse transcription-polymerase chain reaction (rRT-PCR) and cell culture-based assays. Results: In total, 150 samples were tested. Of these, 63 serial samples were obtained from 11 patients with SARS-CoV-2 and 87 from negative controls. Serial respiratory samples were obtained 2 days prior to symptom onset (-2) up to 25 days post-symptom onset. Overall, for rRT-PCR-positive samples (n = 51), the detection sensitivity of Ag rapid FIA and Ag Gold was 74.5% and 53.49%, respectively, with a specificity of 100%; however, for samples with low cycle threshold (Ct) values, Ag rapid FIA and Ag Gold exhibited a sensitivity of 82.61% (Ct ≤ 30, 5.6 log10RNA copies/mL) and 80% (Ct ≤ 25, 6.9 log10RNA copies/mL), respectively. Despite low analytical sensitivity, both Ag-RDTs detected 100% infection in cell culture-positive samples (n = 15) and were highly effective in distinguishing viable samples from those with subgenomic RNA (66.66%). For both Ag-RDTs, all samples that yielded discordant results (rRT-PCR + ve/Ag-RDT -ve) were also negative by culture. Conclusion: The data suggest that Ag-RDTs reliably detect viable SARS-CoV-2; thus, they may serve as an important tool for rapid detection of potentially infectious individuals.

Usefulness of ELISA Using Total Antibody against Plant-Expressed Recombinant Nucleocapsid Protein of SARS-CoV-2.

Here, we aimed to investigate the diagnostic value of a serological assay using the nucleocapsid protein developed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection and evaluated its performance using three commercial enzyme-linked immunosorbent assays (ELISAs), namely, Standard E 2019 novel coronavirus disease (COVID-19) total antibody (Ab) ELISA (SD Biosensor), and EDI novel coronavirus COVID-19 IgG and IgM ELISA. A recombinant nucleocapsid protein (rNP) was expressed from plants and Escherichia coli for the detection of serum total Ab. We prospectively collected 141 serum samples from 32 patients with reverse transcription-PCR (RT-PCR)-confirmed COVID-19 and determined the sensitivity and dynamics of their total Ab response. Specificity was evaluated using 158 prepandemic samples. To validate the assays, we evaluated the performance using two different cutoff values. The sensitivity and specificity for each assay were as follows: 92.91% and 94.30% (plant-rNP), 83.69% and 98.73% (SD Biosensor), 75.89% and 98.10% (E. coli-rNP), 76.47% and 100% (EDI-IgG), and 80.39% and 80% (EDI-IgM). The plant-based rNP showed the highest sensitivity and area under the receiver operating characteristic (ROC) curve (0.980) among all the assays (P < 0.05). The seroconversion rate for total Ab increased sequentially with disease progression, with a sensitivity of 100% after 10 to 12 days of post-symptom onset (PSO) for both rNP-plant-based and SD Biosensor ELISAs. After 2 weeks of PSO, the seroconversion rates were >80% and 100% for EDI-IgM and EDI-IgG ELISA, respectively. Seroconversion occurred earlier with rNP plant-based ELISA (5 days PSO) compared with E. coli-based (7 days PSO) and SD Biosensor (8 days PSO) ELISA. We determined that rNP produced in plants enables the robust detection of SARS-CoV-2 total Abs. The assay can be used for serosurvey and complementary diagnosis of COVID-19. IMPORTANCE At present, the principal diagnostic methods for COVID-19 comprise the identification of viral nucleic acid by genetic approaches, including PCR-based techniques or next-generation sequencing. However, there is an urgent need for validated serological assays which are crucial for the understanding of immune responses against SARS-CoV-2. In this study, a highly sensitive and specific serological antibody assay was developed for the detection of SARS-CoV-2 with an overall accuracy of 93.56% using a recombinant nucleoprotein expressed from plants.

First report of the molecular detection of human pathogen Rickettsia raoultii in ticks from the Republic of Korea.

BACKGROUND: Rickettsial diseases associated with the spotted fever group constitute a growing number of newly identified Rickettsia pathogens and their tick vectors in various parts of the world. At least 15 distinct tick species belonging to six genera have shown the presence of Rickettsia raoultii. Herein, we report the detection of R. raoultii in ticks from the Republic of Korea (ROK). METHODS: Thirty-five ticks were collected from 29 patients with tick bites in Gwangju Metropolitan City, Jeollanam Province, ROK. The ticks were identified using molecular, morphological, and taxonomic characteristics. All samples were screened for presence of Rickettsia species using nested polymerase chain reactions of their outer membrane protein (ompA) and citrate synthase (gltA) genes. The amplified products were sequenced for subsequent phylogenetic analyses. RESULTS: Sequencing data showed the DNA sequences of R. raoultii in three Haemaphysalis longicornis ticks. All three tick samples were 99.4-100% similar to previously reported partial sequences of ompA of R. raoultii strains CP019435 and MF002523, which formed a single clade with the reference strains. CONCLUSIONS: We provide the first description and molecular identification of R. raoultii detected in H. longicornis ticks in the ROK. This observation extends the geographical distribution of R. raoultii. Screening of human samples for this pathogen will provide information about the prevalence of rickettsial infections in this region.

A case of SFTS coinfected with E. coli bacteremia.

BACKGROUND: Severe fever thrombocytopenia syndrome virus (SFTSV) is the causative agent of severe fever thrombocytopenia syndrome (SFTS). SFTS is an emerging infectious disease, characterized by high fever, gastrointestinal symptoms, leukopenia, thrombocytopenia, and a high mortality rate. Until now, little importance has been given to the association of SFTS with leukocytosis and bacterial co-infection. CASE PRESENTATION: A 51-year old man visited our hospital with fever and low blood pressure. He was a farmer by occupation and often worked outdoors. He had a Foley catheter inserted due to severe BPH. Laboratory tests revealed thrombocytopenia, elevated liver function, and elevated CRP levels. He had marked leukocytosis, proteinuria, hematuria, and conjunctival hemorrhage. Initially, we thought that the patient was suffering from hemorrhagic fever with renal syndrome (HFRS). However, we confirmed SFTS through PCR and increasing antibody titer. However, his blood culture also indicated E. coli infection. CONCLUSION: SFTS displays characteristics of fever, thrombocytopenia, elevated liver function, and leukocytopenia. We described a case of SFTS with leukocytosis due to coinfection with E. coli. Since patients with SFTS usually have leukocytopenia, SFTS patients with leukocytosis are necessarily evaluated for other causes of leukocytosis. Here, we report the first case of an SFTS with concurrent E. coli bacteremia.

The complete chloroplast genome of Korean endemic species, Cirsium rhinoceros (H.Lév. & vaniot) Nakai (Asteraceae).

Cirsium rhinoceros (H.Lév. & Vaniot) Nakai has been used a traditional medicine. Complete chloroplast genome of C. rhinoceros is 152,576 bp long and has four subregions: 87,262 bp of large single copy (LSC) and 21,486 bp of small single copy (SSC) regions that are separated by 18,742 bp of inverted repeat (IR) regions including 133 genes (88 protein-coding genes, 8 rRNAs, and 37 tRNAs). The overall GC content of this chloroplast genome is 37.7% and in the LSC, SSC, and IR regions are 36.0%, 31.4%, and 43.8%, respectively. Phylogenetic trees show that Cirsium species are clustered along with their distribution.

Development of chloroplast microsatellite markers for the endangered Maianthemum bicolor (Asparagaceae s.l.).

PREMISE OF THE STUDY: Ten polymorphic chloroplast microsatellite (cpSSR) markers were developed and characterized in an endemic and endangered herb, Maianthemum bicolor (Asparagaceae s.l.), for use in conservation genetics. METHODS AND RESULTS: Primer sets flanking each of the 10 cpSSR loci in noncoding regions of the chloroplast genome of M. bicolor were designed. These cpSSR markers were tested on a total of 33 adult individuals from three natural populations in South Korea. The number of alleles per locus ranged from two to three. The unbiased haplotype diversity per locus ranged from 0.061 to 0.682. All markers were successfully transferred to the congeneric species M. japonicum, M. bifolium, and M. dilatatum with polymorphisms among the species. CONCLUSIONS: The developed cpSSR markers will be useful in assessing the genetic diversity and population structure of M. bicolor and will help to infer its molecular identification, thereby providing a basis for conservation.

The complete plastid genome sequence of Picea jezoensis (Pinaceae: Piceoideae).

The nucleotide sequence of the complete chloroplast genome of P. jezoensis was completed. The total genome size was 124 146 bp, containing a pair of very short inverted repeats (IRa and IRb) of 422 bp, which were separated by large single copy (LSC) and small single copy (SSC) with 66 956 bp and 56 346 bp, respectively. The overall GC contents of the plastid genome were determined as 38.8%. One hundred fifteen genes including 68 peptide-encoding genes, 35 tRNA genes, four rRNA genes, six open-reading frames, and two pseudogenes were annotated. In these genes, 15 genes contained only one or two introns. Phylogenetic analyses using maximum likelihood (ML) methods were performed from fully sequenced Gymnosperms and other species of dataset composed of 69 protein-coding genes.

The complete plastid genome sequence of Abies koreana (Pinaceae: Abietoideae).

The nucleotide sequence of the chloroplast genome from Abies koreana is the first to have complete genome sequence from genus Abies of family Pinaceae. The circular double-stranded DNA, which consists of 121,373 base pairs (bp), contains a pair of very short inverted repeat regions (IRa and IRb) of 264 bp each, which are separated by a small and large single-copy regions (SSC and LSC) of 54,197 and 66,648 bp, respectively. The genome contents of 114 genes (68 peptide-encoding genes, 35 tRNA genes, four rRNA genes, six open reading frames and one pseudogene) are similar to the chloroplast DNA of other species of Abietoideae. Loss of ndh genes was also identified in the genome of A. koreana like other genomes in the family Pinaceae. Thirteen genes contain one (11 genes) or two (rps12 and ycf3 genes) introns. In phylogenetic analysis, the tree confirms that Abies, Keteleeria and Cedrus are strongly supported as monophyletic. Other inverted repeat sequences located in 42-kb inversion points (1186 bp) include trnS-psaM-ycf12- ψtrnG genes.

Alterations of vasoactive intestinal polypeptide receptors in allergic rhinitis.

BACKGROUND: Vasoactive intestinal peptide (VIP) is one of the parasympathetic neurotransmitters involved in the regulation of airway mucus secretion. The biological functions of VIP are mediated through two receptors (vasoactive intestinal peptide receptor type 1 [VPAC1R] and type 2 [VPAC2R]). The purpose of this study was to determine the distribution of VIP receptors and to compare the level of VIP receptor expression in the nasal mucosa of patients with allergic rhinitis and normal controls. METHODS: Inferior turbinate mucosal samples were obtained from 20 normal subjects and 20 patients with allergic rhinitis. VPAC1R and VPAC2R mRNA was extracted from the nasal mucosa, and then a reverse-transcription-polymerase chain reaction was performed. Sections were immunostained using specific antibodies for VIP receptors. Western blot analysis was used to analyze differences in the level of expression of VPAC1R and VPAC2R protein between patients with allergic rhinitis and normal controls. RESULTS: The level of expression of VIP receptor mRNA and protein in patients with allergic rhinitis was significantly increased compared with normal nasal mucosa. VIP receptor immunoreactivity was detected on the nasal epithelium and submucosal glands in nasal specimens from both normal controls and patients with allergic rhinitis. In the epithelium from patients with allergic rhinitis, VIP receptor immunoreactivity was strong, whereas in the nasal epithelium from normal subjects it was faint. CONCLUSION: These results suggest that an increased expression level of VIP receptors is one possible explanation for nasal hyperresponsiveness in patients with allergic rhinitis.

The effect of macrolides on myofibroblast differentiation and collagen production in nasal polyp-derived fibroblasts.

BACKGROUND: Macrolides are known to have anti-inflammatory, immunomodulatory, and tissue reparative effects. The purpose of this study was to determine the effect of macrolides (erythromycin [EM] and roxithromycin [RXM]) on the differentiation of fibroblasts into myofibroblasts and extracellular matrix accumulation in transforming growth factor (TGF) beta1-induced nasal polyp-derived fibroblasts (NPDFs) and to determine if NADPH oxidase (Nox) 4 and reactive oxygen species (ROS) are involved in the aforementioned processes. METHODS: Nasal polyps of six patients (three women and three men; 32.3 ± 5.2 years of age) were acquired during surgery and NPDFs were isolated from surgical tissues. NPDFs were pretreated with macrolides for 2 hours before differentiation induction by TGF-beta1. The mRNA expressions of alpha-smooth muscle actin (SMA), collagen types I and III, and Nox4 were determined by reverse-transcription-polymerase chain reaction, and the expression of alpha-SMA protein was determined by immunocytochemical staining. The amount of total collagen production was analyzed by SirCol collagen dye-binding assay. ROS activity was measured by nitroblue tetrazolium reduction assay and was visualized by fluorescent microscopy. RESULTS: In TGF-beta1-induced NPDFs, EM, and RXM significantly inhibited expressions of alpha-SMA and collagen types I and III mRNA and reduced alpha-SMA and collagen protein levels at concentrations of 5 and 10 µg/mL. EM and RXM also inhibited TGF-ß1-induced ROS production and Nox4 mRNA expression at the same concentrations. CONCLUSION: These results suggest the possibility that EM and RXM may play an important role in inhibiting the development of nasal polyps through their antioxidant effect.

New Record of the Existence of Sarcographa tricosa (Lichenized Ascomycota, Graphidaceace) in Korea.

Lichen genus Sarcographa Fee, a stromatoid Graphidacean taxa, was newly found in Geomun Island, Jeonnam province. The lichen grew on the bark of Camellia japonica and Eurya emarginata along the coastal line of the island. It was identified as Sarcographa tricosa (Ach.) Müll. Arg. for the first time in Korea.

Notes on Some New Records of Macro- and Micro-lichens from Korea.

The paper describes nine new records of macro- and micro-lichens from Korea. A brief taxonomic description and comments are presented for all the studied taxa (Catapyrenium squamellum, Chrysothrix candelaris, Endocarpon pallidulum, Endocarpon petrolepideum, Lecanora oreinoides, Leprocaulon albicans, Parmotrema saccatilobum, Verrucaria glaucina and Xanthoria parietina). The lichen genera Catapyrenium, Chrysothrix and Verrucaria are reported for the first time in this country.

First Report of Heterodermia squamulosa (Lichenized Ascomycota, Physciaceae) in South Korea.

Heterodermia squamulosa (Degel.) W.L. Culb. was found in the mountain of Gariwang, Gangwon province, in 2008. It is characterized by numerous squamules along the margin, decorticate and white lower surface, rhizines along the margin, black and densely squarrosely branched, usually forming a dense mat under the thallus. Apothecia margins densely squamulose, ascospores 12~15 × 25~30 µm. Atranorin and zeorin contained in thallus. This is the first record of this species in South Korea.

Cladonia peziziformis (Lichenized Ascomycota, Cladoniaceae) New to Korea.

Cladonia peziziformis (With.) J.R. Laundon was collected from Baega mountain, Jeonnam Province, Korea in 2008. It is characterized by short and slender podetia with verruculose surface, split along the sides. Apothecia large, pale brown, always growing on the top of the podetia. Primary squamules shell-like, thick, and convex. Fumarprotocetraric acid contained in thallus. This is the first record of this species in Korea.

New Record of Lecanora muralis (Lichenized Fungus) in South Korea.

Lecanora muralis was found on the rock along coastal line during the field trip in Jeju island in 2006. Thallus crustose, placodioid, closely adnate, forming orbicular patches; upper surface grayish green, glossy; central lobes areolate, marginal parts plane, edges thin pruinose; lower surface ecorticate; apothecia sessile, lecanorine type, exciple dense and intact when young, and disc plane, but when mature, exciple laciniate, disc protrudent, yellowish brown to orange, 0.5~1.5 mm in diameter; ascospores ellipsoid, simple, colorless, 12.5~15.0 × 5.0~7.5 µm. Usnic acid and zeorin contained in thallus. This is the first record of this species in South Korea.