High-resolution phylogeographical surveillance of Hantaan orthohantavirus using rapid amplicon-based Flongle sequencing, Republic of Korea.

Orthohantaviruses, etiological agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome, pose a critical public health threat worldwide. Hantaan orthohantavirus (HTNV) outbreaks are particularly endemic in Gyeonggi Province in northern area of the Republic of Korea (ROK). Small mammals were collected from three regions in the Gyeonggi Province during 2017 and 2018. Serological and molecular prevalence of HTNV was 25/201 (12.4%) and 10/25 (40%), respectively. A novel nanopore-based diagnostic assay using a cost-efficient Flongle chip was developed to rapidly and sensitively detect HTNV infection in rodent specimens within 3 h. A rapid phylogeographical surveillance of HTNV at high-resolution phylogeny was established using the amplicon-based Flongle sequencing. In total, seven whole-genome sequences of HTNV were newly obtained from wild rodents collected in Paju-si (Gaekhyeon-ri) and Yeoncheon-gun (Hyeonga-ri and Wangnim-ri), Gyeonggi Province. Phylogenetic analyses revealed well-supported evolutionary divergence and genetic diversity, enhancing the resolution of the phylogeographic map of orthohantaviruses in the ROK. Incongruences in phylogenetic patterns were identified among HTNV tripartite genomes, suggesting differential evolution for each segment. These findings provide crucial insights into on-site diagnostics, genome-based surveillance, and the evolutionary dynamics of orthohantaviruses to mitigate hantaviral outbreaks in HFRS-endemic areas in the ROK.

Comparative Computational Analysis of Spike Protein Structural Stability in SARS-CoV-2 Omicron Subvariants.

The continuous emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with multiple spike (S) protein mutations pose serious threats to current coronavirus disease 2019 (COVID-19) therapies. A comprehensive understanding of the structural stability of SARS-CoV-2 variants is vital for the development of effective therapeutic strategies as it can offer valuable insights into their potential impact on viral infectivity. S protein mediates a virus' attachment to host cells by binding to angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD), and mutations in this protein can affect its stability and binding affinity. We analyzed S protein structural stability in various Omicron subvariants computationally. Notably, the S protein sequences analyzed in this work were obtained directly from our own sample collection. We evaluated the binding free energy between S protein and ACE2 in several complex forms. Additionally, we measured distances between the RBD of each chain in S protein to analyze conformational changes. Unlike most of the prior studies, we analyzed full-length S protein-ACE2 complexes instead of only RBD-ACE2 complexes. Omicron subvariants including BA.1, BA.2, BA.2.12.1, BA.4/BA.5, BA.2.75, BA.2.75_K147E, BA.4.6 and BA.4.6_N658S showed enhanced stability compared to wild type, potentially due to distinct S protein mutations. Among them, BA.2.75 and BA.4.6_N658S exhibited the highest and lowest level of stability, respectively.

Genomic evidence of SARS-CoV-2 reinfection in the Republic of Korea.

As the coronavirus disease 2019 (COVID-19) pandemic continues, reinfection is likely to become increasingly common. However, confirming COVID-19 reinfection is difficult because it requires whole-genome sequencing of both infections to identify the degrees of genetic differences. Since the first reported case of reinfection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the Republic of Korea in April 2020, four additional cases were classified as suspected reinfection cases. We performed whole-genome sequencing of viral RNA extracted from swabs obtained at the initial infection and reinfection stages of these four suspected cases. The interval between initial infection and reinfection of all four suspected cases was more than 3 months. All four patients were young (10-29 years), and they displayed mild symptoms or were asymptomatic during the initial infection and reinfection episodes. The analysis of genome sequences combined with the epidemiological results revealed that only two of the four cases were confirmed as reinfection, and both were reinfected with the Epsilon variant. Due to the prolonged COVID-19 pandemic, the possibility of reinfections with SARS-CoV-2 variants is increasing, as reported in our study. Therefore, continuous monitoring of cases is necessary.

Active Targeted Surveillance to Identify Sites of Emergence of Hantavirus.

BACKGROUND: Endemic outbreaks of hantaviruses pose a critical public health threat worldwide. Hantaan orthohantavirus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS) in humans. Using comparative genomic analyses of partial and nearly complete sequences of HTNV from humans and rodents, we were able to localize, with limitations, the putative infection locations for HFRS patients. Partial sequences might not reflect precise phylogenetic positions over the whole-genome sequences; finer granularity of rodent sampling reflects more precisely the circulation of strains. METHODS: Five HFRS specimens were collected. Epidemiological surveys were conducted with the patients during hospitalization. We conducted active surveillance at suspected HFRS outbreak areas. We performed multiplex polymerase chain reaction-based next-generation sequencing to obtain the genomic sequence of HTNV from patients and rodents. The phylogeny of human- and rodent-derived HTNV was generated using the maximum likelihood method. For phylogeographic analyses, the tracing of HTNV genomes from HFRS patients was defined on the bases of epidemiological interviews, phylogenetic patterns of the viruses, and geographic locations of HTNV-positive rodents. RESULTS: The phylogeographic analyses demonstrated genetic clusters of HTNV strains from clinical specimens, with HTNV circulating in rodents at suspected sites of patient infections. CONCLUSIONS: This study demonstrates a major shift in molecular epidemiological surveillance of HTNV. Active targeted surveillance was performed at sites of suspected infections, allowing the high-resolution phylogeographic analysis to reveal the site of emergence of HTNV. We posit that this novel approach will make it possible to identify infectious sources, perform disease risk assessment, and implement preparedness against vector-borne viruses.

Multiplex PCR-Based Next-Generation Sequencing and Global Diversity of Seoul Virus in Humans and Rats.

Seoul virus (SEOV) poses a worldwide public health threat. This virus, which is harbored by Rattus norvegicus and R. rattus rats, is the causative agent of hemorrhagic fever with renal syndrome (HFRS) in humans, which has been reported in Asia, Europe, the Americas, and Africa. Defining SEOV genome sequences plays a critical role in development of preventive and therapeutic strategies against the unique worldwide hantavirus. We applied multiplex PCR-based next-generation sequencing to obtain SEOV genome sequences from clinical and reservoir host specimens. Epidemiologic surveillance of R. norvegicus rats in South Korea during 2000-2016 demonstrated that the serologic prevalence of enzootic SEOV infections was not significant on the basis of sex, weight (age), and season. Viral loads of SEOV in rats showed wide dissemination in tissues and dynamic circulation among populations. Phylogenetic analyses showed the global diversity of SEOV and possible genomic configuration of genetic exchanges.

Detection of Hantaan virus RNA from anti-Hantaan virus IgG seronegative rodents in an area of high endemicity in Republic of Korea.

Hantaan virus (HTNV), of the family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS) in humans. Although the majority of epidemiologic studies have found that rodents are seropositive for hantavirus-specific immunoglobulin, the discovery of hantavirus RNA in seronegative hosts has led to an investigation of the presence of HTNV RNA in rodents captured in HFRS endemic areas. HTNV RNA was detected in seven (3.8%) of 186 anti-HTNV IgG seronegative rodents in Republic of Korea (ROK) during 2013-2014. RT-qPCR for HTNV RNA revealed dynamic virus-host interactions of HTNV in areas of high endemicity, providing important insights into the epidemiology of hantaviruses.

Neutralization Testing-based Immunogenicity Analysis of Recent Prevalent Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Sublineages.

Although WHO declared the end of the public health emergency for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), XBB lineages continue to evolve and emerge globally. In particular, XBB.1.5 and XBB.1.16 are raising concerns because of their high immune evasion, leading to apprehensions regarding vaccine efficacy reduction and potential reinfection. We aimed to investigate the COVID-19 outbreak in Korea and predict the likelihood of reinfection by testing neutralizing activity against live viruses from the S clade and 19 Omicron sublineages. We found a significant risk of infection with the currently prevalent XBB lineage for individuals who were either vaccinated early or infected during the initial Omicron outbreak. Vaccinated individuals were better equipped than unvaccinated individuals to produce neutralizing antibodies for other SARS-CoV-2 variants upon infection. Therefore, unvaccinated individuals do not easily develop neutralizing activity against other variants and face the highest risk of reinfection by the XBB lineage. Our study provides important information to facilitate the development of strategies for monitoring populations that would be the most susceptible to new COVID-19 outbreaks.

Three distinct ORF1a recombinants of the SARS-CoV-2 Delta variant of concern.

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the emergency of various lineages through mutations and recombination. In the Delta lineage, we identified recombination events in the ORF1a gene, which divided the Delta sublineages into three different genotypes (Delta R1-R3). The regional distributions of Delta R1 and Delta R2 were not correlated, indicating that recombination occurred early in the Delta outbreak. The impact of the ORF1a gene on SARS-CoV-2 transmission remains unclear; however, our findings suggest that recombination may have contributed to the evolution and global spread of the Delta lineage.

Dynamics of SARS-CoV-2 variants during the XBB wave in the Republic of Korea.

As COVID-19 has become endemic, SARS-CoV-2 variants are becoming increasingly diverse, underscoring the escalating importance of global genomic surveillance. This study analyzed 86,762 COVID-19 samples identified in the Republic of Korea from September 2022 to November 2023. The results revealed a consistent increase in the prevalence of the XBB variants following the dominance of BN.1, with various XBB sub-lineages co-circulating in the Republic of Korea. The overall nucleotide diversity (π) among the SARS-CoV-2 genomes was 0.00155. Evolutionary analysis revealed that the average time interval between the first detection and estimated date of the most recent common ancestor of Korean XBB sub-lineages was 47 d, suggesting that the novel variants were efficiently identified in the Korean surveillance system. The mutation rate was determined to be in the range of 5.6 × 10-4 to 9.1 × 10-4 substitutions/site/year. In conclusion, this study provides insights into the genetic diversity and evolutionary interpretation of the XBB sub-lineages during the XBB wave in the Republic of Korea, highlighting the importance of continued genomic surveillance for emerging variants.

Risk factors for SARS-CoV-2 transmission during a movie theater outbreak in Incheon in the Republic of Korea, November 2021: a retrospective study.

BACKGROUND: We examined factors contributing to the transmission of an acute respiratory virus within multi-use facilities, focusing on an outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a movie theater in the Republic of Korea. METHODS: This retrospective cohort study involved a descriptive analysis of 48 confirmed cases. Logistic regression was applied to a cohort of 80 theater attendees to identify risk factors for infection. The infection source and transmission route were determined through gene sequencing data analysis. RESULTS: Of the 48 confirmed cases, 35 were theater attendees (72.9%), 10 were family members of attendees (20.8%), 2 were friends (4.2%), and 1 was an employee (2.1%). Among the 80 individuals who attended the 3rd to 5th screenings of the day, 35 became infected, representing a 43.8% attack rate. Specifically, 28 of the 33 third-screening attendees developed confirmed SARSCoV-2, constituting an 84.8% attack rate. Furthermore, 11 of the 12 cases epidemiologically linked to the theater outbreak were clustered monophyletically within the AY.69 lineage. At the time of the screening, 35 individuals (72.9%) had received 2 vaccine doses. However, vaccination status did not significantly influence infection risk. Multivariate analysis revealed that close contacts had a 15.9-fold higher risk of infection (95% confidence interval, 4.37-78.39) than casual contacts. CONCLUSION: SARS-CoV-2 transmission occurred within the theater, and extended into the community, via a moviegoer who attended the 3rd screening during the viral incubation period after contracting the virus from a family member. This study emphasizes the importance of adequate ventilation in theaters.

Genomic epidemiology of SARS-CoV-2 variants in South Korea between January 2020 and February 2023.

The Korea Disease Control and Prevention Agency (KDCA) has been conducting national genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). To monitor and characterize circulating SARS-CoV-2 variants in South Korea, 102,873 oropharyngeal/nasopharyngeal swab samples collected from patients with confirmed COVID-19 were sequenced, assigned lineages, and phylogenetically analyzed. Each wave followed a pattern of variants emerging first abroad and then spreading domestically. In 2020, B.41 lineage led the first wave, and B.1.497 dominated the second and third waves. In 2021, the fourth wave was driven by Delta (AY.69 and AY.122.5). In 2022, the fifth to seventh waves were dominated by Omicron sub-lineages BA.1/BA.1.1 and BA.2/BA.2.3, BA.5/BA.5.2, and BN.1, sequentially. The KDCA detected and monitored increasing variants in advance prior to large-scale epidemics, but the repeated emergence of new variants could threaten public health again. Therefore, it is important to continue to monitor and characterize emerging and circulating variants through national genomic surveillance.

Draft Genome Sequence of Francisella tularensis subsp. holarctica Strain H0001, Isolated from a Tularemia Patient in the Republic of Korea.

Francisella tularensis is the etiological agent of the zoonosis tularemia. Here, we report the draft genome sequence of F. tularensis subsp. holarctica H0001, which was isolated from a tularemia patient in the Republic of Korea.

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.

Clinical and Immunological Predictors of Hemorrhagic Fever with Renal Syndrome Outcome during the Early Phase.

The ability to accurately predict the early progression of hemorrhagic fever with renal syndrome (HFRS) is crucial for reducing morbidity and mortality rates in severely affected patients. However, the utility of biomarkers for predicting clinical outcomes remains elusive in HFRS. The aims of the current study were to analyze the serum levels of immune function-related proteins and identify novel biomarkers that may help ascertain clinical outcomes of HFRS. Enzyme-linked immunosorbent assay, Luminex, and bioanalyzer assays were used to quantitatively detect 15 biomarkers in 49 serum samples of 26 patients with HFRS. High hemoglobin (HGB) and low urine output (UO) levels were identified as potential biomarkers associated with the acute HFRS. The serum soluble urokinase plasminogen activator receptor (suPAR) and C-X-C motif chemokine ligand 10 (CXCL10) values increased in the early phase of diseases. Elevated suPAR, interleukin-10 (IL-10), CXCL10, and decreased transforming growth factor-beta 3 (TGF-ß3) were representative predictors of the disease severity. Upregulation of the HGB showed a significant correlation with high levels of suPAR and CXCL10. Reduced UO positively correlated with increased suPAR, CXCL10, and TGF-ß2, and decreased vascular endothelial growth factor and TGF-ß3. The changing HGB and UO criteria, high suPAR, IL-10, CXCL10, and low TGF-ß3 of HFRS raise significant awareness for physicians regarding prospective biomarkers for monitoring early warning signs of HFRS. This study provides critical insights into the clinical and immunological biomarkers for disease severity and progression in patients with HFRS to identify early predictions of fatal outcomes.

A Clinical Case of Scrub Typhus in the United States Forces Korea Patient with Eschar and Genetic Identification of Orientia tsutsugamushi Using Multiplex PCR-Based Next-Generation Sequencing.

An epidemiological investigation was conducted for a scrub typhus case reported in a U.S. Forces Korea military patient in the Republic of Korea in November 2018. The patient had a fever, maculopapular rash, and an eschar. The full-length sequence of Orientia tsutsugamushi 56-kDa type-specific antigen (TSA) gene was obtained from eschar tissue by multiplex PCR-based Next Generation Sequencing for genetic identification. Based on the 56-kDa TSA gene, the O. tsutsugamushi aligned most closely with the Boryong strain.

A novel genotype of Hantaan orthohantavirus harbored by Apodemus agrarius chejuensis as a potential etiologic agent of hemorrhagic fever with renal syndrome in Republic of Korea.

BACKGROUND: Orthohantaviruses, causing hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome, pose a significant public health threat worldwide. Despite the significant mortality and morbidity, effective antiviral therapeutics for orthohantavirus infections are currently unavailable. This study aimed to investigate the prevalence of HFRS-associated orthohantaviruses and identify the etiological agent of orthohantavirus outbreaks in southern Republic of Korea (ROK). METHODOLOGY/PRINCIPAL FINDINGS: We collected small mammals on Jeju Island during 2018-2020. We detected the Hantaan virus (HTNV)-specific antibodies and RNA using an indirect immunofluorescence assay test and reverse transcription-polymerase chain reaction on Apodemus agrarius chejuensis (A. chejuensis). The prevalence of anti-HTNV antibodies among rodents was 14.1%. A total of six seropositive mouse harbored HTNV RNA. The amplicon-based next-generation sequencing provided nearly full-length tripartite genomic sequences of six HTNV harbored by A. chejuensis. Phylogenetic and tanglegram analyses were conducted for inferring evolutionary relationships between orthohantaviruses with their reservoir hosts. Phylogenetic analysis showed a novel distinct HTNV genotype. The detected HTNV genomic sequences were phylogenetically related to a viral sequence derived from HFRS patient in southern ROK. Tanglegram analysis demonstrated the segregation of HTNV genotypes corresponding to Apodemus spp. divergence. CONCLUSIONS/SIGNIFICANCE: Our results suggest that A. chejuensis-borne HTNV may be a potential etiological agent of HFRS in southern ROK. Ancestral HTNV may infect A. chejuensis prior to geological isolation between the Korean peninsula and Jeju Island, supporting the co-evolution of orthohantaviruses and rodents. This study arises awareness among physicians for HFRS outbreaks in southern ROK.

Novel Paju Apodemus paramyxovirus 1 and 2, harbored by Apodemus agrarius in the Republic of Korea.

Paramyxoviruses harbored by multiple natural reservoirs pose a potential threat to public health. Jeilongvirus has been proposed as a novel paramyxovirus genus found in rodents, bats, and cats. Paramyxovirus RNA was detected in 108/824 (13.1%) Apodemus agrarius captured at 14 trapping sites in the Republic of Korea. We first present two genetically distinct novel paramyxoviruses, Paju Apodemus paramyxovirus 1 (PAPV-1) and 2 (PAPV-2). The disparity between PAPV-1 (19,716 nucleotides) and -2 (17,475 nucleotides) revealed the presence of the SH gene and length of the G gene in the genome organization. The phylogeny of PAPV-1 and -2 belonged to distinct genetic lineages of Jeilongvirus, respectively, even though these viruses were originated from A. agrarius. PAPV-1 infected human epithelial and endothelial cells, facilitating the induction of type I/III interferons, interferon-stimulated genes, and pro-inflammatory cytokines. Therefore, this study provides insights into the molecular epidemiology, genetic diversity, and virus-host interactions of novel rodent-borne paramyxoviruses.

Multiplex PCR-Based Nanopore Sequencing and Epidemiological Surveillance of Hantaan orthohantavirus in Apodemus agrarius, Republic of Korea.

Whole-genome sequencing of infectious agents enables the identification and characterization of emerging viruses. The MinION device is a portable sequencer that allows real-time sequencing in fields or hospitals. Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius, causes hemorrhagic fever with renal syndrome (HFRS) and poses a critical public health threat worldwide. In this study, we aimed to evaluate the feasibility of using nanopore sequencing for whole-genome sequencing of HTNV from samples having different viral copy numbers. Amplicon-based next-generation sequencing was performed in A. agrarius lung tissues collected from the Republic of Korea. Genomic sequences of HTNV were analyzed based on the viral RNA copy numbers. Amplicon-based nanopore sequencing provided nearly full-length genomic sequences of HTNV and showed sufficient read depth for phylogenetic analysis after 8 h of sequencing. The average identity of the HTNV genome sequences for the nanopore sequencer compared to those of generated from Illumina MiSeq revealed 99.8% (L and M segments) and 99.7% (S segment) identities, respectively. This study highlights the potential of the portable nanopore sequencer for rapid generation of accurate genomic sequences of HTNV for quicker decision making in point-of-care testing of HFRS patients during a hantavirus outbreak.

Discovery and Genetic Characterization of Novel Paramyxoviruses Related to the Genus Henipavirus in Crocidura Species in the Republic of Korea.

Paramyxoviruses, negative-sense single-stranded RNA viruses, pose a critical threat to human public health. Currently, 78 species, 17 genera, and 4 subfamilies of paramyxoviruses are harbored by multiple natural reservoirs, including rodents, bats, birds, reptiles, and fish. Henipaviruses are critical zoonotic pathogens that cause severe acute respiratory distress and neurological diseases in humans. Using reverse transcription-polymerase chain reaction, 115 Crocidura species individuals were examined for the prevalence of paramyxovirus infections. Paramyxovirus RNA was observed in 26 (22.6%) shrews collected at five trapping sites, Republic of Korea. Herein, we report two genetically distinct novel paramyxoviruses (genus: Henipavirus): Gamak virus (GAKV) and Daeryong virus (DARV) isolated from C. lasiura and C. shantungensis, respectively. Two GAKVs and one DARV were nearly completely sequenced using next-generation sequencing. GAKV and DARV contain six genes (3'-N-P-M-F-G-L-5') with genome sizes of 18,460 nucleotides and 19,471 nucleotides, respectively. The phylogenetic inference demonstrated that GAKV and DARV form independent genetic lineages of Henipavirus in Crocidura species. GAKV-infected human lung epithelial cells elicited the induction of type I/III interferons, interferon-stimulated genes, and proinflammatory cytokines. In conclusion, this study contributes further understandings of the molecular prevalence, genetic characteristics and diversity, and zoonotic potential of novel paramyxoviruses in shrews.

Urinary genome detection and tracking of Hantaan virus from hemorrhagic fever with renal syndrome patients using multiplex PCR-based next-generation sequencing.

BACKGROUND: Hantavirus infection occurs through the inhalation of aerosolized excreta, including urine, feces, and saliva of infected rodents. The presence of Hantaan virus (HTNV) RNA or infectious particles in urine specimens of patient with hemorrhagic fever with renal syndrome (HFRS) remains to be investigated. METHODOLOGY/PRINCIPAL FINDINGS: We collected four urine and serum specimens of Republic of Korea Army (ROKA) patients with HFRS. We performed multiplex PCR-based next-generation sequencing (NGS) to obtain the genome sequences of clinical HTNV in urine specimens containing ultra-low amounts of viral genomes. The epidemiological and phylogenetic analyses of HTNV demonstrated geographically homogenous clustering with those in Apodemus agrarius captured in highly endemic areas, indicating that phylogeographic tracing of HTNV genomes reveals the potential infection sites of patients with HFRS. Genetic exchange analyses showed a genetic configuration compatible with HTNV L segment exchange in nature. CONCLUSION/SIGNIFICANCE: Our results suggest that whole or partial genome sequences of HTNV from the urine enabled to track the putative infection sites of patients with HFRS by phylogeographically linking to the zoonotic HTNV from the reservoir host captured at endemic regions. This report raises awareness among physicians for the presence of HTNV in the urine of patients with HFRS.