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.

Molecular diagnosis of patients with hepatitis A virus infection using amplicon-based nanopore sequencing.

High-throughput sequencing is a robust tool used for identifying and tracking pathogen outbreaks. Whole-genome sequencing of hepatitis A virus (HAV) remains poor due to ultra-low viral loads, limitations of next-generation sequencing technology, and its high costs in clinical applications. This study evaluated multiplex polymerase chain reaction (PCR)-based nanopore sequencing to obtain whole-genome sequences of HAV. The HAV genomes were obtained directly from patient specimens for a rapid molecular diagnosis of viral genotypes. Serum and stool samples were collected from six patients with hepatitis A infection. Amplicon-based nanopore sequencing was performed from the clinical specimens to identify HAV genotypes by acquiring nearly complete-genome sequences. TaqMan-based quantitative PCR (qPCR) was conducted to detect and quantify multiple HAV genes. Singleplex-based nanopore sequencing demonstrated high genome coverage rates (90.4-99.5%) of HAV within 8 h, at viral RNA loads of 10 to 105 copies/µL. TaqMan qPCR showed multiplex quantification of HAV genes namely, VP0, VP3, and 3C. This study provides useful insights into rapid molecular diagnosis during hepatitis A outbreaks and may ultimately augment public health disease surveillance in the hospital and epidemiology field.

Whole-genome sequencing and genetic diversity of severe fever with thrombocytopenia syndrome virus using multiplex PCR-based nanopore sequencing, Republic of Korea.

BACKGROUND: Whole-genome sequencing plays a critical role in the genomic epidemiology intended to improve understanding the spread of emerging viruses. Dabie bandavirus, causing severe fever with thrombocytopenia syndrome (SFTS), is a zoonotic tick-borne virus that poses a significant public health threat. We aimed to evaluate a novel amplicon-based nanopore sequencing tool to obtain whole-genome sequences of Dabie bandavirus, also known as SFTS virus (SFTSV), and investigate the molecular prevalence in wild ticks, Republic of Korea (ROK). PRINCIPAL FINDINGS: A total of 6,593 ticks were collected from Gyeonggi and Gangwon Provinces, ROK in 2019 and 2020. Quantitative polymerase chain reaction revealed the presence of SFSTV RNA in three Haemaphysalis longicornis ticks. Two SFTSV strains were isolated from H. longicornis captured from Pocheon and Cheorwon. Multiplex polymerase chain reaction-based nanopore sequencing provided nearly full-length tripartite genome sequences of SFTSV within one hour running. Phylogenetic and reassortment analyses were performed to infer evolutionary relationships among SFTSVs. Phylogenetic analysis grouped SFTSV Hl19-31-4 and Hl19-31-13 from Pocheon with sub-genotype B-1 in all segments. SFTSV Hl20-8 was found to be a genomic organization compatible with B-1 (for L segment) and B-2 (for M and S segments) sub-genotypes, indicating a natural reassortment between sub-genotypes. CONCLUSION/SIGNIFICANCE: Amplicon-based next-generation sequencing is a robust tool for whole-genome sequencing of SFTSV using the nanopore platform. The molecular prevalence and geographical distribution of SFTSV enhanced the phylogeographic map at high resolution for sophisticated prevention of emerging SFTS in endemic areas. Our findings provide important insights into the rapid whole-genome sequencing and genetic diversity for the genome-based diagnosis of SFTSV in the endemic outbreak.

A Portable Diagnostic Assay, Genetic Diversity, and Isolation of Seoul Virus from Rattus norvegicus Collected in Gangwon Province, Republic of Korea.

Seoul virus (SEOV), an etiological agent for hemorrhagic fever with renal syndrome, poses a significant public health threat worldwide. This study evaluated the feasibility of a mobile Biomeme platform for facilitating rapid decision making of SEOV infection. A total of 27 Rattus norvegicus were collected from Seoul Metropolitan City and Gangwon Province in Republic of Korea (ROK), during 2016-2020. The serological and molecular prevalence of SEOV was 5/27 (18.5%) and 2/27 (7.4%), respectively. SEOV RNA was detected in multiple tissues of rodents using the Biomeme device, with differences in Ct values ranging from 0.6 to 2.1 cycles compared to a laboratory benchtop system. Using amplicon-based next-generation sequencing, whole-genome sequences of SEOV were acquired from lung tissues of Rn18-1 and Rn19-5 collected in Gangwon Province. Phylogenetic analysis showed a phylogeographical diversity of rat-borne orthohantavirus collected in Gangwon Province. We report a novel isolate of SEOV Rn19-5 from Gangwon Province. Our findings demonstrated that the Biomeme system can be applied for the molecular diagnosis of SEOV comparably to the laboratory-based platform. Whole-genome sequencing of SEOV revealed the phylogeographical diversity of orthohantavirus in the ROK. This study provides important insights into the field-deployable diagnostic assays and genetic diversity of orthohantaviruses for the rapid response to hantaviral outbreaks in the ROK.

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.

Genotyping and Molecular Diagnosis of Hepatitis A Virus in Human Clinical Samples Using Multiplex PCR-Based Next-Generation Sequencing.

Hepatitis A virus (HAV) is a serious threat to public health worldwide. We used multiplex polymerase chain reaction (PCR)-based next-generation sequencing (NGS) to derive information on viral genetic diversity and conduct precise phylogenetic analysis. Four HAV genome sequences were obtained using multiplex PCR-based NGS. HAV whole-genome sequence of one sample was obtained by conventional Sanger sequencing. The HAV strains demonstrated a geographic cluster with sub-genotype IA strains in the Republic of Korea. The phylogenetic pattern of HAV viral protein (VP) 3 region showed no phylogenetic conflict between the whole-genome and partial-genome sequences. The VP3 region in serum and stool samples showed sensitive detection of HAV with differences of quantification that did not exceed <10 copies/µL than the consensus VP4 region using quantitative PCR (qPCR). In conclusion, multiplex PCR-based NGS was implemented to define HAV genotypes using nearly whole-genome sequences obtained directly from hepatitis A patients. The VP3 region might be a potential candidate for tracking the genotypic origin of emerging HAV outbreaks. VP3-specific qPCR was developed for the molecular diagnosis of HAV infection. This study may be useful to predict for the disease management and subsequent development of hepatitis A infection at high risk of severe illness.

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.

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.

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.

Broad-Spectrum Antiviral Activity of 3D8, a Nucleic Acid-Hydrolyzing Single-Chain Variable Fragment (scFv), Targeting SARS-CoV-2 and Multiple Coronaviruses In Vitro.

The virus behind the current pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the etiology of novel coronavirus disease (COVID-19) and poses a critical public health threat worldwide. Effective therapeutics and vaccines against multiple coronaviruses remain unavailable. Single-chain variable fragment (scFv), a recombinant antibody, exhibits broad-spectrum antiviral activity against DNA and RNA viruses owing to its nucleic acid-hydrolyzing property. The antiviral activity of 3D8 scFv against SARS-CoV-2 and other coronaviruses was evaluated in Vero E6 cell cultures. Viral growth was quantified with quantitative RT-qPCR and plaque assay. The nucleic acid-hydrolyzing activity of 3D8 was assessed through abzyme assays of in vitro viral transcripts and cell viability was determined by MTT assay. We found that 3D8 inhibited the replication of SARS-CoV-2, human coronavirus OC43 (HCoV-OC43), and porcine epidemic diarrhea virus (PEDV). Our results revealed the prophylactic and therapeutic effects of 3D8 scFv against SARS-CoV-2 in Vero E6 cells. Immunoblot and plaque assays showed the reduction of coronavirus nucleoproteins and infectious particles, respectively, in 3D8 scFv-treated cells. These data demonstrate the broad-spectrum antiviral activity of 3D8 against SARS-CoV-2 and other coronaviruses. Thus, it could be considered a potential antiviral countermeasure against SARS-CoV-2 and zoonotic coronaviruses.

Phylogeographic diversity and hybrid zone of Hantaan orthohantavirus collected in Gangwon Province, Republic of Korea.

BACKGROUND: Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius (the striped field mouse), causes hemorrhagic fever with renal syndrome (HFRS) in humans. Viral genome-based surveillance at new expansion sites to identify HFRS risks plays a critical role in tracking the infection source of orthohantavirus outbreak. In the Republic of Korea (ROK), most studies demonstrated the serological prevalence and genetic diversity of orthohantaviruses collected from HFRS patients or rodents in Gyeonggi Province. Gangwon Province is a HFRS-endemic area with a high incidence of patients and prevalence of infected rodents, ROK. However, the continued epidemiology and surveillance of orthohantavirus remain to be investigated. METHODOLOGY/PRINCIPAL FINDINGS: Whole-genome sequencing of HTNV was accomplished in small mammals collected in Gangwon Province during 2015-2018 by multiplex PCR-based next-generation sequencing. To elucidate the geographic distribution and molecular diversity of viruses, we conducted phylogenetic analyses of HTNV tripartite genomes. We inferred the hybrid zone using cline analysis to estimate the geographic contact between two different HTNV lineages in the ROK. The graph incompatibility based reassortment finder performed reassortment analysis. A total of 12 HTNV genome sequences were completely obtained from A. agrarius newly collected in Gangwon Province. The phylogenetic and cline analyses demonstrated the genetic diversity and hybrid zone of HTNV in the ROK. Genetic exchange analysis suggested the possibility of reassortments in Cheorwon-gun, a highly HFRS-endemic area. CONCLUSIONS/SIGNIFICANCE: The prevalence and distribution of HTNV in HFRS-endemic areas of Gangwon Province enhanced the phylogeographic map for orthohantavirus outbreak monitoring in ROK. This study revealed the hybrid zone reflecting the genetic diversity and evolutionary dynamics of HTNV circulating in Gangwon Province. The results arise awareness of rodent-borne orthohantavirus diseases for physicians in the endemic area of ROK.

Genetic diversity and phylogeography of Jeju Orthohantavirus (Hantaviridae) in the Republic of Korea.

Orthohantaviruses are negative-sense, single-stranded RNA viruses harbored by rodents, shrews, moles, and bats. Of the shrew-borne orthohantaviruses in the Republic of Korea (ROK), Jeju orthohantavirus (Jeju virus, JJUV) was found on Jeju Island. This small-scale epidemiologic survey investigated the geographic distribution and molecular phylogeny of JJUV in the ROK. In 32 trapping sites, tissues of 84 Crocidura shantungensis were analyzed for JJUV RNA. JJUV RNA was detected in seven (8.3%) shrews captured on the Korean peninsula. The molecular epidemiologic survey demonstrated the prevalence of JJUV by geographic distribution. The RNA loads of JJUV were evaluated in various tissues. Entire coding sequences of tripartite genomes were recovered from two JJUV strains on the mainland. Phylogenetic relationships of the JJUV revealed a distinct geographic lineage of mainland strains from the strains on Jeju Island. This study sheds light on the molecular epidemiology, phylogeographic diversity, and virus-host co-divergence of JJUV, ROK.

Genomic Epidemiology and Active Surveillance to Investigate Outbreaks of Hantaviruses.

Emerging and re-emerging RNA viruses pose significant public health, economic, and societal burdens. Hantaviruses (genus Orthohantavirus, family Hantaviridae, order Bunyavirales) are enveloped, negative-sense, single-stranded, tripartite RNA viruses that are emerging zoonotic pathogens harbored by small mammals such as rodents, bats, moles, and shrews. Orthohantavirus infections cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome in humans (HCPS). Active targeted surveillance has elucidated high-resolution phylogeographic relationships between patient- and rodent-derived orthohantavirus genome sequences and identified the infection source by temporally and spatially tracking viral genomes. Active surveillance of patients with HFRS entails 1) recovering whole-genome sequences of Hantaan virus (HTNV) using amplicon (multiplex PCR-based) next-generation sequencing, 2) tracing the putative infection site of a patient by administering an epidemiological questionnaire, and 3) collecting HTNV-positive rodents using targeted rodent trapping. Moreover, viral genome tracking has been recently performed to rapidly and precisely characterize an outbreak from the emerging virus. Here, we reviewed genomic epidemiological and active surveillance data for determining the emergence of zoonotic RNA viruses based on viral genomic sequences obtained from patients and natural reservoirs. This review highlights the recent studies on tracking viral genomes for identifying and characterizing emerging viral outbreaks worldwide. We believe that active surveillance is an effective method for identifying rodent-borne orthohantavirus infection sites, and this report provides insights into disease mitigation and preparedness for managing emerging viral outbreaks.

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.

Effects of Panax ginseng extract on human dermal fibroblast proliferation and collagen synthesis.

Current studies of Panax ginseng (or Korean ginseng) have demonstrated that it has various biological effects, including angiogenesis, immunostimulation, antimicrobial and anti-inflammatory effects. Therefore, we hypothesised that P. ginseng may also play an important role in wound healing. However, few studies have been conducted on the wound-healing effects of P. ginseng. Thus, the purpose of this in vitro pilot study was to determine the effects of P. ginseng on the activities of fibroblasts, which are key wound-healing cells. Cultured human dermal fibroblasts were treated with one of six concentrations of P. ginseng: 0, 1, 10 and 100 ng/ml and 1 and 10 µg/ml. Cell proliferation was determined 3 days post-treatment using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, and collagen synthesis was evaluated by the collagen type I carboxy-terminal propeptide method. Cell proliferation levels and collagen synthesis were compared among the groups. The 10 ng/ml to 1 µg/ml P. ginseng treatments significantly increased cell proliferation, and the 1 ng/ml to 1 µg/ml concentrations significantly increased collagen synthesis. The maximum effects for both parameters were observed at 10 ng/ml. P. ginseng stimulated human dermal fibroblast proliferation and collagen synthesis at an optimal concentration of 10 ng/ml.

Expression and characterization of human vascular endothelial growth factor (VEGF165) in insect cells.

Vascular endothelial growth factor (VEGF) is the best characterized multifunctional protein which plays a key role in normal and pathologic angiogenesis. The gene encoding the human VEGF165 was cloned from the ovarian carcinoma cell line (OVCAR3) and expressed in insect cells using the baculovirus expression vector system. The recombinant human VEGF165 (rhVEGF165) protein produced by Sf21 (Spodoptera frugiperda) cells underwent a similar processing compared with mammalian cells, including efficient glycosylation, formation of a disulfide-linked dimer and secretion into the media. The rhVEGF165 had a high affinity for heparin and this characteristic was used to purify this form to homogeneity by heparin affinity, Resource S and Resource RPC columns. The biological activity of the purified 42-kDa homodimer was shown by the induction of the proliferation of human umbilical vein derived endothelial cells. These results demonstrate that an angiogenic growth factor whose normal processing requires glycosylation and disulfide-bridge formation can be efficiently expressed in high concentration (up to 20mg/L) in Sf21 cells.