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Research

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Emerging Infectious Disease and Vector Research

Research fields

  • Research on the infection control of new emerging viruses and vector-borne viruses
  • Research on diagnosis, therapy and vaccine of high risk hemorrhagic fever viruses
  • Research on unknown infectious diseases

Research contents

  • Research on the new emerging respiratory virus and hemorrhagic fever virus
    • Study on diagnosis, therapeutic antibody and vaccine of new respiratory virus, such as MERS-CoV and Henipavirus
    • Study on diagnosis and therapeutic antibody of high risk hemorrhagic fever virus, such as Ebola and Crimean-Congo virus
  • Research on the vector-borne virus
    • Development of differential diagnosis between flavivirus
    • Development of antiviral compounds and vaccine candidates for flavivirus, such as Zika virus and West Nile virus
    • Development of therapeutic agent and relate fundamental research for Severe Fever with Thrombocytopenia Syndrome (SFTS) virus
  • Research on the development of a new emerging virus and a vector-borne virus resource and evaluation system
  • Research on international cooperation of new emerging viruses and arbovirus

Research

Middle East Respiratory Syndrom Coronavirus

    The Middle East respiratory syndrome coronavirus (MERS-CoV), which spreads to humans from zoonotic sources and causes severe acute respiratory diseases with high fatality rates, is of global health concern. In Korea, the first MERS case was identified on May 20, 2015, and a total of 186 cases and 38 deaths due to MERS-CoV were reported. Unfortunately, there is currently no specific vaccine or treatment for MERS-CoV infection. In addition, the potential for the emergence of variants remains unclear, although it has been reported for genetic factors related to human infection. Therefore, we have been developing therapeutic diagnostics and investigating the virologic characteristics of MERS-CoV.

Development of human monoclonal antibodies to neutralize MERS-CoV infection

    B cells from convalescent MERS patients were used to develop therapeutic monoclonal antibodies (mAbs) to neutralize MERS-CoV infection. We screened and selected fully human mAbs with high affinities for the spike (S) protein of MERS-CoV by ELISA. Among MERS-CoV spike-specific antibodies, human antibodies were found to have neutralizing activity against MERS-CoV in plaque reduction neutralization tests (PRNTs). They also had MERS-CoV specificity, as they did not bind to other human coronaviruses in IFAs. These antibodies could be utilized as therapeutic or prophylactic agents in MERS-CoV. Furthermore, ongoing research aims to investigate the physical characteristics of these mAbs and their neutralizing abilities in vivo in a human DPP4 knock-in mouse model.

Genetic and virological variation of MERS-CoV in cell culture passages

    To investigate viral characteristics including genetic changes, we identified two variants of Korean isolates in serial passage in animal cells. To study the effects of these genetic changes on pathogenicity, we analysed the growth kinetics, plaque morphology, and expression patterns of inflammatory cytokines of these viruses (Figure 1). The results implied that truncated genes are not essential for virus replication in vitro and that these proteins may affect inflammation inhibition induced by MERS-CoV infection. Thus, there is the potential for the emergence of MERS-CoV variants and continuous investigation is needed.
    Figure 1. Cytopathic effect of two MERS-CoV mutants from cell-culture passages.
    Figure 1. Cytopathic effect of two MERS-CoV mutants from cell-culture passages.

Characteristics of MERS-CoV escape mutants under selective pressure of neutralizing antibodies

    The receptor binding domain (RBD) of the MERS-CoV S glycoprotein (GP) is a major target of protective immunity in humans. Although a large number of neutralizing antibodies (nAbs) have been developed, escape variants able to undergo viral replication in the presence of a monoclonal antibody (mAb) have emerged. In a previous study, we developed mAb with high MERS-CoVs neutralizing activity. Therefore, to investigate viral mutation under mAb pressure, we isolated escape mutants from the EMC strain and Korean isolate in the presence antibody and analysed their genetic sequences. All escape mutants contained mutations in the RBD region, with reduced antibody neutralizing activity compared to that of the wild-type (Figure 2). These data indicated that MERS-CoVs can sufficiently mutate in the presence of suitable neutralizing antibodies; therefore, neutralizing antibody immunotherapy strategy must consider the prevention of escape variants.

Development of diagnostic resources for emerging respiratory viruses

    Virus isolation is critical for viral characterization. However, the isolation percentage of MERS-CoVs from clinical specimens was low, at approximately 11%. Thus, we improved the virus culture system by using chemicals to enhance virus attachment and fusion. Using this method, additional six MERS-CoV strains from 11 clinical specimens were isolated from MERS patients diagnosed in 2015. Serological assays of MERS-CoVs were conducted to evaluate prior exposure, immunity, and contact investigation. Recombinant ELISA using nucleocapsid (N) and S proteins were generally used to identify the presence of MERS-CoV antibodies. To rapidly prepare a serological assay for emerging infectious diseases such as MERS, we developed and evaluated ELISA for serological diagnosis by producing and purifying the N protein using an in vitro cell-free protein expression system. The system has the advantage of rapid protein expression and readily posttranslational modification for high quality. Moreover, the virus-neutralizing activity of sera has been evaluated using a pseudoviral neutralizing assay (PNA) due to safety concerns, especially for highly pathogenic viruses such as MERS-CoV. Thus, we have developed stable cell lines containing basic genes such as gal, pol, and reporter genes to effectively generate pseudoviruses to develop a PNA for highly pathogenic emerging infectious diseases.

Severe Fever with Thrombocytopenia Syndrome Virus

Molecular genomic characterization of tick- and human-derived severe fever with thrombocytopenia syndrome virus (SFTS) isolates from South Korea

    SFTS is an emerging tick-borne viral disease caused by the SFTS virus (SFTSV) endemic to East Asia. However, the genetic and evolutionary characteristics shared between tick- and human-derived Korean SFTSV strains remain unknown. In a previous surveillance study of SFTSV infection in ticks in South Korea, a single SFTSV strain, KAGWT, was initially isolated from Haemaphysalis longicornis nymphs collected in Samcheok-si, Gangwon Province. We performed whole-genome sequencing of the first tick-derived Korean SFTSV strain and compared it with human SFTSV isolates to explore the genetic variations and relationships to other SFTSV strains. The genome of the KAGWT strain is consistent with 6,368 nucleotides (nt), 3,378 nt, and 1,746 nt of the L, M, and S segments, respectively. Compared to completely sequenced human-derived Korean SFTSVs, the KAGWT strain had the highest nucleotide sequence identity and deduced amino acid level for each segment compared to the KAGWH3 strain isolated from an SFTS patient in the same region, with only a single unique amino acid substitution in the Gn protein (A66S). Phylogenetic analyses of genome sequences revealed that at least four different genotypes of SFTSV are co-circulating in South Korea and that both tick- and human-derived Korean SFTSV in this study are closely related to genotype B strains (Figure 3). These findings provide scientific data for the molecular epidemiology, genetic diversity, and evolution of SFTSV in East Asia. A further research direction is the construction of an infectious full-length clone rescue system of a tick-derived Korean SFTSV strain utilizing reverse genetics. Figure 3. Phylogenetic analysis of SFTS strains
       Figure 3. Phylogenetic analysis based on the complete nucleotide sequences of the L (A), S (B), and M (C) segments of SFTSV strains using the maximum likelihood (ML) method based on the Kimura two-parameter model.

Identification of cellular factors as therapeutic substances against SFTSV

    SFTSV causes severe fever, thrombocytopenia, and vomiting diarrhoea in humans. The regulation of the virus life cycle strongly relies on host cellular proteins. To elucidate the interaction between SFTSV and infected hosts, we employed a high-throughput protein microarray chip composed of approximately 15,000 human proteins to identify the host cellular candidates that interact specifically with the nucleoprotein (NP), nonstructural (NSs), or GP SFTS viral proteins. As a result, we have identified host cellular proteins that interact with the NSs and NP proteins. The gene ontological functions of the identified hits were analysed and a protein map with the identified candidates was constructed. The interaction of these candidates and NP was confirmed by immunoprecipitation assay in co-transfected cells. NP plays important roles in the regulation of viral life cycles including RNA encapsidation, transcription, and replication. To examine the function of the identified NP interactors on SFTSV propagation, specific siRNAs against host proteins were constructed and further exploration of the detailed mechanisms of interaction between NP and host proteins are ongoing. The host candidates identified in this study may be promising targets for the development of efficient antiviral drugs against SFTSV.

Vector-borne Viruses

Evaluation and screening of antiviral compounds against Zika virus

    Zika virus (ZIKV) was first identified from a Rhesus monkey in the Zika Forest of Uganda in 1947. ZIKV is a member of the flaviviridae family and is closely related to other flaviviruses such as the dengue virus (DENV), the West Nile virus (WNV), the yellow fever virus (YFV), and the Japanese encephalitis virus. The extent of the epidemic and the severity of the associated birth defects prompted the World Health Organization (WHO) to declare it a Public Health Emergency of International Concern on February 2016. In Korea, ZIKV disease was designated as a class 4 legal infectious disease on January 29, 2016. Currently, there are no commercial vaccines or therapeutics for ZIKV. At present, research is active globally but is incomplete in Korea. Therefore, we secured type culture strain (MR-766) and Korean isolate strains for the development of potential anti-Zika viral agents. We also calculated the lethal dose 50 values (LD50) in transgenic mice that were inoculated with these ZIKV strains. In addition, we optimized an in vitro cell-based therapeutic-efficacy screening assay using the immunofluorescence-based biosensor for the identification of novel anti-ZIKV agents to identify two candidate therapeutic compounds. In vitro and in vivo efficacy verification of the candidate therapeutic compounds is ongoing.

Development of vaccine candidates against the West Nile virus

    The West Nile virus (WNV), a member of the genus Flavivirus in the family Flaviviridae, contains single-stranded, positive-sense RNA. WNV is genetically and geographically diverse, with a current total of nine reported lineages. WNVs from lineages 1, 2, and 5 cause pathogenicity in humans. Although many studies have been performed, there is no licensed vaccine. This study aims to develop a vaccine candidate. The sequences of the E protein of WNV lineages 1 and 2 were first confirmed to have 94% homology to each other. A total of 18 proteins were then designed in accordance with each E protein and domain. Efficacy analysis will be performed through the evaluation of toxicity and immunogenicity in animal models. In further study, development of a WNV inactivated vaccine requires the adjustment of the chemical concentrations and inactivation times. Both formalin and hydrogen peroxide (H2O2) are used for WNV inactivation. To evaluate the protective effect of vaccine candidates, vaccinated AG129 mice are challenged with 385-99 or B956 strains.

Advanced techniques for laboratory diagnosis of the haemorrhagic fever renal syndrome (HFRS)

    HFRS, caused by hantaviruses (HTNV), is a leading public health problem in Asia and Europe. The clinical symptoms are fever, hypotension, haemorrhage, thrombocytopenia, and renal failure. Because of the similarity in symptoms with other haemorrhagic fever viruses, the accurate and early diagnosis of HFRS is important and required for proper treatment. We established and evaluated HTNV-specific IgG and IgM ELISAs for serological diagnostic testing of HTNV, a major causative agent of Korean HFRS. The 100-aa N-terminal sequence of NP is a major inducer of humoral immune response; thus, a truncated N protein of 1-117 amino acids was designed for the detection of virus-specific antibodies in serum. Next, we employed the truncated protein into IgG or IgM ELISA and evaluated the sensitivity and specificity compared to that of commercial ELISA kits (IgG or IgM). A total of 120 human sera positive for HTNV confirmed by IFA showed the high sensitivity and specificity in the developed HTNV-specific IgG and -IgM ELISAs. The protein did not cross-react with other haemorrhagic fever viruses. As a molecular diagnostic assay, we aimed to develop a real-time RT-qPCR for five HTNV strains. With specific primer/probe sets for the S segment of each virus and synthesized in vitro viral RNA for Korean HTNV, Seoul virus, Imjin virus, Muju virus, and Soochung virus, we determined the sensitivity and specificity of the strain-specific real-time RT-qPCRs. Taken together, the developed assay with serological and gene-based methods could be used as a diagnostic system for the accurate detection of HFRS.
Figure 2. Cytopathic effets
Figure 2. Cytopathic effect of escape mutants cultured in cells with antibodies.
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