Young Ran Ju

Isolation of Tick-Borne Encephalitis Viruses from Wild Rodents, South Korea

To determine whether the tick-borne encephalitis virus (TBEV) is present in vector ticks and mammalian hosts in Korea, we examined two tick species, Haemaphysalis longicornis (n = 548) and Ixodes nipponensis (n = 87), and the lungs or spleens of rodents Apodemus agrarius (n = 24) and wild boars (n = 16). Tick-borne encephalitis virus was detected in samples by reverse transcriptase (RT)-nested polymerase chain reaction (PCR), after which TBEV-positive samples were inoculated into BHK-21 cells and suckling mice. Tick-borne encephalitis virus genes were detected in 4 of 38 tick pools and 5 of 24 wild rodents. Suckling mice inoculated intracerebrally with TBEV-positive rodent samples showed signs of encephalitis at six days post-inoculation. The isolation of TBEV was confirmed by inoculating samples obtained from the brains of sick mice in cell culture. Phylogenetic analysis showed that the E genes of the TBEV isolates were clustered with the Western subtype (98% identity). This study suggests the possible occurrence of tick-borne encephalitis in Korea.

Prevalence of tick-borne encephalitis virus in ticks from southern Korea

The prevalence of tick-borne encephalitis virus (TBEV) in southern Korea was determined by collecting ticks using tick drags. A total of 4,077 of 6,788 ticks collected were pooled (649 pools) according to collection site, species, and developmental stage and assayed for TBEV. The TBEV protein E and NS5 gene fragments were detected using RT-nested PCR in six pools of nymphs collected from Jeju Island (2,491 ticks). The minimum field detection rates for TBEV were 0.17% and 0.14% for Haemaphysalis longicornis and Haemayphysalis flava nymphs, respectively. The 252 bp NS5 and 477 bp protein E gene amplicons were sequenced. Phylogenetic analysis showed that the NS5 and protein E genes of the Jeju strain were clustered with Western subtype (98.0% and 99.4% identity, respectively). The Western subtype of TBEV is endemic in Korea, including Jeju Island. The study of vector and zoonotic host susceptibility to TBEV is required to better understand its potential impact on public health.

Characterizing affinity epitopes between prion protein and β-amyloid using an epitope mapping immunoassay.

Cellular prion protein, a membrane protein, is expressed in all mammals. Prion protein is also found in human blood as an anchorless protein, and this protein form is one of the many potential sources of misfolded prion protein replication during transmission. Many studies have suggested that β-amyloid1–42 oligomer causes neurotoxicity associated with Alzheimer’s disease, which is mediated by the prion protein that acts as a receptor and regulates the hippocampal potentiation. The prevention of the binding of these proteins has been proposed as a possible preventative treatment for Alzheimer’s disease; therefore, a greater understanding of the binding hot-spots between the two molecules is necessary. In this study, the epitope mapping immunoassay was employed to characterize binding epitopes within the prion protein and complementary epitopes in β-amyloid. Residues 23–39 and 93–119 in the prion protein were involved in binding to β-amyloid1–40 and 1–42, and monomers of this protein interacted with prion protein residues 93–113 and 123–166. Furthermore, β-amyloid antibodies against the C-terminus detected bound β-amyloid1–42 at residues 23–40, 104–122 and 159–175. β-Amyloid epitopes necessary for the interaction with prion protein were not determined. In conclusion, charged clusters and hydrophobic regions of the prion protein were involved in binding to β-amyloid1–40 and 1–42. The 3D structure appears to be necessary for β-amyloid to interact with prion protein. In the future, these binding sites may be utilized for 3D structure modeling, as well as for the pharmaceutical intervention of Alzheimer’s disease.

Analysis of the Envelope (E) Protein Gene of Tick-Borne Encephalitis Viruses Isolated in South Korea

We determined the nucleotide and deduced amino acid sequences of the complete envelope (E) protein gene of the five tick-borne encephalitis virus (TBEV) strains KrM 93, KrM 213, KrM 215, KrM 216, and KrM 219, isolated from wild rodents in South Korea. We analyzed genetic variability within the isolates and compared them with 13 other TBEV strains. The complete E protein genes were amplified by reverse transcription polymerase chain reaction (RT-PCR), cloned into pGEM-T vectors, and sequenced. The five isolates were similar to the Western subtype in nucleotide and deduced amino acid sequences (97%-99% identity) and by phylogenetic analysis. The deduced amino acid alignments had 13 unique amino acids, as in the Western subtypes. Among the signature amino acids, those at positions 206 and 317 were unique to each subtype. We were also able to identify amino acid substitutions in each of the three domains when comparing the 5 Korean isolates with the 13 other TBEV strains. Thus, we confirmed that the 5 Korean isolates belong to the Western subtype. These data will provide useful information for the development of an effective recombinant vaccine.

Prion Diseases as Transmissible Zoonotic Diseases

Abstract Prion diseases, also called transmissible spongiform encephalopathies (TSEs), lead to neurological dysfunction in animals and are fatal. Infectious prion proteins are causative agents of many mammalian TSEs, including scrapie (in sheep), chronic wasting disease (in deer and elk), bovine spongiform encephalopathy (BSE; in cattle), and Creutzfeldt–Jakob disease (CJD; in humans). BSE, better known as mad cow disease, is among the many recently discovered zoonotic diseases. BSE cases were first reported in the United Kingdom in 1986. Variant CJD (vCJD) is a disease that was first detected in 1996, which affects humans and is linked to the BSE epidemic in cattle. vCJD is presumed to be caused by consumption of contaminated meat and other food products derived from affected cattle. The BSE epidemic peaked in 1992 and decreased thereafter; this decline is continuing sharply owing to intensive surveillance and screening programs in the Western world. However, there are still new outbreaks and/or progression of prion diseases, including atypical BSE, and iatrogenic CJD and vCJD via organ transplantation and blood transfusion. This paper summarizes studies on prions, particularly on prion molecular mechanisms, BSE, vCJD, and diagnostic procedures. Risk perception and communication policies of the European Union for the prevention of prion diseases are also addressed to provide recommendations for appropriate government policies in Korea.

Prevalence of Tick-Borne Encephalitis Virus in Ixodid Ticks Collected from the Republic of Korea During 2011–2012

Objectives In this study, we demonstrated that TBEV-infected ticks have been distributed in the ROK, combined with our previous results. These results suggest that TBEV may exist in the ROK, and H. longicornis, H. flava, and I. nipponensis may be potential vectors of TBEV. In addition, these results emphasize the need for further epidemiological research of TBEV. Methods We examined for the presence of RNA of TBEV by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) using ixodid ticks captured in 25 localities of 10 provinces. Ticks were collected by the flagging and dragging method or using sentinel BG traps at forests, grass thickets, and grassland. A total of 13,053 ticks belonging to two genera and four species were collected and pooled (1292 pools), according to collection site, species of tick, and developmental stage. Results Among 1292 pools, the envelope (E) protein gene of TBEV was detected using RT-nested PCR in 10 pools (3 pools of the 1,331 adult ticks and 7 pools of the 11,169 nymph ticks) collected from Gangwon-do province, Jeonrabuk-do province, and Jeju Island. The minimum infection rates for TBEV of Haemaphysalis longicornis, Haemaphysalis flava, and Ixodes nipponensis were 0.06%, 0.17%, and 2.38%, respectively. Phylogenetic analysis based on the partial E protein gene was performed to identify relationships between the TBEV strains. This showed that 10 Korean strains clustered with the Western subtype. Conclusion In this study, we investigated the prevalence of tick-borne encephalitis virus (TBEV) in ixodid ticks from various regions of the Republic of Korea (ROK) during 2011–2012 to identify whether TBEV is circulating and to determine the endemic regions of TBEV.

Historical review and surveillance of Japanese encephalitis, Republic of Korea, 2002–2004

Japanese encephalitis virus (JEV), transmitted by culicine mosquitoes, is endemic throughout much of South‐East Asia, extending to the Korean Peninsula. The zoonotic cycle is from large water birds to culicine mosquitoes, with swine as an amplifying host and man as an incidental host. Culex tritaeniorhynchus, the primary JEV vector in the Republic of Korea, populations peak in late August through to early September when most cases of Japanese encephalitis (JE) are reported. Cx. tritaeniorhynchus were observed near the Demilitarized Zone in each of the years that mosquitoes were assayed for JEV. Each year that vector mosquitoes were assayed for JEV, minimum field infection rates (number of JEV positive mosquites/1000 Cx. tritaeniorhynchus assayed) ranged from 0.31 to 3.27. The epidemiology of JE has been recorded in Korea for more than half a century, from 1949 to 2005. During a major epidemic in 1949, there were 5616 cases and 2729 deaths reported, with levels persisting near epidemic levels of 1000 cases annually thereafter until 1969. Following the introduction and government mandated mass immunization in 1971, JE decreased dramatically. Since 1984, 0–6 cases of JE have been reported each year. However, continued evidence of mosquitoes positive for JEV indicates that JE continues to be a civilian and military health threat to immunocompromised persons in Korea, as well as non‐immune US soldiers, civilians and their family members.

Review: Laboratory diagnosis and surveillance of Creutzfeldt–Jakob disease

Creutzfeldt–Jakob disease (CJD) is a representative human transmissible spongiform encephalopathy associated with central nervous system degeneration. Prions, the causative agents of CJD, are composed of misfolded prion proteins and are able to self‐replicate. While CJD is a rare disease affecting only 1–1.5 people per million worldwide annually, it has attracted both scientific and public attention as a threatening disease since an epidemic of variant CJD (vCJD) cases appeared in the mid‐1990s. Due to its unconventional transmission and invariable fatality, CJD poses a serious risk to public health. The hundreds of sporadic, genetic, and iatrogenic CJD cases as well as potential zoonotic transmission suggest that CJD is an ongoing concern for the field of medicine. Nevertheless, treatment aimed at clinical prevention and treatment that reverses the course of disease does not exist currently. Active surveillance and effective laboratory diagnosis of CJD are, therefore, critical. In this report, the surveillance systems and laboratory tests used currently to diagnose CJD in different countries are reviewed. The current efforts to improve surveillance and diagnosis for CJD using molecular and biochemical findings are also described. J. Med. Virol. 87: 175–186, 2015.

Identification of Dengue Type 1 Virus (DENV-1) in Koreans Traveling Abroad

Objectives To date, no indigenous dengue virus (DENV) transmissions have been reported in Korea. However, imported dengue infections have been diagnosed in travelers returning from endemic areas. This study presents the first virological evidence of travel-associated DENV importation into South Korea. Methods From January 2004 to June 2006, a total of 278 serum samples from 245 patients with suspected dengue fever were tested using the Panbio Dengue Duo IgM/IgG Rapid Strip Test. We selected 11 of the early symptomatic-phase sera that were negative for IgM and retrospectively studied them by virus isolation and reverse transcription-polymerase chain reaction. Results All 11 serum samples were found to be DENV positive by reverse transcription-polymerase chain reaction and viruses were successfully isolated from seven of the 11 serum samples. All the isolates were identified as DENV serotype-1. Conclusion We successfully isolated seven DENV serotype-1 strains for the first time in South Korea from imported infections. Considering that the vector mosquito, Aedes albopictus, already exists in South Korea, we propose that a vector surveillance program for dengue is urgently needed.

Development and field evaluation of a nested RT-PCR kit for detecting Japanese encephalitis virus in mosquitoes

A novel nested reverse transcription-polymerase chain reaction (RT-PCR)-based kit is described for detecting Japanese encephalitis virus (JEV), especially for genotype 1 and 3 strains. The assay consists of a first round RT-PCR and a subsequent nested PCR amplification. It has unique features such as the use of a premix system in which all reagents are lyophilized in reaction tubes and the inclusion of control RNA in each reaction to monitor false negative results. In addition, an automatic tissue homogenizer and a RNA extraction system are used concurrently for assay standardization and increasing throughput. The assay using the kit proved specific for JEV with no amplification of other JEV-related flaviviruses. The detection limits were approximately 0.1 PFU/ml and 1 PFU/ml for JEV genotypes 1 and 3, respectively. The assay protocol has been validated in large-scale field trials in South Korea during the 2008-2009 surveillance seasons. Nineteen of 1136 pools of mosquitoes (54,583 mosquitoes total) were identified as JEV positive. This nested RT-PCR kit combined with control RNA and an automatic RNA extraction system should be suitable for routine JEV surveillance programs.