Genetic Characterization of Japanese Encephalitis Virus Genotype 5 Isolated from Patient, South Korea, 2015.

We isolated Japanese encephalitis virus genotype 5 from human specimens in South Korea. Whole-genome analysis showed 90.4% identity with other genotype 5 viruses from humans. This virus had a unique insertion in the NS4A gene. However, the envelope protein contained Lys 84, which was specific to strains of genotype 5 viruses from South Korea.

Viral and serological kinetics in Zika virus-infected patients in South Korea.

Zika virus is a mosquito-borne flavivirus that causes clinical symptoms similar to those observed in dengue and chikungunya virus infections. The Korea Centers for Disease Control and Prevention initiated laboratory testing using a real-time reverse transcription-polymerase chain reaction in January 2016. More than 1,000 suspected cases of infection were tested and nine were confirmed as imported cases of Zika virus infection from January to July 2016. The travel destinations of the infected individuals were Brazil, Philippines, Viet Nam, Guatemala, Puerto Rico, and the Dominican Republic. Phylogenetic analysis based on the partial envelope gene indicated that the viruses belonged to the Asian genotype circulating in South America. We further investigated the duration for which the viral RNA and virus-specific antibodies were detectable after the symptom onset. After the day of symptom onset, Zika virus was detectable until 6 days in serum, 14 days in urine and saliva, and 58 days in semen. Immunoglobulin M against Zika virus was detected as early as 2 days after the symptom onset and was maintained at these levels until 41 days, whereas Immunoglobulin G was detectable from 8 days after the symptom onset and was maintained until 52 days. These findings would help diagnostic laboratories improve their testing programs for Zika virus infection.

First complete genomic characterization of two tick-borne encephalitis virus isolates obtained from wild rodents in South Korea.

We determined for the first time the complete genome sequences of two Korean strains of the tick-borne encephalitis virus (TBEV), designated KrM 93 and KrM 213, isolated from the lung tissues of wild rodents in 2006. The genomes are 11,097 nucleotides (nt) in length and consist of a 132 nt 5'-noncoding region (NCR), a 10,245 nt open reading frame (ORF) containing 10 viral protein-coding regions (3,415 amino acids), and a 720 nt 3'-NCR. Compared with the 31 fully sequenced TBEV strains currently available, KrM 93 and KrM 213 show genomic nucleotide (and deduced amino acid) sequence divergences ranging from 1.8 (0.7) to 19.2 (26.6)% and 1.9 (0.8) to 19.3 (26.7)%, respectively. Phylogenetic and recombination analyses based on the complete genome sequence were performed to identify genetic variations and relationships between the TBEV strains. These showed that the Korean TBEV strains clustered with the Western subtype rather than with Far-Eastern or Siberian subtypes, and phylogenetic trees derived from capsid (C), envelope (E), nonstructural (NS) 4B and NS5 regions represented the same branching pattern shown by the complete genome-based tree. Although no recombination events were identified in these two Korean strains, 11 putative recombination events were identified within the NS5 regions or in the 3'-NCRs of TBEV strains in general. The results provide insight into the genetics of TBEV strains to understand the molecular epidemiology, genetic diversity, and evolution of TBEV.

Molecular epidemiology of Japanese encephalitis virus circulating in South Korea, 1983-2005.

We sequenced the envelope (E) gene of 17 strains of the Japanese encephalitis virus (JEV) isolated in South Korea in 1983-2005 and compared the sequences with those from previously reported strains. Our results show the remarkable genetic stability of the E gene sequence in Korean JEV strains. Five pairs of E gene sequences from 10 Korean strains were identical, despite geographical differences and a maximum five-year time span. Sequence comparisons with other Asian strains revealed that the Korean strains are closely related to those from China, Japan, and Vietnam. Genotype 3 strains were predominant in Korea before 1993, when genotype 1 strain K93A07 was first isolated. The two genotypes were detected simultaneously in 1994 but since then, only genotype 1 has been isolated in South Korea. Thus, the genotype change occurred according to the year of isolation rather than the geographical origin.

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.

Comparison of the Epidemiological Aspects of Imported Dengue Cases between Korea and Japan, 2006-2010.

To compare the epidemiological characteristics of dengue cases imported by travelers or immigration in both Korea and Japan, we determined dengue incidence and related risk factors. During 2006-2010, 367 and 589 imported dengue cases were reported in Korea and Japan, respectively. In Korea, the presumptive origins for the dengue infections were Southeast Asia (82.6%), Southern Asia (13.9%), Eastern Asia (1.1%), South America (0.3%), Central America (0.3%), Africa (0.3%), and other countries (1.6%). In Japan, the origins of the infections were Southeast Asia (69.8%), Southern Asia (20.0%), Eastern Asia (1.7%), South America (2.5%), Central America (1.2%), Africa (1.2%), Oceania (2.4%), and other countries (1.2%). In both countries, more dengue cases were reported for men than for women (p < 0.01), and those aged 20-30 years accounted for > 60% of the total cases. The frequency of imported cases in summer and autumn (∼70% of total cases) was similar in both countries. This study demonstrates that there is a similar pattern of imported dengue cases in Korea and Japan. Therefore, there is a risk of an autochthonous dengue outbreak in Korea, as indicated by the recent outbreak in Japan in 2014.

Prevalence of Neutralizing Antibodies to Japanese Encephalitis Virus among High-Risk Age Groups in South Korea, 2010.

After an extensive vaccination policy, Japanese encephalitis (JE) was nearly eliminated since the mid-1980s in South Korea. Vaccination in children shifted the affected age of JE patients from children to adults. However, an abrupt increase in JE cases occurred in 2010, and this trend has continued. The present study aimed to investigate the prevalence of neutralizing antibodies to the JE virus (JEV) among high-risk age groups (≥40 years) in South Korea. A plaque reduction neutralization test was conducted to evaluate the prevalence of neutralizing antibodies to JEV in 945 subjects within four age groups (30-39, 40-49, 50-59, and 60-69 years) in 10 provinces. Of the 945 enrolled subjects, 927 (98.1%) exhibited antibodies against JEV. No significant differences were found in the prevalence of neutralizing antibodies according to sex, age, or occupation. However, there were significant differences in the plaque reduction rate according to age and occupation; oldest age group had a higher reduction rate, and subjects who were employed in agriculture or forestry also had a higher value than the other occupations. We also found that three provinces (Gangwon, Jeonnam, and Gyeongnam) had a relatively lower plaque reduction rate than the other locations. In addition, enzyme-linked immunosorbent assays were conducted to determine recent viral infections and 12 (1.3%) subjects were found to have been recently infected by the virus [corrected]. In conclusion, the present study clearly indicated that the prevalence of neutralizing antibodies has been maintained at very high levels among adult age groups owing to vaccination or natural infections, or both. In the future, serosurveillance should be conducted periodically using more representative samples to better understand the population-level immunity to JE in South Korea.

Complete Genome Sequences of Three Clinical Isolates of Dengue Virus Serotype 1 from South Korean Travelers.

In this study, we report the complete genome sequences of three clinical isolates of dengue virus serotype 1 isolated from South Korean travelers returning from different countries in Southeast Asia. The nucleotide sequence identities ranged from 91.5 to 92.2%, while the amino acid sequence identities ranged from 97.5 to 97.9% among the three clinical isolates.

Detection of Japanese encephalitis virus genotype V in Culex orientalis and Culex pipiens (Diptera: Culicidae) in Korea.

Japanese encephalitis virus (JEV) causes significant viral encephalitis and is distributed throughout the Asian countries. The virus is known to be transmitted by Culex tritaeniorhynchus, which mainly breeds in rice paddies in Korea. In this study, we investigated the presence of other mosquito species that can transmit JEV as a second or regional vector. We selected five cities where patients have experienced JE in the last 5 years as mosquito-collecting locations and subdivided them into four collection sites according to the mosquito habitats (cowshed, downtown area, forest, and swamp). Mosquitoes were caught using the BG-Sentinel trap, CDC black-light trap, Fay-Prince trap, and Gravid trap. A total of 993 pools from 22,774 mosquitoes were prepared according to their species, collection date, and site. We performed a SYBR Green 1-based real-time RT-PCR assay to detect JEV from the mosquito pools. A total of six JEV-positive pools were detected from Culex orientalis and Culex pipiens caught in the Gangwon-do and Gyeonngi-do provinces. All the detected JEVs were revealed as genotype V by phylogenetic analysis of the envelope gene. Our findings confirm that a new genotype of JEV was introduced in Korea and suggest that two mosquito species may play a role in JEV transmission.

A novel immunochromatographic test applied to a serological survey of Japanese encephalitis virus on pig farms in Korea.

Among vertebrate species, pigs are a major amplifying host of Japanese encephalitis virus (JEV) and measuring their seroconversion is a reliable indicator of virus activity. Traditionally, the hemagglutination inhibition test has been used for serological testing in pigs; however, it has several limitations and, thus, a more efficient and reliable replacement test is required. In this study, we developed a new immunochromatographic test for detecting antibodies to JEV in pig serum within 15 min. Specifically, the domain III region of the JEV envelope protein was successfully expressed in soluble form and used for developing the immunochromatographic test. The test was then applied to the surveillance of Japanese encephalitis (JE) in Korea. We found that our immunochromatographic test had good sensitivity (84.8%) and specificity (97.7%) when compared with an immunofluorescence assay used as a reference test. During the surveillance of JE in Korea in 2012, the new immunochromatographic test was used to test the sera of 1,926 slaughtered pigs from eight provinces, and 228 pigs (11.8%) were found to be JEV-positive. Based on these results, we also produced an activity map of JEV, which marked the locations of pig farms in Korea that tested positive for the virus. Thus, the immunochromatographic test reported here provides a convenient and effective tool for real-time monitoring of JEV activity in pigs.

Cloning and Expression of Recombinant Tick-Borne Encephalitis Virus-like Particles in Pichia pastoris.

OBJECTIVES: The purpose of this study was to verify the feasibility of using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promotor based Pichia pastoris expression system to produce tick-borne encephalitis virus (TBEV) virus-like particles (VLPs). METHODS: The complementary DNA encoding the TBEV prM signal peptide, prM, and E proteins of TBEV Korean strain (KrM 93) was cloned into the plasmid vector pGAPZɑA, then integrated into the genome of P. pastoris, under the control of the GAP promoter. Expression of TBEV VLPs was determined by Western blotting using monoclonal antibody against TBEV envelope (E) protein. RESULTS: Recombinant TBEV VLPs consisting of prM and E protein were successfully expressed using the GAP promoter-based P. pastoris expression system. The results of Western blotting showed that the recombinant proteins were secreted into the culture supernatant from the P. pastoris and glycosylated. CONCLUSION: This study suggests that recombinant TBEV VLPs from P. pastoris offer a promising approach to the production of VLPs for use as vaccines and diagnostic antigens.

Comparison of four serological tests for detecting antibodies to Japanese encephalitis virus after vaccination in children.

OBJECTIVES: Several different methods are currently used to detect antibodies to Japanese encephalitis virus (JEV) in serum samples or cerebrospinal fluid. These methods include the plaque reduction neutralization test (PRNT), the hemagglutination inhibition (HI) test, indirect immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA). The purpose of this study was to compare the performance of each method in detecting vaccine-induced antibodies to JEV. METHODS: The study included 29 children who had completed a primary immunization schedule with an inactivated vaccine against JEV derived from mouse brain (n = 15) or a live attenuated SA14-14-2 vaccine (n = 14). Serum samples were collected between 3 months and 47 months after the last immunization. The serum samples were tested by performing the PRNT, HI test, in-house IFA, and commercial ELISA. The antibody detection rates were compared between tests. RESULTS: All 29 serum samples were positive with the PRNT, showing antibody titers from 1:20 to 1:2560. The HI test showed positive rates of 86.7% (13/15) and 71.4% (10/14) in the inactivated and live attenuated vaccine groups, respectively. The results of the IFA for immunoglobulin (Ig)G were positive in 53.3% (8/15) of children in the inactivated vaccine group and 35.7% (5/14) in the live attenuated vaccine group. Neither the IFA nor ELISA detected JEV IgM antibodies in any of the 29 children. CONCLUSION: These results show that detection rates of vaccine-induced antibodies to JEV have a wide range (0-100%) depending on the testing method as well as the time since immunization and individual differences between children. These findings are helpful in interpreting serological test results for the diagnosis of Japanese encephalitis in situations where vaccines are widely administered.

Travel-Associated Chikungunya Cases in South Korea during 2009-2010.

OBJECTIVES: Chikungunya (CHIK) has been classified as a communicable disease group IV in South Korea since late 2010. Based on this, we investigated the extent of imported cases of CHIK in dengue-suspected individuals returning from dengue-endemic regions. METHODS: A total of 486 dengue-suspected serum samples were screened for CHIK by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Further RT-PCR-positive samples were used for the viral culture, and CHIK was subsequently confirmed by sequence analysis of the culture samples. RESULTS: Five out of 107 dengue-positive samples were found to be positive for CHIK and 15 out of 379 dengue-negative samples were found to be positive for CHIK by immunoglobulin M ELISA. Further, a CHIK virus was isolated from one of the two RT-PCR-positive sera by cell culture and confirmed by sequence analysis. CONCLUSION: The present study documents the first evidence of travel-associated CHIK infection in South Korea. Considering the intense international traffic between countries, our finding emphasizes the urgent need for active patient and vector surveillance for timely response to reduce the introduction of CHIK in Korea.

Epidemiologic features of animal bite cases occurring in rabies-endemic areas of Korea, 2005 to 2009.

OBJECTIVES: Human rabies is a reemerging infectious disease in Korea. There was no human rabies case for 14 years until the disease had reoccurred in 1999. To prevent occurrence of human rabies, surveillance for animal bite patients in rabies endemic areas in Korea was conducted since 2005 as a part of a human rabies control program. The animal bite cases were analyzed to determine whether patients were treated according to the post-exposure prophylaxis (PEP) guideline of the Korea Centers for Disease Control and Prevention. METHODS: Information of animal bite cases that occurred from 2005 to 2009 in rabies high-risk regions were collected by cooperation with Regional Public Health Centers in 18 cities/districts of rabies endemic areas. RESULTS: A total of 2458 animal bite cases were reported. Dogs accounted for 86% of animal bites and 67% of the animals were not vaccinated against rabies virus. For PEP, among rabies-vaccinated animals, 92.7% were observed for clinical signs and 1.4% underwent necropsy. Among unvaccinated animals, 72.7% were observed for clinical signs and 4.1% underwent necropsy. The remaining animals were not available for examination. Of the animal bite patients, 32.5% received PEP and 51.6% were treated by first aid or by washing the wound. CONCLUSIONS: Given that no human rabies cases were reported since 2005 and animal rabies was continuously reported in endemic areas of Korea, the human rabies control program implemented in 2005 appears to have a significant role in the prevention and control of human rabies.

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.

Serum MicroRNA Expression Profiling in Mice Infected with Rabies Virus.

OBJECTIVES: Serum or plasma microRNAs (miRNAs) are potential biomarkers for the diagnosis for cancer and prenatal diseases. This study was conducted to investigate whether rabies virus causes a change in serum miRNA expression. METHODS: ICR mice were intramuscularly inoculated with rabies virus and were sacrificed weekly to collect serum and brain tissue for 4 weeks postinoculation. Mice were assigned to four groups based on the results of indirect immunofluorescent assays, enzyme-linked immunosorbent assay, and nested reverse transcription-polymerase chain reaction and the expression profiles of serum miRNAs were compared using a commercial mouse miRNA expression profiling assay. RESULTS: The expression levels of miRNAs changed significantly with the different stages of the disease. The expression level of 94 serum miRNAs in infected mice changed at least twofold. Seven microRNAs of them were significantly upregulated or downregulated in all infected mice regardless of disease status. The number of miRNAs with an expression level change decreased with the progression of the disease. In a hierarchical cluster analysis, infected mice clustered into a group separate from uninfected control mice. CONCLUSIONS: Based on the relationship of miRNAs to gene expression regulation, miRNAs may be candidates for the study of viral pathogenesis and could have potential as biomarkers.

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.

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.