Emergence of Japanese encephalitis virus genotype V in the Republic of Korea

  • Ratree Takhampunya1,

    Affiliated with

    • Heung-Chul Kim2,

      Affiliated with

      • Bousaraporn Tippayachai1,

        Affiliated with

        • Ampornpan Kengluecha1,

          Affiliated with

          • Terry A Klein3,

            Affiliated with

            • Won-Ja Lee4,

              Affiliated with

              • John Grieco5 and

                Affiliated with

                • Brian P Evans1Email author

                  Affiliated with

                  Virology Journal20118:449

                  DOI: 10.1186/1743-422X-8-449

                  Received: 8 August 2011

                  Accepted: 23 September 2011

                  Published: 23 September 2011

                  Abstract

                  Background

                  Japanese encephalitis virus (JEV) genotype V reemerged in Asia (China) in 2009 after a 57-year hiatus from the continent, thereby emphasizing a need to increase regional surveillance efforts. Genotypic characterization was performed on 19 JEV-positive mosquito pools (18 pools of Culex tritaeniorhynchus and 1 pool of Cx. bitaeniorhynchus) from a total of 64 positive pools collected from geographically different locations throughout the Republic of Korea (ROK) during 2008 and 2010.

                  Findings

                  Two regions of the JEV genome were sequenced from 19 pools; the envelope gene and the nonstructural protein 5 (NS5)/3'-untranslated region (UTR). Eighteen pools of Culex tritaeniorhynchus and one pool of Cx. bitaeniorhynchus were positive for genotype I and genotype V, respectively. Sequence alignment of the complete E gene from Cx. bitaeniorhynchus showed high amino acid similarity (98.8%) to the Muar strain, characterized as the first report of genotype V, isolated from an encephalitis patient in Malaysia in 1952.

                  Conclusion

                  This study represents the first report of JEV genotype V in the ROK. The reemergence of genotype V in Asia (China and ROK) after more than a half-century and its discovery in Cx. bitaeniorhynchus, a mosquito species previously unknown to carry JEV in the ROK, emphasizes the need for enhanced JE surveillance to monitor the dynamics of JEV strains within the region. Future findings may have implications with regard to JEV vaccination/prevention strategies.

                  Keywords

                  Japanese encephalitis virus genotype I genotype V Culex tritaeniorhynchus Culex bitaeniorhynchus Muar

                  Background

                  Japanese encephalitis virus (JEV) is a mosquito-borne member of the family Flaviviridae, genus Flavivirus, and a primary cause of viral encephalitis in humans within its range [1]. The positive-sense RNA viral genome is approximately 11 kb in length and is translated into three structural proteins [Capsid (C), Membrane (M), and Envelope (E)] and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) with untranslated regions (UTR) at the 5' and 3' ends of the genome [2]. Historically, Culex tritaeniorhynchus was implicated as the primary vector of JEV in the Republic of Korea (ROK) and much of Asia [3, 4]. However, JEV has since been detected in additional culicine species throughout its range, including Cx. bitaeniorhynchus from the ROK [5]. JEV strains are generally classified into five genotypes (genotypes I, II, III, IV, and V) based on similarities in the E gene nucleotide sequence [6]. Previously, only genotype I was detected on the Korean peninsula [7]. Therefore, we characterized JEV-positive pools of Cx. tritaeniorhynchus and Cx. bitaeniorhynchus to determine whether the unexpected finding of JEV in Cx. bitaeniorhynchus in the ROK may have coincided with the appearance of an additional genotype.

                  Materials and Methods

                  Nineteen JEV-positive mosquito pools, from a total of 64 JEV-positive pools collected during 2008 and 2010 in the ROK (18 pools of Cx. tritaeniorhynchus and 1 pool of Cx. bitaeniorhynchus), and one JEV culture received from USAMRIID (United States Army Medical Research Institute of Infectious Diseases, USA) were genotypically characterized (Table 1, Figure 1). Total RNA was extracted from mosquito homogenate using Trizol reagent (Invitrogen, USA) in accordance with the manufacturer's instructions and was resuspended in 50 μl of RNase-free water containing 10 units of RNasin® Plus RNase Inhibitor (Promega, USA). RNA was used as the template for cDNA synthesis using the SuperScript III first strand synthesis system (Invitrogen, USA) with a random hexamer primer. The synthesized cDNA was then used for PCR amplification using iProof™ High-Fidelity DNA polymerase (Bio-Rad, USA). The NS5 gene/3' UTR and envelope (E) gene of 19 JEV-positive pools were amplified using EMF1/VD8 primers [8] and 940S/1720A primers [9], respectively. Products were purified using the QIAquick PCR purification kit (Qiagen, USA) and sequenced by AITBiotech Company (AITbiotech, Singapore).
                  Table 1

                  Locations and collection dates of JEV-positive mosquito pools analyzed in this study

                  Collection Serial No.

                  Collection Date

                  Collection Sites (US Military Bases, Villages/Cities)

                  Province

                  Species

                  Accession no.

                  A8.789

                  29-Jul-08

                  Haenam

                  Jeonnam

                  Cx. tritaeniorhynchus

                  JN587257, JN587261

                  A10.825

                  28-Sep-10

                  Changnyeong

                  Gyeongnam

                  Cx. tritaeniorhynchus

                  JN587255, JN587259

                  A10.881

                  21-Oct-10

                  Jinju

                  Gyeongnam

                  Cx. tritaeniorhynchus

                  JN587256, JN587260

                  10-1742

                  1-Sep-10

                  Warrior Base* (Munsan)

                  Gyeonggi

                  Cx. tritaeniorhynchus

                  JN587241

                  10-1748

                  1-Sep-10

                  Warrior Base* (Munsan)

                  Gyeonggi

                  Cx. tritaeniorhynchus

                  JN587242

                  10-1728

                  31-Aug-10

                  Daeseongdong

                  Gyeonggi

                  Cx. tritaeniorhynchus

                  JN587240

                  10-1937

                  11-Sep-10

                  Daeseongdong

                  Gyeonggi

                  Cx. tritaeniorhynchus

                  JN587245

                  10-2044

                  14-Sep-10

                  Daeseongdong

                  Gyeonggi

                  Cx. tritaeniorhynchus

                  JN587248

                  10-2097

                  21-Sep-10

                  Daeseongdong

                  Gyeonggi

                  Cx. tritaeniorhynchus

                  JN587249

                  10-2130

                  21-Sep-10

                  Daeseongdong

                  Gyeonggi

                  Cx. tritaeniorhynchus

                  JN587250

                  10-2357

                  13-Oct-10

                  Daeseongdong

                  Gyeonggi

                  Cx. tritaeniorhynchus

                  JN587252

                  10-1827

                  8-Sep-10

                  Daeseongdong

                  Gyeonggi

                  Cx. bitaeniorhynchus

                  JN587243, JN587258

                  10-1835

                  8-Sep-10

                  CP Humphreys* (Pyeongtaek)

                  Gyeonggi

                  Cx. tritaeniorhynchus

                  JN587244

                  10-1291

                  5-Aug-10

                  Gunsan Air Base* (Gunsan)

                  Jeonbuk

                  Cx. tritaeniorhynchus

                  JN587239

                  10-2204

                  8-Sep-10

                  Gunsan Air Base* (Gunsan)

                  Jeonbuk

                  Cx. tritaeniorhynchus

                  JN587251

                  10-1990

                  30-Aug-10

                  Gwangju Air Base* (Gwangju)

                  Jeonnam

                  Cx. tritaeniorhynchus

                  JN587246

                  10-1992

                  30-Aug-10

                  Gwangju Air Base* (Gwangju)

                  Jeonnam

                  Cx. tritaeniorhynchus

                  JN587247

                  10-2378

                  2-Sep-10

                  Gwangju Air Base* (Gwangju)

                  Jeonnam

                  Cx. tritaeniorhynchus

                  JN587253

                  10-2397

                  27-Sep-10

                  Gwangju Air Base* (Gwangju)

                  Jeonnam

                  Cx. tritaeniorhynchus

                  JN587254

                  Locations are presented in Figure 1.

                  * US military training site or installation.

                  http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-8-449/MediaObjects/12985_2011_1563_Fig1_HTML.jpg
                  Figure 1

                  Locations of JEV-positive mosquito pools collected during 2008 and 2010 in the Republic of Korea. Daeseongdong is a village near the military demarcation line (MDL) (center of the 4-Km wide demilitarized zone separating North and South Korea); Warrior Base training area is approximately 5 km north of Munsan; Camp Humphreys is in a rural area of Pyeongtaek; Gunsan Air Base is located near the small city of Gunsan; Gwangju Air Base is located near the metropolitan city of Gwangju; Haenam, Jinju, and Changnyeong sites are beef/swine farms near the small cities. Pool = number of sequenced samples/total JEV-positive samples.

                  The sequences were edited and assembled using the Sequencer program v4.1.4 (Applied Biosystems, USA). Multiple sequence alignments and phylogenetic analysis were performed using ClustalX version 2.0.11 and MEGA version 5 programs [10, 11]. Percent sequence similarity/divergence was calculated using the MegAlign program found in the Lasergene v.8 software (DNASTAR, Inc., Madison, WI, USA). Phylogenetic analysis of the partial E gene (705 bp) was performed using the neighbor-joining method and Tamura-Nei model of nucleotide substitution. The maximum likelihood (ML) tree was constructed from the NS5/3'UTR nucleotide sequences (550 bp) by PhyML software v 3.0 [12] using the best fit model with aLRT branch support [13]. The ML tree for the complete E gene used the Tamura-Nei model with bootstrap analysis (2, 000 replicates) for testing the reliability of the tree using the MEGA5 (version 5) program (The Biodesign Institute, Tempe, Arizona) [11].

                  Results

                  The phylogenetic relationships among 19 JEV strains and JEV sequences retrieved from GenBank representing genotypes I-V were analyzed. The ML tree for the NS5/3'UTR (550 bp) and the neighbor-joining tree for the partial E gene (705 bp) showed similar branching patterns with high bootstrap support. Therefore, the ML tree is only presented in this report (Figure 2). Two genotypes were identified among the 19 JEV strains. JEV strains from 18 Cx. tritaeniorhynchus mosquitoes grouped into genotype I. These genotype I strains were closely-related to strains isolated from China, Korea, Japan, Vietnam, and Thailand from the early 1980s to the present (Figure 2). The remaining strain from Cx. bitaeniorhynchus (10-1827) grouped into genotype V together with the Muar strain which was isolated from an encephalitis patient in Malaysia in 1952.
                  http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-8-449/MediaObjects/12985_2011_1563_Fig2_HTML.jpg
                  Figure 2

                  Maximum likelihood tree of JEV strains from the ROK using NS5/3'UTR base-sequence homologies. Phylogenetic analysis was performed using the GTR+G4+I model of nucleotide substitution (ln(L) = -3090.1 571) and aLRT branch support (indicated at major nodes). The phylogenetic tree has been rooted at its midpoint. Scale bar represents substitutions per site.

                  The complete E gene was sequenced from a subset of strains in genotype I (A10.825, A10.881, A8.789) and genotype V (10-1827). The ML tree constructed from the complete E gene of these strains together with representative JEV genotype I-V sequences is shown in Figure 3. This ML tree supports the phylogenetic analysis results performed on the NS5/3'UTR (Figure 2) and the partial E gene previously mentioned. The ML tree in Figure 3 shows that the 10-1827 strain grouped with the Muar strain with 79% bootstrap support, while the remaining sequences clustered in genotype I together with K01-JN and K05-GS strains that were isolated from Cx. tritaeniorhynchus in the ROK in 2001 and 2005, respectively.
                  http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-8-449/MediaObjects/12985_2011_1563_Fig3_HTML.jpg
                  Figure 3

                  Maximum likelihood tree of JEV strains from the ROK using complete E gene nucleotide sequence homologies. Phylogenetic analysis was performed using the MEGA5 program by the Tamura-Nei model of nucleotide substitution (ln(L) = -7791.2). Murray Valley encephalitis virus strain (MVE-1-51) was used as an outgroup. Bootstrap values at each major node were calculated using 2, 000 replicates. Scale bar represents the number of nucleotide substitutions per site.

                  Sequence analysis of 18 strains shows minimal sequence variation among viruses in genotype I, with nucleotide sequence similarity of 97.5-100% for the NS5/3'UTR (Figure 2) and 99.6-100% for the E gene (Figure 3). In an earlier study, genetic stability was also observed among JEV strains isolated from mosquito vectors in the ROK between 1994 and 2005 [7]. Examination of the complete E sequence of 10-1827 strain (genotype V) showed less similarity to the other genotypes, with nucleotide similarity approximately 77.3% (91.3% for amino acids) to genotype I (K01-JN, K05-GS), 78.1% (91.0% for amino acids) to genotype II (FU strain), 77.7% (90.4% for amino acids) to genotype III (Nakayama), and 77.8% (91.0% for amino acids) to genotype IV (JKT6468) (Table 2). However, nucleotide and amino acid similarities to the Muar strain were 90.0% and 98.8%, respectively (Table 2). Likewise, the XZ0934 strain, a JEV genotype V recently isolated from China (2009), showed E gene nucleotide and amino acid sequence similarities to the Muar strain of 86.0% and 93.2%, respectively [14].
                  Table 2

                  Nucleotide sequence similarity and divergence of the complete E gene from ROK mosquito pools

                   

                  A10.825

                  A10.881

                  A8.789

                  JE_USAMRIID

                  K01-JN

                  K05-GS

                  FU

                  Nakayama

                  JKT6468

                  10-1827

                  Muar

                  A10.825 (1)

                   

                  99.4

                  99.3

                  94.3

                  98.2

                  99.3

                  89.0

                  87.8

                  81.8

                  77.2

                  76.8

                  A10.881 (1)

                  0.6

                   

                  99.3

                  94.1

                  98.1

                  99.2

                  89.0

                  88.0

                  82.2

                  77.4

                  76.5

                  A8.789 (1)

                  0.7

                  0.7

                   

                  94.3

                  98.2

                  99.3

                  89.1

                  87.8

                  82.1

                  77.2

                  76.4

                  JE_USAMRIID (1)

                  6.0

                  6.3

                  6.0

                   

                  94.1

                  94.3

                  88.0

                  86.6

                  82.1

                  77.2

                  76.6

                  K01-JN (1)

                  1.8

                  1.9

                  1.8

                  6.2

                   

                  98.3

                  89.1

                  87.6

                  81.8

                  77.3

                  76.8

                  K05-GS (1)

                  0.7

                  0.8

                  0.7

                  6.0

                  1.8

                   

                  89.4

                  88.0

                  81.8

                  77.4

                  76.6

                  FU (2)

                  12.2

                  12.2

                  12.1

                  13.6

                  12.2

                  11.7

                   

                  88.1

                  81.9

                  78.1

                  77.0

                  Nakayama (3)

                  13.8

                  13.5

                  13.7

                  15.3

                  14.1

                  13.6

                  13.3

                   

                  83.0

                  77.7

                  77.5

                  JKT6468 (4)

                  21.8

                  21.1

                  21.3

                  21.3

                  21.7

                  21.7

                  21.5

                  20.1

                   

                  77.8

                  77.1

                  10-1827 (5)

                  27.6

                  27.4

                  27.7

                  27.6

                  27.5

                  27.3

                  26.3

                  26.9

                  26.8

                   

                  90.0

                  Muar (5)

                  28.2

                  28.6

                  28.7

                  28.4

                  28.1

                  28.5

                  27.9

                  27.2

                  27.7

                  11.1

                   

                  The upper triangle represents similarity while the lower triangle represents divergence. A8.789, A10.825, A10.881, and 10-1827 represent the ROK mosquito pools. JEV reference strains are shown for 5 genotypes: I (K01-JN, K05-GS), II (FU), III (Nakayama), IV (JKT6468), and V (Muar). Percent similarity/divergence was computed using the MegAlign program (Lasergene v.8 software, USA). Numbers in parentheses represent the JEV genotype.

                  Figure 4 shows the amino acid sequence alignment of the complete E gene derived from strains A10.825, A10.881, A8.789, and 10-1827 and reference sequences (Muar, K01-JN, K05-GS). The E protein of the strains in genotype I is very conserved with few amino acid changes detected: A10.825 (from S = serine to N = asparagine at position 123) and A8.789 (from L = leucine to M = methionine at position 371). The alignment reveals differences in 6 amino acid residues between the Muar and 10-1827 strains (Figure 4). The eight Muar signature amino acid residues in domain III comprising a putative receptor binding region [15] were also identified in the 10-1827 strain along with the critical amino acid residue thought to be involved in receptor binding activity (Q = glutamine at position 327) [16]. Table 3 provides a complete listing of the strains that are referenced in this study.
                  http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-8-449/MediaObjects/12985_2011_1563_Fig4_HTML.jpg
                  Figure 4

                  Amino acid sequence alignment of the full-length envelope gene from ROK JEV strains. ROK mosquito pools collected during 2008 and 2010 were aligned with the reference sequences GV (Muar) and GI (K01-JN, K05-GS). Dots indicate consensus. Differences in amino acids between Muar and 10-1827 strains are underlined. Residues enclosed by boxes represent 8 Muar signature amino acids in domain III.

                  Table 3

                  Origin of 30 JEV strains referenced in this study

                  Strain

                  Location

                  Year

                  Host

                  Genotype

                  Accession no.

                  1070/82_Subin

                  Thailand

                  1982

                  Human

                  1

                  GQ902059

                  90VN70

                  Vietnam

                  1990

                  Human

                  1

                  HM228921

                  B-0860/82

                  Thailand

                  1985

                  Swine

                  1

                  GQ902058

                  Beijing-1

                  China

                  1949

                  Mosquito

                  3

                  L48961, FJ872376

                  Bennett

                  Korea

                  before 1951

                  Human

                  2

                  FJ515927

                  FU

                  Australia

                  1995

                  Human

                  2

                  AF217620

                  GZ56

                  China

                  2008

                  Human

                  1

                  HM366552

                  Ishikawa

                  Japan

                  1998

                  Mosquito

                  1

                  AB051292

                  JEV/sw/Mie/40/2004

                  Japan

                  2004

                  Swine

                  1

                  AB241118

                  JEV40783

                  Korea

                  before 1971

                  Human

                  3

                  FJ515923

                  JKT6468

                  Indonesia

                  1981

                  Mosquito

                  4

                  AY184212

                  JKT7003

                  Indonesia

                  1981

                  Mosquito

                  4

                  U70408

                  JX61

                  China

                  2008

                  Swine

                  1

                  GU556217

                  K01-JN

                  Korea

                  2001

                  Mosquito

                  1

                  FJ938222

                  K87P39

                  Korea

                  1987

                  Mosquito

                  3

                  AY585242

                  K88A07

                  Korea

                  1988

                  Mosquito

                  3

                  FJ938227

                  K91P55

                  Korea

                  1991

                  Mosquito

                  -

                  U34928

                  K94P05

                  Korea

                  1994

                  Mosquito

                  1

                  AF045551

                  KO5-GS

                  Korea

                  2005

                  Mosquito

                  1

                  FJ938223

                  KPP82-39-214CT

                  Thailand

                  -

                  Mosquito

                  3

                  GQ902063

                  KV1899

                  Korea

                  1999

                  Swine

                  1

                  AY316157

                  Muar

                  Malaysia

                  1952

                  Human

                  5

                  HM596272

                  MVE-1-51

                  Australia

                  1951

                  Human

                  -

                  AF161266

                  Nakayama

                  Japan

                  1935

                  Human

                  3

                  EF571853

                  SC04-17

                  China

                  2004

                  Mosquito

                  1

                  GU187972

                  SH17M

                  China

                  2007

                  Mosquito

                  1

                  EU429297

                  T1P1

                  Taiwan

                  1997

                  Mosquito

                  3

                  AF254453

                  XJ69

                  China

                  2007

                  Mosquito

                  1

                  EU880214

                  XJP613

                  China

                  2007

                  Mosquito

                  1

                  Eu693899

                  XZ0938

                  China

                  2009

                  Mosquito

                  1

                  HQ652538

                  All strains are JEV, with the exception of MVE-1-51, a strain of Murray Valley encephalitis virus.

                  Conclusion

                  This study is the first report of JEV genotype V in the ROK and represents the third report of genotype V in Asia, with the most recent findings from Cx. tritaeniorhynchus collected in Tibet, China (2009) [14]. The fact that JEV genotype V, first reported from an encephalitis patient in Malaysia in 1952 (Muar strain), came long before the discovery of its reemergence in China in 2009 and now its subsequent appearance in the ROK may mark the beginning of a genotypic shift in JEV within the region. Additionally, the emergence of this strain in Cx. bitaeniorhynchus, a mosquito species previously unknown to carry JEV in the ROK, underscores the need to step-up surveillance efforts within the ROK. The reemergence of this genotype after 57 years may have future implications with regard to JEV vaccination effectiveness and policy among civilian and military populations, as well as with preventive strategies designed to reduce the health impact and incidence of JEV among at risk Asian populations.

                  Declarations

                  Acknowledgements

                  This work was supported through the joint partnership between the Uniformed Services University (Bethesda, MD), the Armed Forces Health Surveillance Center-Global Emerging Infections Surveillance and Response System (Silver Spring, MD), the National Center for Medical Intelligence (Fort Detrick, MD), the Korea National Institute of Health (Osong, Chungbuk Province, ROK), the Armed Forces Research Institute of Medical Sciences (Bangkok, Thailand), and the 65th Medical Brigade (ROK). The mention of trade names or commercial products does not constitute endorsement or recommendation for use by the Department of the Army, the Department of Defense, or the Korea National Institute of Health. The opinions and assertions contained herein are those of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.

                  Authors’ Affiliations

                  (1)
                  Department of Entomology, United States Army Medical Component, Armed Forces, Research Institute of Medical Sciences
                  (2)
                  5th Medical Detachment, 168th Multifunctional Medical Battalion, 65th Medical Brigade, Unit 15247
                  (3)
                  Force Health Protection and Preventive Medicine, 65th Medical Brigade/USAMEDDAC-Korea, Unit 15281
                  (4)
                  National Institute of Health, Korea Centers for Disease Control and Prevention
                  (5)
                  Department of Preventive Medicine and Biometrics, Uniformed Services University of Health Sciences

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                  Copyright

                  © Takhampunya et al; licensee BioMed Central Ltd. 2011

                  This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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