Open Access

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

  • Ratree Takhampunya1,
  • Heung-Chul Kim2,
  • Bousaraporn Tippayachai1,
  • Ampornpan Kengluecha1,
  • Terry A Klein3,
  • Won-Ja Lee4,
  • John Grieco5 and
  • Brian P Evans1Email author
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.

https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-8-449/MediaObjects/12985_2011_Article_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.
https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-8-449/MediaObjects/12985_2011_Article_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.
https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-8-449/MediaObjects/12985_2011_Article_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.
https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-8-449/MediaObjects/12985_2011_Article_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)
Unit 15247
(3)
Force Health Protection and Preventive Medicine, 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 article is published under license to BioMed Central Ltd. 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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