Open Access

Insights into the evolutionary history of Japanese encephalitis virus (JEV) based on whole-genome sequences comprising the five genotypes

Contributed equally
Virology Journal201512:43

https://doi.org/10.1186/s12985-015-0270-z

Received: 1 September 2014

Accepted: 23 February 2015

Published: 14 March 2015

Abstract

Background

Japanese encephalitis virus (JEV) is the etiological agent of Japanese encephalitis (JE), one of the most serious viral encephalitis worldwide. Five genotypes have been classified based on phylogenetic analysis of the viral envelope gene or the complete genome. Previous studies based on four genotypes have reported that in evolutionary terms, genotype 1 JEV is the most recent lineage. However, until now, no systematic phylogenetic analysis was reported based on whole genomic sequence of all five JEV genotypes.

Findings

In this study, phylogenetic analysis using Bayesian Markov chain Monte Carlo simulations was conducted on the whole genomic sequences of all five genotypes of JEV. The results showed that the most recent common ancestor (TMRCA) for JEV is estimated to have occurred 3255 years ago (95% highest posterior density [HPD], −978 to−6125 years). Chronologically, this ancestral lineage diverged to produce five recognized virus genotypes in the sequence 5, 4, 3, 2 and 1. Population dynamics analysis indicated that the genetic diversity of the virus peaked during the following two periods: 1930–1960 and 1980–1990, and the population diversity of JEV remained relatively high after 2000.

Conclusions

Genotype 5 is the earliest recognized JEV lineage, and the genetic diversity of JEV has remained high since 2000.

Keywords

Japanese encephalitis virus Genotype Genetic diversity

Findings

Japanese encephalitis virus (JEV) is the prototype member of the JEV serogroup within the genus Flavivirus, family Flaviviridae. JEV comprises five genotypes (G1-G5) [1-3]. In previous studies, the phylogenetic characteristics of JEV were analyzed and the most recent common ancestor (TMRCA) was estimated. The TMRCA of JEV was estimated to be 1690 years when calculations were based on the complete sequence of four genotypes (G1-G4) [4], whereas, analysis of JEV using a limited number of whole genomic sequences from five genotypes indicated that TMRCA of JEV appeared approximately 460 years [5]. More recently, however, G5 strain XZ0934 isolated in 2009, which had not been included in earlier analyses, was shown to be significantly different from the G5 Muar isolate [6]. Therefore, in order to improve our understanding of the evolutionary progress and population diversity of JEV, a comprehensive dataset was established for evolutionary analysis of JEV in this study. In the dataset, 100 whole genomic sequences of JEV representing all five genotypes of JEV, isolated from various hosts (humans, pigs and bats) and vectors (mosquitoes and midges) were collected and analyzed.

Two G5 JEV full-length genome sequences (Muar and XZ0934) were downloaded from GenBank (GB No. HM596272 and JF915894, respectively) and added to the database established in a previous report [4], forming a new database for analysis (Table 1). The new JEV sequence database was analyzed using Bayesian Markov chain Monte Carlo (MCMC) method. The General Time Reversible (GTR) model + Invariant (I) + Gamma (G) model was selected using MrModelTest [7]. The nucleotide substitution rates and divergence times of the most recent common ancestor (TMRCA) were estimated using the relaxed (uncorrelated lognormal) molecular clock model in the BEAST software package [8]. Demographic histories of JEV were inferred based on Bayesian skyline reconstruction. The analysis was run through 1,000,000,000 generations to ensure sufficient mixing. Finally, the maximum clade credibility (MCC) tree was built using TreeAnnotator with 10% burn-in (http://beast.bio.ed.ac.uk/).
Table 1

Information of JEV isolates analyzed in this study

Strain

Date

Country

Host a

Genotype

GenBank accession no.

47

1950’s

China:Heilongjiang

CSF

3

JF706269

14178

2001

India

-

3

EF623987

57434

2005

India

-

3

EF623988

04940-4

2002

India

-

3

EF623989

B58

1989

China:Yunnan

Bat

3

FJ185036

Beijing-1

1949

China

Human brain

3

L48961

BL06-50

2006

China:Guangxi

Culex tritaeniorhynchus

1

JF706270

BL06-54

2006

China:Guangxi

Culex tritaeniorhynchus

1

JF706271

CBH

1954

China:Fujian

CSF

3

JN381860

CH-13

1957

China:Sichuan

CSF

3

JN381870

CH1392

1990

Taiwan

Culex tritaeniorhynchus

3

AF254452

CTS

1955

China:Fujian

CSF

3

GQ429184

CZX

1954

China:Fujian

CSF

3

JN381865

DH107

1989

China:Yunnan

Aedes lineatopennis

3

JN381873

DL04-29

2004

China:Yunnan

Culex theileri

3

JF706272

DL04-45

2004

China:Yunnan

Ar. Subalbatus & Mansonia uniform

3

JN381854

Fj02-29

2002

China:Fujian

CSF

3

JF706273

Fj02-76

2002

China:Fujian

Human blood

3

JN381867

FJ03-39

2003

China:Fujian

Human blood

3

JN381859

FJ03-94

2003

China:Fujian

Human blood

3

JN381858

FU

1995

Australia

Human serum

2

AF217620

G35

1954

China:Fujian

Mosquito pool

3

GQ429185

GB30

1997

China:Yunnan

Murina aurata brain tissue

3

FJ185037

GP78

1978

India

Human brain

3

AF075723

GS07-TS11

2007

China:Gansu

Culex tritaeniorhynchus

1

JN381843

GSBY0801

2008

China:Gansu

Culex tritaeniorhynchus

1

JF706274

GSBY0804

2008

China:Gansu

Culex tritaeniorhynchus

1

JN381844

GSBY0810

2008

China:Gansu

Culex tritaeniorhynchus

1

JN381840

GSBY0816

2008

China:Gansu

Culex tritaeniorhynchus

1

JN381842

GSBY0827

2008

China:Gansu

Culex tritaeniorhynchus

1

JN381845

GSBY0861

2008

China:Gansu

Culex tritaeniorhynchus

1

JN381833

GSS

1960’s

China:Beijing

CSF

3

JF706275

GX0519

2005

China:Guanxi

Culex tritaeniorhynchus

1

JN381835

GX0523/44

2005

China:Guanxi

Culex tritaeniorhynchus

1

JN381832

GZ04-2

2004

China:Guizhou

Armigeres

3

JN381857

GZ56

2006

China:GuiZhou

CSF

1

HM366552

Ha-3

1960’s

China:Heilongjiang

CSF

3

JN381872

HB49

1990

China:Yunnan

Rousettus leschenaulti blood

3

JF706284

HB97

1990

China:Yunnan

Rousettus leschenaulti blood

3

JF706285

HLJ02-134

2002

China:Heilongjiang

Genus culicoides

3

JF706276

HN04-11

2004

China:Henan

Culex

1

JN381831

HN04-21

2004

China:Henan

Culex

1

JN381841

HN06129

2006

China:Henan

Armigeres

1

JF706277

HN0621

2006

China:Henan

Culex

1

JN381830

HN0626

2006

China:Henan

Culex

1

JN381837

HVI

1965

Taiwan

Mosquito

3

AF098735

HYZ

1979

China:Yunnan

Patient blood

3

JN381853

Ishikawa

1994

Japan

Culex tritaeniorhynchus

1

AB051292

JaGAr 01

1959

Japan

Cluex

3

AF069076

JaOArS982

1982

Japan

Mosquito

3

M18370

JaOH0566/Japan/1966/human

1966

Japan

Human

3

AY508813

JEV/sw/Mie/40/2004

2004

Japan

Swine serum

1

AB241118

JEV/sw/Mie/41/2002

2002

Japan

Swine serum

1

AB241119

JH04-18

2004

China:Yunnan

Whitmorei & Anophelessinensis

3

JN381855

JKT6468

1981

Indonesia

Mosquito

4

AY184212

K87P39

1987

South Korea

Mosquito

3

AY585242

KV1899

1999

Korea

Swine

1

AY316157

LFM

1955

China:Fujian

Human blood

3

JN381863

Ling

1965

Taiwan

Human brain

3

L78128

LN02-102

2002

China:liaoning

Culex modestus

1

JF706278

LN0716

2007

China:Liaoning

Culex tritaeniorhynchus

1

JN381849

LYZ

1957

China:Fujian

CSF

3

JN381869

M28

1977

China:Yunnan

Culex pseudovishnui

1

JF706279

Nakayama

1935

Japan

Human brain

3

EF571853

P3

1949

China:Beijing

Human brain

3

U47032

RP-2 ms

1985

Taiwan

Mosquito

3

AF014160

RP-9

1985

Taiwan

Mosquito

3

AF014161

SA14

1954

China

Mosquito

3

U14163

SC04-12

2004

China:Sichuan

Culex

1

JN381839

SC04-15

2004

China:Sichuan

Culex tritaeniorhynchus

1

JN381838

SD0810

2008

China:Shandong

Culex tritaeniorhynchus

1

JF706286

SH03-103

2003

China:Shanghai

Culex tritaeniorhynchus

1

JN381847

SH03-105

2003

China:Shanghai

Culex tritaeniorhynchus

1

JN381846

SH04-10

2004

China:Shanghai

Culex tritaeniorhynchus

3

JN381856

SH04-5

2004

China:Shanghai

Culex tritaeniorhynchus

3

JN381866

SH17M-07

2007

China

-

1

EU429297

SH-3

1987

China:Shanghai

CSF

3

JN381864

SH-53

2001

China:Shanghai

Culex tritaeniorhynchus

1

JN381850

SH-80

2001

China:Shanghai

Culex tritaeniorhynchus

1

JN381848

T1P1

1997

Taiwan

Armigeres subalbatus

3

AF254453

TLA

1971

China:Liaoning

CSF

3

JN381868

Vellore P20778

1958

India

Human brain

3

AF080251

XJ69

2007

China

Culex pipiens pallens

1

EU880214

XJP613

2007

China

Culex tritaeniorhynchus

1

EU693899

XZ0938

2009

China:Xizang

Culex tritaeniorhynchus

1

HQ652538

YLG

1955

China:Fujian

CSF

3

JF706280

YN

1954

China:Yunnan

CSF

3

JN381871

YN05124

2005

China:Yunnan

Culex tritaeniorhynchus

1

JF706281

YN05155

2005

China:Yunnan

Culex tritaeniorhynchus

1

JN381852

YN0623

2006

China:Yunnan

Culex tritaeniorhynchus

1

JN381836

YN0911

2009

China:Yunnan

Culex tritaeniorhynchus

1

JF706267

YN0967

2009

China:Yunnan

Culex tritaeniorhynchus

1

JF706268

YN79-Bao83

1979

China:Yunnan

Culex tritaeniorhynchus

1

JN381851

YN82-BN8219

1982

China:Yunnan

Mosquito

1

JN381834

YN83-Meng83-54

1983

China:Yunnan

Lasiohelea taiwana Shiraki

1

JF706282

YN98-A151

2003

China:Yunnan

Mosquitoes

3

JN381861

ZMT

1955

China:Fujian

CSF

3

JF706283

ZSZ

1955

China:Fujian

CSF

3

JN381862

Muar

1952

Malaysia

Human brain

5

HM596272

XZ0934

2009

China:Tibet

Culex tritaeniorhynchus

5

JF915894

a- Information not available.

Based on Bayesian Markov chain Monte Carlo (MCMC) analysis, the maximum clade credibility (MCC) tree for the whole genomic sequences of JEV was established (Figure 1). Representatives of the five distinct lineages were included in the analysis. The posterior probability values for the nodes of each lineage were >0.95, indicating their robustness. JEV was estimated to have emerged 3255 years ago (95% HPD: −978 to −6125 years) and subsequently diverged at least five times to produce the 5 recognized genotypes. In chronological order, they diverged in the order G5, G4, G3, G2 and G1. Thus, G5 represents the most ancestral lineage among genotypes 1–5.
Figure 1

Maximum clade credibility (MCC) tree for 100 whole-genome sequences of JEV. Five distinct lineages were identified: G1 (red), G2 (yellow), G3 (blue), G4 (green) and G5 (orange). Estimated TMRCAs of these lineages (with their 95% HPD values in parentheses) are shown, G1: 155(104–315), G2: 530(235–1131), G3: 880(420–1855), G4: 1653(765–3372), and G5: 3255(978–6125).

The mean rate of nucleotide substitution for the whole genomic sequences of 100 JEV strains isolated from a variety of hosts worldwide, estimated using a Bayesian MCMC approach, was 1.01 × 10−4 nucleotide substitutions per site per year (95% HPD values, 4.37 × 10−5, 1.56 × 10−4). This is similar to previous estimates based on analysis of four JEV genotypes [4].

The population dynamics of JEV are shown in Figure 2. The skyline plot showed that the JEV population had experienced complicated changes during the process of evolution. However, the virus population remained relatively stable during the first 2700 years (Figure 2A), followed by a period of rapid decline from the 1700s, reaching a minimum in the 1900s. It then increased rapidly from the 1930s until the 1960s and formed the first peak. The second peak appeared in the 1980-1990s and subsequently the populations of JEV remained high after 2000 (Figure 2B).
Figure 2

Bayesian skyline plots for JEV. Highlighted areas correspond to 95% HPD intervals. (A) Populations during the whole evolutionary history; (B) Populations during the later evolutionary history since 1800.

The findings in this study have similarities with previous studies [5]. For example, the divergence pattern of the genotypes occurred in the order G5, G4, G3, G2 and G1, and the mean rate of nucleotide substitution was similar to previous estimates. However, the occurrence time of TMRCA determined in this study (~3255 years ago) was quite different compared with that measured in the report (~460 years ago) of Mohammed et al. [5]. The reason for this discrepancy could be attributed to the dataset used for analysis. In Mohammed’s study, only 35 whole genomic sequences were used and the only G5 representative included was the Muar strain. Therefore, since our new dataset includes two G5 representatives with robust sequences, the occurrence time of TMRCA (~3255 years ago) obtained in this study should reflect more precisely the evolutionary patterns and diversity of JEV.

Two main peak periods in population dynamics were identified in this study, 1930–1960 and 1980-1990s, respectively. These fluctuations were reflections of the virus activity in the sylvatic environment. Since the 1930s, JEV strains belonging to G3 emerged and were isolated from Asian countries. G5 was first identified in 1952 [2,4]. This was a good interpretation of the first peak. Subsequently, G2 and G4 strains were isolated during the 1980s. Importantly, the G1 genotype emerged during the 1980s onwards [4]. Therefore, the virus population diversity peaked in the 1980-1990s. Since 2000, G1 JEV has become the dominant genotype in most endemic regions [4], and although a relatively small decrease was observed, the virus remains the most active and G5 reemerged.

Interestingly, although G5 is estimated to be the most ancestral JEV lineage, this virus showed a highly active dispersal capacity following its reemergence. Indeed, this new G5 strain was isolated from mosquitoes collected in southern region of the Asian continent (Tibet, China) in 2009 [2] and northeast region of Asia (South Korea) during the same year [9]. Thus, G5 now appears to be dispersing widely in Asia. A recent study showed that genotype 1 JEV originated in Southeastern Asia and spread to the entire Asian continent [10]. Based on these observations, it seems likely that G5 will follow a dispersal pattern similar to that of G1 JEV, and has dispersed or will disperse over the entire Asian continent. Clearly, G5 should be monitored closely throughout JEV endemic regions.

Finally, the available inactivated and live attenuated JE vaccines are derived from G3 JEVs [11]. Thus, the level of cross protection of the current vaccines against G5 JEV is likely to be sub-optimal and should therefore be analyzed carefully since the reemergence of G5 and its widespread dispersal, and significant genetic variation could impact on its epidemiology. This possibility is emphasized by the fact that Muar (the first G5 JEV) strain was isolated from a patient with severe viral encephalitis [12]. Thus, there is the realistic possibility that the newly isolated G5 viruses could be highly virulent. Thus, the potential disease burden of viral encephalitis caused by G5 JEV requires careful reassessment.

Notes

Abbreviations

JEV: 

Japanese encephalitis virus

JE: 

Japanese encephalitis

E: 

Envelope

TMRCA: 

The most recent common ancestor

HPD: 

Highest posterior density

G: 

Genotype

MCMC: 

Markov chain Monte Carlo

MCC: 

Maximum clade credibility

Ser: 

Serine

ORF: 

Open reading frame

Declarations

Acknowledgements

We appreciate Dr. Ernest A Gould from Aix Marseille University for his important revisions and comments on the manuscript. This work was supported by grants from the National Natural Science Foundation of China (81290342), the Ministry of Science and Technology, China (2011CB504702), and State Key Laboratory for Infectious Disease Prevention and Control (2014SKLID03).

Authors’ Affiliations

(1)
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention
(2)
Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases
(3)
School of Life Sciences, Shandong University of Technology

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© Gao et al.; licensee BioMed Central. 2015

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