Conceived and designed the experiments: M-HL H-YW G-DL. Performed the experiments: M-HL S-HF W-XC. Analyzed the data: M-HL S-HF H-YW G-DL. Contributed reagents/materials/analysis tools: Y-HG Q-YL Y-XL H-ML WD DZDJ X-MY. Wrote the paper: M-HL G-DL.
The authors have declared that no competing interests exist.
Japanese encephalitis (JE) is a global public health issue that has spread widely to more than 20 countries in Asia and has extended its geographic range to the south Pacific region including Australia. JE has become the most important cause of viral encephalitis in the world. Japanese encephalitis viruses (JEV) are divided into five genotypes, based on the nucleotide sequence of the envelope (E) gene. The Muar strain, isolated from patient in Malaya in 1952, is the sole example of genotype V JEV. Here, the XZ0934 strain of JEV was isolated from
Japanese encephalitis virus (JEV) is a mosquito-borne virus that causes Japanese encephalitis (JE) with significant morbidity and mortality. Five genotypes (genotype I–V) have been identified based on the nucleotide sequence of viral envelope (E) gene of JEV. To date, the only known strain of genotype V is Muar strain, isolated from patient in Malaya in 1952. Since then, no genotype V JEV has been detected in the world. In this study, the JEV strain, XZ0934, was isolated from mosquito samples collected in China in 2009. The full-length genome sequences of the XZ0934 strain was determined and founded to be the second strain of genotype V JEV based on the phylogenetic analysis using the complete genome and structural gene sequences. This suggests that genotype V JEV is re-emerging after 57 years (1952–2009). Therefore, increased surveillance and more effective diagnosis for cases of JE caused by genotype V JEV are needed.
Japanese encephalitis (JE), which is caused by JE virus (JEV), is one of the most important viral encephalitis in the world
JEV has a zoonotic transmission cycle between mosquitoes (principally of the genus
JEV is a member of the genus
JEVs have been divided into five genotypes (genotype I, II, III, IV, V), based on nucleotide sequence of E gene
C6/36 (
An arbovirus survey was conducted in Tibet in the summer of 2009. Mosquitoes were collected in Mainling County (altitude 2900 m) and Medog County (altitude 1000 m) in the Nyingchi area of Tibet. Mosquito samples were collected using mosquito-trapping lamps (Wuhan Lucky Star Environmental Protection Tech Co., Ltd., Hubei, China) in the evening. Collection locations were proximal to sites of frequent human activity. Collection nets containing mosquitoes were frozen for 30 min at −20°C and transferred onto an ice plate for determination of mosquito species (blood-fed and male mosquitoes were discarded). Female mosquitoes were identified to species level by morphologic characteristics and sorted into pools of 100 specimens according to species. The pools were put into collection tubes individually and stored in liquid nitrogen
Mosquito pools were added to 1.5 ml minimal essential medium (HyClone), supplemented with 2 mM glutamine, 0.12% NaHCO3, and 100 U ml−1 penicillin and streptomycin, followed by grinding in a pre-cooled sterile plastic grinding tube using a TissueLyser (GIAGEN, Germany). Homogenized samples were centrifuged at 17,000× g in a microcentrifuge for 20 min at 4°C, and the clarified supernatants were used to inoculate monolayers of BHK-21 and C6/36 cells and incubated at 37°C and 28°C, respectively. The cells were observed daily to check for development of cytopathic effects (CPE). A sample was regarded as virus-positive if it caused CPE in successive cell passages
Viral supernatants were applied to six-well plates (Corning, USA) of confluent BHK-21 cells and incubated for one hour. Plates were first overlaid with medium containing 75% agarose and then with medium containing neutral red vital stain after three days incubation at 37°C in a 5% CO2 incubator. Plaques of different sizes and shape were shattered in 500 ul MEM medium after being picked out using a sterile pipette tip. As described previously
Viral RNA was extracted from 140 ul supernatant from virus-infected BHK-21 cell cultures using a Viral RNA Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions. cDNA was synthesized using a Ready-to-Go You-Prime First-Strand Beads Kit (GE healthcare, UK) and random hexanucleotide primers. PCR amplification using universal primers specific for flaviviruses, alphaviruses and bunyaviruses was conducted for identification of virus isolates
Primer | Sequence (5′–3′) | Position | Orientation |
JEV-V-1 |
|
1–21 | Sense |
JEV-V-2 |
|
868–887 | Antisense |
JEV-V-3 |
|
563–582 | Sense |
JEV-V-4 |
|
1495–1512 | Antisense |
JEV-V-5 |
|
1080–1097 | Sense |
JEV-V-6 |
|
2951–2968 | Antisense |
JEV-V-7 |
|
2490–2508 | Sense |
JEV-V-8 |
|
3755–3772 | Antisense |
JEV-V-9 |
|
3480–3497 | Sense |
JEV-V-10 |
|
4674–4692 | Antisense |
JEV-V-11 |
|
4553–4571 | Sense |
JEV-V-12 |
|
5301–5320 | Antisense |
JEV-V-13 |
|
5261–5278 | Sense |
JEV-V-14 |
|
6149–6168 | Antisense |
JEV-V-15 |
|
5988–6007 | Sense |
JEV-V-16 |
|
7107–7126 | Antisense |
JEV-V-17 |
|
6677–6696 | Sense |
JEV-V-18 |
|
7876–7895 | Antisense |
JEV-V-19 |
|
7538–7555 | Sense |
JEV-V-20 |
|
8802–8821 | Antisense |
JEV-V-21 |
|
8333–8350 | Sense |
JEV-V-22 |
|
10465–10484 | Antisense |
JEV-V-23 |
|
9972–9990 | Sense |
JEV-V-24 |
|
10964–10983 | Antisense |
The full-length genome of the XZ0934 strain was compiled using SeqMan in the Lasergene software package (DNASTAR). Nucleotide and amino acid sequence alignments were generated by ClustalX version 2.0.9
Full-length nucleotide sequences of 32 selected JEV strains of varying genotype isolated from different locations and sources, and across a number of years, were downloaded from GenBank (
No. | Genotype | Strain | Year | Location | Source | GenBank accession no. |
1 | I | K94P05 | 1994 | Korea | Mosquito | AF045551 |
2 | I | KV1899 | 1999 | Korea | Pig | AY316157 |
3 | I | Ishikawa | 1998 | Japan | Mosquito | AB051292 |
4 | I | JEV/sw/Mie/41/2002 | 2002 | Japan | Pig | AB241119 |
5 | I | JEV/sw/Mie/40/2004 | 2004 | Japan | Pig | AB241118 |
6 | I | SC04-17 | 2004 | China | Mosquito | GU187972 |
7 | I | HEN0701 | 2007 | China | Pig | FJ495189 |
8 | I | XJ69 | 2007 | China | Mosquito | EU880214 |
9 | I | XJP613 | 2007 | China | Mosquito | EU693899 |
10 | I | SH17M-07 | 2007 | China | Mosquito | EU429297 |
11 | I | JX61 | 2008 | China | Pig | GU556217 |
12 | II | FU | 1995 | Australia | Human | AF217620 |
13 | III | Vellore P20778 | 1958 | India | Human | AF080251 |
14 | III | GP78 | 1978 | India | Human | AF075723 |
15 | III | 014178 | 2001 | India | Human | EF623987 |
16 | III | 04940-4 | 2002 | India | Mosquito | EF623989 |
17 | III | 057434 | 2005 | India | Human | EF623988 |
18 | III | Nakayama | 1935 | Japan | Human | EF571853 |
19 | III | JaGAr01 | 1959 | Japan | Mosquito | AF069076 |
20 | III | JaOH0566 | 1966 | Japan | Human | AY508813 |
21 | III | JaOArS982 | 1982 | Japan | Mosquito | M18370 |
22 | III | K87P39 | 1987 | Korea | Mosquito | AY585242 |
23 | III | p3 | 1949 | China | Human | U47032 |
24 | III | Beijing-1 | 1949 | China | Human | L48961 |
25 | III | SA14-14-2 | 1954 | China | Vaccine strain | AF315119 |
26 | III | HW | 1988 | China | Pig | AY849939 |
27 | III | WHe | 1988 | China | Pig | EF107523 |
28 | III | SH0601 | 2006 | China | Pig | EF543861 |
29 | III | Ling | 1965 | Taiwan | Human | L78128 |
30 | III | CH1392 | 1990 | Taiwan | Mosquito | AF254452 |
31 | III | T1P1 | 1997 | Taiwan | Mosquito | AF254453 |
32 | IV | JKT6468 | 1981 | Indonesia | Mosquito | AY184212 |
To better understand the phylogenetic relationship between genotype V JEV and other flaviviruses, full-length nucleotide sequences of previously published JEV strains and other flaviviruses were downloaded from GenBank, including sequences from Murray Valley encephalitis virus (MVEV), West Nile virus (WNV), Kunjin virus (KUNV), St. Louis encephalitis virus (SLEV), Dengue virus 1 (DENV1, Dengue virus 2 (DENV2), Dengue virus 3 (DENV3), Dengue virus 4 (DENV4), Yellow fever virus (YFV), Powassan virus (POWV), Langat virus (LANV), Louping ill virus (LIV), Tick-borne encephalitis virus (TBEV) and Culex flavivirus (
Virus | Strain | GenBank accession no. |
Murray Valley encephalitis virus (MVEV) | MVE-1-51 | NC_000943 |
West Nile virus (WNV) | ArB3573/82 | DQ318020 |
Kunjin virus (KUNV) | MRM61C | D00246 |
St. Louis encephalitis virus (SLEV) | Kern217 | DQ525916 |
Dengue virus 1 (DENV1) | SG(EHI)D1227Y03 | FJ469909 |
Dengue virus 2 (DENV2) | D2/SG/05K4155DK1/2005 | EU081180 |
Dengue virus 3 (DENV3) | D3/H/IMTSSA-MART/1999/1243 | AY099337 |
Dengue virus 4 (DENV4) | 341750 | GU289913 |
Yellow fever virus (YFV) | 17D/Tiantan | FJ654700 |
Powassan virus (POWV) | Spassk-9 | EU770575 |
Langat virus (LANV) | TP21 | NC_003690 |
Louping ill virus (LIV) | 369/T2 | NC_001809 |
Tick-borne encephalitis virus (TBEV) | Toro-2003 | DQ401140 |
Culex flavivirus | Tokyo | AB262759 |
After homogenized supernatants were inoculated onto monolayers of BHK-21 and C6/36 cells, a single pool containing 100 specimens of
Viral RNA was extracted and amplified by PCR using primers specific for flaviviruses, alphaviruses and bunyaviruses. XZ0934 was positive when primers specific for flaviviruses (FU1/cFD2)
Recent reports have suggested that JEVs currently circulating in China belong to genotypes I and III
Because the structural gene sequence of genotype V (Muar) has been reported
Genotype | Strain | Percentage homology of nucleotides (amino acids) | |||
C | PrM | M | E | ||
G I | Ishikawa (AB051292) | 79.5% (77.2%) | 78.3% (87.0%) | 84.0% (93.3%) | 77.0% (89.4%) |
G II | FU (AF217620) | 81.4% (78.7%) | 74.3% (84.8%) | 81.8% (94.7%) | 77.5% (90.6%) |
G III | p3 (U47032) | 81.4% (78.0%) | 76.8% (85.9%) | 83.6% (93.3%) | 77.4% (90.2%) |
G IV | JKT 6468 (AY184212) | 78.2% (72.4%) | 71.7% (81.5%) | 80.0% (85.3%) | 77.5% (90.6%) |
G V | Muar (Hasegawa et al.(25)) | 88.5% (85.8%) | 84.1% (90.2%) | 95.6% (100.0%) | 86.0% (93.2%) |
To establish the phylogenetic relationship between XZ0934 and other JEV strains, a phylogenetic tree was constructed using the complete genome sequences of XZ0934 and 32 selected JEV strains (genotypes I–IV). Murray Valley encephalitis virus (MVEV) was used as an outgroup. Five distinct phylogenetic groups were identified. The XZ0934 strain, which was isolated from China, formed a branch divergent from other genotype I–IV JEV strains (
A) complete genome; B) C gene; C) PrM gene; D) M gene; E) E gene. Phylogenetic analyses were performed by the neighbor-joining method using MEGA version 4.0.2 software package (
To study their phylogenetic relationship, a phylogenetic tree was constructed using the reported structural gene nucleotide sequences of Muar
A phylogenetic tree was also constructed using genomic nucleotide sequences in order to understand the phylogenetic relationship between XZ0934 and other flaviviruses. Data indicated that XZ0934 was indeed a JEV rather than any of the other 14 flaviviruses (
Phylogenetic analyses were performed by the neighbor-joining method using MEGA version 4.0.2 software package (
In recent years, the sequence of the JEV viral envelope (E) gene has been used by various authors to perform phylogenetic analyses
Primers designed for JEV genotypes I and III were used for full-length amplification of XZ0934. Of these, only a few (4/32 genotype I and 10/48 genotype III) resulted in successful amplification. This suggests a low whole genome sequence homology between XZ0934 and genotype I and I JEV isolates. In order to further understand the differences between XZ0934 and other JEV strains (genotype I–IV), an identity analysis was conducted using the full-length nucleotide sequences of XZ0934 and 62 known JEV isolates (genotypes I–IV) in Genbank. Data suggested that XZ0934 and the genotype I–IV JEV strains were dissimilar. The nucleotide sequence identity varied from 78.6% to 79.7% and amino acid sequence identity from 90.0% to 91.6%. Indeed, the sequence divergence ranged from 20.3% to 21.4% (nt) and 8.4%–10.0% (aa). It has been suggested that the nucleotide sequence divergence between different JEV genotypes is ∼10%
Four viral encephalitis cases were reported in Malaya (n = 1) and Singapore (n = 3) in the summer of 1952. All patients exhibited high fever, vomiting, headache, disturbance of consciousness, stiff neck and deep coma with rapid progression to death by respiratory failure. Four virus strains were isolated from brain tissue specimens and identified as JEV by neutralization test using the Japanese Nakayama JEV strain
Many factors may contribute to spread of JEV
We would like to thank Hong-Yue Jiang and Jing-Lin Wang in State Key Laboratory for Infectious Disease Prevention and Control, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention for technical assistance.