• View in gallery

    Map of Vietnam showing locations of the 11 mosquito collection sites in this study.

  • View in gallery

    Distribution of the eight species of Culex mosquitoes collected in four provinces in the highlands of Vietnam in June and July 2007. The “other Culex species” group contains three species: Cx. bitaeniorhynchus, Cx. quinquefasciatus and Cx. pseudovishnui.

  • View in gallery

    Neighbor-joining dendrogram showing phylogenetic relationships of the nucleotide sequences of the E gene of 47 JEV strains. Bootstrap values correspond to 1,000 replications. Bar denotes the nucleotide similarity distance. The shaded area marks JEV genotype I strains. GenBank accession numbers for sequences used in the phylogenetic analysis are in Table 2. The five JEV strains isolated in this study (VNHT/05/2006, VNHT/07/2006, VNKT/479/2007, VNKT/486/2007, and VNTN/04/2008) are in bold.

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Surveillance of Japanese Encephalitis Virus Infection in Mosquitoes in Vietnam from 2006 to 2008

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  • Department of Medical Entomology, National Institute of Infectious Diseases, Tokyo, Japan; Department for Training and Research Management, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam; Department of Virology, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam; Department of Medical Entomology and Zoology, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam; Department of Medical Entomology and Zoology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan; National Institute of Hygiene and Epidemiology, Hanoi, Vietnam

Japanese encephalitis virus (JEV) infection in mosquitoes was monitored in Vietnam from 2006 to 2008. A total of 15,225 mosquitoes, identified as 26 species in five genera were collected and 12,621 were grouped into 447 pools for examination of JEV infection by assays for cytopathic effects in C6/36 cells and by RT-PCR to detect flavivirus RNA. Three JEV strains were isolated from Culex tritaeniorhynchus Giles collected in northern and southern Vietnam and two JEV strains were isolated from Culex vishnui Theobald collected in the highlands of Vietnam. Genetic and phylogenetic analyses, based on complete E gene nucleotide sequences, revealed that the five JEV strains were classified into the genotype I group and six amino acid differences were found in these five strains. These results indicated that multiple JEV genotype I populations are circulating countrywide in Vietnam, transmitted by bites of their Cx. tritaeniorhynchus and Cx. vishnui.

Introduction

Japanese encephalitis (JE) is a leading cause of viral encephalitis, an important human infectious disease, and is endemic in many Asian countries. Approximately 30,000–50,000 clinical JE cases, with 10,000 deaths, have been reported annually.1,2 Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has been found throughout Asia3,4 and relatively recently spread to south India, Sri Lanka, and northern Australia.57 The JEV is transmitted by paddy-breeding mosquitoes of the Culex vishnui subgroup, primarily Culex tritaeniorhynchus Giles, and amplified by infection of pigs and/or most likely Ardeidae birds in nature. Hence, JE distribution is significantly linked to irrigated rice production combined with pig rearing.

Several studies have previously analyzed the phylogentic relatedness of JEV strains isolated in Asian countries.810 The JEVs have been divided into five genotypes (genotype I to V) based on the nucleotide sequence of the viral envelope (E) gene.11 The only reported genotype V JEV is the Muar strain, first isolated from Malaysia in 1952, but genotype V JEV re-emerged in China in 2009.12 The JEV genotype I first appeared in Taiwan in 2008, but genotype III is still dominant in Taiwan.13,14 Genotype III was the major epidemic JEV genotype throughout the tropical and temperate regions of Asia before the 1990s.15 However, genotype III disappeared in northern Vietnam, Korea, and Japan during the early to mid-1990s and was replaced by genotype I.8,16 Genotype I has increased gradually and is now recognized as the dominant strain in most of the JEV-endemic areas in Asia.17 A molecular epidemiological study of JEV in Vietnam, China, and Japan divided genotype I into two subgenotypes (1-A and 1-B), and subgenotype 1-A into eight subclusters (1-A-1 to 1-A-8).18 These diverse JEV genotype I populations have been circulating in East Asia, with some being carried a long distance by migrating infected birds and/or mosquitoes.9 Vietnam has been suggested to be an important site on the flyway of birds and/or mosquitoes for the dispersal and consequent outbreaks of JEV throughout Asia.

In Vietnam, JEV has been circulating predominantly in rural environments and is recognized as one of the most significant mosquito-borne flaviviruses.19 The first JE case in Vietnam was reported in 1951 and JE epidemics then increased up to the late 1970s.2022 Clinical cases subsequently decreased after vaccination was introduced in 1997,19 However, JE is still endemic countrywide in Vietnam with an annual incidence of 1,000–3,000 cases in 2002.2,23 Epidemics of acute encephalitis syndrome (AES), on the other hand, have been frequently reported in Vietnam since the 1960s, with reported incidences as high as 22/100,000 population.20 A large number of AES cases were reported recently, however laboratory tests showed that 52% of these were JEV infections.19 Both JE and AES infections have been recognized as important public health problems in Vietnam.

Mosquito surveys were carried out in Ha Tay province in northern Vietnam in 2003, and the distribution of important JE vectors, Culex vishnui subgroup and Culex gelidus Theobald, were investigated.24 Arbovirus and mosquito surveillance was subsequently conducted from 2004 to 2006 and four virus species (i.e., Sagiyama, Getah, Oya, and Akabane viruses), but not JEV, were isolated from mosquitoes.25 However, these surveillance studies were mostly conducted in northern Vietnam, around the city of Hanoi. Therefore, in the current study, we focused on JEV infection in mosquitoes throughout Vietnam, not only in northern areas, to investigate the current distribution of JEV in Vietnam. It has recently been reported that there are amino acid variations in the E protein among JEV genotype I strains2629; we also carried out genetic and phylogenetic analyses of viral E gene sequences to study the relationships between Vietnamese JEVs and JEVs from other areas of Asia.

Materials and Methods

Study sites.

Mosquito collection was carried out at 11 sites in four districts of Vietnam from May 2006 to June 2008 (Figure 1). The sites were in Ha Tay province (the current administrative section is Hanoi city); Hai Phong city in northern Vietnam; Quang Binh province in central Vietnam; Kon Tum, Gia Lai, Dak Lak, and Dak Nong provinces in the highlands of Vietnam; and Tay Ninh province in southern Vietnam. Cat Que and Quang Trung communes in Ha Tay province (N21°13′ E105°49′ and N20°58′ E105°46′, respectively) are in mountainous and hilly areas, whereas Do Son town in Hai Phong city (N20°43′ E106°45′) is in a coastal area. Two communes, Tay Trach and Trung Trach, in Quang Binh province (N17°45′ E106°26′ and N17°31′ E106°31′, respectively) are near the coast. Each two collection sites in four provinces are located in the highlands, which are dry and cool during June and July. They were Hoa Binh in Kon Tum province (N14°16′ E107°59′), Jab Lang in Gia Lai province (N13°39′ E108°04′), Ebuk in Dak Lak province (N12°50′ E108°02′), and Tam Trang in Dak Nong province (N12°36′ E107°54′). Two communes in Tay Ninh province (N11°16′ E106°06′ and N11°21′ E106°11′, respectively) are in southwestern Vietnam, which has a tropical climate. Most commune inhabitants were rice farmers. Each site targeted for mosquito collection was located at a pig farm, a cattle farm, or a rice field within approximately a 10 m radius of a house with a confirmed human case of JEV.

Figure 1.
Figure 1.

Map of Vietnam showing locations of the 11 mosquito collection sites in this study.

Citation: The American Society of Tropical Medicine and Hygiene 88, 4; 10.4269/ajtmh.12-0407

Mosquito collection.

Adult mosquitoes were collected using several methods: Centers for Disease Control and Prevention (CDC) dry ice-baited traps,30 sweeping nets, aspirators, and animal nets24 based on the situation at each collection site. Sweeping nets and aspirators were used at all collection sites. In particular, animal net (cattle-baited) traps were used for mosquito collection in Quang Binh and Tay Ninh provinces, because it was difficult to supply dry ice for collection at these sites. Briefly, a stock stay was double covered with pieces of white nets to attract mosquitoes from livestock. Mosquitoes attracted to the outside of the inner net were caught by sweeping nets and/or aspirators from the area between the inner and outer nets. Female mosquitoes collected by this method did not have a blood meal in their body. Mosquito collection was generally carried out for 3 hours per night between 6:00 pm and 9:00 pm.

Identification of mosquito species.

All field-collected mosquitoes were transported on ice to the local provincial health office for species identification according to established identification keys.3133 Blood-fed females were kept in mosquito cages for a few days before species identification, because blood in the mosquito gut must be digested before being used for virus isolation. After species identification, mosquitoes were sorted by species and sex, pooled in 2 mL microtubes (Eppendorf, Hamburg, Germany), with a maximum of 50 adults per pool, and kept in a vapor shipper (MVZ, Wheaton, CA) during transportation to the National Institute of Hygiene and Epidemiology (NIHE), Hanoi. For Anopheles mosquitoes, species identification was supplemented with molecular procedures using internal transcribed spacer (ITS) 2 sequences according to the previously described method.34,35 Mosquitoes were sorted into pools containing 20–50 adults and stored at −80°C

Virus isolation.

The mosquito C6/36 cell line, derived from Aedes albopictus Skuse (Health Science Research Resources Bank (HSRRB), Osaka, Japan), was used for virus isolation as described previously.36,37 Briefly, pools of mosquitoes were homogenized in 500 μL ice-cold Eagle's minimum essential medium (MEM, Sigma-Aldrich, St. Louis, MO) containing 2% heat-inactivated fetal bovine serum (FBS, MP Biomedicals, Costa Mesa, CA), 2% non-essential amino acids (NEAA, Sigma-Aldrich), 200 U penicillin/mL, 200 μg streptomycin/mL, and 10 μL Fungizone (Gibco BRL, Gaithersburg, MD)/mL using a Mixer Mill (Model MM300, Retsch GmbH, Haan, Germany). The homogenates were clarified by centrifugation, and the resulting supernatants passed through sterile 0.45 μm filters (Ultrafree MC, Millipore, Bedford, MA). The filtrates were diluted 10-fold with the same medium and 50 μL of these were inoculated onto monolayers of C6/36 cells in 24-well culture plates. The plates were incubated for 2 hr at 28°C to allow virus adsorption. After addition of 500 μL fresh medium, the cell cultures were incubated under the same conditions for ∼7 d. Observations were made daily by phase-contrast microscopy for cytopathic effects. Culture supernatants were collected after at least three blind passages and used as virus stocks. The virus stocks were stored at −80°C.

Reverse transcription-polymerase chain reaction (RT-PCR) and sequence analysis of viral RNA.

Viral RNA was extracted from cell culture supernatants using a High Pure Viral RNA Kit (Roche Diagnostics, Mannheim, Germany) or a QIAamp Viral RNA Mini Kit (QIAGEN Inc., Valencia, CA). The RT-PCR was conducted using a TaKaRa One Step RT-PCR Kit (Takara Bio, Shiga, Japan). Flavivirus universal primer sets for fragments of the NS3 gene (Fla-U5004/Fla-L5457)38 and NS5 gene (FU1/cFD2 and FU2/cFD3)39 were used. The RT-PCR was carried out according to the manufacturer's instructions. The amplified products were purified by agarose gel electrophoresis, followed by fragment extraction using a QIAEXII Gel Extraction Kit (QIAGEN). Purified DNA fragments were directly cycle-sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Kit v.1.1 (Applied Biosystems, Foster City, CA) and ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Sequence analyses were performed using the GENETYX-WIN v.10 program (Genetyx Corp., Tokyo, Japan).

Phylogenetic analysis of JEV.

To analyze the phylogenetic relationships of the JEV isolates, we reconstructed a phylogenetic tree based on complete E gene nucleotide sequences of the five JEV strains isolated in this study and 42 JEV strains in the GenBank database. The sequences were aligned by CLUSTALX ver. 2.0.840 and the aligned matrix data were analyzed by a neighbor-joining (NJ) algorithm using MEGA ver. 4.1.41 The statistical significance of the resulting NJ tree was evaluated using a bootstrap test with 1,000 replications.

Results

Mosquito collection.

A total of 15,225 mosquitoes were collected during a 3-year survey in Vietnam (data not shown). Of the 26 species collected in Vietnam in this study, Cx. tritaeniorhynchus was the predominant species (34.4% of the mosquitoes collected), followed by Cx. quinquefasciatus Say (28.2%), Cx. vishnui Theobald (11.0%), and Cx. gelidus (7.0%). Because 497 mosquitoes in the Cx. vishnui subgroup, consisting of Cx. tritaeniorhynchus, Cx. vishnui, and Cx. pseudovishnui Colless, were unidentified (3.3%), the actual prevalence of both Cx. tritaeniorhynchus and Cx. vishnui could be higher. Of these 496 Anopheles mosquitoes collected (3.3%), a total of 485 specimens were classified (3.2%) in five species: An. peditaeniatus Leicester, An. sinensis Wiedemann, An. subpictus Grassi, An. tessellates Theobald, and An. vagus Doenitz. The determined sequences by ITS2 sequence analysis were deposited in the GenBank database (accession nos.: AB731654–AB731658, respectively).

Species composition of Culex mosquitoes in the highlands of Vietnam.

A total of 6,724 adult mosquitoes were collected in the four highlands provinces, with 93.2% identified as belonging to eight species of Culex mosquitoes (Figure 2). Culex tritaeniorhynchus was the most abundant species in the four highlands provinces followed by Cx. vishnui (50.8% and 26.5% of the total, respectively) (data not shown). After Cx. tritaeniorhynchus and Cx. vishnui, Cx. fuscocephala Theobald was the most abundant species in Kon Tum province, Cx. whitmorei Giles in Gia Lai province and Cx. gelidus in Dak Lak and Dak Nong provinces. Culex whitmorei was also collected in Kon Tum and Gia Lai provinces, and Cx. gelidus was also collected in Dak Lak and Dak Nong provinces. Significant differences in species compositions were found between June and July. The number of Cx. tritaeniorhynchus collected was significantly higher in June than in July in Kon Tum, Gia Lai, and Dak Lak provinces (χ2 test, P < 0.0001), but not in Dak Nong province. In contrast, the number of Cx. vishnui collected was significantly higher in July than in June (χ2 test, P < 0.0001) in Kon Tum, Dak Lak, and Dak Nong provinces, but not in Gia Lai province, although the significance of the difference was low (χ2 test, P > 0.4). The number of Cx. fuscocephala collected was higher in July than in June in Kon Tum, Gia Lai, and Dak Nong provinces (χ2 test, P < 0.0001), but not in Dak Lak province.

Figure 2.
Figure 2.

Distribution of the eight species of Culex mosquitoes collected in four provinces in the highlands of Vietnam in June and July 2007. The “other Culex species” group contains three species: Cx. bitaeniorhynchus, Cx. quinquefasciatus and Cx. pseudovishnui.

Citation: The American Society of Tropical Medicine and Hygiene 88, 4; 10.4269/ajtmh.12-0407

Virus isolation from mosquitoes.

A total of 12,621 mosquitoes (10,407 females and 2,214 males) in 447 pools were analyzed for flaviviruses using C6/36 cells (Table 1). Three of the 131 pools of Cx. tritaeniorhynchus collected from Ha Tay province in May 2006 and from Tay Ninh province in June 2008, and two of the 46 pools of Cx. vishnui collected from Kon Tum province in July 2007 were positive for JEV. All five JEV isolates were confirmed by their nucleotide sequences.

Table 1

Mosquitoes from Vietnam processed for JEV isolation from 2006 to 2008

SpeciesNo. mosquitoes testedNo. pools testedNo. JEV isolates
TotalFemaleMale
Aedes aegypti45223022223 
Ae. albopictus85612421 
Anopheles vagus429429013 
Armigeres spp.57282913 
Culex bitaeniorhynchus4402 
Cx. fuscocephala472472020 
Cx. gelidus1,0179774045 
Cx. infula1101 
Cx. pseudovishnui878706 
Cx. quinquefasciatus3,6931,8211,872101 
Cx. sitiens1101 
Cx. tritaeniorhynchus4,1994,182171313
Cx. vishnui1,5421,5420462
Cx. vishnui subgroup20020007 
Cx. whitmorei345345011 
Mansonia annulifera100101 
Ma. dives1101 
Ma. indiana7701 
Ma. uniformis191903 
Total12,62110,4072,2144475

The minimum infection rate (MIR) of JEV is defined as (number of JEV-positive pools/number of mosquitoes tested) × 1,000. From the data in Table 1, the MIR was 0.71 (3 positive pools/4,199 mosquitoes tested) for JEV in Cx. tritaeniorhynchus and 1.30 (2 positive pools/1,542 mosquitoes tested) in Cx. vishnui.

Genetic and phylogenetic analysis of JEV.

The complete nucleotide sequences of the E genes of the five JEVs isolated in this study have been submitted to the DNA Data Bank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL), and GenBank databases (Table 2). The details of the three JEV strains isolated from Cx. tritaeniorhynchus (designated VNHT/05/2006, VNHT/07/2006, and VNTN/04/2008) and the two JEV strains isolated from Cx. vishnui (designated VNKT/479/2007 and VNKT/486/2007) are shown in Table 2. An NJ tree was constructed based on 1,500 complete nucleotide sequences of the E genes with 42 corresponding strains from the GenBank database (Table 2). The NJ tree showed that the five JEV strains isolated in this study were in the JEV genotype I branch, with four strains in clade 1 of genotype I, and one strain in clade 2 of genotype I (Figure 3). Clade 1 strains of JEV are widespread throughout China, Korea, and Japan, and in the northern and highlands areas of Vietnam. Clade 1 can be divided into two subclusters, clade 1a and 1b. The four JEV strains of clade 1 isolated in this study were in subcluster clade 1a. Clade 2 contained JEV strains from southern Vietnam and Thailand, including one of the JEV strains isolated in this study. Clade 3 contained JEV isolates from Thailand and Australia, but no Vietnamese JEV isolated in this study.

Table 2

JEV strains used for phylogenetic analysis*

StrainYearLocation SourceGenotypeGenBank accession no.
TS002000AustraliaBadu IslandSwine serumIAF289814
SH-962001ChinaShanghaiCx. tritaeniorhynchusIAY555760
LN02-1022002ChinaLiaoningCx. tritaeniorhynchusIDQ404085
SH03-1282003ChinaShanghaiCx. tritaeniorhynchusIDQ404102
SC04-252004ChinaSichuanCulex spp.IDQ404094
GX05232005China Cx. tritaeniorhynchusIFJ161968
GX05582005China Cx. tritaeniorhynchusIFJ161969
XJP6132007China Cx. tritaeniorhynchusIEU693899
JX612008China Pig serumIGU556217
Mie/41/20022002JapanMieSwine bloodIAB112709
JaNAr01022002JapanNagasakiSwine bloodIAY377577
JEV-eq-Tottori2003JapanKurayoshiHorse cerebrumIAB213007
Mie/40/20042004JapanMieSwine bloodIAB231463
Kagawa-352004JapanKagawaSwine bloodIAB231464
JaNAr14-072007JapanNagasakiMosquitoIFJ185147
JaNAr06-072007JapanNagasakiMosquitoIFJ185143
K95P051994KoreaNagasakiCx. tritaeniorhynchusIU34929
KV18991999KoreaGyeonggiSwine bloodIAY316157
ThCMAr44921992ThailandChiang MaiMosquitoIDQ084229
JE_RT_362003ThailandRatchaburiSwine bloodIDQ087975
JE_KK_802004ThailandKhon KhenSwine bloodIDQ111784
JE_PK522004ThailandPhuketCx. quinquefasciatusIDQ084229
JE_CM_11962005ThailandChiang MaiSwine bloodIDQ238602
JE_KK_11162005ThailandKhon KhenSwine bloodIDQ343290
JE_KK_5802005ThailandKhon KhenSwine bloodIDQ238600
TPC0806c2008Taiwan Cx. tritaeniorhynchusIGQ260635
YL08062008Taiwan Cx. tritaeniorhynchusIGQ260633
VN882001VietnamHa NamSwine bloodIAY376464
VN222002VietnamHa TaySwine bloodIAY376465
VN342002VietnamHa TayMosquitoIAY376466
VN782002VietnamHa TayMosquitoIAY376467
VN1052002VietnamHa NamMosquitoIAY376468
LA_H07-052005VietnamLong AnSwine bloodIFJ185154
LAH_2079-052005VietnamLong AnSwine bloodIFJ185155
LA_H06-052005VietnamLong AnSwine bloodIFJ185153
VNHT/05/20062006VietnamHa TayCx. tritaeniorhynchusIAB728497
VNHT/07/20062006VietnamHa TayCx. tritaeniorhynchusIAB728498
VNKT/479/20072007VietnamKon TumCx. vishnuiIAB728499
VNKT/486/20072007VietnamKon TumCx. vishnuiIAB728500
VNKT/04/20082008VietnamTay NinhCx. tritaeniorhynchusIAB728501
WTP1970Malaysia MosquitoIIU70421
JKT54411981IndonesiaBali IslandAn. vagusIIU70406
FU1995Australia HumanIIAF217620
T1P11997TaiwanLiu-Chiu isletAr. subalbatusIIIAF254453
VN1181979VietnamHo Chin MinhCx. fatigansIIIU70420
TN2072002Taiwan MosquitoIIIEU683895
NJ20082008China  IIIGQ918133

The data in bold mark the five JEVs isolated in this study.

Figure 3.
Figure 3.

Neighbor-joining dendrogram showing phylogenetic relationships of the nucleotide sequences of the E gene of 47 JEV strains. Bootstrap values correspond to 1,000 replications. Bar denotes the nucleotide similarity distance. The shaded area marks JEV genotype I strains. GenBank accession numbers for sequences used in the phylogenetic analysis are in Table 2. The five JEV strains isolated in this study (VNHT/05/2006, VNHT/07/2006, VNKT/479/2007, VNKT/486/2007, and VNTN/04/2008) are in bold.

Citation: The American Society of Tropical Medicine and Hygiene 88, 4; 10.4269/ajtmh.12-0407

The amino acid sequences of the E genes of 14 JEV strains isolated in Vietnam and one JEV strain (Mie/41/2002, AB241119) isolated in Japan were compared. There were nine amino acid differences among these JEVs, at residues 10, 34, 36, 65, 83, 123, 159, 363, and 469 (Table 3). For the five JEV strains isolated in this study, the two isolates from Ha Tay province (VNHT/05/2006 and VNHT/07/2006) had the same amino acid sequence, but the other three strains (VNKT/479/2007, VNKT/486/2007, and VNTN/04/2008) had several amino acid differences. Namely, six amino acid differences were found in these five Vietnamese JEV strains.

Table 3

Amino acid differences in the E genes of JEV strains from Vietnam*

YearGenotypeStrainAmino acid at position in the E gene
1034366583123159363469
1979IIIVN118DMDVERATW
2002IMie/41/2002DMSVESATW
2001IVN88DMNVESATW
2001IVN78DMNVENATW
2002IVN105DMNVESATW
2002IVN22DMNVESATW
2002IVN34DMNVENATW
2005ILA_H06-05DMSVESATW
2005ILA_H07-05DMNVESATW
2005ILAH_2079-05DMSIDNVTW
2006IVNHT/05/2006DMNVENATW
2006IVNHT/07/2006DMNVENATW
2007IVNKT/479/2007DMNVESAAW
2007IVNKT/486/2007NSNVESATR
2008IVNKT/04/2008DMSVESATW

The data in bold mark the five JEVs isolated in this study.

Data for amino acids 1–500 in the E gene.

Data for the Mie/41/2002 strain has been submitted to the GenBank database (accession no. AB241119).

Discussion

The paddy-breeding mosquitoes, Culex vishnui subgroup, consists of Cx. tritaeniorhynchus, Cx. vishnui, and Cx. pseudovishnui, with Cx. tritaeniorhynchus being the primary vector of JEV throughout Asia.4 In this study, five JEV strains were isolated, two from pools of Cx. tritaeniorhynchus collected in Ha Tay province in northern Vietnam, one from a pool of Cx. tritaeniorhynchus in Tay Ninh province in southern Vietnam, and two from pools of Cx. vishnui collected in Kon Tum province in the highlands of Vietnam. A previous study carried out during June of 2002 and July–August 2004 in Ha Tay province found no JEV in any mosquito species,25 although the species composition in that report and this study were similar, except a difference in the investigation period. Because, our Ha Tay strains of JEV were obtained from Cx. tritaeniorhynchus collected in May. Therefore, we may conclude that JEV has been endemic and circulating throughout Vietnam, transmitted by Cx. tritaeniorhynchus and Cx. vishnui bites.

In the highland provinces of Vietnam, there was a seasonal difference between Cx. tritaeniorhynchus and Cx. vishnui, with Cx. tritaeniorhynchus predominant in June and Cx. vishnui predominant in July. Either species was in the majority depending on the season of the highlands of Vietnam. In particular, the MIR was 2.22 for the two JEV strains isolated from two pools containing a total of 899 Cx. vishnui collected in Kon Tum province, but was 1.3 for the two JEV strains isolated from two of the 46 pools containing a total of 1,542 Cx. vishnui collected throughout Vietnam (data not shown). However, JEV may be circulating in Vietnam by other Culex species. In Thailand, Cx. quinquefasciatus is an important JEV vector, probably because this species is predominantly an urban mosquito and the most common domestic mosquito species in semi-urban and rural areas.42 Hence, the two JEVs were probably isolated from Cx. vishnui in Kon Tum province in July 2007 because this was the most abundant Culex species in that province at that time. These results suggested that, in addition to Cx. tritaeniorhynchus, Cx. vishnui was an important vector for JEV transmission in Vietnam.

The JEV has been isolated from a number of mosquito species and Cx. tritaeniorhynchus to date; e.g., Cx. quinquefasciatus in Thailand; Cx. sitiens Wiedemann, Cx. rubithoracis Leicester, and Aedes vexans Theobald in Taiwan; Cx. annulirostris Skuse in Australia; and Armigeres subalbatus Coquillett in China.4245 Among these species, at least Cx. tritaeniorhynchus, Cx. pseudovishnui, Cx. gelidus, Cx. annulirostris, Cx. sitiens, and Cx. fuscocephala have been suggested to be efficient vectors in the laboratory.4648 One JEV isolate was recovered from Cx. vishnui captured in 1993 in northern Thailand.49 Culex vishnui should be recognized as the vector for JEV, although the vector competence of this species has been reported to date. In our study, a total of 497 mosquitoes that belonged in the Cx. vishnui subgroup have still not been unidentified. Distinguishing the three species in the Cx. vishnui subgroup has frequently been problematic,25,50,51 because there are too few morphological differences among them, in particular in the adult stage. Therefore, it has been necessary to distinguish these species using PCR-based techniques. Culex vishnui is endemic in all tropical regions and is the predominant species in most agricultural areas in Vietnam.24 The bionomics and vector competence of the members of the Cx. vishnui subgroup need to be closely monitored, because the vector competence of Cx. vishnui and Cx. pseudovishnui may be higher than has been recognized. The prevalence of Cx. quinquefasciatus has increased because of the urbanization surrounding Hanoi city and a large number of Cx. quinquefasciatus have been collected near Hanoi; e.g., in Ha Tay province and Hai Phong city (data not shown). Although Cx. quinquefasciatus carrying JEV have not been found, JEV transmission by this species should be monitored in Vietnam and in Thailand.42

The NJ phylogenetic tree reconstructed with the five JEV strains isolated in this study and 42 other JEV strains showed that the JEV genotype I strains were divided into three subgenotypes (clade 1, 2, and 3 in Figure 3). Clade 1 strains of JEV are widely spread throughout China, Korea, and Japan, and northern Vietnam and the highlands of Vietnam. In particular, the four JEV strains isolated from Ha Tay and Kon Tum provinces in this study were in the clade 1a subcluster. Clade 1a is probably the same as the previously reported subcluster 1-A-1 based on other criteria.18 Clade 1b probably includes five different subclusters, previously described as 1-A-2 to 1-A-6,18 but no JEV strain isolated in Vietnam in this study was in clade 1b. Subgenotype clade 2 contained JEV isolates from Thailand and southern Vietnam. Although three JEV isolates recovered from Long An province in southern Vietnam (LA_H07-05, LAH_2079-05, and LA_H06-05) were not previously classified in any subcluster,18 one isolate from Tay Ninh province in this study (VNTN/04/2008) formed a new subcluster with the three Long An isolates. The remaining subgenotype, clade 3, contained Australian and Thailand JEV strains, but no Vietnamese strain. Clade 3 has been suggested to be equivalent to subgenotype 1-B based on previous criteria.18 This phylogenetic tree showed that the classification of each JEV strain was not related to its vector species (Cx. tritaeniorhynchus or Cx. vishnui), but to the geography of its collection sites.

It has been reported that there are amino acid variations in the E protein among JEV genotype I strains.2629 Single amino acid substitutions at several E protein positions have been suggested to be associated with JEV virulence. For example, a Glu to Lys substitution at amino acid 138 attenuates JEV virulence,26,27 and a Met to Lys substitution at amino acid 279 increases JEV virulence in mice.28 In addition, a Ser to Arg substitution at amino acid 123 affects JEV growth and pathogenicity.29 Alignment of JEV E gene nucleotide sequences in the GenBank database showed that the great majority of JEV strains had Ser, not Arg, at residue 123.29 The five Vietnamese JEV isolates in this study had amino acid differences at nine E protein positions. The amino acid differences in the three JEV strains from the highlands and southern Vietnam (VNKT/479/2007, VNKT/486/2007, and VNTN/04/2008) appeared similar to those in the JEV strains previously isolated in Long An province in southern Vietnam (LA_H06-05, LA_H07-05, and LAH_2079-05). A Ser to Asp substitution in amino acid 123 has been found in JEV isolated from mosquitoes in Vietnam since the 2000s. Both JEV strains with Ser at amino acid 123 and JEV strains with Asp at amino acid 123 have been isolated in Vietnam. Further data are needed to explain the function and mechanism of the E protein in JEV infection, and on other structural and non-structural JEV proteins (e.g., prM and NS4A5254) and on the JEV 5′ and 3′ NTRs.29,55

This study has identified multiple JEV populations in Vietnam, and the nucleotide sequences of these JEVs were similar to those isolated in other area of Asia. This suggested that some JEV strains have migrated between different areas in Asia by vector mosquitoes. In addition, a large number of AES cases have been reported in Vietnam, and over 50% of these AES cases had laboratory evidence of recent JEV infection.19,23 The effects of nucleotide substitutions on JEV virulence and pathogenesis may be related to the high prevalence of AES cases in Vietnam. Accurate JE surveillance data has recently been reported from Vietnam, Japan, and other Asian countries.4,12,13,43,51 Hence, ecological and epidemiological JEV surveillance in humans and mosquitoes should be carried out in JEV-endemic areas to provide data for understanding JEV transmission and infection.

ACKNOWLEDGMENTS

We thank Trang Huynh T.T. of the Department of Medical Entomology and Zoology, Pasteur Institute in Ho Chi Minh City, Vietnam, the staff members of Department of Medical Entomology and Zoology, National Institute of Hygiene and Epidemiology, Vietnam, and the Department of Medical Entomology of National Institute of Infectious Diseases, Japan, for their kind arrangements for field work and helpful criticisms. We also thank K. Morita of the Department of Virology of Institute of Tropical Medicine, Nagasaki University, Japan, for his helpful discussion.

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Author Notes

* Address correspondence to Kyoko Sawabe, Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. E-mail: sawabe@nih.go.jp† These authors contributed equally to this work.

Financial support: This work was partially supported by grant-in-aids awarded by the Ministry of Health, Labor and Welfare (H21-Shinko-Ippan-005), JSPS KAKENHI Grant (no. 21406012) and by Japan initiative for global Research network on infectious diseases (J-grid) (Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases (2005–2009).

Authors' addresses: Ryusei Kuwata, Keita Hoshino, Haruhiko Isawa, Toshinori Sasaki, Yoshio Tsuda, Mutsuo Kobayashi, and Kyoko Sawabe, Department of Medical Entomology, National Institute of Infectious Diseases, Tokyo, Japan, E-mails: ryusei@nih.go.jp, khoshino@nih.go.jp, hisawa@nih.go.jp, tsasaki@nih.go.jp, tsudayso@nih.go.jp, mutsuo@nih.go.jp, and sawabe@nih.go.jp. Phan Thi Nga, Department for Training and Research Management, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam, E-mail: pnga_arboviruses@yahoo.com. Nguyen Thi Yen and Tran Vu Phong, Department of Medical Entomology and Zoology, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam, E-mails: yenanihe@yahoo.com and tranvuphong@yahoo.com. Yukiko Higa and Masahiro Takagi, Department of Medical Entomology and Zoology Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan, E-mails: yukko@nagasaki-u.ac.jp and mstakagi@nagasaki-u.ac.jp. Nguyen Vet Hoang, Bui Minh Trang, and Do Phuong Loan, Department of Virology, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam, E-mails: hoangyhn2000@gmail.com, trangminhbui3007@gmail.com, and loankobe@yahoo.com.

Reprint requests: Kyoko Sawabe, Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan, Tel: 81-3-5285-1111, Fax: 81-3-5285-1178, E-mail: sawabe@nih.go.jp.

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