• View in gallery

    Survey sites for virus isolation in Toyama Prefecture, Japan, during 2004–2009. Stars indicate sites of pigpens where pig serum samples were obtained. Other symbols indicate corresponding sites as indicated in the box where mosquitoes were obtained for virus isolation. Filled marks indicate sites where Japanese encephalitis virus–positive specimens were obtained.

  • View in gallery

    Survey sites for detecting the seasonal changes in the number of female Culex tritaeniorhynchus mosquitoes in Toyama Prefecture, Japan. Triangle, circles, and diamond indicate farms where mosquitoes were obtained and counted. Mosquitoes were obtained at six sites during 2004–2009. Mosquitoes were obtained at three sites only in certain years: the numbers near the three circles indicate years of collection. Mosquitoes were collected at seven sites every year.

  • View in gallery

    The minimum infection rate (MIR) of Japanese encephalitis virus (JEV) and number of female Culex tritaeniorhynchus mosquitoes, Toyama Prefecture, Japan. The MIRs were calculated by using the formula (JEV-positive pool number/number of mosquitoes tested) × 1,000. Numbers at the top left of each graph indicate years. A, The MIR of JEV of female Cx. tritaeniorhynchus at the pigpen in Nanto (Figure 1) and at the cattle shed in Toyama (Figure 1), and number of female Cx. tritaeniorhynchus at seven farms (Figure 2). Numbers of female Cx. tritaeniorhynchus are shown as averages. Average numbers of mosquitoes were calculated from the weekly collection numbers and excluded maximum and minimum values among the seven farms to remove anomalous data. Virus isolation was not performed at the pigpen in Nanto in 2004. B, The MIR of JEV of female Cx. tritaeniorhynchus in pigpens and cattle sheds and average number of female Cx. tritaeniorhynchus during 1966–1972. Data were obtained from reports of previous studies conducted in Toyama Prefecture.10,18 Average numbers of female Cx. tritaeniorhynchus were calculated as described in A among 4–10 farms. For the MIR, first and last dates of investigation and dates when JEV was detected from mosquitoes are plotted. The first date in 1970 was May 25th and is not plotted in this graph.

  • View in gallery

    Phylogenetic tree of envelope (A) and capsid/premembrane (B) genes of Japanese encephalitis virus (JEV) isolates. Japanese encephalitis virus isolates from Toyama Prefecture, Japan, are shown as Toyama/year (isolate numbers) or isolate name. Isolate names are given to the JEVs isolated in this study as indicated by Mo (mosquitoes) or Sw (swine)/Toyama (prefecture)/sample no. and inoculated cell (c = C6/36 and v = Vero)/year. GI–GIV indicates JEV genotypes. Reference strains are shown by accession no./strain name/country/prefecture/year. Sequence of Murray Valley encephalitis (MVE) virus was used as an outgroup. Scale bar indicates genetic distance in nucleotide substitutions per site. Numbers at branches indicate bootstrap values (%) > 50%. Bootstrap replications were performed 1,000 times.

  • View in gallery

    Alignment of nucleotide sequences of the 3′-untranslated regions (UTRs) of Toyama isolates and the reference strains of Japanese encephalitis virus (JEV), Toyama Prefecture, Japan. Japanese encephalitis virus isolates in Toyama Prefecture are shown as Toyama/year (number of isolates) in bold letters. Reference strains are shown by strain name (accession no.). GI–GIV indicates JEV genotypes. Deletions are indicated by hyphens. The stop codons are underlined. Novel deletion sites are in boxes. Nucleotides of strains isolated in this study that were different from Toyama/2005 (19 isolates) are indicated by asterisks.

  • View in gallery

    Changes in Japanese encephalitis virus (JEV) in Toyama Prefecture, Japan, during 2005–2009. The capsid/premembrane (C/prM) and envelope (E) genes are shown by cluster or subcluster names. The 3′ untranslated regions (UTRs) are shown by the deletion types indicated in Figure 5 Ishikawa type indicates the same deletion as Ishikawa (accession no. AB051292) and Sw/Mie/40/2004 (accession no. AB241118). Ishikawa + Kagawa type indicates the same deletion as Sw/Kagawa/35/2004 (accession no. AB231627). Ishikawa + Novel type indicates the novel deletion type observed in 2008 and 2009.

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Continuity and Change of Japanese Encephalitis Virus in Toyama Prefecture, Japan

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  • Toyama Institute of Health, Toyama, Japan; Department of Medical Entomology, National Institute of Infectious Diseases, Tokyo, Japan; Toyama-Airport Detached Office of Niigata Quarantine Station, Toyama, Japan; Laboratory of Public Health, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan

To determine the mechanisms of maintenance and evolution of Japanese encephalitis virus (JEV) in a temperate zone, we attempted to isolate JEV from mosquitoes and pigs in Toyama Prefecture, Japan. A total of 87 JEVs were isolated from female Culex tritaeniorhynchus mosquitoes and pigs during 2005–2009. The prevalence of JEV in Toyama Prefecture was seasonally late in comparison with that of the virus during 1966–1972. Furthermore, JEVs were isolated after the peak in the number of female Cx. tritaeniorhynchus. Among JEV strains isolated in this study, two distinct groups were observed within genotype I of the phylogeny generated from nucleotide sequence information derived from the envelope and capsid/premembrane genes: strains belonging to the major type were isolated during 2005–2009, and strains from the minor type were isolated only in 2007. The major type has exhibited gradual change in its envelope and capsid/premembrane genes, and all isolates obtained in 2008 and 2009 had a novel deletion of seven nucleotides in the variable region of the 3′-untranslated region.

Introduction

Japanese encephalitis virus (JEV) belongs to the family Flaviviridae and genus Flavivirus. The JEV genome is a single-stranded, positive-sense RNA molecule of approximately 11 kb, which comprises 5′- and 3′-untranslated regions (UTRs) and a single open reading frame.1 The open reading frame encodes structural proteins (capsid [C], premembrane [prM], and envelope [E]) and nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).2,3 On the basis of nucleotide sequence information for the E gene, JEV has been divided into five genotypes.4,5

Japanese encephalitis virus is a mosquito-borne virus and Culex tritaeniorhynchus mosquitoes are the most important vector of JEV in Japan. The virus exists in an enzootic cycle between mosquitoes and either pigs or birds.1 Pigs are the major amplifier and reservoir for JEV.6,7

Japanese encephalitis virus causes severe encephalitis in humans and has caused epidemics in eastern and southern Asia. In Japan, hundreds to thousands of cases of JEV infection in human were reported every year until the 1960s.8 Since 1992, less than 10 cases/year have been reported because of vaccinations that were introduced in Japan in 1954 and environmental changes, such as the separation of houses from pigpens. However, after the use of the JEV vaccination was discouraged in 2005 because of the occurrence of acute disseminated encephalomyelitis after vaccination, herd immunity against JEV in persons in Japan has decreased9 and the threat of an outbreak of JEV has increased.

Environmental conditions in Toyama Prefecture support the enzootic cycle for JEV because there are pigs, the amplifying host of JEV, and many rice fields where the larvae of Cx. tritaeniorhynchus can develop. Since the 1970s, human cases of JEV infection have been reported in 1982 and 1997 in Toyama Prefecture.10 Since 1965, antibodies against JEV in pig serum have been investigated in Toyama Prefecture.9,10 The finding that the seroprevalence of many newly born pigs has exceeded 50% almost every year suggests that JEV is still prevalent. Conversely, we predicted that certain factors, such as the method of breeding pigs and control of rice fields, affect the prevalence of mosquitoes10,11 and JEV. Because small pigpens gradually decreased in number and large ones increased, the total number of pigpens decreased from the 1960s to the 1970s.10,11 Furthermore, pigpens have moved from near rice fields and houses on the plains to hillsides in recent years. As a result, the likelihood that pigs and humans are bitten by Cx. tritaeniorhynchus might have decreased.

In recent reports, researchers have discussed from where and how JEV came to Japan.12,13 It has also been considered necessary to clarify how JEV strains were maintained in local areas after the most frequently isolated genotype changed from III to I in the 1990s14 in Japan. A previous study of genetic change and variation in JEV genotype III in Taiwan15 suggested that JEV isolates fall into three clusters by area and are genetically stable in Taiwan.

In this study, we isolated JEVs from mosquitoes and pigs in Toyama Prefecture and performed genetic analysis to determine how JEV maintains genetic continuity or undergoes genetic changes locally. Furthermore, to assess the effect of environmental changes such as the method of breeding pigs and control of rice fields, we investigated the relationship between the prevalence of JEV and that of mosquitoes and compared these findings with data described in previous reports.

Materials and Methods

Mosquitoes.

To isolate viruses, mosquitoes were obtained once a week by using CO2 traps during 2004–2009 at 21 sites, which included six farms (three pigpens, two cattle sheds, and one horse stable), seven gardens of private houses, four wooded areas, one airport, and three harbors (Figure 1). The traps were battery-operated light traps (Inokuchi-Tekko, Nagasaki, Japan), CDC Miniature Light Traps (John W. Hock Company, Gainesville, FL), 12 volt battery-operated light traps (FHK, Fujihira Industry Co. Ltd., Tokyo, Japan), or 6 volt battery-operated traps (Rakuno Gakuen University, Hokkaido, Japan), which were set with dry ice or a CO2 refill and left overnight. Some mosquitoes were obtained by using a net on planes at an airport. Mosquitoes were classified according to collection site, date of collection, species, and sex. Mosquitoes were then pooled into groups that consisted of a maximum of 50 individuals and stored at −80°C.

Figure 1.
Figure 1.

Survey sites for virus isolation in Toyama Prefecture, Japan, during 2004–2009. Stars indicate sites of pigpens where pig serum samples were obtained. Other symbols indicate corresponding sites as indicated in the box where mosquitoes were obtained for virus isolation. Filled marks indicate sites where Japanese encephalitis virus–positive specimens were obtained.

Citation: The American Society of Tropical Medicine and Hygiene 84, 5; 10.4269/ajtmh.2011.10-0188

To study the seasonal changes of the female Cx. tritaeniorhynchus population, mosquitoes were captured by using light traps at seven farms (one pigpen, five cattle sheds, and one horse stable) during June–October 2004–2009 (Figure 2). The traps were set overnight once a week. Mosquitoes were classified and counted as described above. The average numbers of mosquitoes were calculated from the weekly collected numbers and excluded the maximum and the minimum values for the seven farms.

Figure 2.
Figure 2.

Survey sites for detecting the seasonal changes in the number of female Culex tritaeniorhynchus mosquitoes in Toyama Prefecture, Japan. Triangle, circles, and diamond indicate farms where mosquitoes were obtained and counted. Mosquitoes were obtained at six sites during 2004–2009. Mosquitoes were obtained at three sites only in certain years: the numbers near the three circles indicate years of collection. Mosquitoes were collected at seven sites every year.

Citation: The American Society of Tropical Medicine and Hygiene 84, 5; 10.4269/ajtmh.2011.10-0188

Minimum infection rate.

To estimate mosquito infection rates, the minimum infection rate (MIR) was calculated. The MIR of JEV is defined as (JEV-positive pool number/number of mosquitoes tested) × 1,000.

Pig serum sample.

A total of 1,451 serum samples were obtained from pigs approximately six months of age in four areas (Nanto, Oyabe, Kamiich, and Kurobe) in Toyama Prefecture (Figure 1) during July–October 2005–2009.

Virus isolation.

Pools of mosquitoes were homogenized in a 0.5–1.0 mL of maintenance medium (Eagle's minimum essential medium containing 2% fetal bovine serum or 0.11% bovine serum albumin fraction V) and centrifuged at 5,867 × g for 5 minutes. The supernatants were passed through 0.45-μm filters (Ultrafree MC; Millipore Corp., Bedford, MA). The filtrates were diluted 10-fold with the medium and inoculated onto monolayers of C6/36 (no. IFO50010; Health Sciences Research Resources Bank, Osaka, Japan) and Vero (no. JCRB9013; Health Sciences Research Resources Bank) cells. These cultures were incubated at either 28°C (C6/36) or 35°C (Vero) for 2 hours in an atmosphere of 5% CO2. After maintenance medium was added, these cells were incubated for 6–8 days. Pig serum samples were diluted 10-fold with the medium and inoculated onto the cell monolayers as described above. Two or three cell passages were usually performed and culture media were obtained when cytopathic effects appeared.

RNA extraction.

Viral RNA was extracted from culture supernatants with a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA) in accordance with the manufacturer's instructions.

Reverse transcription–polymerase chain reaction.

Reverse transcription–polymerase chain reaction (RT-PCR) was carried out with either the TaKaRa One Step RNA PCR Kit (AMV) (TaKaRa Bio Inc., Otsu, Japan) or Ready-To-Go RT-PCR Beads (GE Healthcare, Piscataway, NJ). The E gene was amplified with primers JE955f (5′-TGYTGGTCGCTCCGGCTTA) and JE2536r (5′-AAGATGCCACTTCCACAYCTC).12 The mixture was incubated at 50°C for 45 minutes; 94°C for 2 minutes; 45 cycles at 94°C for 1 minute, 50°C for 1 minute, and 72°C for 2 minutes; and 72°C for 10 minutes. The C/prM gene was amplified with primers JE-prM-FW (5′-CGYCGTGAACAAGCGGGGCARAAA) and JE-prM-RV (5′-TGCAGCGACCATAYTGSACGTAGA) (Hoshino Y. and others, unpublished data). The 3′ UTR was amplified with primers JE10141f (5′-TGGATTGAAGAAAATGAATGGATG) and JE10965r (5′-AGATCCTGTGTTCTTCCTCTC).12 The mixture was incubated at 53°C for 40 minutes; 40 cycles at 92°C for 1 minute, 53°C for 1 minute, and 72°C for 1 minute; and 72°C for 5 minutes.

Sequencing analysis.

After purification of the amplicons, the E, C/prM, and 3′UTR gene sequences were determined by using the BigDye Terminator v1.1 or v3.1 Cycle Sequencing Kit and ABI 3100 or 3130 sequencer (Applied Biosystems, Branchburg, NJ). Nucleotide sequences were edited and aligned by using Sequencher (version 4.7) software (Gene Codes Co., Ann Arbor, MI).

Phylogenetic analysis.

The nucleotide sequences of the reference strains of JEV were obtained from GenBank and 1,500-basepair sequences of the E region or 240 of 299 basepair sequences of the C/prM region were analyzed by using MEGA 3.1 software.16 A phylogenetic tree was constructed by using the neighbor-joining method, and genetic distances were calculated according to Kimura's two-parameter method.17 The reliability of the tree was estimated by performing 1,000 bootstrap replications, and bootstrap values ≥ 50% were considered statistically significant for a grouping. A phylogenetic tree was also constructed by using the maximum-likelihood method in PhyML 3.0 (http://atgc.lirmm.fr/phyml/) and NJplot (http://pbil.univ-lyon1.fr/software/njplot.html).

Japanese encephalitis virus sequences generated in this study have been submitted to GenBank under accession numbers AB538601–AB538852 and AB543738–AB543746.

Results

Mosquitoes and pig serum.

Japanese encephalitis virus isolation was performed to investigate the species of mosquitoes and the sites where JEV is prevalent. In total, 51,265 mosquitoes (2,740 pools), representing 15 species, were used for virus isolation, which included 45,190 Cx. tritaeniorhynchus (88.1%), 4,590 Culex pipiens group (9.0%), 1,333 Aedes albopictus (2.6%), and other mosquitoes from 12 species (Table 1). Most of the Cx. tritaeniorhynchus were captured on farms, whereas Cx. pipiens group and Ae. albopictus were usually captured at other survey sites (Table 2).

Table 1

Number of mosquitoes used for virus isolation classified by species, Toyama Prefecture, Japan

Species200420052006200720082009Total
No. sampledNo. in poolNo. sampledNo. in poolNo. sampledNo. in poolNo. sampledNo. in poolNo. sampledNo. in poolNo. sampledNo. in poolNo. sampledNo. in pool
Culex tritaeniorhynchus2,6771398,2333053,14719213,37033713,8513043,9129445,1901,371
Culex pipiens group9141651,47523376218568512526668488504,590826
Aedes albopictus18482318115381127643328145105341,333436
Culex orientaris1155198137113922
Tripteroides bambusa322810323414
Aedes japonicus5510955332322
Culex infantulus4453551412
Aedes flavopictus108221210
Culex bitaeniorhynchus1144112121108
Uranotaenia novobscura65111187
Armigeres subalbatus11222255
Anopheles sinensis11221144
Aedes nipponicus1111
Lutzia vorax1111
Culex modestus inatomii1111
Total3,79740610,0616834,35053514,14651414,4024214,50918151,2652,740
Table 2

Number of Culex tritaeniorhynchus, Culex pipiens group, and Aedes albopictus mosquitoes used for virus isolation classified by sites, Toyama Prefecture, Japan

SiteCulex tritaeniorhynchusCulex pipiens groupAedes albopictus
No. sampledNo. in poolNo. sampledNo. in poolNo. sampledNo. in pool
Farm42,3819791,427129138
House2,3262331,835320755251
Wood407109936260182101
Airport5341102638123
Harbor2392905430253
Total45,1901,3714,5908261,333436

A total of 51 of 1,371 pools of Cx. tritaeniorhynchus harbored JEV through the investigation period (Table 3). All mosquitoes positive for JEV were females and obtained on farms (Table 3). Of these 51 pools, 35 were obtained near a pigpen and 16 were obtained in a cattle shed. Japanese encephalitis virus was not isolated in 2004 or 2006 from mosquitoes. Samples for virus isolation were simultaneously applied to both C6/36 and Vero cells because the viruses derived from the same sample but isolated by different culture cells often have different nucleotide sequences. In total, 77 JEVs were isolated from mosquito samples, of which 51 viruses were isolated in C6/36 cells and 26 viruses were isolated in Vero cells. Japanese encephalitis viruses were isolated from nine pig serum samples (Table 3). Two of these nine serum samples were obtained in September 2005, six were obtained during September–October in 2007, and one was obtained in September 2008. Japanese encephalitis virus was not isolated in 2006 or 2009 from pig serum samples Ten JEVs were isolated from pig serum samples, of which seven viruses were isolated in C6/36 cells and three viruses were isolated in Vero cells.

Table 3

Number of pools of Culex tritaeniorhynchus mosquitoes or pig serum samples from which Japanese encephalitis virus was isolated, Toyama Prefecture, Japan*

Sites200420052006200720082009Total
Cx. tritaeniorhynchus(No. in pool)Farm0/4111/1540/12110/29227/2863/8551/979
House0/750/990/59NTNTNT0/233
Wood0/230/440/80/34NTNT0/109
AirportNT0/80/40/110/110/70/41
HarborNTNTNTNT0/70/20/9
Total0/13911/3050/19210/33727/3043/9451/1,371
Pig serum sampleNantoNT0/930/1241/1780/900/601/545
OyabeNT2/800/1012/1700/850/604/496
KamiichiNTNT0/453/800/750/603/260
KurobeNTNTNTNT1/900/601/150
TotalNT2/1730/2706/4281/3400/2409/1,451

Values are no. positive/no. tested. NT = not tested.

The farms where JEV-positive mosquitoes and pig serum samples were obtained were located in rural areas and suburbs of Toyama Prefecture (Figure 1). These sites were distributed in both western and eastern areas of Toyama Prefecture and were not concentrated in any particular place. Japanese encephalitis viruses were simultaneously isolated from mosquitoes and pig serum samples at a pigpen in Nanto (Figure 1).

To clarify the correlation between seasonal change in mosquito numbers and prevalence of JEV, the average numbers of female Cx. tritaeniorhynchus mosquitoes obtained on seven farms (Figure 2) were determined during 2004–2009. Their seasonal changes were compared with the MIR of JEV at two survey sites, the pigpen in Nanto and the cattle shed in Toyama (Figure 3A and Figure 1).

Figure 3.
Figure 3.

The minimum infection rate (MIR) of Japanese encephalitis virus (JEV) and number of female Culex tritaeniorhynchus mosquitoes, Toyama Prefecture, Japan. The MIRs were calculated by using the formula (JEV-positive pool number/number of mosquitoes tested) × 1,000. Numbers at the top left of each graph indicate years. A, The MIR of JEV of female Cx. tritaeniorhynchus at the pigpen in Nanto (Figure 1) and at the cattle shed in Toyama (Figure 1), and number of female Cx. tritaeniorhynchus at seven farms (Figure 2). Numbers of female Cx. tritaeniorhynchus are shown as averages. Average numbers of mosquitoes were calculated from the weekly collection numbers and excluded maximum and minimum values among the seven farms to remove anomalous data. Virus isolation was not performed at the pigpen in Nanto in 2004. B, The MIR of JEV of female Cx. tritaeniorhynchus in pigpens and cattle sheds and average number of female Cx. tritaeniorhynchus during 1966–1972. Data were obtained from reports of previous studies conducted in Toyama Prefecture.10,18 Average numbers of female Cx. tritaeniorhynchus were calculated as described in A among 4–10 farms. For the MIR, first and last dates of investigation and dates when JEV was detected from mosquitoes are plotted. The first date in 1970 was May 25th and is not plotted in this graph.

Citation: The American Society of Tropical Medicine and Hygiene 84, 5; 10.4269/ajtmh.2011.10-0188

At the pigpen, the MIR of JEV peaked during September–October and the number of female Cx. tritaeniorhynchus mosquitoes on the seven farms showed two peaks in August and September (Figure 3A), indicating that the MIR of JEV increased after the peak in the number of female mosquitoes. Japanese encephalitis virus isolation from mosquitoes in this pigpen was not performed in 2004. Conversely, the MIR at the cattle shed peaked during August–early September in 2005, 2007, and 2008, when most mosquitoes were captured (Figure 3A). In 2009, the MIR of JEV at the cattle shed followed the peak in the number of mosquitoes and peaked in early October. In 2006, only a few mosquitoes were captured at the two survey sites (Figure 3A). Thus, JEV was not isolated from either mosquitoes or pigs (Figure 3A and Table 3).

The number of female Cx. tritaeniorhynchus mosquitoes and the MIR of JEV among them during 1966–197218 are shown in Figure 3B. Japanese encephalitis viruses were isolated from the end of July to early September when the MIR and the number of mosquitoes peaked, with the exception of 1968. These data suggest that the JEV isolation period has been delayed in recent years compared with that in 1966–1972.

Phylogenetic analysis.

To estimate how JEVs underwent genetic change or showed continuity in Toyama Prefecture over several years, phylogenetic analysis was performed for 87 isolates (77 from mosquitoes and 10 from pig serum samples) in Toyama. All JEV isolates were classified into genotype I on the basis of sequencing analyses of E (Figure 4A) and C/prM genes (Figure 4B). Genotype I of JEV became the dominantly isolated genotype in Japan in the 1990s. Before that time, genotype III was the most frequently isolated genotype in Japan.14

Figure 4.
Figure 4.

Phylogenetic tree of envelope (A) and capsid/premembrane (B) genes of Japanese encephalitis virus (JEV) isolates. Japanese encephalitis virus isolates from Toyama Prefecture, Japan, are shown as Toyama/year (isolate numbers) or isolate name. Isolate names are given to the JEVs isolated in this study as indicated by Mo (mosquitoes) or Sw (swine)/Toyama (prefecture)/sample no. and inoculated cell (c = C6/36 and v = Vero)/year. GI–GIV indicates JEV genotypes. Reference strains are shown by accession no./strain name/country/prefecture/year. Sequence of Murray Valley encephalitis (MVE) virus was used as an outgroup. Scale bar indicates genetic distance in nucleotide substitutions per site. Numbers at branches indicate bootstrap values (%) > 50%. Bootstrap replications were performed 1,000 times.

Citation: The American Society of Tropical Medicine and Hygiene 84, 5; 10.4269/ajtmh.2011.10-0188

Using the E gene sequences, we subdivided the isolates in this study into three clusters: A, B, and C (Figure 4A). There were 19–35 nucleotide differences among clusters A, B, and C (Table 4). With the exception of 16 isolates in cluster A, viruses in each cluster had the same amino acid sequences (Table 5). Cluster A was further divided into three subclusters: A-1, A-2, and A-3 (Table 4). These subclusters differed from each other by 2–13 nucleotides. Subcluster A-1 was composed of 21 isolates in 2005. Fifteen isolates in 2007, seven isolates in 2008, and three isolates in 2009 belonged to subcluster A-2. Subcluster A-3 was composed of 35 isolates in 2008. The phylogenetic tree of the JEV isolates in this study and the reference strains is shown in Figure 4A. The isolates that belonged to cluster A were similar to the reference strains isolated in Hyogo, Japan, 2008 (accession no. AB481224), Sw/Mie/40/2004 (isolated in Mie, Japan, 2004), 01VN88 (isolated in HaTay, Vietnam, 2001), SC04-16 (isolated in Sichuan, China, 2004), and 02VN105 (isolated in HaNam, Vietnam, 2002). Three isolates in 2007 belonged to cluster B (Table 4) and their nucleotide sequences matched 100% with strain JaNAr07-04 (isolated in Nagasaki, Japan, 2004) and were similar to SH03-124 (isolated in China, 2003) (Figure 4A). The other three isolates that belonged to cluster C (Table 4) were similar to strains Sw/Kagawa/35/2004 (isolated in Kagawa, Japan, 2004), YN86-B8639 (isolated in Yunnan, China, 1986), and SH03-127 (isolated in Shanghai, China, 2003) (Figure 4A).

Table 4

Nucleotide sequence differences within envelope (E) gene among strains of Japanese encephalitis virus isolated, Toyama Prefecture, Japan

No. isolatesClusterSubclusterNucleotide no. in the E region3615305487105120156201204207228240261291294435438481510522541570594642654690692753765795841849865894934
2005200720082009ConsensusTTATCCAAACTAACCCTATGTTGCCTCCCTTGATCCA
1AA-1
1Y
1Y
1T
1T
1
1WYY
1
2TTCT
2TTT
2TT
2
5TTT
1AA-2GTCR
1GTC
2GCTCG
2GCTCG
9GTC
2GTTC
2GATC
3GTC
1GTC
2GTC
1AA-3GTTTC
1GTTC
1GTTC
1TGTTC
1GTTC
2GTTC
2GTTCC
2GATTC
24GTTC
3BCGCTGTTCCTTT
1CCCGTGTCGCACTTA
2CCGTGTCGCACTTA
No. isolatesClusterSubclusterNucleotide no. in the E region9579679699849939961011102810351055105910681071107710041006111611201134114911521164117611851194121212181233126313231341138913981425142914341480
2005200720082009ConsensusTGCTTTTCAGGAACGTCACCGGTCACTGTTTACAGGC
1AA-1AY
1CAR
1RR
1R
1A
1AT
1RY
1
2
2Y
2GA
2A
5
1AA-2A
1AC
2TAG
2TA
9A
2A
2GAA
3A
1CTA
2TA
1AA-3AG
1ATG
1RAG
1AGR
1CAG
2TAG
2AG
2AAAG
24AG
3BCTTTCCT
1CTCCGGTCTGTCAAT
2CCGGTCTGTCAAT

Y = C or T; W = A or T; R = A or G. Isolates that had superimposed signals in the nucleotide sequence are indicated in gray.

Table 5

Amino acid sequence differences within envelope (E) gene among strains of Japanese encephalitis virus isolated, Toyama Prefecture, Japan*

No. isolatesClusterSubclusterAmino acid no. in the E region161181231281312323343365366374477
2005200720082009ConsensusAGTTKVASSMD
1AA-1S or P
1
1
1
1
1
1
1
2
2
2V
2
5
1AA-2K or E
1
2AV
2AV
9
2M
2S
3
1
2
1AA-3
1
1V or I
1D or N
1
2
2
2TN
24
3B
1C
2

Isolates that have amino acid differences from another isolate(s) are indicated in gray.

For the C/prM gene, isolates in this study were further divided into three clusters: A′, B′, and C′ (Figure 4B). There were 4–9 nucleotide differences among these three clusters (Table 6). Cluster B′ had one amino acid difference from clusters A′ and C′. All the isolates classified into clusters A, B, and C on the basis of the E gene corresponded with those classified into clusters A′, B′, and C′ on the basis of the C/prM gene, respectively. Cluster A′ was further divided into three subclusters: A′-1, A′-2, and A′-3 (Table 6). These subclusters were different from each other by 1–5 nucleotides. Cluster A′-3 had one amino acid difference from clusters A′-1 and A′-2. All 21 isolates in 2005, four isolates in 2007, and all 42 isolates in 2008 belonged to subcluster A′-1 (Table 6). Subclusters A′-2 and A′-3 were composed of 11 isolates in 2007 and 3 isolates in 2009, respectively. The isolates that belonged to cluster A′ were similar to strains SC04-16 and Sw/Mie/40/2004 (Figure 4B) and were the same isolates that were in cluster A in the E gene phylogeny. Clusters B′ and C′ were each composed of three isolates in 2007 (Table 6). The three isolates in cluster B′ were not similar to existing strains (Figure 4B). The three isolates in cluster C′ were similar to strains YN86–B8639 and SH03-127 and were the same isolates that were in cluster C in the E gene phylogeny.

Table 6

Nucleotide sequence differences within capsid (C)/premembrane (prM) gene among strains of Japanese encephalitis virus isolated, Toyama Prefecture, Japan

No. isolatesClusterSubclusterNucleotide no.C regionprM region
32434536537212318193102126153169210213216
2005200720082009ConsensusCTCTACCACTTGCTG
20441A'A'-1
1C
1C
10A'A'-2T
1TG
2A′A′-3CT
1CGT
3B′TTTTCA
3C′TGTCTA

We also generated a phylogenetic tree using maximum-likelihood and found that the isolates were divided into clusters A, B, and C (E gene) or A′, B′, and C′ (C/prM) as observed using Kimura's two-parameter method (Figure 4A and B). Among 21 JEVs isolated in 2007, 11 isolates that were obtained from mosquitoes on September 25 or October 1 in the pigpen in Nanto belonged to clusters A′/A, B′/B, and C′/C. Therefore, JEV strains of three types (clusters A′/A, B′/B, and C′/C) co-circulated from the end of September to early October 2007 in the pigpen in Nanto. Two isolates belonging to different clusters were occasionally obtained from the same pool by using two different cell types for isolation. Furthermore, superimposed signals in the nucleotide sequence were observed for the E gene in 10 isolates (Table 4). This finding indicates that these isolates contained at least two different strains.

All the isolates were divided into either eight or three types according to the nucleotide sequences or deletions, respectively, in the 3′ UTR (Figure 5). All isolates in 2005 and 18 isolates in 2007 were shown to have the same deletion (nucleotide no. 5–6, 14–26, 35, 46, and 58–59) as the Ishikawa strain19 and the Sw/Mie/40/2004 strain.12 The other three isolates in 2007 had an additional nine-nucleotide deletion (nucleotide no. 34–43) similar to Sw/Kagawa/35/2004. These isolates were found to constitute the clusters C and C′ by phylogenetic analysis of the E and C/prM genes, as indicated in Figure 4A and B. All the isolates in 2008 and 2009 had a novel additional deletion of seven nucleotides (nucleotide no. 44–51), although they were divided into two different subclusters (A-2 and A-3) according to the E gene (Table 4) and subclusters A′-1 and A′-3 according to the C/prM gene (Table 6).

Figure 5.
Figure 5.

Alignment of nucleotide sequences of the 3′-untranslated regions (UTRs) of Toyama isolates and the reference strains of Japanese encephalitis virus (JEV), Toyama Prefecture, Japan. Japanese encephalitis virus isolates in Toyama Prefecture are shown as Toyama/year (number of isolates) in bold letters. Reference strains are shown by strain name (accession no.). GI–GIV indicates JEV genotypes. Deletions are indicated by hyphens. The stop codons are underlined. Novel deletion sites are in boxes. Nucleotides of strains isolated in this study that were different from Toyama/2005 (19 isolates) are indicated by asterisks.

Citation: The American Society of Tropical Medicine and Hygiene 84, 5; 10.4269/ajtmh.2011.10-0188

On the basis of these results, changes in JEV in Toyama during 2005–2009 are summarized in Figure 6. In 2005, strains of “A′-1 (C/prM)/A-1 (E)/Ishikawa type (3′UTR)” predominated. In 2007, strains of “A′-1/A-2/Ishikawa type” and “A′-2/A-2/Ishikawa type” emerged. Minor strains of “B′/B/Ishikawa type” and “C′/C/Ishikawa + Kagawa type” appeared but disappeared in 2008. In 2008, strains of “A′-1 (C/prM)/A-2 (E)” still circulated although the 3′UTR of these strains had a novel deletion. Major strains were “A′-1/A-3/Ishikawa + Novel type” in 2008. In 2009, strains were “A′-3/A-2/Ishikawa + Novel type”. The results show that predominant strains in cluster “A′/A” changed from year to year but certain subcluster strains “A′-1/A-2” remained circulating during 2007–2008. Minor strains “B′/B” and “C′/C” were present only in one year (2007) and disappeared in later years.

Figure 6.
Figure 6.

Changes in Japanese encephalitis virus (JEV) in Toyama Prefecture, Japan, during 2005–2009. The capsid/premembrane (C/prM) and envelope (E) genes are shown by cluster or subcluster names. The 3′ untranslated regions (UTRs) are shown by the deletion types indicated in Figure 5 Ishikawa type indicates the same deletion as Ishikawa (accession no. AB051292) and Sw/Mie/40/2004 (accession no. AB241118). Ishikawa + Kagawa type indicates the same deletion as Sw/Kagawa/35/2004 (accession no. AB231627). Ishikawa + Novel type indicates the novel deletion type observed in 2008 and 2009.

Citation: The American Society of Tropical Medicine and Hygiene 84, 5; 10.4269/ajtmh.2011.10-0188

Virus replication characteristics in tissue culture of Vero and C6/36 cells were examined among these isolates. Culture supernatants were collected one, two, three, and six days after infection of Vero (multiplicities of infection [MOI] were 0.01 and 0.001) and C6/36 cells (MOI = 0.001 and 0.0001) and virus titers in culture fluids were determined. Virus titers peaked at 2–3 days in Vero cells and at six days in C6/36 cells. The range of the peak virus titers was approximately 5 × 107–5 × 108 focus-forming units (FFU)/mL (MOI = 0.01 in Vero cells), 108–109 FFU/mL (MOI = 0.001 in Vero cells), 109–1010 FFU/mL (MOI = 0.001 in C6/36 cells) and 108–109 FFU/mL (MOI = 0.0001 in C6/36 cells). Among several isolates belonging to different clusters and having different deletions in the 3′UTR, virus replication did not correlate with the different clusters or deletion status.

Discussion

There has been much discussion concerning how JEV appears every summer in Japan. One possible explanation is that the virus is introduced from tropical or subtropical zones of other countries in Asia every year. Another explanation is that JEV overwinters in Japan and re-emerges in early summer.

This study was performed to investigate how JEV maintains genetic continuity or undergoes genetic change locally for several years. Japanese encephalitis virus isolation and genetic characterization were performed in Toyama Prefecture, Japan, during 2005 to 2009.

Overall, strain “A′/A” seems to have remained in Toyama Prefecture and changed gradually. This fact may indicate that this type of JEV is a predominant strain that is endemic locally. The novel deletion in the 3′UTR might be an additional change. Conversely, strains “B′/B” and “C′/C” might be sporadically introduced to Japan and did not become predominant strains. Overwintering might be one of the factors for maintenance of predominant strain. Japanese encephalitis virus in Japan is considered to be a mixture of the overwintering type and a type from overseas because one subcluster was isolated only in Japan, and another type of JEV was also isolated elsewhere, such as in China and Vietnam.13 Japanese encephalitis virus has not been isolated from overwintering mosquitoes in Japan, and JEV was shown to overwinter locally in Hokkaido, Japan because outbreaks of abortion in pigs caused by JEV were observed in early June, the interepidemic period of JEV.20 In another report, JEV was maintained during winter in lizards experimentally, although it was not isolated from wild ones.21 Therefore, the overwintering mechanism of JEV in Japan is still unclear. Conversely, JEV was isolated from overwintering mosquitoes in South Korea.22 In Taiwan, one of the subtypes was shown to have been present for at least 11 years.15 Because Japan, like Taiwan, consists of islands, some subtypes of JEV may also be maintained for several years in Japan. Although many isolates of JEV in Japan are considered to originate from southeast Asia, overseas migration of virus might be a rare event. Even if the viruses migrated from outside Japan, most of them might have been extinct. Our results support this hypothesis.

All JEV isolates in Toyama Prefecture were similar to strains in China and Vietnam, and these reference strains had already been isolated before strains in this study were isolated. This result also supports the theory that JEV was introduced from southeast Asia and continental eastern Asia to Japan.13 Furthermore, strains of genotype III isolated before 1990 in Japan were similar to those in South Korea and Taiwan. All strains isolated in South Korea after 1991 belonged to genotype I, as did those in Japan. However, in Taiwan, strains of genotype III were isolated until 2002. These results indicate that the movement of JEV may be linked between Japan and South Korea. It is important to determine whether recent isolates in South Korea are similar to isolates in Toyama once nucleotide sequences of recent isolates in South Korea become available.

Strains isolated in 2005 and 2007 had the same deletions in the 3′UTR as the Ishikawa strain, and three strains in cluster C in 2007 had the same deletions as Sw/Kagawa-35/2004 strain. Japanese encephalitis virus strains isolated in 2008 and 2009 had an additional novel deletion in the 3′UTR, although they belonged to the same cluster as strains isolated in 2005 and 2007 on the basis of the E and C/prM genes. This novel deletion might have occurred in the JEV maintained in Toyama Prefecture or originated in another area and spread to Toyama Prefecture. Because JEV strains with this novel deletion have so far only been found in Toyama Prefecture, the former hypothesis seems to be likely. However, further analysis of isolates in other areas may clarify this issue.

Conversely, strains of “B′/B/Ishikawa type” and “C′/C/Ishikawa + Kagawa type” most likely migrated from other regions and then became extinct. They were detected in 2007 but were not isolated in 2008 and 2009. They might have disappeared in 2008 because they did not acclimate to local environmental conditions or they competed with other types of strains, or were not detected because of their low prevalence compared with those of prevalent strains.

All JEVs isolated from female Cx. tritaeniorhynchus mosquitoes were obtained on farms and belonged to genotype I. This result confirms that Cx. tritaeniorhynchus mosquitoes are the major vector of genotype I of JEV in Toyama Prefecture. Japanese encephalitis viruses were not only isolated from pigpens but also from a cattle shed. Because JEV is not known to cause viremia in cattle,23,24 mosquitoes harboring JEV might have flown from other places, such as a pigpen 2 km away, to this shed. The fact that antibody nor JEV was not detected in seven cattle less than one year of age in 2009 supports the above hypothesis. In a previous report, cattle acquired antibody after experimental infection with JEV.24 Because Cx. tritaeniorhynchus mosquitoes were few in number in 2009 (Figure 3A), there might have been little opportunity for the infection of cattle.

Although JEVs were isolated during August–October in 2005–2009 in this study, they were mainly isolated from the end of July through early September in Toyama Prefecture in 1966–1972 (Figure 3B),18 during July–August in Nagasaki Prefecture in 1964–1973,25,26 and during July–early September in Osaka Prefecture in 1968–1997.27 Conversely, the number of Cx. tritaeniorhynchus mosquitoes at the end of July in the 1970s, and from the end of August to the beginning of September after the 1990s in Toyama Prefecture.10 Thus, the late isolation of JEV in this study seems to correlate with the late increase in the number of Cx. tritaeniorhynchus mosquitoes. Although the reason for the late peak in the number of mosquitoes is not clear, it might be caused by the way that insecticides are applied and/or the method of water control of rice fields.

From the late 1960s to the 1970s, rice fields were filled with water in May and Cx. tritaeniorhynchus mosquitoes developed during June–July. The growth of Cx. tritaeniorhynchus mosquitoes was suppressed by the first application of insecticide from a helicopter at the end of July, the second application in early August, and the drying of rice fields at the end of August before harvest time.10,11 The application of insecticide by using a helicopter was stopped in 1995 because it disturbed ecologic systems around rice fields. Recently, because rice fields had been filled with water relatively late in the season, insecticide had been applied onto rice seedlings, and rice fields had been dried in June, Cx. tritaeniorhynchus mosquitoes did not develop in June. However, insecticides are not used frequently, except on seedlings. After the refilling of rice fields with water, Cx. tritaeniorhynchus mosquitoes may develop and peak during August–September. These factors may also affect the late prevalence of Japanese encephalitis, which has recently occurred mainly in September,9 despite occurring in August in the past.8

Japanese encephalitis virus was isolated from mosquitoes in a pigpen after the peak in the number of female Cx. tritaeniorhynchus mosquitoes. This finding is in contrast to a previous report showing that JEV infections of mosquitoes occurred before or at the peak in the number of mosquitoes (Figure 3B).18 At temperatures higher than 24°C, JEV reproduction in Cx. tritaeniorhynchus mosquitoes was found to be faster and virus titer was higher and peaked earlier after infection than at lower temperatures.28 In the 1960s and 1970s, the number of Cx. tritaeniorhynchus mosquitoes peaked in July when the temperature was high. Japanese encephalitis virus might effectively reproduce in mosquitoes and peak in number at the time of the peak in the number of mosquitoes. Recently, the number of Cx. tritaeniorhynchus mosquitoes peaked in August and September, a period with a lower temperature. As a result, JEV might not have effectively reproduced in mosquitoes. Thus, the peak in the MIR of JEV followed the peak in the number of Cx. tritaeniorhynchus mosquitoes. This finding also means that recently the MIR for JEV in mosquitoes has not been high during August–September when the number of mosquitoes peaks. Therefore, the risk of infection in humans that are bitten by mosquitoes may now be lower in late summer and autumn than in summer. Furthermore, mosquitoes have more difficulty biting persons in autumn than in summer because people wear long sleeves. These factors might be related to the recent decrease in the prevalence of Japanese encephalitis in Japan, in addition to the effects of human vaccinations.

In conclusion, JEV still circulates between mosquitoes and pigs in Toyama Prefecture and is correlated with the prevalence of mosquitoes. However, the peak level of JEV circulation occurs later in the year than in the past. On the basis of the nucleotide sequence information derived from the E and C/prM genes, all isolates belong to genotype I. The major type of JEV might have remained in Toyama Prefecture and gradually changed over five years, and two types of JEV might have migrated from other countries and then become extinct. Japanese encephalitis virus isolates in 2008 and 2009 had a novel deletion in the 3′UTR.

ACKNOWLEDGMENTS:

We thank Miyuki Maekawa for technical assistance; volunteers, Toyama-Airport Detached Office of Niigata Quarantine Station, Takaoka Health Center, and Toyama Prefectural Meat Inspection Center for surveillance of mosquitoes and pigs; and the Department of Virology 1 and Department of Medical Entomology, National Institute of Infectious Diseases, for advice on virus isolation.

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Author Notes

*Address correspondence to Mayumi Obara, Toyama Institute of Health, 17-1 Nakataikoyama, Imizu, Toyama 939-0363, Japan. E-mail: mayumi.obara@pref.toyama.lg.jp

Financial support: This study was supported by a Health Labor Sciences Research Grant in Research on Emerging and Re-emerging Infectious Disease (H17-shinkou-ippan-018, H20-shinkou-ippan-015) from the Japanese Ministry of Health, Labor and Welfare.

Authors' addresses: Mayumi Obara, Takeo Yamauchi, Sumiyo Hasegawa, Masae Iwai, Eiji Horimoto, Takeshi Kurata, and Takenori Takizawa, Toyama Institute of Health, 17-1 Nakataikoyama, Imizu, Toyama 939-0363, Japan, E-mails: mayumi.obara@pref.toyama.lg.jp, takeo.yamauchi@pref.toyama.lg.jp, sumiyo.hasegawa@pref.toyama.lg.jp, masae.iwai@pref.toyama.lg.jp, eiji.horimoto@pref.toyama.lg.jp, takeshi.kurata@pref.toyama.lg.jp, and takenori.takizawa@pref.toyama.lg.jp. Mamoru Watanabe, Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan, E-mail: tabanus-wata@titan.ocn.ne.jp. Yasufumi Ueda and Kentaro Matsuno, Toyama-Airport Detached Office of Niigata Quarantine Station, 191 Akigashima, Toyama, Toyama 939-8252, Japan, E-mails: ueda-yasufumi@keneki.go.jp and matsuno-kentarou@keneki.go.jp. Hiroaki Kariwa and Ikuo Takashima, Laboratory of Public Health, Graduate School of Veterinary Medicine, Hokkaido University, Kita-18, Nishi-9, Kitaku, Sapporo, Hokkaido 060-0818, Japan, E-mails: kariwa@vetmed.hokudai.ac.jp and takasima@vetmed.hokudai.ac.jp.

Reprint requests: Mayumi Obara, Toyama Institute of Health, 17-1 Nakataikoyama, Imizu, Toyama 939-0363, Japan, E-mail: mayumi.obara@pref.toyama.lg.jp.

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