Molecular characterization of the first Australian isolate of Japanese encephalitis virus, the FU strain

David T. Williams1,2, Lin-Fa Wang1, Peter W. Daniels1 and John S. Mackenzie2

CSIRO, Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, Victoria 3220, Australia1
Department of Microbiology and Parasitology, University of Queensland, Brisbane, Queensland 4072, Australia2

Author for correspondence: David Williams at CSIRO. Fax +61 3 5227 5555. e-mail david.williams{at}dah.csiro.au


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The complete genomic and predicted amino acid sequence of the Japanese encephalitis virus (JEV) FU strain, a human isolate recovered from the first outbreak of Japanese encephalitis in Australian territory, was determined. Comparison of the FU genome with 15 fully sequenced JEV genomes revealed high levels of sequence identity, ranging from 88·7% (GP78) to 89·7% (K94P05) for nucleotides and 96·8% (K94P05) to 98·0% (JaGAr01) for amino acid sequences. A total of 39 unique amino acid differences were found in the FU strain polyprotein. Phylogenetic analyses were performed on all available full-length JEV genomes and a selection of 64 E gene sequences from temporally and geographically diverse JEV strains. For comparison with the E gene phylogeny, phylogenetic analysis using cognate prM gene sequences was also carried out. The FU strain was found to be most closely related to Korean isolate K94P05 in the full-length analysis and to Southeast Asian strains in the E and prM gene analyses. The E gene analysis corresponded well with the prM gene analysis and with previous genotyping studies using the prM gene. The epidemiological implications of this investigation are discussed.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The mosquito-borne flavivirus Japanese encephalitis is the most important cause of virus encephalitis in eastern and southern Asia, with over 40000 cases occurring annually. Of these cases, around 25% are fatal while 50% result in permanent neuropsychiatric sequelae (Burke & Leake, 1988 ). Japanese encephalitis virus (JEV) exists in a zoonotic cycle between mosquitoes and pigs and/or water birds (Monath & Heinz, 1996 ).

Until recently, the south-eastern limit of JEV activity was considered to lie in western Indonesia (Olson et al., 1985 ; Wuryadi & Suroso, 1989 ). However, in 1995 human infections of the disease occurred in Australian territory, in residents of the Torres Strait Islands between the Cape York Peninsula and Papua New Guinea (PNG; Hanna et al., 1996 ). In subsequent years, continued JEV activity was demonstrated in the Torres Strait Islands (Shield et al., 1996 ; Mackenzie et al., 1997 ), and in 1998 two more human cases were reported, one of which was the first to occur on the Australian mainland (Hanna et al., 1999 ). More recently, JEV activity in PNG (Johansen et al., 1997 ) and Irian Jaya (Spicer et al., 1999 ) has been demonstrated, providing evidence for the source of virus activity in Australia.

The JEV genome, like that of all flaviviruses, is a positive-sense single-stranded RNA molecule approximately 11 kb in length. It is capped at its 5' end and contains a single open reading frame (ORF) encoding a polyprotein. The viral structural proteins are encoded by the 5' one-third of the ORF and consist of the capsid (C), membrane (M; formed by proteolytic cleavage of its precursor protein prM) and envelope (E) proteins. The non-structural proteins (NS1 to NS5) are encoded in the remaining 3' region. The ORF is flanked by 5' and 3' untranslated regions (UTRs) approximately 95 and 582 nt long, respectively (Sumiyoshi et al., 1987 ; Hashimoto et al., 1988 ).

The genome of the Torres Strait Island FU strain, a human isolate from a subclinical infection during the 1995 outbreak, has previously been partially sequenced (Poidinger et al., 1996 ). We have determined the nucleotide and predicted amino acid sequence of the full-length genome of the FU strain in order to characterize more fully this virus at the molecular level and to establish its relationships with other sequenced JEV strains. We have also attempted an extensive genetic analysis of JEV isolates using E gene sequence information from a wide range of geographically and temporally diverse JEV strains. In previous studies, phylogenetic analyses have focused on limited (240 nt) but highly variable sequence information from the prM gene (Chen et al., 1990 , 1992 ; Huong et al., 1993 ; Ali & Igarashi, 1997 ) that divided the JE viruses into four genotypes. Only limited phylogeny has been attempted for the E gene (Ni & Barrett, 1995 ; Nam et al., 1996 ; Paranjpe & Banerjee, 1996 ; Wu et al., 1998 ; Mangada & Takegami, 1999 ). Here we compare JEV phylogeny generated from the E gene with that generated from cognate prM gene sequence information.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus growth and purification.
The JEV FU strain was originally isolated from the serum of an asymptomatic human resident of Badu Island in the Torres Strait in 1995 (Hanna et al., 1996 ). Virus was propagated in C6-36 cells and purified as previously described (Gould & Clegg, 1985 ). Briefly, following infection of confluent cell monolayers, virus was harvested and cellular debris was removed by centrifugation (2000 g, 15 min). Virions were then precipitated from the supernatant by incubation with polyethylene glycol 6000 (10 mM) and NaCl (400 mM). After overnight incubation at 4 °C, the mixture was centrifuged (5000 g, 60 min) and the pellet containing virus was resuspended in Tris–saline–EDTA buffer. For convenience of handling, virus stocks were then gamma-irradiated (30 kGy) to inactivate the virus. Virus was further purified by centrifugation (25000 r.p.m., 180 min) through a discontinuous 50–17% sucrose gradient.

{blacksquare} RT–PCR.
Viral RNA was extracted from 140 µl of purified virus culture stocks using the QIAamp viral RNA extraction kit (QIAGEN) according to the manufacturer’s instructions and stored at -80 °C until use. Purified RNA was used as template for cDNA synthesis using the Superscript one-step RT–PCR system (GIBCO/BRL Life Technologies) and primers designed from the K94P05 strain genome sequence. RT–PCR reaction conditions were as follows: 2 min RNA denaturation at 94 °C followed by cDNA synthesis at 50 °C for 30 min; this was immediately followed by 35 cycles of PCR using 94 °C denaturation for 1 min, 55 °C primer annealing for 1 min, and 72 °C primer extension for 2 min.

{blacksquare} Nucleotide sequencing.
RT–PCR amplicons were purified using the QIAGEN gel extraction kit. Approximately 30–60 ng of purified cDNA template was used in direct cycle sequencing using the BigDye terminator cycle sequencing reaction kit (Perkin Elmer/Applied Biosystems). Products were purified by ethanol precipitation, according to the manufacturer’s instructions, and analysed using an automated Applied Biosystems DNA sequencer.

{blacksquare} Multiple alignments and sequence analyses.
The full-length FU strain genome was compiled using SeqMan in the Lasergene software package (DNASTAR). Multiple sequence alignments were carried out using ClustalX (Thompson et al., 1997 ) with minor manual adjustments to produce optimal nucleotide alignment for later use in phylogenetic analyses. Percentage similarities between aligned nucleotide or amino acid sequences were calculated using ClustalX.

{blacksquare} Phylogenetic analyses.
A list of the JEV strains used in phylogenetic analyses showing their host, year and country of isolation and GenBank accession numbers is presented in Table 1. These include the JEV FU strain, 16 strains for which full-length genomic sequences are available, selected E gene sequences of 49 additional strains and, where available, their prM gene sequences. A total of 122 E gene sequences were evaluated for analysis. The final selection was based on relevance of candidate strains to the literature and degree of sequence identity among candidate strains isolated from the same country (i.e. in instances where strains isolated in the same country showed very high levels of nucleotide sequence identity, a representative sequence was selected).


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Table 1. Details of JEV strains used in this study

 
Phylogenetic trees were constructed using the neighbour-joining (NJ) method of Saitou & Nei (1987) , the maximum parsimony (MP) method, and the Fitch & Margoliash (1967) and Least Squares methods (Kitsch). The Kunjin (KUN) virus genome (Coia et al., 1988 ) was used as an outgroup in all analyses. ClustalX was used for construction of the NJ trees and PHYLIP (Felsenstein, 1993 ) for construction of MP and Kitsch trees. Constructed NJ trees were subject to bootstrap analysis using 1000 replicates and nucleotide distances were estimated from bootstrapped datasets using Kimura’s-two-parameter method (Kimura, 1980 ).

For construction of MP and Kitsch trees, multiple sequence alignments were first subjected to bootstrap analysis using the SEQBOOT program and 1000 replicates. For MP analysis, character state parsimony algorithms in DNAPARS were used on bootstrapped datasets to generate the most parsimonious trees. The CONSENSE program was then employed to build a strict consensus bootstrapped tree. For Kitsch analysis, nucleotide distances were first estimated by Kimura’s-two-parameter method (Kimura, 1980 ) from bootstrapped datasets in DNADIST. Fitch & Margoliash (1967) and Least Squares methods with evolutionary clock analyses were then carried out on these estimates in the KITCH program to produce the final phylogenetic tree. All trees were drawn using TreeView software (Page, 1996 ).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Full-length nucleotide and amino acid sequence analysis
Both strands of overlapping cDNA PCR products generated from the FU strain RNA genome were sequenced at least once with an overall average redundancy of 4·8. Comparison of the assembled FU genome with 16 fully sequenced JEV strains revealed high levels of similarity, ranging from 88·7% (GP78) to 89·7% (K94P05) for nucleotides and 96·8% (K94P05) to 98·0% (JaGAr01) for the corresponding amino acid sequences. A total of 39 unique amino acid differences were found for the FU strain, of which 18 were non-conservative (Table 2). The prM gene contained the highest percentage of these amino acid differences (3·3%), followed by the C gene, NS1 and NS2a (2·4% each). No nucleotide changes were found in the 5'-UTR of the FU strain; however, nine unique differences were observed in the 3'-UTR.


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Table 2. Amino acid substitutions in the JEV FU strain relative to available full-length JEV genomes

 
The 11 bp deletion immediately downstream of the ORF stop codon previously reported by Poidinger et al. (1996) was confirmed here. This deletion, found in all available sequenced Australian and PNG JEV isolates, was hitherto considered a potential molecular fingerprint for Australasian strains (Mackenzie, 1999 ). Here, however, it was also found in the Korean K94P05 strain (data not shown). This 11 bp deletion was not found in any other JEV sequence for which 3'-UTR information is available.

E gene nucleotide and amino acid sequence analysis
Comparison of the FU strain E gene with 63 E gene sequences from temporally and geographically diverse JEV strains (Table 1) was performed. Highest sequence similarities with FU were 94·7% (WTP-70-22) and 98·6% (Sagiyama, SA14, ML-117) for nucleotide and amino acid sequences, respectively. The Muar strain showed the lowest nucleotide (78·4%) and amino acid (90·6%) identity compared to FU, followed by JKT7003 (82·5% nucleotide and 94·8% amino acid sequence identity). The 12 cysteine residues reported to form six disulfide bridges (Nowak & Wengler, 1987 ) are conserved for FU, as is the putative receptor binding motif Arg-Gly-Asp (RGD) at E387–389 (Rey et al., 1995 ).

Overall, 18 unique nucleotide changes were found for FU, the majority of which were transitions located at the third position of the codons resulting in silent mutations. Four nucleotide changes resulted in three unique, non-conservative amino acid changes at E208 (Ser to Pro), E308 (Phe to Ser) and E311 (Ala to Arg). Two other shared amino acid differences of significance were also revealed: E108 (Ser to Phe), shared with JKT5441, is located on the putative membrane fusion peptide (Roehrig et al., 1989 ; Rey et al., 1995 ); and E307 (Lys to Asn), shared with JKT1724 and R53567, which together with E308 and E311 makes up a group of non-conservative changes located on domain III of the E protein, a domain predicted to participate in cell receptor attachment (Rey et al., 1995 ). The amino acid changes at E108 and E307, E308 and E311 also correspond to residues in predicted continuous and conformational epitopes, respectively, of the JEV E protein (Kolaskar & Kulkarni-Kale, 1999 ).

Phylogenetic analyses
NJ, MP, Fitch & Margoliash (1967) and Least Squares (Kitsch) methods were employed to establish the genetic relationship between: (i) the full-length genomic sequence of the FU strain and available full-length JEV sequences; (ii) the E gene of FU and the 63 JEV strains described above; and (iii) the prM gene of FU and the 49 available JEV strains cognate to those used in the E gene analysis. All tree-building methods produced trees of similar topology for each analysis with differences predominantly confined to bootstrap support.

A phylogenetic tree produced by the NJ method for the full-length sequences is shown in Fig. 1. Two distinct phylogenetic groups were evident; one contained the FU and K94P05 strains, while the second comprised isolates from India, China, Taiwan and Japan. Three separate clusters defined the second group. The first of these contained early isolates from China (Beijing), India (P20778) and Taiwan (Ling); the second contained the Japanese immunotype JaGAr01 (Okuno et al., 1968 ) along with several Taiwanese isolates; while isolates from Japan, India and China – including SA14 and its vaccine derivatives – made up the third cluster.



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Fig. 1. Phylogenetic relationships of JEV strains predicted from available full-length nucleotide sequences. Trees were constructed using the neighbour-joining method in ClustalX (Thompson et al., 1997 ). The scale at the bottom of the trees indicates the number of nucleotide substitutions per site. Horizontal branch lengths are proportional to genetic distance while vertical branch lengths have no significance. The numbers above each branch represent the percentage of 1000 bootstrap replicate support. The tree was rooted using sequence information of the Kunjin (KUN) virus, a member of the JEV serocomplex.

 
The E gene phylogenetic tree, produced by the NJ method and comprising a total of 65 virus strains (including the KUN outgroup), is shown in Fig. 2(a). Four distinct phylogenetic groupings could be identified that corresponded to the four genotypes (GI to GIV) described previously (Chen et al., 1990 , 1992 ; Ali & Igarashi, 1997 ). Three strains, Muar, JKT6468 and K82P01, fell outside these major groupings.




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Fig. 2. Phylogenetic relationships of JEV strains predicted from (a) selected E gene sequences; and (b) cognate prM gene sequences, where available. Phylogenetic groupings corresponding to the genotyping classification of Chen et al. (1990 , 1992 ) and Ali & Igarashi (1997) are indicated (GI to GIV). Strains that differ in their location within the E gene tree compared to the prM tree, and vice versa, are shaded grey. For details of the trees, see legend to Fig. 1.

 
The majority of strains analysed in this study grouped into GIII, which comprised four distinct clusters. For the purpose of discussion only, these clusters have been designated subgroups A–D. The first of these, subgroup A, was made up of predominantly early isolates from central and eastern Asia, including the prototype strains. This group also contained both Vietnamese isolates included in the analyses and the two representative immunotypes of JEV, JaGAr01 and Nakayama (Okuno et al., 1968 ). Subgroup B consisted of relatively recent isolates from India, Sri Lanka, Japan and Taiwan, along with single isolates from the Philippines (PhAn1242) and northern Thailand (Chiang Mai). The remaining subgroups in GIII consisted of isolates from Indonesia and Nepal (subgroup C) and Korea and Japan (subgroup D).

A second major phylogenetic grouping consisted of two distinct clusters which corresponded to GI and GII. Strains isolated in Korea, northern Thailand and Cambodia made up GI, while south-east Asian strains made up GII. The Australian FU strain grouped into this latter cluster, consistent with previous reports (Hanna et al., 1996 ; Mackenzie et al., 1997 ). The Indonesian strains JKT7003 and JKT9092 formed the final cluster, which corresponded to genotype IV.

The remainder of the phylogenetic tree consisted of three separate branches: the Indonesian strain JKT6468 fell between GIV and GII; the Korean strain K82P01 formed an outlying branch from GIII; while the Muar strain, isolated in Singapore in 1952, occupied the most divergent branch of the tree within the JEV groupings, consistent with previous reports (Hasegawa et al., 1994 ; Paranjpe & Banerjee, 1996 ; Mangada & Takegami, 1999 ).

For comparison with the E gene phylogeny, phylogenetic analysis using cognate prM gene sequence information was carried out. The prM phylogenetic tree produced here (Fig. 2b) was virtually identical to those produced in previous studies using the prM gene (Chen et al., 1990 , 1992 ; Ali & Igarashi, 1997 ), with four distinct and corresponding phylogenetic groups identified. When compared to the E gene tree (Fig. 2a), an overall similar tree topology was observed. However, in addition to several differences in clustering at the terminal nodes, a number of strains moved from either intermediate positions on the E gene phylogenetic tree to a distinct genotypic group in the prM tree (Muar, JKT6468 and K82P01), or from one genotype in the E gene tree to another in the prM gene tree (K91P55 and JKT1724).


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Genetic variation among JEV strains isolated from widely different time periods and geographical regions has been reported in several studies (Chen et al., 1990 , 1992 ; Ni & Barrett, 1995 ; Nam et al., 1996 ; Paranjpe & Banerjee, 1996 ; Ali & Igarashi, 1997 ; Tsuchie et al., 1997 ; Wu et al., 1998 ; Mangada & Takegami, 1999 ). This variation has been linked with patterns of virus activity and has been suggested to be attributable to regional differences in virulence (Chen et al., 1990 , 1992 ). Phylogenetic analyses of JEV have previously focused on limited (240 nt) but highly variable sequence information from the prM gene (Chen et al., 1990 , 1992 ; Huong et al., 1993 ; Ali & Igarashi, 1997 ), dividing the JE viruses into four genotypes based on their geographical origin. It has been observed, however, that the functional significance of the prM genetic variation remains to be established (Ni & Barrett, 1995 ). Furthermore, the dangers of using short sequences (<300 nt) in phylogenetic analyses of flaviviruses has been highlighted and cautioned against because of a propensity for these analyses to produce unreliable information (Westaway & Blok, 1997 ; Kuno et al., 1998 ).

In recent years, the viral envelope (E) gene and its corresponding protein have become established phylogenetic markers for JEV (Ni & Barrett, 1995 ; Nam et al., 1996 ; Paranjpe & Banerjee, 1996 ; Wu et al., 1998 ; Mangada & Takegami, 1999 ). The E protein plays an important role in both induction of protective immune responses and in the biology of the virus (Rice, 1996 ; McMinn, 1997 ). To date, phylogenetic analyses using the E gene have used relatively low numbers of isolates and thus provided only partial information. A more conclusive genetic analysis of the JE viruses using E gene sequence information was therefore attempted. As part of this analysis, comparative phylogeny using analogous prM gene sequences was also performed.

Analysis of the E gene of 64 JEV strains revealed four distinct phylogenetic groupings, reflecting broad geographical and temporal relationships (Fig. 2a). These groupings corresponded to those identified in the prM phylogenetic tree (Fig. 2b) which, in turn, was consistent with the classification of Chen et al. (1990 , 1992 ). However, because bootstrap support for the major nodes defining these groupings was relatively poor, they should not be considered fully justified at this stage. The tree topology produced from the E gene analysis in this study was similar to those produced using the prM gene (Fig. 2b; Chen et al., 1990 , 1992 ; Ali & Igarashi, 1997 ). There were, however, major exceptions, with several strains found to occupy a different genotype or branching position within the two trees. These differences may be an effect of poor bootstrap support at the relevant branching points for each tree. Alternatively, the observed differences may be a consequence of the much shorter length of sequence information (240 nt) used in the prM analyses.

Phylogenetic analysis of the full-length FU genome and 16 fully sequenced JEV isolates from Korea, Japan, China, India and Taiwan (Fig. 1) revealed two distinct genetic groups, which broadly reflected the E and prM gene phylogenetic analyses. The FU strain clustered with the Korean K94P05 strain and although these strains were isolated at around the same time (1995 and 1994 respectively), no other temporal or geographical correlations were found within the tree. This analysis represents the largest known phylogenetic analysis of full-length JEV strains attempted; the only previous full-length study used just six virus strains (Vrati et al., 1999 ). Additional full-length sequence information will help to provide more accurate estimates of phylogeny than single gene analysis, thereby establishing clearer relationships between JEV genomes.

Two distinct patterns of JEV activity occur in nature: epidemic and endemic. In temperate areas of Asia such as Japan, China, Taiwan, Korea, Nepal, India and Sri Lanka, and the northern parts of Thailand and Vietnam, epidemic activity has been observed (Burke & Leake, 1988 ; Huong et al., 1993 ). In tropical equatorial regions such as Indonesia, Malaysia, southern Thailand, southern Vietnam, Cambodia and the Philippines, JEV is considered endemic (Burke & Leake, 1988 ; Chen et al., 1990 ). Previous studies by Chen et al. (1990 , 1992 ) have indicated that significant genetic differences exist between virus strains circulating in epidemic and endemic regions. The majority of strains used in the present study were isolated from regions of epidemic virus activity. Despite this, our findings were consistent with those of Chen et al. (1990 , 1992 ), with strains considered as either epidemic or endemic grouping predominantly into separate clusters. Epidemic isolates were found to group together in GI and GIII, while endemic strains grouped together in clusters forming GII and GIV.

Significantly, exceptions to this pattern were demonstrated. The Philippine isolate PhAn1492, the Indonesian isolates Indonesia and JKT1724 (E gene analysis only), and the southern Vietnamese isolates VN118 and Saigon all clustered in GIII, a genotype containing predominantly epidemic strains. The relative positions of the Philippine and Vietnamese isolates are consistent with analyses using the prM gene (Chen et al., 1990 , 1992 ; Ali & Igarashi, 1997 ), while those for Indonesia and Saigon are supported by E gene analyses (Paranjpe & Banerjee, 1996 ; Mangada & Takegami, 1999 ). These latter strains may represent isolates introduced from epidemic regions which have subsequently become established in regions of endemic JEV activity.

The reverse may be true of the FU strain, found to be most closely related to virus strains from Malaysia and Indonesia – regions of endemic JEV activity. Until now, JEV activity in tropical northern Australasia has followed a seasonal pattern indicative of epidemic strains (Mackenzie, 1999 ). This conflicts with the previous observation that tropical regions are associated with endemic JEV activity (Burke & Leake, 1988 ; Monath & Heinz, 1996 ). Thus, although there exists a correlation between genotype and virus activity, the examples cited above suggest that the epidemic or endemic pattern of virus activity may be more a function of climate, geography and the immune status of host populations rather than genetic potential of the virus per se. The perception that genotype correlates with epidemic potential is a major confounding factor, since place of isolation also correlates with genotype (Chen et al., 1992 ).

The possibility that JEV could become established on the northern Australian mainland in enzootic cycles is a major public health concern. Given the existence of suitable vector mosquitoes and vertebrate amplifier hosts in northern Australia (Mackenzie et al., 1997 ; Mackenzie, 1999 ), the question of whether JEV can become established in endemic cycles may be influenced most significantly by prevailing climatic conditions. Other factors such as the presence of susceptible hosts and competition with enzootic flaviviruses will also affect this outcome.

Characterization of the FU strain at the molecular level is an important step towards identifying those properties of the virus that may aid in disease prevention and control. Future studies may be directed towards delineating the effects, if any, of the high number of unique amino acid differences observed in the FU genome, particularly those in the E protein, on the biology and immunogenicity of this virus. With respect to the latter, future studies may also be aimed at investigating the efficacy of existing vaccines against the FU strain and other Australasian JEV strains.


   Acknowledgments
 
This study was supported by the Australian Quarantine Inspection Services. We thank Mr Ross Lunt, Australian Animal Health Laboratory (AAHL), Geelong, for the growth and purification of the virus stocks used in this study. The assistance of Mr Ian Pritchard (AAHL, Geelong) is also gratefully acknowledged for instruction in the use of phylogenetic programs.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 3 March 2000; accepted 13 June 2000.