Japanese encephalitis virus antigenic variants with characteristic differences in neutralization resistance and mouse virulence

ELSEVIER

Virus Research 51 (1997) 173-181

Virus Research

Japanese encephalitis virus antigenic variants with characteristic differences in neutralization resistance and mouse virulence Suh-Chin Wu a,b,*, Wei-Cheng Lian a, Li-Ching Hsu a, Ming-Yi Liau a National Institute of Preventive Medicine, Taipei, Taiwan b Department of LiJb Science, National Tsing Hua University, Hsinehu 30043, Taiwan

Received 19 March 1997; received in revised form 7 August 1997

Abstract

Two different plaque variants of Japanese encephalitis virus were selected from a wild-type Taiwanese isolate using Vero cells. One variant was found to exhibit small plaque morphology with retarded virus replication kinetics in Vero cells, and was demonstrated to be resistant to monoclonal antibody (mAb) E3.3 neutralization. The other variant showed large plaque morphology, was sensitive to mAb E3.3 neutralization, and manifested reduced virulence in mice on both intracranial and intraperitoneal inoculations. These two variants propagated in Vero cells retained high levels of infectivity but had relatively low HA titers as compared with the parent strain. The envelope sequences of these two variants showed four amino acid differences at residues E-85 (Glu/Arg), E-306 (Glu/Gly), E-331 (Ser/Arg), and E-387 (Met/Arg). Our results indicated the neutralizing epitope of Japanese encephalitis virus did not overlap with virus virulence determinant. © 1997 Elsevier Science B.V. Keywords: Japanese encephalitis virus; Antigenic variants; Neutralization resistance; Mouse virulence

I. Introduction

Japanese encephalitis virus (JEV) is a mosquitoborne virus belonging to the family Flaviviridae, genus flavivirus (Westaway et al., 1985). Like other flaviviruses, JEV contains a single-stranded R N A genome of approximately 11 kb in size. The virus particle contains three structural proteins * Corresponding author. Tel.: + 886 3 5742906; fax: + 886 3 5715934; e-mail: [email protected]

(capsid (C), transmembrane (M), and envelope (E)) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) (Chambers et al., 1990). The E protein contains approximately 500 amino acids and has six disulfide bonds which are required to maintain its conformational structure. The E protein is believed to be involved in viral attachment, fusion, penetration, hemagglutination, neutralization and protective immunity, host range and cell tropism, and virus virulence and attenuation. Most studies of E

0168-1702./97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S01 68-1 702(97)00098-1

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protein function were based on the analysis of neutralization-resistant (nt-resistant) mutants against monoclonal antibodies (mAbs) (Mason et al., 1989; Holzmann et al., 1990; Cecilia and Gould, 1991; Hasegawa et al., 1992; Jiang et al., 1993; Lin et al., 1994; Gao et al., 1994; McMinn et al., 1995; Hiramatsu et al., 1996). Recently, the three-dimensional structure of tick-borne encephalitis virus E protein, determined by X-ray crystallography, showed that the dimeric form of E protein, which presents on the virion surface, contains three antigenic domains: a central •]-barrel (domain I), an elongated dimerization region (domain II), and a C-terminal immunoglobulinlike module (domain III) (Rey et al., 1995). The actual functional sites of these three antigenic domains of E protein have not yet been completely determined. In this paper we selected two JEV variants from a wild-type Taiwanese isolate CH2195 using a neutralizing mAb E3.3. These two variants exhibited differences in phenotypes for plaque size, virus growth kinetics, mAb neutralization and mouse virulence. The envelope gene sequences of these two variants were also determined.

2. Materials and Methods

2.1. Viruses and cells The wild-type JEV isolate CH2195 (provided by Ying-Chang Wu, Division of Epidemiology, National Institute of Preventive Medicine, Taiwan) was isolated from the mosquito Culex tritaeniorhynchus in Taiwan in 1994, and subsequently cultured in C6/36 cells for 3 passages and Vero cells for 6-8 passages. Two JEV vaccine strains, Beijing-1 and Nakayama-NIH, were maintained in mouse brain for preparation of the inactivated vaccines used for Taiwan Government Immunization Program and further grown in Vero cell for 2-3 passages. C6/36 cells were grown in Eagles minimal essential medium with 10% fetal bovine serum (FBS). Vero cells were grown in M199 medium with 5% bovine calf serum (CS).

2.2. Monoclonal antibody MAb E3.3 was prepared by fusing NS1 myeloma cells with spleen cells from BALB/c mice immunized with the Beijing-1 strain, following a standard protocol (Harlow and Lane, 1988). The hybridoma cells were cloned by limiting dilution and screened by indirect immunofluorescence. Pristane-primed BALB/c mice (4-weeks old) were injected with hybridoma cells to obtain high titer mAb E3.3 in ascitic fluid. The specificity of mAb E3.3 on JEV E protein was determined by Western blotting under non-reducing condition. MAb E3.3 (ascitic fluid) showed > 95% plaque neutralization against the Beijing-1 (homologous) strain and blocked virus hemagglutination at the reciprocal titer of 320. The isotype of mAb E3.3 was characterized as IgG2,Kby use of a commercial kit (Mouse MonoAb ID EIA, Zymed).

2.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PA GE) and Western blotting Virus samples were solubilized in 2% SDS, boiled for 3 min under non-reducing or reducing (i.e. 5 or 10% fl-mercaptoethanol) conditions, and electrophoresed on 12% SDS-polyacrylamide gels. Western blotting was performed by transferring proteins from SDS-PAGE gels to nitrocellulose membranes using a semi-dry system (SEMIPHOR, Hoefer Scientific, San Francisco, CA). The nitrocellulose membranes were blotted with 1% BSA for 30 min, and incubated with mAbcontaining ascitic fluids diluted 1:50 100 or hybridoma supernatants diluted 1:20 at 4°C overnight. Then, the membranes were incubated at room temperature with goat anti-mouse IgG (H + L) biotin conjugated antibody diluted 1:10000 for 1 h, and streptavidin alkaline phosphotase-conjugated antibody diluted 1:3000 for another 1 h. Between each step above, three-time washes of PBST (PBS + 0.01% Tween 20) were performed to remove non-specific bindings to the nitrocellulose membranes. The membranes were finally treated with BCIP/TNBT substrate for color development.

s.-c. Wu et al./Virus Research 51 (1997) 173-181 2.4. Selection o f viral variants and plaque neutralization assay

Viral variants were selected from cultured Vero cells in the presence of mAb E3.3. CH2195 viral stocks were mixed with E3.3 ascitic fluid diluted 1:10 at 4°C for 18-21 h. The mixtures were then transferred to monolayer Vero cells for 1.5 h at 37°C and replaced with 2 ml of medium 199 containing 5% CS and 0.8% agarose for 2 days. Plaques were stained by adding 1 ml of the same medium with 0.02% neutral red. Each viral variant was picked from each individual plaque and subjected to a second time of plaque purification in the presence (for nt-resistant variants) or absence (for nt-sensitive variants) of mAb E3.3. Plaque neutralization assay was performed using cultured BHK-21 cells. Virus stocks containing a final titer of 100 PFU per well were mixed with mouse ascitic fluid diluted 1: 10, 1: 20, 1: 40, 1:160 and incubated at 4°C for 18-21 h. Then, 1 ml of the mixture was added to monolayer BHK21 cells for 1 h, followed by overlaying with medium containing 1.1% methylcellulose for 3 days incubation. Plaques were stained with naphthol blue black dye and counted. Percentage of neutralization was determined by the reduction of plaque formation with respect to control (no mAb used). 2.5. Hemagglutination assay

Hemagglutination (HA) assay was performed by a modification of the standard method (Clarke and Casals, 1958). Viral samples were diluted by 2-fold serial dilution. Then, 0.05 ml of each dilution was mixed with an equal volume of 0.33% goose erythrocytes in U-shape microplates for 1 h incubation at 37°C. HA titer was determined as the reciprocal titer of the highest dilution to induce hemagglutination of goose erythrocytes by visual inspection. 2.6. Virus replication kinetics and virus adsorption assay

Virus replication kinetics was examined in Vero cells (3 × 106 cells in T 25 flask) infected with two

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variant viruses at MOI = 1. The medium was removed and replaced with 10 ml fresh medium after 2 h incubation at 37°C. Samples were taken from the culture supernatants at 8, 20, 48 days post infection and assayed for infectivity in BHK21 cells. Virus adsorption assay was performed in Vero cells grown in 6 well plates. Cells were first incubated at 4°C for 1 h, then 0.5 ml diluted viral samples at a concentration of approximately 200 PFU per well was added. At different time periods (i.e. 5, 15, 30, 60 and 120 min), the culture supernatants were removed, and the cells were washed 2 times with phosphate buffer solution (PBS) to remove unbound viruses. Methylcellulose overlay medium was then added and the plaque numbers were determined after 3 days incubation. 2. 7. Mouse neurovirulence and neuroinvasion

Groups of 8-10 ICR mice aged 3 weeks were used to determine virus neurovirulence and neuroinvasion. Serial diluted virus samples in PBS (pH = 7.2) were inoculated intracranially at 0.03 ml per mouse. Mortality was observed daily and recorded after 14 days. Virus neurovirulence was determined based on the mortality to calculate its lethal dose 50 value (LDs0) using an LDso computer program (provided by John Spouge, NCBI, National Institutes of Health, USA). Virus neuroinvasion was determined by intraperitoneal inoculation at 0.5 ml virus dilution per mouse (i.e. 3 × 10 6, 3 × 105, 3 × 10 4 PFU/ mouse). The mortality rates were recorded daily for 21 days. In some cases, the surviving mice were challenged with 100 LDs0 parent strain virus CH2195 and observed for another 14 days. 2.8. Reverse transcriptase-polymerase chain reaction ( R T - P C R ) and nucleotide sequence analysis

Viral genomic RNA was extracted from infected Vero cell culture supernatants and purified using a commercial kit (QIAamp HCV Kit, QIAGEN, Germany). RT-PCR was conducted to obtain viral cDNA using pfu polymerase (Strategen,

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Viru~s Research 51 (1997) 173 181

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pairs of primer sets: (l) 5 ' T C A C T G A T C G T G G G T G G G 3 ' and 5 ' C C C A C C C A C G A T C A G T G A 3'; (2) 5'AAACAGAGAACTCCTC3' and 5'GAG G A G T T C T C T G T T T 3 ' ; (3) 5 ' C T C A A A G G T G C T G G T C G A G A T G Y and 5'CATCTCGACCAG C A C C T T T G A G 3 ' . Sequences were determined by incorporating fluorescent dye-labeled ddNTPs and read by an automatic fluorescent sequencer.

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1:160 1':40 1:20 Concentration of mAb E3.3 Ascitic Fluid

1:10

Fig. 1. Neutralization titration curves of mAb E3.3 concentration (ascitic fluid) in three Japanese encephalitis viruses: CH2195 (O), Beijing-1 (~7,) and Nakayama-N1H (I;).

USA) and the oligonucleotide primers designed based on Beijing-1 sequence (Hashimoto et al., 1988) plus two additional restriction sites of B a m H I and EcoRI in both ends. The sequences of the forward and reverse primers, respectively, were 5 ' A T G C G C G G A T C C A T G C T T G G C A G T A A C A A C G G T C A A C A C A Y and 5'GGTTACAC GTACGAC TGTGAA TCCTTAAGCGCGTA3'. PCR was carried out for 25 cycles of 95°C (1 rain), 55°C (2 min), and 72°C (3 min) in a DNA Thermocycler. The RT-PCR products were cloned into pUC18 (Promega, Madison, Wiscosin) for two selected variants. Direct sequencing was used for the parent strain CH2195. Sequencing reactions were performed by the dideoxynucleotide method using a sequencing kit (ABL3370, Applied Biosystems, USA) with three additional

CH2195

3. Results

3.1. Selection o f nt-resistant and sensitive variants

The titration curves of mAb E3.3 neutralization against a Taiwanese isolate (CH2195) and two vaccine strains (Beijing-1 and Nakayama-NIH) were demonstrated in Fig. 1. An approximately 40% fraction of CH2195 was not neutralized by mAb E3,3 at the titers over 1:40, while there was of > 95% reduction by mAb E3.3 neutralization for the two vaccine strains. Loss of the nt-resistant characteristics was found when these samples were tested using a pooled human polyclonal sera (data not shown). To further investigate the nt-resistant characteristics of CH2195, plaque isolation procedures were conducted to obtain different antigenic variants. Our results showed the plaque morphology of CH2195 in Vero cells was heterogeneous, containing different viral plaque sizes (Fig. 2). Based on the plaque morphology, we selected two variants: (1) a large-plaque variant CH2195LA and

CH2195SA

CH2195LA

Fig. 2. Plaque morphology of the parent strain, CH2195 and two selected variants: CH2195SA (small-plaque variant) and CH2195LA (large-plaque variant).

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(2) a small-plaque variant CH2195SA (Fig. 2). When applying in vitro neutralization tests to these two variants, we found CH2195LA was completely sensitive but CH2195SA was completely resistant to mAb E3.3. The percentage of plaque number reduction by mAb E3.3 neutralization was 60% for CH2195, 100% for CH2195LA, 0% for CH2195SA, 100% for Beijing1, and 100% for Nakayama-NIH. Therefore, CH2195LA, Beijing-1, and N a k a y a m a - N I H were nt-sensitive, and CH2195SA was nt-resistant to mAb E3.3. In addition, we also investigated other four selected variants (three with small plaque morphology and one with large plaque morphology), of which all showed small-plaque variants were nt-resistant (data not shown). 3.2. Western blot analysis o f mAb E3.3 binding to viral E protein

To investigate the antigenic determinant of mAb E3.3 neutralization, CH2195, CH2195LA, CH2195SA, Beijing-1, and N a k a y a m a - N I H were subjected to Western blot analysis. Our results showed that under non-reducing condition the E proteins of CH2195, CH2195LA, Beijing-1, and N a k a y a m a - N I H reacted with mAb E3.3 (MW = 52 kD) but not CH2195SA (Fig. 3). The epitope determining mAb E3.3 resistance was thus correlated with the loss of viral E protein binding in Western blots. Furthermore, the E-protein bindings of mAb E3.3 to CH2195, CH2195LA, Beijing-1, and N a k a y a m a - N I H were lost when tested under reducing conditions (data not shown).

105 8249332919-

Fig. 3. Western blot analysis of mAb E3.3 binding to viral E protein. 12% SDS-PAGE gels were conducted under non-reducing condition. Marker proteins were indicated with molecular sizes in kilodaltons on the left. The position of E protein was shown on the right.

1024 and 4096, respectively). CH2195SA showed retarded replication kinetics in Vero cells as compared to CH2195LA (see Fig. 4). At 24 h post-infection, an approximately 10-fold lower titer of extracellular virus was observed for CH2195LA (i.e. (1.05 _+ 0.01)× 108 PFU/ml) compared with CH2195SA (i.e. (1.25 _+ 0.04) × 107 PFU/ml). At 48 h post-infection, the extracellular virus titer of

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3.3. Viral in vitro and in vivo properties

2

To evaluate viral in vitro and in vivo properties, CH2195LA and CH2195SA were studied in parallel in Vero cell and mouse model systems. Viral production from these two variants achieved similar peak levels of viral infectivity (see Table 1). However, the HA titers of these two variants were much lower (HA = 128-256) than the parent strain (CH2195) ( H A = 2 0 4 8 ) and two vaccine strains (Beijing-1 and Nakayama-NIH) ( H A =

__= lO 5

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i

104 o

10

20

30

40

50

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Fig. 4. Replication kinetics of CH2195LA (e) and CH2195SA (©) in Vero cells. Each point represented as the mean value with its standard deviation (error bar) from two separated experiments.

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Table l In vitro and in vivo viral properties of CH2195, CH2195LA, CH2195SA, Beijing-1 and Nakayama-NIH Virus strain

Infectivity (log pfu/ml)

HA~

Neurovirulence (pfu/LDs~)

3 × 106 pfu

Neuroinvasion mortality (%) 3 × 105 pfu

3×

CH2195 CH2195LA CH2195SA Beijing-1 Nakayama-NIH

7.90 7.74 7.83 7.52 7.84

2048 128 256 4096 1024

4.0 55.4 4.3 1.5 1.9

75 l0 55 78 61

75 5 35 50 24

61 10 15 61 28

10 4

pfu

~'HA, reciprocal titer of the highest dilution to induce agglutination of goose erythrocytes. CH2195SA increased to its maximum titer, similar to that of CH2195LA. In addition, we did not observe differences in virus adsorption kinetics between these two variants and the parent strain (data not shown). However, the in vivo viral characteristics of these two variants were different in mice. Virus neurovirulence determined by intracranial inoculation into mouse brains showed the nt-resistant variant CH2195SA did not significantly differ from the parent strain (CH2195) and two vaccine strains (Beijing-1 and N a k a y a m a - N I H ) . Interestingly, the nt-sensitive large-plaque variant CH2195LA showed reduced mouse neurovirulence (CH2195LA = 55.4 PFU/LDs0) as compared with the others ( C H 2 1 9 5 S A = 4 . 3 PFU/LDso, C H 2 1 9 5 = 4 . 0 PFU/LDso, Beijing-I = 1.5 P F U / LDso, and N a k a y a m a - N I H = 1.9 PFU/LDs0 ). Virus neuroinvasion determined by intraperitoneal inoculation at three different concentrations (3 x 10 6, 3 × 105, and 3 × 10 4 P F U per mouse) also indicated that CH2195LA was less neuroinvasive for mice (see Table 1). Only 10% mortality was observed at 3 x 106 P F U inoculation for CH2195LA while other viruses or variants tested induced much higher mortality (55-78%). Those mice which survived in CH2195LA at this concentration were further challenged with the parent strain CH2195 at 100 LDs0 and were shown to be protected. Therefore, CH2195LA was less virulent and could induce protective immunity against CH2195 parent virus infection. 3.4. N u c l e o t i d e s e q u e n c e a n a l y s i s

The JEV envelope protein E sequences of the

parent strain CH2195 and two selected variants CH2195LA and CH2195SA were determined. The results were compared and summarized in Table 2. The large-plaque variant CH2195LA showed to differ from the parent strain at the amino acid resides E-13 (Glu to Lys), E-85 (Glu to Arg), E-117 (Lys to Cys), and E-118 (Phe to Cys). The small-plaque variant CH2195SA differed from the parent strain at residues E-13 (Glu to Lys), E-117 (Lys to Cys), E-118 (Phe to Cys), E-306 (Gly to Glu), E-331 (Arg to Ser), and E 387 (Arg to Met). These two selected variants only differed from each other at four amino acid residues E-85, E-306, E-331, and E387. Furthermore, the amino acids at these seven residues (see Table 2) were found to be the same for two vaccine strains: Beijing-1 (Hashimoto et al., 1988) and N a k a y m a - N I H (McAda et al., 1987). The large-plaque variant CH2195LA only differed from two vaccine strains at residues E-85 and E-387, while the small-plaque variant CH2195SA differed at residues E-306 and E-331. Therefore, the neutralizing epitope against m A b E3.3 could be located at the residues E-306 and E-331 by comparing these sequences.

4. Discussion

The resistant feature of the Taiwanese wild-type isolate CH2195 against m A b E3.3 neutralization, as compared to two vaccine strains (Beijing-1 and N a k a y a m a - N I H ) , was investigated by obtaining antigenic variants with different plaque morp-

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Table 2 Differences of JEV envelopeprotein sequences for CH2195, CH2195LA, CH2195SA and the comparisons with two vaccinestrains (Beijing-l, and Nakayama-NIH) Amino acid position (number in E protein)

CH2195

CH2195LAa

CH2195SA b

Beijing-1

Nakayama-NIH

13 85 117 118 306 331 387

AAA (Lys) CGA (Arg) TGC (Cys) TGC (Cys) GAA (Glu) AGT (Ser) ATG (Met)

GAA (Glu) CAA (Glu) AAA (Lys) TTC (Phe) GAA (Glu) AGT (Ser) ATG (Met)

GAA (Glu) CGA (Arg) AAA (Lys) TTC (Phe) GGA (Gly) AGA (Arg) AGG (Arg)

GAA (Glu) CGA (Arg) AAA (Lys) TTC (Phe) GAA (Glu) AGT (Ser) AGG (Arg)

GAA (Glu) CGA (Arg) AAA (Lys) TTC (Phe) GAA (Glu) AGT (Ser) AGG (Arg)

aGenBank accession number U92644. bGenBank accession number U92643.

hology. We isolated two variants, one with large and the other with small plaque morphology. The small-plaque variant exhibited nt-resistant phenotype for mAb E3.3 and displayed retarded virus replication kinetics in Vero cells. The large-plaque variant was nt-sensitive and showed significant reductions of virus neurovirulence and neuroinvasion in mice. Nucleotide sequence analysis revealed four deduced amino acids of E protein were different for these two variants. The antigenic variants of CH2195 were selected based on plaque sizes and demonstrated to be either nt-resistant or sensitive to mAb E3.3. These two variants propagated in Vero cells still retained high levels of infectivity but had relatively low H A titers as compared with the parent strain and two vaccine strains. Although it has been reported that some nt-resistant variants lacking high H A activity exhibited decreased virulence in mice (Cecilia and Gould, 1991; McMinn et al., 1995), the nt-resistant variant did not significantly affect virus attenuation as compared with the nt-sensitive variant. Obtaining the attenuated variant CH2195LA did not depend on the selection by mAb E3.3, since the variant still exhibited the nt-sensitive phenotype. More interestingly, the manifestation of large plaque morphology in Vero cells by CH2195LA did not agree with other reports that large-plaque variants are associated with stronger virulence (Eckels et al., 1988; Sumiyoshi et al., 1995; Chen et al., 1996). This

disagreement of in vitro and in vivo viral properties indicates JEV replication in mice requires other viral or host cell-specific factors not present in Vero cell infection. Our results of viral E protein sequences showed seven amino acids were different between the parent strain and two plaque-purified variants. Only four amino acids (at residue E-85, E-306, E-331, and E-387) were different between these two plaque-purified variants. The viral epitope recognized by mAb E3.3 and responsible for nt-resistant phenotype could be further localized to the region at residues E-306 and E-331 based on the sequences of these two variants. The other two amino acids at residues E-85 and E-387 were not related since two nt-sensitive vaccine strains (Beijing-1 and Nakayama-NIH) had the same amino acid residues. The former two amino acids likely responsible for nt-resistant phenotype are located in the domain III structure (residues 303-395), as demonstrated by X-ray crystallographic studies on the tick-born encephalitis E protein (Rey et al., 1995). Other reports on flavivirus neutralizing epitopes located in the domain III included JEV at residue 333 (Cecilia and Gould, 1991), louping ill virus at residues 308, 310, 311 (Jiang et al., 1993), and dengue virus at residues 307 (Lin et al., 1994) and 383-393 (Hiramatsu et al., 1996). Since the binding of mAb E3.3 was sensitive to the reducing condition, the neutralizing epitope was dependent on the disulfide bond-maintained conformational structure. These two residues (E-306 and E-331)

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were within or near to the AxCx sheet in the domain III of three dimensional structure of E protein (Rey et al., 1995), which was maintained by the Cys Cys disulfide bond located at residues E-304 and E-335. Moreover, the reduced neurovirulence and neuroinvasion observed in CH2195LA, as compared to CH2195SA, suggests the neutralizing epitope of mAb E3.3 located at residue E-306 and/or E-331 appears to be less affiliated with the attenuation of virus virulence in mice. The other two amino acids at residues E-85 and E-387 may thus be responsible, or in part, for causing virus attenuation of CH2195LA observed in mice. Although other factors except E protein may also affect virus virulence, our findings of the amino acid change from Glu to Gly at residue E-306 did not correlate with the increased JEV neuroinvasion observed between P3 and SA,4-14 2 strains as previously reported (Ni and Barrett, 1996), however the difference may arise from other strainspecific virulence factors. On the other hands, the attenuated properties of CH2195LA observed in mice may be caused by the change of amino acid from Arg to Met at residue 387, where the Arg3s7Glu388-Asp389 (or R3~7-G3s~_D3s9) sequence motif was previously reported as an epitope determining flavivirus pathogenicity (Lobigs et al., 1990). To further understand the exact molecular mechanism of CH2195LA virus attenuation, the complete nucleotide sequence is currently undergoing in our studies.

Acknowledgements The authors would like to thank Drs. Chi-Byi Horng (Director) and Ying-Chang Wu (Head of Epidemiology Division), National Institute of Preventive Medicine, Taiwan, for their support to this work. This research was supported by the Department of Health of Taiwan (Grants DOH86-PM-008 and DOH87-TD-1020).

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