Laboratory of Public Health, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, 060-0818, Japan
Correspondence
Ikuo Takashima
takasima{at}vetmed.hokudai.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences described in this paper are AB185914AB185917.
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INTRODUCTION |
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The WN virus endemic in NY was characterized by large-scale mortality in wild birds, a phenomenon that had not been observed in other areas prior to the NY outbreak (Garmendia et al., 2001). Therefore, the pathogenicity of the WN virus strain that was isolated in NY (NY strain) appears to differ from that of previously isolated strains. Elucidation of the pathogenesis of the NY strain is important for the development of new vaccines and therapies; however, studies of this type have been limited to date.
The flavivirus envelope (E) protein is an important structural protein in viruscell interactions and is a major target of host antibody responses (Seligman & Bucher, 2003; Wang et al., 2001a
). The E proteins of many flaviviruses have one or two potential N-linked glycosylation sites (Chambers et al., 1990
). Glycosylation of the Japanese encephalitis virus E protein, for example, is essential to the native conformation of the epitopes in this protein (Lad et al., 2000
). Of the many WN virus sequences that are listed in GenBank, some viruses, including the NY strain, contain the N-linked glycosylation motif (N-Y-T/S) at residues 154156 of the E protein, whereas others lack the glycosylation site due to amino acid substitutions (Table 1
). Halevy and coworkers isolated variants WN25 and WN25A from the WN virus strain that was isolated in Israel in 1952 and found that these variants had glycosylated E proteins and were not neuroinvasive in mice that were infected intraperitoneally (i.p.) (Chambers et al., 1998
; Halevy et al., 1994
). The authors concluded that E protein glycosylation was not directly responsible for viral attenuation, because other glycosylated variants of the same virus strain were neuroinvasive when administered i.p. to mice. However, Scherret et al. (2001)
reported that the glycosylated clone of the Kunjin virus, which is a subtype of WN virus, produced 10- to 100-fold more virus in cell culture than non-glycosylated Kunjin virus. Therefore, the relationship of E protein glycosylation with WN virus pathogenicity remains controversial.
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METHODS |
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Sequence analysis.
Viral RNA was extracted by using a MagExtractor Viral RNA Purification kit (Toyobo), according to the manufacturer's instructions. First-strand cDNA was synthesized by using M-MLV reverse transcriptase (TaKaRa Bio). In preparation for DNA sequencing, the full-length viral sequence was divided into seven regions and amplified by using Platinum Taq High Fidelity DNA polymerase (Invitrogen) and specific primers. The amplified DNA fragments were purified by using a QIAquick PCR Purification kit (Qiagen) and then sequenced directly in both directions or cloned into pT7Blue-2 T vector (Novagen) before subsequent sequencing.
The 3' non-coding region (NCR) was sequenced by using a previously reported method (Kolykhalov et al., 1996; Yun et al., 2003
). Briefly, ddATP was incorporated into the 5'-phosphorylated primer (T: 5'-CCAGTGTTGTGGCCTGCAGGGCGAATT-3') with terminal deoxynucleotidyltransferase (TdT) to prevent intramolecular and intermolecular ligation of the primers. The following protocol was used: 500 pmol T primer was mixed with 15 U TdT (TaKaRa Bio), 40 U RNase inhibitor (TaKaRa Bio), 20 nmol ddATP (TaKaRa Bio), 2 µl 10 mM CoCl2, 2 µl 0·1 % BSA and 4 µl 5x TdT buffer; diethyl pyrocarbonate (DEPC)-treated water (Ambion) was added to give a final volume of 20 µl. The mixture was incubated at 37 °C for 1 h and the T primer was extracted with phenol, precipitated with ethanol and resuspended in DEPC-treated water. The modified T primer was ligated into the 3' end of the viral genomic RNA by mixing viral genomic RNA with the modified T primer, 40 U T4 RNA ligase (TaKaRa Bio), 5 µl 10x T4 RNA ligase buffer, 3 µl 0·1 % BSA, 40 U RNase inhibitor and 12·5 µl 50 % PEG 6000 (Wako Pure Chemical Industries); DEPC-treated water was added to give a final volume of 50 µl. The mixture was incubated at 16 °C for 1216 h and then used directly for first-strand cDNA synthesis. Reverse transcription was performed with the TR15 primer (5'-AATTCGCCCTGCAGG-3') and M-MLV reverse transcriptase. PCR amplification was performed with the forward primer (5'-AAATGGAGTGACGTCCCATA-3', nt 1018910208 of WN virus strain NY99-6922) and TR20 primer (5'-AATTCGCCCTGCAGGCCACAACA-3') by using Platinum Taq High Fidelity DNA polymerase. The amplified fragment was purified by using a QIAquick PCR purification kit, ligated into the pT7Blue-2 T vector and sequenced.
All sequencing analyses were performed by using a BigDye Terminator cycle sequencing kit and an ABI PRISM 310 genetic analyser (Applied Biosystems).
To examine the influence of substitutions in the 3' NCR, RNA structure models of the 3' terminus were constructed by using GENETYX-WIN software (GENETYX).
Western blot analysis.
BHK cell monolayers were infected with each of the virus variants. After incubation for 48 h, supernatants were collected and mixed with lysis buffer (1 % Triton X, 50 mM Tris/HCl, 150 mM NaCl, 5 mM EDTA). The supernatants of the extracts were immunoprecipitated with the anti-flavivirus mAb D1-4G2 (ATCC) and protein GSepharose (Amersham Biosciences). The samples were then separated by SDS-PAGE under non-reducing conditions and transferred to a nitrocellulose membrane (Millipore). To detect the glycoproteins, the membranes were treated with biotinConA (Honen Corporation) and then with peroxidase-conjugated streptavidin (Sigma). To confirm that the D1-4G2 antibody had precipitated the WN virus protein, the membranes were probed by using D1-4G2 as the primary antibody and peroxidase-conjugated anti-mouse IgG as the secondary antibody (Sigma). The membranes were developed with diaminobenzidine.
Virus infection and sample collection.
To assay virus growth in vitro, BHK cell monolayers were inoculated with each of the virus variants at an m.o.i. of 1. Supernatant samples were collected at the indicated times and titrated as described below.
For the in vivo analysis, 6-week-old male or female BALB/c mice (Japan SLC) were used. To investigate the viral virulence and distribution in mice, the indicated doses of viruses were inoculated subcutaneously (s.c.) into the axilla or injected intracerebrally (i.c.) into the left hemisphere and the survival rates of the mice were monitored. Virus-inoculated mice were euthanized by ether overdose and tissue samples were collected at the indicated number of days post-infection (p.i.). Tissue samples were homogenized in minimum essential medium (MEM; Nissui Pharmaceutical) to yield a 10 % suspension and then centrifuged at 5000 r.p.m. for 15 min at 4 °C. Supernatants were collected and stored at 80 °C until used.
Viral titration.
Viruses in the working stocks and collected samples were titrated by plaque assay on BHK cells. BHK cell monolayers were grown in 12-well plates and inoculated with serial dilutions of the virus solutions. After 60 min virus adsorption, the virus solution was aspirated and the cells were washed twice with PBS. An overlay consisting of MEM containing 1·5 % carboxymethylcellulose (CMC; Wako) and 2 % fetal calf serum (CMC-MEM) was added to the cells and the plates were incubated at 37 °C in a CO2 incubator. After 4 days cell culture, the CMC-MEM was aspirated, the cells were washed twice with PBS and then fixed and stained with a solution of 0·1 % crystal violet and 10 % formalin in PBS under UV light. After staining for 2 h, the cells were washed with water and dried and plaques were counted. The virus titre was calculated from the virus dilution that produced 10100 plaques per well and expressed as p.f.u.
Statistical analysis.
Statistically significant differences were determined by using the unpaired t-test or MannWhitney test. To determine the statistical significance of the survival curves, the log-rank and generalized Wilcoxon tests were performed. Values of P<0·05 were considered to be statistically significant.
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RESULTS |
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Western blotting
Western blotting analysis was performed to verify the glycosylation status of the viral E proteins. The WN virus proteins were immunoprecipitated by the mAb D1-4G2 (Fig. 1c). As predicted by the sequencing results, the 6-LP and B-LP variants contained glycosylated E proteins and the 6-SP and B-SP variants did not contain glycosylated E proteins (Fig. 1d
). To avoid contamination of the membranes with the heavy chain of IgG, which was used for the immunoprecipitation, the electrophoresis procedure was performed under non-reducing conditions; therefore, the precise molecular masses of the viral proteins could not be deduced. However, the non-glycosylated E proteins migrated faster than the glycosylated forms.
Virus replication in tissue culture
The kinetics of virus replication were examined by using BHK cells (Fig. 2). The 6-LP and 6-SP variants exhibited similar kinetics; however, the titre of 6-LP was 10-fold higher than that of 6-SP at 24 h p.i. (P<0·05). Variant B-SP replicated slowly, whereas B-LP replicated very rapidly. The titre of B-LP was approximately 1000-fold higher than that of B-SP at 24 h p.i. (P<0·01). Thus, the titres of the glycosylated viruses tended to be higher than those of the non-glycosylated viruses during the early period of replication in vitro.
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Changes in body weight of the mice after infection were also determined (Fig. 3). Body weights of the infected mice began to decrease when the mice showed clinical signs of disease; in mice infected with either 6-LP or B-LP, weight loss began 6 days p.i. In contrast, body weights of mice infected with either 6-SP or B-SP increased during the time-course of infection. Eight days after infection, differences in the body weights between the 6-LP- and 6-SP-infected animals and between the B-SP- and B-LP-infected animals were statistically significant (P<0·05).
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DISCUSSION |
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We isolated the 6-LP and 6-SP strains from NY99-6922 and the B-SP and B-LP strains from BC787 by plaque purification. Sequence analysis detected an amino acid difference between 6-LP and 6-SP; this difference was in the glycosylation site of the E protein. On the other hand, in addition to a difference in the amino acid sequence at the glycosylation site, two other amino acid differences between B-LP and B-SP were detected. A nucleotide change from A to T at position 287 (nt 5470) of the NS3 protein was seen in B-SP. The NS3 protein of flaviviruses is multifunctional and includes serine proteinase, RNA helicase and nucleoside triphosphatase activities (Falgout et al., 1991; Li et al., 1999
). The RNA helicase region is classified into subfamilies, based on the sequence of motif II (Matusan et al., 2001
), and the DExH motif exists at positions 285288 of the NS3 protein of WN virus. The substitution detected in the NS3 protein of B-SP was in the x position in the DExH motif, which suggested that this amino acid substitution would not affect the function of the NS3 protein. The B-LP variant stock contained two viral types with different amino acids at position 86 of the NS1 protein (substitutions at nt 2725). The precise role of the flavivirus NS1 protein remains unclear; however, it is strongly immunogenic (Timofeev et al., 2004
; Xu et al., 2004
). Therefore, a mutation at this position of NS1 may influence the host immune response, but the role of this mutation is unclear. In addition, two or three nucleotide substitutions were identified in the 3' NCR of 6-LP and 6-SP and one substitution was seen in that of B-LP and B-SP, as compared with the 3' NCR sequence of the 382-99 strain. The 3' NCR of all sequenced flavivirus genomes can be folded into similar 3'-terminal structures that are formed by approximately 100 nt near the 3' end of the genome, consisting of a large stemloop (SL) followed by a smaller SL (Shi et al., 1996
; Tilgner & Shi, 2004
). In addition, the conserved sequences (CS) CS1, CS2, repeated CS2 (RCS2), CS3 and RCS3 exist upstream of the 3' SL structure of mosquito-borne flaviviruses (Lo et al., 2003
). These SL structures and the CS region are important in genomic RNA synthesis (Lo et al., 2003
; Shi et al., 1996
; Tilgner & Shi, 2004
). In our isolated variants, the nucleotide substitutions at positions 10754 and 10851 were upstream of the 3' SL structure, but were not in the CS region. Therefore, we considered that these substitutions would not have a major influence on pathogenicity. The nucleotide substitution at position 10956 of variant 6-LP was in the SL region. However, when we performed an analysis of the predicted RNA structure by using GENETYX-WIN software and compared the results with published data, we found that the SL structure of 6-LP was not altered at all by the substitution at position 10956. These findings suggested that none of the substitutions in the 3' NCR that were identified in variants were critical to the known functions of the viral RNA.
As described above, a number of nucleotide substitutions were detected between the LP and SP variants. However, our analyses suggested that most of the nucleotide and amino acid substitutions were not critical and that the determining difference between the LP and SP variants was that the E protein of the LP variants was glycosylated, whereas that of the SP variants was not.
The kinetics of replication of the descendent variants were determined by using BHK cell cultures. Although the titres of the four variants were similar during the final stages of infection, glycosylated viruses exhibited more rapid replication than non-glycosylated viruses during the early stages of infection. Consistent with our results, Scherret et al. (2001) reported that a glycosylated clone of Kunjin virus produced 10- to 100-fold more virus than a non-glycosylated clone. Together, these findings indicate that glycosylation of the E protein leads to enhanced virus replication.
In our animal-infection studies, when mice were infected by the s.c. route, only viruses with glycosylated E protein were neuroinvasive, suggesting that glycosylation of the E protein is a molecular determinant of viral neuroinvasiveness. Virus with non-glycosylated E protein was detected in blood and spleen samples from mice infected via the s.c. route; however, replication of non-glycosylated virus in the peripheral organs was inefficient, as compared to that of glycosylated virus. This suggests that the efficiency of WN virus replication in peripheral organs may correlate with neuroinvasiveness.
Chambers et al. (1998) suggested that E protein glycosylation might not be directly responsible for the attenuation of WN viruses, as the non-glycosylated E protein variant of the Israel strain of the WN virus showed neuroinvasiveness; however, our results differed from those findings, possibly due to differences in the inoculation routes. The authors of the previous study used i.p. injection as the peripheral infection route. In the present study, we used s.c. injection as the peripheral infection route, to simulate the natural mode of infection. In animal models of JE serocomplex virus infection, the outcome of peripheral infection with some viruses differs depending on the route of infection. In our studies, mice infected by the s.c. route with the WN variants exhibited mortality rates that were independent of dose. Similar results were shown for s.c. infection of 8- to 12-week-old C57BL/6 mice with a WN virus strain isolated in NY in 2000 (Diamond et al., 2003
). In addition, it has been reported that intravenous (i.v.) infection of mice with WN virus and Murray Valley encephalitis virus leads to dose-independent mortality (Licon Luna et al., 2002
; Wang et al., 2003b
). On the other hand, i.p. infection of mice with WN virus showed dose-dependent mortality and the LD50 value could be estimated (Beasley et al., 2002
; Wang et al., 2001b
, 2003a
). The reason for this difference is unclear; however, the i.p. inoculation route differs from other peripheral infection routes in that the virus enters directly into the peritoneal tissue. Taking into consideration the natural route of WN virus infection, the s.c. inoculation route may be useful for the elucidation of the pathogenesis of WN virus infection. In addition, the results of experiments using the s.c. and i.v. routes suggest that the quantity of virus inoculated into the mouse does not correlate directly with eventual mortality; only the secondary virus replication in the peripheral organs was related to viral invasion of the central nervous system (CNS).
In this study, all four WN variants induced lethality when inoculated into mice by the i.c. route. This result suggests that E protein glycosylation is not a molecular determinant of neurovirulence for WN virus. In addition, the viruses that were isolated from infected horses, B-LP and B-SP, were strongly neurovirulent (as compared to the viruses that were isolated from mosquitoes). Three major amino acid differences [MI at position 11 (nt 498) of the preM protein; S
P at position 557 (nt 6280) of the NS3 protein; and T
A at position 165 (nt 7408) of the NS4B protein] were detected in B-LP and B-SP when compared to the sequence of the type strain NY99-flamingo 382-99. The amino acid substitutions in NS3 and NS4B were also observed in strain NY99-equi (GenBank accession no. AF260967), which was isolated from a horse in NY in 1999. However, WN Italy 1998-equi (accession no. AF404757) and PaAn001 (accession no. AY268132), which were isolated from horses in Europe, did not have these mutations. Further studies are needed to elucidate the relationship between viral virulence and the substitutions detected in the NY strain isolated from a horse.
The results of this study suggest that glycosylation of the E protein is a molecular determinant of neuroinvasiveness of the NY strains of WN virus. However, additional factors involved in the pathogenesis of WN viruses remain to be elucidated. It has been reported that the initial target of infection for dengue virus, a mosquito-borne flavivirus, are dendritic cells (Wu et al., 2000) and that dendritic cells migrate to the local lymph nodes following arbovirus infection (Johnston et al., 2000
). However, subsequent events leading to viral invasion of the CNS remain unclear. To further the development of effective therapies and new vaccines against WN virus infections, additional studies are needed to elucidate the pathogenesis of WN virus infection.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Beasley, D. W. C., Li, L., Suderman, M. T. & Barrett, A. D. T. (2002). Mouse neuroinvasive phenotype of West Nile virus strains varies depending upon virus genotype. Virology 296, 1723.[CrossRef][Medline]
Chambers, T. J., Hahn, C. S., Galler, R. & Rice, C. M. (1990). Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44, 649688.[CrossRef][Medline]
Chambers, T. J., Halevy, M., Nestorowicz, A., Rice, C. M. & Lustig, S. (1998). West Nile virus envelope proteins: nucleotide sequence analysis of strains differing in mouse neuroinvasiveness. J Gen Virol 79, 23752380.[Abstract]
Diamond, M. S., Shrestha, B., Marri, A., Mahan, D. & Engle, M. (2003). B cells and antibody play critical roles in the immediate defense of disseminated infection by West Nile encephalitis virus. J Virol 77, 25782586.
Falgout, B., Pethel, M., Zhang, Y.-M. & Lai, C.-J. (1991). Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol 65, 24672475.[Medline]
Garmendia, A. E., Van Kruiningen, H. J. & French, R. A. (2001). The West Nile virus: its recent emergence in North America. Microbes Infect 3, 223229.[CrossRef][Medline]
Halevy, M., Akov, Y., Ben-Nathan, D., Kobiler, D., Lachmi, B. & Lustig, S. (1994). Loss of active neuroinvasiveness in attenuated strains of West Nile virus: pathogenicity in immunocompetent and SCID mice. Arch Virol 137, 355370.[Medline]
Hamman, M. H., Delphine, H. C. & Winston, H. P. (1965). Antigenic variation of West Nile virus in relation to geography. Am J Epidemiol 82, 4055.
Hubálek, Z. & Halouzka, J. (1999). West Nile fever a reemerging mosquito-borne viral disease in Europe. Emerg Infect Dis 5, 643650.[Medline]
Johnston, L. J., Halliday, G. M. & King, N. J. C. (2000). Langerhans cells migrate to local lymph nodes following cutaneous infection with an arbovirus. J Invest Dermatol 114, 560568.
Kolykhalov, A. A., Feinstone, S. M. & Rice, C. M. (1996). Identification of a highly conserved sequence element at the 3' terminus of hepatitis C virus genome RNA. J Virol 70, 33633371.[Abstract]
Lad, V. J., Shende, V. R., Gupta, A. K., Koshy, A. A. & Roy, A. (2000). Effect of tunicamycin on expression of epitopes on Japanese encephalitis virus glycoprotein E in porcine kidney cells. Acta Virol 44, 359364.[Medline]
Li, H., Clum, S., You, S., Ebner, K. E. & Padmanabhan, R. (1999). The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids. J Virol 73, 31083116.
Licon Luna, R. M., Lee, E., Müllbacher, A., Blanden, R. V., Langman, R. & Lobigs, M. (2002). Lack of both Fas ligand and perforin protects from flavivirus-mediated encephalitis in mice. J Virol 76, 32023211.
Lo, M. K., Tilgner, M., Bernard, K. A. & Shi, P.-Y. (2003). Functional analysis of mosquito-borne flavivirus conserved sequence elements within 3' untranslated region of West Nile virus by use of a reporting replicon that differentiates between viral translation and RNA replication. J Virol 77, 1000410014.
Matusan, A. E., Pryor, M. J., Davidson, A. D. & Wright, P. J. (2001). Mutagenesis of the Dengue virus type 2 NS3 protein within and outside helicase motifs: effects on enzyme activity and virus replication. J Virol 75, 96339643.
Scherret, J. H., Mackenzie, J. S., Khromykh, A. A. & Hall, R. A. (2001). Biological significance of glycosylation of the envelope protein of Kunjin virus. Ann N Y Acad Sci 951, 361363.
Seligman, S. J. & Bucher, D. J. (2003). The importance of being outer: consequences of the distinction between the outer and inner surfaces of flavivirus glycoprotein E. Trends Microbiol 11, 108110.[CrossRef][Medline]
Shi, P.-Y., Brinton, M. A., Veal, J. M., Zhong, Y. Y. & Wilson, W. D. (1996). Evidence for the existence of a pseudoknot structure at the 3' terminus of the flavivirus genomic RNA. Biochemistry 35, 42224230.[CrossRef][Medline]
Shirato, K., Mizutani, T., Kariwa, H. & Takashima, I. (2003). Discrimination of West Nile virus and Japanese encephalitis virus strains using RT-PCR RFLP analysis. Microbiol Immunol 47, 439445.[Medline]
Tilgner, M. & Shi, P.-Y. (2004). Structure and function of the 3' terminal six nucleotides of the West Nile virus genome in viral replication. J Virol 78, 81598171.
Timofeev, A. V., Butenko, V. M. & Stephenson, J. R. (2004). Genetic vaccination of mice with plasmids encoding the NS1 non-structural protein from tick-borne encephalitis virus and dengue 2 virus. Virus Genes 28, 8597.[CrossRef][Medline]
Wang, T., Anderson, J. F., Magnarelli, L. A., Bushmich, S., Wong, S., Koski, R. A. & Fikrig, E. (2001a). West Nile virus envelope protein: role in diagnosis and immunity. Ann N Y Acad Sci 951, 325327.
Wang, T., Anderson, J. F., Magnarelli, L. A., Wong, S. J., Koski, R. A. & Fikrig, E. (2001b). Immunization of mice against West Nile virus with recombinant envelope protein. J Immunol 167, 52735277.
Wang, T., Scully, E., Yin, Z. & 7 other authors (2003a). IFN--producing
T cells help control murine West Nile virus infection. J Immunol 171, 25242531.
Wang, Y., Lobigs, M., Lee, E. & Müllbacher, A. (2003b). CD8+ T cells mediate recovery and immunopathology in West Nile virus encephalitis. J Virol 77, 1332313334.
Wu, S.-J. L., Grouard-Vogel, G., Sun, W. & 14 other authors (2000). Human skin Langerhans cells are targets of dengue virus infection. Nat Med 6, 816820.[CrossRef][Medline]
Xu, G., Xu, X., Li, Z., He, Q., Wu, B., Sun, S. & Chen, H. (2004). Construction of recombinant pseudorabies virus expressing NS1 protein of Japanese encephalitis (SA14-14-2) virus and its safety and immunogenicity. Vaccine 22, 18461853.[CrossRef][Medline]
Yun, S.-I., Kim, S.-Y., Rice, C. M. & Lee, Y.-M. (2003). Development and application of a reverse genetics system for Japanese encephalitis virus. J Virol 77, 64506465.
Received 1 May 2004;
accepted 23 August 2004.