Discrimination of West Nile Virus and Japanese Encephalitis Virus Strains Using RT-PCR RFLP Analysis

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1 Discrimination of West Nile Virus and Japanese Encephalitis Virus Strains Using RT-PCR RFLP Analysis Kazuya Shirato, Tetsuya Mizutani, Hiroaki Kariwa, and Ikuo Takashima* Laboratory of Public Health, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido , Japan Received November 18, 2002; in revised form, March 12, Accepted March 14, 2003 Abstract: West Nile (WN) virus is a mosquito-borne flavivirus that induces lethal encephalitis in humans and horses. Since an outbreak of WN encephalitis in humans and horses occurred in New York City in late August 1999, the possibility exists that WN virus will invade regions that have close links with the United States, such as Japan. We developed a genetic diagnostic method that discriminates between strains of WN virus and Japanese encephalitis (JE) virus. The method involves RT-PCR restriction fragment length polymorphism (RFLP) analysis with a RT-PCR primer set, a nested PCR primer set, and a restriction enzyme. We detected WN and JE viruses in experimentally infected animal brain, spleen, and serum samples. Our method is useful in distinguishing WN viruses from the endemic background of JE viruses, and in discriminating the highly virulent WN strain, which was isolated in New York in 1999, from other WN virus strains. Key words: West Nile virus, RT-PCR RFLP Microbiol. Immunol., 47(6), , 2003 *Address correspondence to Dr. Ikuo Takashima, Laboratory of Public Health, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Kita-18 Nishi-9, Kita-ku, Sapporo, Hokkaido , Japan. Fax: takasima@vetmed.hokudai. ac.jp The West Nile (WN) virus belongs to the genus Flavivirus of the family Flaviviridae, and is a member of the Japanese encephalitis (JE) virus serocomplex. WN virus is a mosquito-borne flavivirus that induces lethal encephalitis in humans and horses. The WN virus is distributed geographically in Africa, the Middle East, western and central Asia, India, and Europe (1, 5, 7). An outbreak of WN encephalitis in humans and horses occurred in New York City (NY) in late August 1999, which was the first case of WN virus encephalitis in the western hemisphere (4). Currently, the WN virus epidemic is spreading throughout the United States (3). The genomic sequence of the WN viral strain isolated in NY in 1999 (NY99) is similar to that of a strain isolated in Israel in 1998, which suggests that strain NY99 was transported from Israel to the United States by infected humans, birds, mosquitoes, or other hosts (4, 9, 10). Therefore, the possibility exists that strain NY99 will invade regions that have close links with the United States, such as Japan. Since strain NY99 causes largescale mortality of wild birds, a feature of the virus that was not evident during the endemic spread of WN virus in Romania and the Czech Republic (3), the pathogenesis of strain NY99 appears to be different from that of previously identified WN strains. Therefore, it is important in terms of epidemic prevention to be able to distinguish the WN strains. On the other hand, viruses of the JE virus serocomplex have antigenic cross-reactivity with each other, and they are not readily differentiated by serological methods (13). Therefore, diagnostic methods to differentiate WN virus from JE virus are required to facilitate preventive measures against WN viruses in an endemic background of other JE serocomplex viruses. In this study, we developed a genetic diagnostic method that can be used to distinguish strain NY99 from other WN virus strains, and which also differentiates the WN virus from the JE virus. RT-PCR has been used to develop sensitive and specific assays for the identification of the WN virus (14). Igarashi et al. reported that WN and JE virus genes could be detected Abbreviations: JE, Japanese encephalitis; MVE, Murray Valley encephalitis; NY99, West Nile viral strain isolated in New York City in 1999; RELP, restriction fragment length polymorphism; RT-PCR, reverse transcription-polymerase chain reaction; TBE, tick-borne encephalitis; WN, West Nile. 439

2 440 K. SHIRATO ET AL by RT-PCR (8). However, strain NY99 had not yet been identified when that study was performed. RT- PCR restriction fragment length polymorphism (RFLP) analysis is useful in the detection of point mutations in genes, and it is simple and cost-effective. Since RFLP analysis is widely used for virus research (15, 17), we adopted this method to distinguish WN virus strains. Materials and Methods Viruses. WN virus strains NY99A-301 (CDC ID# M28568), BC787 (CDC ID# M28583W), NY (CDC ID# M28623W), and Eg101 (CDC ID# M and GenBank accession No. AF260968) were kindly provided by Dr. Duane Gubler, Center for Disease Control and Prevention (CDC, Fort Collins, Colo., U.S.A). The WN virus strain FCG (GenBank accession No. #M12294) was kindly provided by Dr. Akira Oya, National Institute of Infectious Diseases, Japan. Various other flaviviruses, including JE viruses (JaGAr01, Ishikawa (19), and Muar (6) strain), Murray Valley encephalitis (MVE) virus, tick-borne encephalitis (TBE) virus far-eastern subtype [Sofjin strain, and Oshima 5-10 strain (18)], Langat virus, and Powassan virus, were used in this study. The Getah virus (Togaviridae) was also used. All of the viruses were propagated in the brains of suckling mice. Briefly, one-day-old suckling mice were inoculated intracerebrally with virus. When clinical symptoms appeared (3 4 days post-infection), the mice were euthanized by ether overdose. The brains were collected, homogenized in a 10% phosphatebuffered saline that contained 10% FCS and antibiotics, and the virus stocks stored at 80 C. Viruses were titrated using the standard plaque assay with BHK cell. Primers. The primers for RT-PCR were designed based on the nucleotide sequences of WN virus strain NY99-flamingo (382-99) (GenBank accession No. AF196835) and JEV strain JaGAr01 (GenBank accession No. AF069076). We tried to distinguish WN and JE virus with RT-PCR using several sets of each specific primer, but it was not successful. Therefore, we selected common sequences of nonstructural protein (NS)1 and NS2A as primers in order to amplify the RNA of both WN and JE virus. The primer set that was used in RT-PCR to amplify 957 bp region, and the primer set that was used in nested PCR to amplify 921 bp region, are shown in Table 1. Since the amplified regions facilitated the discrimination between strains of WN virus and JE virus using a restriction enzyme, these primer sets are convenient. The primers were used at a concentration of 10 pmol/µl for RT-PCR and nested PCR. RNA extraction. Viral RNAs were prepared from virus stocks or sera of virus-infected mice using ISO- GEN-LS (Nippon Gene Co., Japan), and RNA samples were prepared from virus-infected mice using ISOGEN (Nippon Gene Co.) according to the manufacturer s protocol. RT-PCR. RT-PCR was performed using One Step RNA PCR Kit (AMV) (TaKaRa Shuzo Co., Japan). The protocol was as follows: the indicated volume of viral RNA was mixed with 5 µl of 10 One Step RNA PCR Buffer, 10 µl of 25 mm MgCl 2, 5 µl of 10 mm dntp, 1 µl of RNase Inhibitor (40 U/µl), 1 µl of AMV RTase XL (5 U/µl), 1 µl of AMV-Optimized Taq (5 U/µl), 1 µl of sense primer, and 1 µl of anti-sense primer, and RNase-free water was added to give a final volume of 50 µl. The mixture was incubated at 50 C for 30 min, and then at 94 C for 2 min, in accordance with the instructions. The mixture was then subjected to 40 amplification cycles (denaturation at 94 C for 30 s, annealing at 62 C for 30 s, extension at 72 C for 1 min). Aliquots (5 µl) of the amplified products were detected by ethidium bromide staining after separation using 2% agarose gel electrophoresis. Nested PCR. The RT-PCR-amplified products were purified using the QIAquick PCR Purification Kit (QIA- GEN), and nested PCR was performed in a 50 µl of reaction mixture that contained 1 µl of purified RT-PCR product, 5 µl of 10 high-fidelity PCR buffer, 4 µl of 2.5 mm dntp mixture, 2 µl of 50 mm MgSO 4, 0.5 µl of PLATINUM Taq High Fidelity (Invitrogen), 1 µl of Table 1. Oligonucleotide primers used in one-step RT-PCR and nested PCR Primers Genome Position Sequence Product size One-step RT-PCR Sense '-GACTGGAGCATCAAATGTGGGAAGC-3' 957 bp JaGAr Anti-sense '-TCCACCTCTTGCGAAGGAC-3' JaGAr Nested PCR Sense '-GCATCAAATGTGGGAAGC-3' 921 bp JaGAr Anti-sense '-CCAAGAACACGACCAGAAGGC-3'

3 RT-PCR RFLP FOR WNV AND JEV 441 sense primer, 1 µl of anti-sense primer, and 35.5 µl of distilled water. The mixture was subjected to 40 amplification cycles (denaturation at 94 C for 30 s, annealing at 60 C for 30 s, extension at 68 C for 1 min). The PCR was terminated with a final elongation step at 68 C for 10 min. Aliquots (5 µl) of the amplified products were detected by ethidium bromide staining after separation using 2% agarose gel electrophoresis. Control mixtures that lacked the template were used to detect potential cross-contamination. RFLP analysis. The RT-PCR- or nested PCR-amplified products were separated by 2% agarose gel electrophoresis, then stained with ethidium bromide, and bands were visualized by under ultraviolet illumination. Bands of the appropriate sizes (957 bp for RT-PCR and 921 bp for nested PCR) were gel-purified using the QIAquick Gel Purification Kit (QIAGEN), and then 5 µl aliquots of the purified amplicons were digested with the restriction enzyme TaqI (TaKaRa Shuzo Co., Japan). Twenty units of TaqI, 2 µl of TaqI buffer, and 2 µl of 0.1% BSA were mixed, and sterile distilled water was added to give a final volume of 20 µl. The samples were then incubated at 65 C for 2 hr and the products were subjected to electrophoresis in a 2% low-melting temperature agarose gel, stained with ethidium bromide, and visualized by ultraviolet illumination. Sequence analysis. The RT-PCR amplicons were gel-purified using QIAquick Gel Purification Kit (QIA- GEN). The purified amplicons were sequenced using the BigDye Terminator Cycle Sequencing Kit and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Virus infection. Six- to seven-week-old female BALB/c mice were infected subcutaneously (s.c.) with 10 5 PFU of NY , FCG, or JE virus. The mice, which lacked clinical signs of disease, were euthanized by ether overdose 3 7 days post-infection (p.i.), and serum samples were collected. The brains and spleens were collected from virus-infected mice that succumbed to infection. The collected tissues were stored at 80 C until assayed by RT-PCR. Results RT-PCR One-step RT-PCR was performed using 1 µg of total RNA as the template to confirm that WN and JE viruses were amplified by the primer set used in this study. Although the one-step RT-PCR sense primer was not identical to the sequences of WN virus strains FCG and Eg101, the primer set was used successfully to detect all of the WN and JE virus strains. MVE virus, which is one of the JE serocomplex viruses, was also amplified. Other flaviviruses, such as TBE viruses (Sofjin and Oshima 5-10 strains), Langat virus, and Powassan virus, and viruses of other families, such as Getah virus, were not amplified by this primer set (Fig. 1A). To increase the sensitivity of detection, we performed nested PCR with the RT-RCR amplicon as a template. Although the sequence of the nested anti-sense primer was not identical to JE and MVE virus genomes, products of the expected size were detected for all of WN virus strains, JE, and MVE virus (Fig. 1B). To measure the sensitivity of the onestep RT-PCR and nested PCR, RNA was extracted from 10 8 PFU of NY or JaGAr01, and one-step RT- PCR was performed using serially diluted RNA as the template. At least 10 3 to 10 4 PFU/µl of virus was required for amplicon detection by one-step RT-PCR (Fig. 2A), whereas nested PCR amplicons were detected at viral concentrations of 10 0 PFU/µl (Fig. 2B). Fig. 1. Visualization of (A) the one-step RT-PCR product (957 bp), (B) the nested PCR product in JE virus serocomplex viruses (921 bp), and (C) the nested PCR product in other serocomplex flaviviruses and Getah virus. The amplicons were detected by staining with ethidium bromide after 2% agarose gel electrophoresis. (A) Lane 1, NY ; lane 2, NY99-A301; lane 3, BC787; lane 4, Eg101; lane 5, FCG; lane 6, JE JaGAr 01; lane 7, MVE virus; lane 8, TBE virus Sofjin strain; lane 9, TBE virus Oshima 5-10 strain; lane 10, Langat virus; lane 11, Powassan virus; lane12, Togaviridae genus Alphavirus Getah virus; lane M, Smart Ladder (Nippon Gene) molecular size marker. (B) Lane 1, NY ; lane 2, NY99-A301; lane 3, BC787; lane 4, Eg101; lane 5, FCG; lane 6, JE JaGAr01; lane 7, MVE virus; lane 8, negative control (H 2O); lane M, Smart marker. (C) Lane 1, TBE virus Sofjin strain; lane 2, TBE virus Oshima 5-10 strain; lane 3, Langat virus; lane 4, Powassan virus; lane5, Getah virus; lane M, Smart Ladder.

4 442 K. SHIRATO ET AL Fig. 2. Sensitivities of (A) the one-step RT-PCR, and (B) the nested PCR. RNA samples were extracted from 10 8 PFU of NY or JaGAr01, and serially diluted RNA samples were subjected to one-step RT-RCR and nested PCR, as described in Materials and Methods. The amplicons were detected by staining with ethidium bromide after 2% agarose gel electrophoresis. Left side, NY ; right side, JaGAr01. (A) Lane 1, 10 6 (PFU/µl); lane 2, 10 5 ; lane 3, 10 4 ; lane 4, 10 3 ; lane 5, 10 2 ; lane 6, 10 1 ; lane M, Smart Ladder. (B) Lane 1, 10 3 (PFU/µl); lane 2, 10 2 ; lane 3, 10 1 ; lane 4, 10 0 ; lane 5, 10 1 ; lane 6, 10 2 ; lane M, Smart Ladder. RFLP Analysis To distinguish WN virus strains from JE virus strains, RT-PCR amplicons were digested with the restriction enzyme TaqI, as described in Materials and Methods. TaqI cleaved the NY99 DNA into 357 bp, 289 bp, and 311 bp fragments, the Eg101 DNA into 526 bp, 120 bp, and 311 bp fragments, the FCG DNA into 67 bp, 153 bp, 137 bp, 277 bp, 187 bp, 37 bp, and 99 bp fragments, and the DNA of JE virus JaGAr01 strain into 280 bp and 677 bp fragments. However, the MVE virus amplicon was not cleaved by TaqI (Fig. 3). The TaqI sites were also conserved in nested-pcr products (Fig. 3). The NY99 amplicon sequences (i.e., NY , NY99-A301, and BC787) were identical to those of the strain, and the amplicon sequences of Eg101, JaGAr01, and MVE were identical to the sequences listed in the GenBank database. Although two point mutations were detected in the sequence of the FCG amplicon compared to the published sequence (position 3338: A to G, and position 3559: A to C), the TaqI sites were conserved (Fig. 4). The WN virus strain Fig. 3. Visualization of (A) RFLP analysis of the one-step RT-PCR (left) and nested PCR (right) products in JE serocomplex viruses, and (B) RFLP analysis of the nested PCR products in other serocomplex viruses. The amplicons were digested with TaqI, as described in Materials and Methods, and then visualized by staining with ethidium bromide after electrophoresis in a 2% low-melting-temperature agarose gel. (A) Lane 1, NY ; lane 2, NY99-A301; lane 3, BC787; lane 4, Eg101; lane 5, FCG; lane 6, JE JaGAr01; lane 7, MVE virus; Lane M, Smart Ladder. (B) Lane 1, TBE virus Sofjin strain; lane 2, TBE virus Oshima 5-10 strain; lane 3, Langat virus; lane 4, Powassan virus; lane M, Smart Ladder.

5 RT-PCR RFLP FOR WNV AND JEV 443 from USA, Connecticut 2741 (GenBank accession No. AF206518) also had TaqI sites that were identical to those of the NY99 strain, and another lineage 2 virus (GenBank accession No. NC_001563) had TaqI sites that were identical to those of strain FCG. Kunjin virus is classified into lineage I of WN virus (16). Based on the amplified region sequence of Kunjin virus (Gen- Bank accession No, D00246), Kunjin virus was predicted to be cleaved into 451 bp, 178 bp, and 328 bp fragments. JE virus was divided into five genotypic groups based on the pre-m and envelope region sequences (2, 20). Based on the alignment of the amplified region sequences of twenty genotype 3 JE viruses that were obtained from GenBank, the TaqI sites were conserved among almost all genotype 3 viruses. Two genotype 3 viruses, including Beijing strain, are predicted to be cleaved into 280 bp, 425 bp, and 252 bp fragments, but this pattern is different from that of WN strains. We also compared the alignment of the amplified region sequences of three genotype 2 viruses. Two genotype 2 viruses are predicted to be cleaved into the patterns different from that of JaGAr01, but the patterns are different from that of WN strains. Additionally, we tested the JE virus Ishikawa strain, which was genotype 1 virus (19), and the Muar strain (6), which was genotype 5 virus, in RFLP analysis of RT-PCR amplicon, and the fragment patterns were identical to that of JaGAr01 strain (data not shown). Genotype 4 viruses and their sequence data of target region were not available to us. We also performed nested PCR with the RT-PCR amplicons of other serocomplex flaviviruses and Getah virus as templates. We detected amplified products around 900 bp except in the case of the Getah virus (Fig. 1C), but the sum total of bands exceeded 921 bp. Therefore, we thought that all of the amplicons were cleaved into two or more patterns simultaneously by TaqI enzyme (Fig. 3B), and the amplicons were not specific target sequences, but sets of non-specific products of some kinds. We suppose that it is possible to distinguish the viruses of JE serocomplex by comparing the cutting pattern by TaqI enzyme, even when a certain product from the viruses of other serocomplex appears. Fig. 4. Sequence analysis of the one-step RT-PCR products. The RT-PCR amplicons were gel-purified using the QIAquick Gel Purification Kit (QIAGEN). The purified amplicons were sequenced using the BigDye terminator cycle sequencing kit and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Asterisk, one-step RT-PCR primer; bar underneath sequence, nested PCR primer; thick gray bar, TaqI site (TCGA); bold bar underneath sequence, nucleotide substitutions in the FCG strain at positions 3338 (A to G) and 3559 (A to C).

6 444 K. SHIRATO ET AL Table 2. Detection of WN virus and JE virus in experimentally infected mouse tissues using RT-PCR and nested PCR Infecting virus Survival (days) Tissue Sample number RT-PCR Nested PCR RFLP NY a) spleen 3 0/3 3/3 NY99 brain 3 3/3 NY99 WNV-FCG a) spleen 3 0/3 2/3 FCG brain 3 3/3 FCG JaGAr01 9 spleen 1 b) 0/1 1/1 JEV brain 1 b) 1/1 JEV a) The data are represented as the mean survival days standard deviation. b) Since the mortality of JEV-infected mice (s.c.) was very low, we could not obtain sufficient numbers of samples. Table 3. Detection of WN and JE virus in the sera of experimentally infected mice using RT-PCR and nested PCR Infected virus Days p.i. Sample number RT-PCR Nested PCR RFLP NY /3 0/ /3 1/3 NY99 WNV-FCG 3 3 0/3 2/3 FCG 7 3 0/3 1/3 FCG JaGAr /3 0/ /3 0/3 Detection of Viral Genes in Virus-Infected Mice Since Japan is a WN virus-free country, we were unable to obtain clinical field samples. Therefore, to verify that primer set used in this study could detect viral genes in animal tissues, we performed RT-PCR and nested PCR on RNA from tissue samples of mice that were infected experimentally with NY , FCG, or JE virus. We used the s.c. inoculation route to simulate the natural mode of infection. As shown in Tables 2 and 3, viral RNA sequences were detected in the brains of dead mice using the one-step RT-PCR method. We could not detect any viral RNA in mouse sera or spleen by one-step RT-PCR. However, some of these samples were positive according to nested PCR, and showed the expected results in RFLP analysis. Discussion In this report, we describe a diagnostic method for distinguishing WN virus strains from JE virus strains. We successfully detected WN virus and JE virus in experimentally infected animal tissues using one-step RT-PCR and nested PCR, and we were able to discriminate between these viruses using RFLP analysis. We detected WN virus not only in the spleens and brains, but also in the sera of virus-infected animals. In our method, a result does not come out until the RT- PCR products are digested with a restriction enzyme, even if a certain product is seen by RT-PCR or nested PCR. But, WN virus can be certainly classified from other flaviviruses by digesting RT-PCR products with restriction enzyme finally. RT-PCR is precise compared to serological diagnostic methods, such as immunoglobulin M (IgM)-capture enzyme-linked immunoabsorbent assay (ELISA), and RT-PCR results can be obtained more rapidly than the results of virus isolation methods (14). It has been reported that RT-PCR can be used to detect WN virus gene in homogenized tissue samples of virus-infected animals (11, 12) and in the cerebrospinal fluids of human with acute encephalitis (8). Recently, diagnostic techniques with increased sensitivity for WN virus, such as the TaqMan RT-PCR assay (11) and the nucleic acid sequence-based amplification (NASBA) assay (12) have been developed. These techniques are more sensitive than RT-PCR (11, 12). However, the TaqMan RT-PCR assay is very expensive to perform. The cost of the NASBA assay is lower than that of the TaqMan RT- PCR assay (12) and a commercial kit can be easily developed, but amplicon detection in the NASBA assay requires specific techniques and instruments. Although the sensitivity of RT-PCR is inferior to that of NASBA assay, RT-PCR method is performed with ease in many laboratories. In addition, in case of diagnosis of an unknown virus, the sequencing analysis of the RT-PCR amplicons is useful, but in case of diagnosis of an already known virus, RT-PCR RFLP analysis is superior to the sequencing analysis on the points of cost benefit and rapid results. Therefore, we believe that RT-PCR is a useful method for detecting WN virus genes in clinical

7 RT-PCR RFLP FOR WNV AND JEV 445 samples from animals and humans. In summary, our method distinguishes WN virus from the endemic background of JE virus, and discriminates the highly virulent NY99 strain from other WN virus strains. We will develop genetic diagnostic methods that have higher sensitivity and that are simple and inexpensive to perform. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. We are grateful for Dr. H. Hasegawa, Ehime University and Dr. T. Takegami, Kanazawa Medical University for providing Japanese encephalitis virus strains. References 1) Anderson, J.F., Vossbrink, C.R., Andreadis, T.G., Iton, A., Beckwith W.H., III, and Mayo, D.R A phylogenetic approach to following West Nile virus in Connecticut. Proc. Natl. Acad. Sci. U.S.A. 98: ) Chen, W.R., Tesh, R.B., and Hesse, R.R Genetic variation of Japanese encephalitis virus in nature. J. Gen. Virol. 71: ) Garmendia, A.E., Van Kruiningen, H.J., and French, R.A The West Nile virus: its recent emergence in North America. Microbes Infect. 3: ) Giladi, M., Metzkor-Cotter, E., Martin, D.A., Siegman-Igra, Y., Korczyn, A.D., Rosso, R., Berger, S.A., Campbell, G.L., and Lanciotti, R.S West Nile encephalitis in Israel, 1999: the New York connection. Emerg. Infect. Dis. 7: ) Hamman, H.M., Clarke, D.H., and Price, W.H Antigenic variation of West Nile virus in relation to geography. Am. J. Epidemiol. 82: ) Hasegawa, H., Yoshida, M., Fujita, S., and Kobayashi, Y Comparison of structural proteins among antigenically different Japanese encephalitis virus strains. Vaccine 12: ) Hubálek, Z., and Halouzka, J West Nile fever a reemerging mosquito-borne viral disease in Europe. Emerg. Infect. Dis. 5: ) Igarashi, A., Tanaka, M., Morita, K., Takasu, T., Ahmed, A., Ahmed, A., Akram, D.S., and Wagar, M.A Detection of West Nile and Japanese encephalitis viral genome sequences in cerebrospinal fluid from acute encephalitis cases in Karachi, Pakistan. Microbiol. Immunol. 38: ) Jia, X.Y., Briese, T., Jordan, I., Rambaut, A., Chi, H.C., Mackenzie, J.S., Hall, R.A., Scherret, J., and Lipkin, W.L Genetic analysis of West Nile New York 1999 encephalitis. Lancet 354: ) Lanciotti, R.S., Roehrig, J.T., Deubel, V., Smith, J., Parker, M., Steele, K., Crise, B., Volpe, K.E., Crabtree, M.B., Scherret, J.H., Hall, R.A., MacKenzie, J.S., Cropp, C.B., Panigrahy, B., Ostlund, E., Shumitt, B., Malkinson, M., Banet, C., Weissman, J., Komar, N., Savage, H.M., Stone, W., McNamara, T., and Gubler, D.J Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 286: ) Lanciotti, R.S., Kerst, A.J., Nansi, R.S., Godsey, M.S., Mitchell, C.J., Savage, H.M., Komar, N., Panella, N.A., Allen, B.C., Volpe, K.E., Davis, B.S., and Roehrig, J.T Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-pcr assay. J. Clin. Microbiol. 38: ) Lanciotti, R.S., and Kerst, A.J Nucleic acid sequencebased amplification assays for rapid detection of West Nile virus and St. Louis encephalitis virus. J. Clin. Microbiol. 39: ) Martin, D.A., Muth, D.A., Brown, T., Johnson. A.J., Karabatsos, N., and Roehrig, J Standardization of immunoglobulin M capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections. J. Clin. Microbiol. 38: ) Porter, K.R., Summers, P.L., Dubois, D., Puri, B., Nelson, W., Henchal, E., Oprandy, J.J., and Hayes, C.G Detection of West Nile virus by the polymerase chain reaction and analysis of nucleotide sequence variation. Am. J. Trop. Med. Hyg. 48: ) Saito, R., Oshitani, H., Masuda, H., and Suzuki, H Detection of amantadine-resistant influenza A virus strains in nursing homes by PCR-restriction fragment length polymorphism analysis with nasopharyngeal swabs. J. Clin. Microbiol. 40: ) Scherret, J.H., Poidinger, M., Mackenzie, J.S., Broom, A.K., Deubel, V., Lipkin, W.I., Briese, T., Gould, E.A., and Hall, R.A The relationship between West Nile and Kunjin viruses. Emerg. Infect. Dis. 7: ) Siafakas, N., Markoulatos, P., and Stanway, G Variability in molecular typing of coxackie A virus by RFLP analysis and sequencing. J. Clin. Lab. Anal. 16: ) Takashima, I., Morita, K., Chiba, M., Hayasaka, D., Sato, T., Takezawa, C., Igarashi, A., Kariwa, H., Yoshimitsu, K., Akikawa, J., and Hashimoto, N A case of tick-borne encephalitis in Japan and isolation of the virus. J. Clin. Microbiol. 35: ) Takegami, T., Ishak, H., Miyamoto, C., Shirai, Y., and Kamimura, K Isolation and molecular comparison of Japanese encephalitis virus in Ishikawa, Japan. Jpn. J. Infect. Dis. 53: ) Uchil, P.D., and Satchidanandam, V Phylogenetic analysis of Japanese encephalitis virus: envelope gene based analysis reveals a fifth genotype, geographic clustering, and multiple introductions of the virus into the Indian subcontinent. Am. J. Trop. Med. Hyg. 65: