Specimens from a patient of the 2000 Israel West Nile virus epidemic were analyzed by reverse transcription-polymerase chain reaction. Products corresponding to E, NS3, and NS5 sequences were amplified from cerebellar but not from cortical samples. Phylogenetic analyses indicated a closer relationship of this isolate to 1996 Romanian and 1999 Russian than to 1998-99 Israeli or 1999 New York isolates

Andrew Rambaut

Berthet

Briese

Calisher

Goldblum

Hindiyeh

Hoogstraal

Hubálek

Hurlbut

Jihad Bishara

Jordan

Lanciotti

Lvov

Marberg

McIntosh

Melissa Pathmajeyan

Miller

Miriam Weinberger

Savage

Silvio Pitlik

Taylor

Thomas Briese

W. Ian Lipkin

Emerging Infectious Diseases

English

PubMed

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Phylogenetic Analysis of a Human Isolate from the 2000 Israel West Nile virus Epidemic

Briese, Thomas

Rambaut, Andrew

Pathmajeyan, Melissa

Bishara, Jihad

Weinberger, Miriam

Pitlik, Silvio

Lipkin, W. Ian

eScholarship - University of California

UC IrvineUC Irvine Previously Published WorksTitlePhylogenetic Analysis of a Human Isolate from the 2000 Israel West Nile virus EpidemicPermalinkhttps://escholarship.org/uc/item/3rs870vjJournalEmerging Infectious Diseases, 8(5)ISSN1080-6059AuthorsBriese, ThomasRambaut, AndrewPathmajeyan, Melissaet al.Publication Date2002-05-01 Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaDISPATCHESPhylogeneticAnalysis of a HumanIsolate from the2000 Israel WestNile virus EpidemicThomas Briese,* Andrew Rambaut,† Melissa Pathmajeyan,* Jihad Bishara,‡ Miriam Weinberger,‡ Silvio Pitlik,‡ and W. Ian Lipkin*Specimens from a patient of the 2000 Israel West Nile virus epi-demic were analyzed by reverse transcription-polymerasechain reaction. Products corresponding to E, NS3, and NS5sequences were amplified from cerebellar but not from corticalsamples. Phylogenetic analyses indicated a closer relationshipof this isolate to 1996 Romanian and 1999 Russian than to1998-99 Israeli or 1999 New York isolates.est Nile fever is typically a mild febrile illness charac-terized by headache, myalgias, lymphadenopathy, and amaculopapular rash; West Nile fever occurs sporadicallythroughout endemic areas of northeastern Africa and tropicalAsia (1-5). The causative agent, West Nile virus (WNV), is amember of the genus Flavivirus (family Flaviviridae), whichis grouped together with Alfuy virus, Cacipacore virus, Kou-tango virus, Japanese encephalitis virus, Kunjin virus, MurrayValley encephalitis virus, St. Louis encephalitis virus, Usutuvirus, and Yaounde virus in the Japanese encephalitis virusantigenic complex (6,7). Two different lineages (I and II) ofWNV are characterized genetically (8). Whereas lineage IIviruses are isolated in endemic areas, lineage I viruses are iso-lated during epidemic outbreaks of WNV infection and maycause severe encephalitis in the elderly or immunosuppressedpersons. Epidemics of West Nile fever were first reported inthe 1950s in Israel (2) and the 1970s in South Africa (9). Sitesof notable recent outbreaks include Romania (1996, 1997),Italy (1998), Russia (1999), France (2000), United States(1999, 2000) and Israel (2000) (5,10). Sequence analysis ofthe WNV responsible for the United States outbreak in 1999(WNV-NY1999) showed a close phylogenetic relationship to aWNV isolated from a goose in Israel in 1998 (WNV-ISR1998)(11,12). WNV is transmitted mainly by mosquito vectors, althoughit has also been isolated in several tick species (3,5,13). Birdsare an important WNV reservoir. In several avian species,virus replication generates serum titers sufficient to sustainarthropod transmission (4,5). Birds, during seasonalW*University of California at Irvine, Irvine, California, USA; †University ofOxford, Oxford, UK; and ‡Rabin Medical Center–Beilinson Campus,Petah Tikva, Israel528 Emerging Infectious Diseasmigrations, are also believed to be instrumental in the geo-graphic spread of WNV (3-5,14). The virus is only occasion-ally transmitted to humans or other mammals. Viremia inmammals is low level; thus, mammals are considered to bedead-end hosts.The StudyFrom July through November 2000, a WNV epidemicoccurred in central and northern Israel. More than 430 peoplewere diagnosed with WNV infection; 29 of these patients hadfatal encephalitis. We report phylogenetic analysis of WNVsequences isolated from the brain of an encephalitis patientfrom the 2000 Israel epidemic. A 72-year-old woman with a history of recurrent meningi-oma of the sphenoidal ridge, dementia, and depression washospitalized because of fever and general deterioration of 5days’ duration. On admission, the patient was responsive onlyto painful stimuli and had generalized muscle stiffness andlimb tremors. Clinical and laboratory values were consistentwith viral encephalitis; thus, the patient was initially treatedwith intravenous acyclovir for presumptive herpes simplexencephalitis. When polymerase chain reaction (PCR) analysisof cerebrospinal fluid (CSF) showed no evidence of herpessimplex virus infection, and WNV antibodies were detected inserum and CSF, acyclovir was discontinued and ribavirin wasinitiated at an oral dosage of 2.4 g per day. The patient’s clini-cal status continued to deteriorate with aspiration pneumoniaand intermittent generalized seizures. Intravenous immunoglo-bulin was added (35 g/d for 2 days) without improvement.Approximately one month after onset of illness, the patientdied of respiratory failure. Postmortem examination of the brain showed multiplemeningiomas, generalized atrophy, and surgical resection ofthe right parietal lobe. Histology was remarkable for neu-rofibrillary plaques consistent with Alzheimer’s disease, andscattered microglial nodules and perivascular lymphocyticinflammation in the medulla, pons, and midbrain were consis-tent with viral encephalitis.RNA was extracted from frontal cortex and cerebellumwith TRI-Reagent (Molecular Research Center, Cincinnati,OH). Four micrograms of total RNA from each brain regionwere used as a template for reverse transcription-polymerasechain reaction (RT-PCR) with primer sets representing threeregions of sequence conservation in flavivirus genomes: NS3-1 (EDL/Fla-U5004, 5´- GGA ACD TCM GGH TCN CCH ATand EDL/Fla-L5457, 5´- GTG AAR TGD GCY TCR TCCAT), NS5-1.1 (EDL/Fla-U9093, 5´- AGY MGR GCH ATHTGG TWY ATG TGG and EDL/Fla-L9279, 5´- TCC CAVCCD GCK GTR TCA TC), and NS5-2 (EDL/Fla-U9954, 5´-GSS AAA KCH TAY GCN CAV ATG TGG and EDL/Fla-L10098 5´- AGC ATR TCT TCH GTN GTC ATC CA)(15,16). Amplification products were obtained with RNAderived from the cerebellum in reactions with all three primersets; no amplification products were obtained with RNA fromthe cortex. These amplification products were cloned into thees  •  Vol. 8, No. 5, May 2002DISPATCHESpGEM-Teasy vector (Promega, Madison, WI) and subjected toautomated dideoxy sequencing (ABI Prism Model 377, FosterCity, CA). Sequences were submitted to GenBank (NS3, Gen-Bank accession no. AF394218; NS5, GenBank accession nos.AF394219 and AF394220).Signal of cerebellar amplification products in ethidiumbromide-stained gels was reduced in comparison with similarstudies performed with brain materials from four patients ofthe 1999 New York City outbreak (data not shown; New Yorkpatients were 75 years to 80 years of age, 3 male, 1 female,who died of severe WNV encephalitis during the 1999 out-break [16]). The 5´-nuclease real-time RT-PCR analysis (17)indicated a relative virus load of 140 copies/200 ng RNA inthe Israeli sample, compared with a range of 20 to 7000 cop-ies/200 ng RNA in specimens analyzed from the New YorkCity outbreak (Table). However, since the virus load of thesample from Israel was within the range of virus loadsobserved with the New York samples, this result for a singleIsraeli sample may not indicate a strain difference. Quantita-tive analysis was restricted to the NS5 target because no signalwas obtained with primer/probe set prNS3 (fwd, 5´- GCa CTGAGA GGA CTG CCc AT; probe, 5´-6FAM-TAc CAG ACATCc GCA GTG cCC AGA-T-TAMRA; rev, 5´- TGg GTGAGG GTa GCA TGa CA), because of point mutations in WNV-ISR2000 sequence that prevented efficient hybridization withthe primer and probe oligonucleotides (fwd - 2 mismatches,probe – 3 mismatches, rev - 3 mismatches; given above inlower case). Sensitivity was not substantially reduced in assayswith primer/probe set prNS5, which had one mismatch in the3′-terminal sequence of the probe oligonucleotide (Table). Table. Real Time reverse transcription-polymerase chain reaction (RT-PCmens using primer set NY1999-NS5NS5 standard NY1999 Armored RNAArmored RNA extract NY199Amounta CTb Dil.c Amountd Dil.e Amountd Patient no.2.5x106 16.4 1:101 4.5x106 1:101 4.4x105 12.5x105 20.0 1:102 3.4x105 1:102 4.0x104 22.5x104 23.6 1:103 3.1x104 1:103 4.3x103 32.5x103 27.2 1:104 3.1x103 1:104 6.0x102 52.5x102 31.1 1:105 3.4x102 1:105 9.8x1012.5x101 34.9 1:106 3.1x101 1:10 2.1x1002.5x100 36.8h 1:107 3.0x100 h 1:107 n.di0 >45 0 0 0 0a Plasmid DNA p88-D-21 was quantitated spectrophotometrically, and dilutions containing th(PCR) assay.b CT , Cycle number at which signal crosses threshold.c Armored RNA West Nile virus (HNY1999) standard (Ambion, Austin, TX) was diluted 1:1lent to the indicated dilution of the stock (5 µL).d Amount calculated based on calibration curve obtained with NS5 Standard NY1999 (colume Dilutions of Armored RNA West Nile virus (HNY1999) standard (Ambion) were extracted PCR to result in amounts per assay equivalent to the indicated dilution of the stock (5 µL).f Plasmid DNA pISR-Dfrag-D6 was quantitated spectrophotometrically, and dilutions contaig Amount calculated based on calibration curve obtained with NS5 Standard ISR2000 (columh Poisson effects take place at low template concentration; duplicate assay deviations: 36.2 / i n.d. ,  not determined.Emerging Infectious DiseasSequence analysis of the cloned NS3 and NS5 gene frag-ments indicated similarity to completely sequenced Romanianand Russian WNV isolates WNV-RO97-50-1996 and WNV-RUS-VLG4-1999, respectively; thus, to facilitate detailedphylogenetic analysis, E gene sequence from the Israel humanbrain sample was amplified. An E gene sequence of 1,509nucleotides (GenBank accession number AF394217) wasamplified from total RNA by using GeneChoice UNIPOLpolymerase (PGC Scientific, Gaithersburg, MD) and primersEDL/E-U1006 (5´- GGA GTG TCT GGA GCA ACA TGGGT) or EDL/E-U1476 (5´- TCC TGC GGC GCC TTC AT) andEDL/E-L2244 (5´- CCC CTC CAA CTG ATC CAA AGT CC)or EDL/E-L2538 (5´- TCC ATC CAA GCC TCC ACA TCA),respectively. Sequence analysis of this fragment confirmeddata from NS3 and NS5 sequence analyses, indicating a closerrelationship of WNV-ISR-hISR2000 sequence to Romanianand Russian isolates than to the 1997/98/99 Israeli and theWNV-NY1999 isolates (Figure).The extent to which this WNV genotype contributed tohuman disease in the 2000 epidemic remains to be determined.WNV-ISR-hISR2000 may have been carried into Israel bymigrating birds from reservoirs in southeastern Europe or res-ervoirs in northeastern Africa, where a highly related viruswas isolated in 1998 (WNV-KEN-KN3829-1998) (18). The2000 Israel isolates in birds (and pigs, strains ISR-00GooMaSand ISR-00PigC) were different from the previous Israeli iso-lates (1997/98/99; strains ISR-97Goo1, ISR-98Goo1, ISR-IS98St1, ISR-99Goo, and ISR-99Gull [Figure]), but similar tothe human isolate. Nonetheless, precedent exists for implicat-ing more than one genotypic variant in a WNV outbreak.R) analysis of RNA extracts from 2000 Israel West Nile patient speci-9 specimensNS5 standard ISR2000ISR2000 specimensCT Amountd Amountf CT Sample CT Amountg29.3 6.9x102 2.5x106 16.5 cereb. 31.1 1.4x10225.6 7.3x103 2.5x105 20.0 cortex 36.4h 1.9x10030.0 4.6x102 2.5x104 23.134.8 2.3 x101 2.5x103 26.72.5x102 30.32.5x101 34.12.5x100 37.3 h0 >45e indicated copy number of target sequence were added to each polymerase chain reaction 0, boiled, reverse transcribed, and then diluted to result in amounts per PCR assay equiva-n 1).with TRI-Reagent (Molecular Research Center, Cincinnati, OH) and then subjected to RT-ning the indicated copy number of target sequence were added to each PCR assay.n 5).37.4, NY1999; 6.0 x100 / 0, armored RNA; 37.4 / 37.1, ISR2000; and 36.4 / >45, cortex.es  •  Vol. 8, No. 5, May 2002 529DISPATCHES530 Emerging Infectious DiseasFigure. Phylogenetic analysis of WNV-hISR2000 E gene sequence. Phy-logenetic analysis of the sequences listed below was performed withPAUP (Phylogenetic analysis using parsimony) 4.0b8 (Sinaur Associ-ates, Sunderland, MA). A neighbor-joining tree was constructed usingmaximum likelihood distances with the HKY85 model of substitution andallowing different rates of substitution at each codon position. Bootstrapvalues are the result of 1000 neighbor-joining replicates under this samemodel. Only relevant bootstrap values are shown. WNV-Eg101-1951(human, H), AF260968; WNV-RUS-HP94-1963, AF237565; WNV-RUS-A1628-1967 (bird, B), AF237563; WNV-ISR-TL443-1952 (H), AF205881;WNV-RSA-H442, AF205880; WNV-Palestine-1998, V. Deubelunpub.data; WNV-RO96-1030-1996 (H), AF130363; WNV-FRA-PaH651-1965 (H), AF001560; WNV-ROM96(0334)-1996, AF208579;WNV-RUS-T1304; AF237566; WNV-ISR-99Goo-1999 (B), AY033391;WNV-ISR-99Gull-1999 (B), AY033390; WNV-ISR-97Goo1-1997 (B),AF380663; WNV-USA-NY99eqhs-1999 (equus, E), AF260967; WNV-USA-CT99-2741-1999 (mosquito, M), AF206518; WNV-USA-hNY1999-1999 (H), AF202541; WNV-USA-NY99Flamingo382-99-1999 (B),AF196835; WNV-ISR-IS98ST1-1998 (B), AY033389; WNV-ISR-00Eq1-2000 (E), AF380669; WNV-ISR-98Goo1-1998 (B), AF205882; WNV-ISR-00GooN-2000 (B), AF380665; WNV-RUS-ASTR986-1999 (H),AF237562; WNV-RO97-50-1996 (M), AF260969; WNV-RUS-VLG4-1999 (H), AF317203; WNV-KEN-KN3829-1998 (M), AF146082; WNV-ISR-00GooMaS-2000 (B), AF380667; WNV-ISR-00PigC-2000 (pig, P);WNV-SEN-ArD93548-1993 (M), AF001570; WNV-ISR-hISR2000-2000(H), AF394217; WNV-CAR-HB6343-1989 (H), AF001558; WNV-ALG-ArDjanet-1968 (M), AF001567; WNV-SEN-AnD27875-1979 (primate, P),AF001569; WNV-CAR-ArB310-1967 (M), AF001566; WNV-I.C.-ArA3212-1981 (M), AF001561; KUNV-AUS-MRM61c-1960 (M), D00246;KUNV-AUS-Boort-1984 (E), AF196519; KUNV-AUS-P1553-1994 (M),AF196495; WNV-IND-G2266-1955 (M), AF196525; WNV-IND-804994-1980 (H), AF196526; WNV-IND-G16919-1955, AF205885; WNV-WEN-GLER, M12294; WNV-UGA-B956-1937 (H), AF394221; WNV-SEN-ArD78016-1990 (M), AF001556; WNV-UGA-Ent63134, AF001573;WNV-UGA-MP22-1959 (M), AF001562; WNV-RCA-AnB3507-1972 (B),AF001563; WNV-CAR-HB83P55-1983 (H), AF001557; WNV-RCA-ArB3573-1972 (M), AF001565; WNV-MAD-ArMg956-1986 (M),AF001564; WNV-KEN-Na1047 (M), AF001571; WNV-MAD-ArMg978-1988 (M), and AF001574.During the 1999 outbreak in Volgograd, Russia, two differentgenotypes were isolated: WNV-RUS-ASTR986-1999 (similarto 1997/98/99 Israeli and the WNV-NY1999 isolates, geno-type lineage I subtype 2a) and WNV-VLG22889/WNV-RUS-VLG4-1999 (similar to WNV-ISR-hISR2000, subtype 2b[Figure]) (19). Indeed, even more divergent genotypes wereidentified during the 1996-97 WNV outbreak in Romania(WNV-RO97-50-1996 similar to WNV-ISR-hISR2000, geno-type lineage I subtype 2b; WNV-RO96-1030-1996 and WNV-ROM96(0334)-1996, belonging to a different subtype, subtype1 [Figure]) (20). The fact that no such divergence of genotypesof WNV isolates was observed during the 1999 New York epi-demic (Figure) was interpreted as being compatible with a sin-gle, new introduction of this virus to the Western Hemisphere.While this manuscript was under review, another groupreported WNV sequences from four patients of the 2000 Israeloutbreak: two isolates most closely related to WNV-R097-50-1996 and two identical to the WNV-NY1999 isolates (21).Analysis of additional isolates from the Israel 2000 and otheroutbreaks, including isolates obtained in 2000, 2001, and sub-sequent years in the USA, will be required to establish theextent to which avian migration and viral mutation contributeto the epidemiology of WNV-related disease.This study was supported by the National Institutes of Health(NS-29425).Dr. Briese is associate director of the Emerging Diseases Labora-tory and assistant professor at the University of California, Irvine.  Insummer 2002, he will join the faculty in the Mailman School of Pub-lic Health at Columbia University as an associate professor of Epide-miology.References  1. Goldblum N, Sterk VV, Paderski B. West Nile fever. The clinical featuresof the disease and the isolation of West Nile virus from the blood of ninehuman cases. Am J Hyg 1954;59:89-103.  2. Marberg K, Goldblum N, Sterk VV, Jasinska-Klingberg W, KlingbergMA. The natural history of West Nile fever. I. Clinical observations dur-ing an epidemic in Israel. Am J Hyg 1956;64:259-65.  3. Taylor RM, Work TH, Hurlbut HS, Rizk F. A study of the ecology ofWest Nile virus in Egypt. Am J Trop Med Hyg 1956;5:579-620.  4. Monath TP, Heinz FX. Flaviviruses. In: Fields BN, Knipe DM,  HowleyPM, editors. Virology. Philadelphia: Lippincott-Raven; 1996. p.961-1034.  5. Hubálek Z, Halouzka J. West Nile fever—a reemerging mosquito-borneviral disease in Europe. Emerg Infect Dis 1999;5:643-50.  6. Heinz FX, Collett MS, Purcell RH, Gould EA, Howard CR, Houghton M,et al. Family Flaviviridae. In: Van Regenmortel MH, Fauquet CM, BishopDHL, Carstens E, Estes MK, Lemon S, et al., editors. Virus Taxonomy.7th report of the International Committee for the Taxonomy of Viruses.San Diego: Academic Press; 2000. p.859-78.  7. Calisher CH. Antigenic classification and taxonomy of flaviviruses (fam-ily Flaviviridae) emphasizing a universal system for the taxonomy ofviruses causing tick-borne encephalitis [see comments]. Acta Virol1988;32:469-78.  8. Berthet FX, Zeller HG, Drouet MT, Rauzier J, Digoutte JP, Deubel V.Extensive nucleotide changes and deletions within the envelope glyco-protein gene of Euro-African West Nile viruses. J Gen Virol1997;78:2293-7.es  •  Vol. 8, No. 5, May 2002DISPATCHES  9. McIntosh BM, Jupp PG, Dos Santos I, Meenehan GM. Epidemics of WestNile and Sindbis viruses in South Africa with Culex (Culex) univittatusTheobald as vector. S Afr J Sci 1976;72:295-300.10. Jordan I, Briese T, Fischer N, Lau JY-N, Lipkin WI. Ribavirin inhibitsWest Nile virus replication and cytopathic effect in neural cells. J InfectDis 2000;182:1214-7.11. Jia XY, Briese T, Jordan I, Rambaut A, Chi HC, Mackenzie JS, et al.Genetic analysis of West Nile New York 1999 encephalitis virus. Lancet1999;354:1971-2.12. Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, et al.Origin of the West Nile virus responsible for an outbreak of encephalitisin the northeastern United States. Science 1999;286:2333-7.13. Hurlbut HS. West Nile virus infection in arthropods. Am J Trop Med Hyg1956;5:76-85.14. Hoogstraal H, Kaiser MN, Gaber S, Traylor MA, Guindy E. Ticks(Ixodoidea) on birds migrating from Africa to Europe and Asia. BullWorld Health Organ 1961;24:197-212.15. Lipkin WI, Briese T. West Nile-like virus: PCR primers and protocols.ProMed. October 13, 1999. Accessed at: http://www.promedmail.org,archive number: 19991013.1826.16. Briese T, Jia XY, Huang C, Grady LJ, Lipkin WI. Identification of a Kun-jin/West Nile-like flavivirus in brains of patients with New York encepha-litis. Lancet 1999;354:1261-2.Emerging Infectious Diseas17. Briese T, Glass WG, Lipkin WI. Detection of West Nile virus sequencesin cerebrospinal fluid. Lancet 2000;355:1614-5.18. Miller BR, Nasci RS, Godsey MS, Savage HM, Lutwama JJ, LanciottiRS, et al. First field evidence for natural vertical transmission of WestNile virus in Culex univittatus complex mosquitoes from Rift Valleyprovince, Kenya. Am J Trop Med Hyg 2000;62:240-6.19. Lvov DK, Butenko AM, Gromashevsky VL, Larichev VP, GaidamovichSY, Vyshemirsky OI, et al. Isolation of two strains of West Nile virus dur-ing an outbreak in southern Russia, 1999. Emerg Infect Dis 2000;6:373-6.20. Savage HM, Ceianu C, Nicolescu G, Karabatsos N, Lanciotti R,Vladimirescu A, et al. Entomologic and avian investigations of an epi-demic of West Nile fever in Romania in 1996, with serologic and molecu-lar characterization of a virus isolate from mosquitoes. Am J Trop MedHyg 1999;61:600-11.21. Hindiyeh M, Shulman LM, Mendelson E, Weiss L, Grossman Z, Bin H.Isolation and characterization of West Nile virus from the blood ofviremic patients during the 2000 outbreak in Israel. Emerg Infect Dis2001;7:748–50.Address for correspondence: T. Briese, Emerging Diseases Laboratory,Departments of Neurology, Microbiology and Molecular Genetics, 3107Gillespie Neuroscience Building, University of California at Irvine, Irvine,California 92697-4292, USA; fax: 949-824-1229; e-mail: tbriese@uci.edues  •  Vol. 8, No. 5, May 2002 531

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DISPATCHESPhylogeneticAnalysis of a HumanIsolate from the2000 Israel WestNile virus EpidemicThomas Briese,* Andrew Rambaut,† Melissa Pathmajeyan,* Jihad Bishara,‡ Miriam Weinberger,‡ Silvio Pitlik,‡ and W. Ian Lipkin*Specimens from a patient of the 2000 Israel West Nile virus epi-demic were analyzed by reverse transcription-polymerasechain reaction. Products corresponding to E, NS3, and NS5sequences were amplified from cerebellar but not from corticalsamples. Phylogenetic analyses indicated a closer relationshipof this isolate to 1996 Romanian and 1999 Russian than to1998-99 Israeli or 1999 New York isolates.est Nile fever is typically a mild febrile illness charac-terized by headache, myalgias, lymphadenopathy, and amaculopapular rash; West Nile fever occurs sporadicallythroughout endemic areas of northeastern Africa and tropicalAsia (1-5). The causative agent, West Nile virus (WNV), is amember of the genus Flavivirus (family Flaviviridae), whichis grouped together with Alfuy virus, Cacipacore virus, Kou-tango virus, Japanese encephalitis virus, Kunjin virus, MurrayValley encephalitis virus, St. Louis encephalitis virus, Usutuvirus, and Yaounde virus in the Japanese encephalitis virusantigenic complex (6,7). Two different lineages (I and II) ofWNV are characterized genetically (8). Whereas lineage IIviruses are isolated in endemic areas, lineage I viruses are iso-lated during epidemic outbreaks of WNV infection and maycause severe encephalitis in the elderly or immunosuppressedpersons. Epidemics of West Nile fever were first reported inthe 1950s in Israel (2) and the 1970s in South Africa (9). Sitesof notable recent outbreaks include Romania (1996, 1997),Italy (1998), Russia (1999), France (2000), United States(1999, 2000) and Israel (2000) (5,10). Sequence analysis ofthe WNV responsible for the United States outbreak in 1999(WNV-NY1999) showed a close phylogenetic relationship to aWNV isolated from a goose in Israel in 1998 (WNV-ISR1998)(11,12). WNV is transmitted mainly by mosquito vectors, althoughit has also been isolated in several tick species (3,5,13). Birdsare an important WNV reservoir. In several avian species,virus replication generates serum titers sufficient to sustainarthropod transmission (4,5). Birds, during seasonalW*University of California at Irvine, Irvine, California, USA; †University ofOxford, Oxford, UK; and ‡Rabin Medical Center–Beilinson Campus,Petah Tikva, Israel528 Emerging Infectious Diseasmigrations, are also believed to be instrumental in the geo-graphic spread of WNV (3-5,14). The virus is only occasion-ally transmitted to humans or other mammals. Viremia inmammals is low level; thus, mammals are considered to bedead-end hosts.The StudyFrom July through November 2000, a WNV epidemicoccurred in central and northern Israel. More than 430 peoplewere diagnosed with WNV infection; 29 of these patients hadfatal encephalitis. We report phylogenetic analysis of WNVsequences isolated from the brain of an encephalitis patientfrom the 2000 Israel epidemic. A 72-year-old woman with a history of recurrent meningi-oma of the sphenoidal ridge, dementia, and depression washospitalized because of fever and general deterioration of 5days’ duration. On admission, the patient was responsive onlyto painful stimuli and had generalized muscle stiffness andlimb tremors. Clinical and laboratory values were consistentwith viral encephalitis; thus, the patient was initially treatedwith intravenous acyclovir for presumptive herpes simplexencephalitis. When polymerase chain reaction (PCR) analysisof cerebrospinal fluid (CSF) showed no evidence of herpessimplex virus infection, and WNV antibodies were detected inserum and CSF, acyclovir was discontinued and ribavirin wasinitiated at an oral dosage of 2.4 g per day. The patient’s clini-cal status continued to deteriorate with aspiration pneumoniaand intermittent generalized seizures. Intravenous immunoglo-bulin was added (35 g/d for 2 days) without improvement.Approximately one month after onset of illness, the patientdied of respiratory failure. Postmortem examination of the brain showed multiplemeningiomas, generalized atrophy, and surgical resection ofthe right parietal lobe. Histology was remarkable for neu-rofibrillary plaques consistent with Alzheimer’s disease, andscattered microglial nodules and perivascular lymphocyticinflammation in the medulla, pons, and midbrain were consis-tent with viral encephalitis.RNA was extracted from frontal cortex and cerebellumwith TRI-Reagent (Molecular Research Center, Cincinnati,OH). Four micrograms of total RNA from each brain regionwere used as a template for reverse transcription-polymerasechain reaction (RT-PCR) with primer sets representing threeregions of sequence conservation in flavivirus genomes: NS3-1 (EDL/Fla-U5004, 5´- GGA ACD TCM GGH TCN CCH ATand EDL/Fla-L5457, 5´- GTG AAR TGD GCY TCR TCCAT), NS5-1.1 (EDL/Fla-U9093, 5´- AGY MGR GCH ATHTGG TWY ATG TGG and EDL/Fla-L9279, 5´- TCC CAVCCD GCK GTR TCA TC), and NS5-2 (EDL/Fla-U9954, 5´-GSS AAA KCH TAY GCN CAV ATG TGG and EDL/Fla-L10098 5´- AGC ATR TCT TCH GTN GTC ATC CA)(15,16). Amplification products were obtained with RNAderived from the cerebellum in reactions with all three primersets; no amplification products were obtained with RNA fromthe cortex. These amplification products were cloned into thees  •  Vol. 8, No. 5, May 2002DISPATCHESpGEM-Teasy vector (Promega, Madison, WI) and subjected toautomated dideoxy sequencing (ABI Prism Model 377, FosterCity, CA). Sequences were submitted to GenBank (NS3, Gen-Bank accession no. AF394218; NS5, GenBank accession nos.AF394219 and AF394220).Signal of cerebellar amplification products in ethidiumbromide-stained gels was reduced in comparison with similarstudies performed with brain materials from four patients ofthe 1999 New York City outbreak (data not shown; New Yorkpatients were 75 years to 80 years of age, 3 male, 1 female,who died of severe WNV encephalitis during the 1999 out-break [16]). The 5´-nuclease real-time RT-PCR analysis (17)indicated a relative virus load of 140 copies/200 ng RNA inthe Israeli sample, compared with a range of 20 to 7000 cop-ies/200 ng RNA in specimens analyzed from the New YorkCity outbreak (Table). However, since the virus load of thesample from Israel was within the range of virus loadsobserved with the New York samples, this result for a singleIsraeli sample may not indicate a strain difference. Quantita-tive analysis was restricted to the NS5 target because no signalwas obtained with primer/probe set prNS3 (fwd, 5´- GCa CTGAGA GGA CTG CCc AT; probe, 5´-6FAM-TAc CAG ACATCc GCA GTG cCC AGA-T-TAMRA; rev, 5´- TGg GTGAGG GTa GCA TGa CA), because of point mutations in WNV-ISR2000 sequence that prevented efficient hybridization withthe primer and probe oligonucleotides (fwd - 2 mismatches,probe – 3 mismatches, rev - 3 mismatches; given above inlower case). Sensitivity was not substantially reduced in assayswith primer/probe set prNS5, which had one mismatch in the3′-terminal sequence of the probe oligonucleotide (Table). Table. Real Time reverse transcription-polymerase chain reaction (RT-PCmens using primer set NY1999-NS5NS5 standard NY1999 Armored RNAArmored RNA extract NY199Amounta CTb Dil.c Amountd Dil.e Amountd Patient no.2.5x106 16.4 1:101 4.5x106 1:101 4.4x105 12.5x105 20.0 1:102 3.4x105 1:102 4.0x104 22.5x104 23.6 1:103 3.1x104 1:103 4.3x103 32.5x103 27.2 1:104 3.1x103 1:104 6.0x102 52.5x102 31.1 1:105 3.4x102 1:105 9.8x1012.5x101 34.9 1:106 3.1x101 1:10 2.1x1002.5x100 36.8h 1:107 3.0x100 h 1:107 n.di0 >45 0 0 0 0a Plasmid DNA p88-D-21 was quantitated spectrophotometrically, and dilutions containing th(PCR) assay.b CT , Cycle number at which signal crosses threshold.c Armored RNA West Nile virus (HNY1999) standard (Ambion, Austin, TX) was diluted 1:1lent to the indicated dilution of the stock (5 µL).d Amount calculated based on calibration curve obtained with NS5 Standard NY1999 (colume Dilutions of Armored RNA West Nile virus (HNY1999) standard (Ambion) were extracted PCR to result in amounts per assay equivalent to the indicated dilution of the stock (5 µL).f Plasmid DNA pISR-Dfrag-D6 was quantitated spectrophotometrically, and dilutions contaig Amount calculated based on calibration curve obtained with NS5 Standard ISR2000 (columh Poisson effects take place at low template concentration; duplicate assay deviations: 36.2 / i n.d. ,  not determined.Emerging Infectious DiseasSequence analysis of the cloned NS3 and NS5 gene frag-ments indicated similarity to completely sequenced Romanianand Russian WNV isolates WNV-RO97-50-1996 and WNV-RUS-VLG4-1999, respectively; thus, to facilitate detailedphylogenetic analysis, E gene sequence from the Israel humanbrain sample was amplified. An E gene sequence of 1,509nucleotides (GenBank accession number AF394217) wasamplified from total RNA by using GeneChoice UNIPOLpolymerase (PGC Scientific, Gaithersburg, MD) and primersEDL/E-U1006 (5´- GGA GTG TCT GGA GCA ACA TGGGT) or EDL/E-U1476 (5´- TCC TGC GGC GCC TTC AT) andEDL/E-L2244 (5´- CCC CTC CAA CTG ATC CAA AGT CC)or EDL/E-L2538 (5´- TCC ATC CAA GCC TCC ACA TCA),respectively. Sequence analysis of this fragment confirmeddata from NS3 and NS5 sequence analyses, indicating a closerrelationship of WNV-ISR-hISR2000 sequence to Romanianand Russian isolates than to the 1997/98/99 Israeli and theWNV-NY1999 isolates (Figure).The extent to which this WNV genotype contributed tohuman disease in the 2000 epidemic remains to be determined.WNV-ISR-hISR2000 may have been carried into Israel bymigrating birds from reservoirs in southeastern Europe or res-ervoirs in northeastern Africa, where a highly related viruswas isolated in 1998 (WNV-KEN-KN3829-1998) (18). The2000 Israel isolates in birds (and pigs, strains ISR-00GooMaSand ISR-00PigC) were different from the previous Israeli iso-lates (1997/98/99; strains ISR-97Goo1, ISR-98Goo1, ISR-IS98St1, ISR-99Goo, and ISR-99Gull [Figure]), but similar tothe human isolate. Nonetheless, precedent exists for implicat-ing more than one genotypic variant in a WNV outbreak.R) analysis of RNA extracts from 2000 Israel West Nile patient speci-9 specimensNS5 standard ISR2000ISR2000 specimensCT Amountd Amountf CT Sample CT Amountg29.3 6.9x102 2.5x106 16.5 cereb. 31.1 1.4x10225.6 7.3x103 2.5x105 20.0 cortex 36.4h 1.9x10030.0 4.6x102 2.5x104 23.134.8 2.3 x101 2.5x103 26.72.5x102 30.32.5x101 34.12.5x100 37.3 h0 >45e indicated copy number of target sequence were added to each polymerase chain reaction 0, boiled, reverse transcribed, and then diluted to result in amounts per PCR assay equiva-n 1).with TRI-Reagent (Molecular Research Center, Cincinnati, OH) and then subjected to RT-ning the indicated copy number of target sequence were added to each PCR assay.n 5).37.4, NY1999; 6.0 x100 / 0, armored RNA; 37.4 / 37.1, ISR2000; and 36.4 / >45, cortex.es  •  Vol. 8, No. 5, May 2002 529DISPATCHES530 Emerging Infectious DiseasFigure. Phylogenetic analysis of WNV-hISR2000 E gene sequence. Phy-logenetic analysis of the sequences listed below was performed withPAUP (Phylogenetic analysis using parsimony) 4.0b8 (Sinaur Associ-ates, Sunderland, MA). A neighbor-joining tree was constructed usingmaximum likelihood distances with the HKY85 model of substitution andallowing different rates of substitution at each codon position. Bootstrapvalues are the result of 1000 neighbor-joining replicates under this samemodel. Only relevant bootstrap values are shown. WNV-Eg101-1951(human, H), AF260968; WNV-RUS-HP94-1963, AF237565; WNV-RUS-A1628-1967 (bird, B), AF237563; WNV-ISR-TL443-1952 (H), AF205881;WNV-RSA-H442, AF205880; WNV-Palestine-1998, V. Deubelunpub.data; WNV-RO96-1030-1996 (H), AF130363; WNV-FRA-PaH651-1965 (H), AF001560; WNV-ROM96(0334)-1996, AF208579;WNV-RUS-T1304; AF237566; WNV-ISR-99Goo-1999 (B), AY033391;WNV-ISR-99Gull-1999 (B), AY033390; WNV-ISR-97Goo1-1997 (B),AF380663; WNV-USA-NY99eqhs-1999 (equus, E), AF260967; WNV-USA-CT99-2741-1999 (mosquito, M), AF206518; WNV-USA-hNY1999-1999 (H), AF202541; WNV-USA-NY99Flamingo382-99-1999 (B),AF196835; WNV-ISR-IS98ST1-1998 (B), AY033389; WNV-ISR-00Eq1-2000 (E), AF380669; WNV-ISR-98Goo1-1998 (B), AF205882; WNV-ISR-00GooN-2000 (B), AF380665; WNV-RUS-ASTR986-1999 (H),AF237562; WNV-RO97-50-1996 (M), AF260969; WNV-RUS-VLG4-1999 (H), AF317203; WNV-KEN-KN3829-1998 (M), AF146082; WNV-ISR-00GooMaS-2000 (B), AF380667; WNV-ISR-00PigC-2000 (pig, P);WNV-SEN-ArD93548-1993 (M), AF001570; WNV-ISR-hISR2000-2000(H), AF394217; WNV-CAR-HB6343-1989 (H), AF001558; WNV-ALG-ArDjanet-1968 (M), AF001567; WNV-SEN-AnD27875-1979 (primate, P),AF001569; WNV-CAR-ArB310-1967 (M), AF001566; WNV-I.C.-ArA3212-1981 (M), AF001561; KUNV-AUS-MRM61c-1960 (M), D00246;KUNV-AUS-Boort-1984 (E), AF196519; KUNV-AUS-P1553-1994 (M),AF196495; WNV-IND-G2266-1955 (M), AF196525; WNV-IND-804994-1980 (H), AF196526; WNV-IND-G16919-1955, AF205885; WNV-WEN-GLER, M12294; WNV-UGA-B956-1937 (H), AF394221; WNV-SEN-ArD78016-1990 (M), AF001556; WNV-UGA-Ent63134, AF001573;WNV-UGA-MP22-1959 (M), AF001562; WNV-RCA-AnB3507-1972 (B),AF001563; WNV-CAR-HB83P55-1983 (H), AF001557; WNV-RCA-ArB3573-1972 (M), AF001565; WNV-MAD-ArMg956-1986 (M),AF001564; WNV-KEN-Na1047 (M), AF001571; WNV-MAD-ArMg978-1988 (M), and AF001574.During the 1999 outbreak in Volgograd, Russia, two differentgenotypes were isolated: WNV-RUS-ASTR986-1999 (similarto 1997/98/99 Israeli and the WNV-NY1999 isolates, geno-type lineage I subtype 2a) and WNV-VLG22889/WNV-RUS-VLG4-1999 (similar to WNV-ISR-hISR2000, subtype 2b[Figure]) (19). Indeed, even more divergent genotypes wereidentified during the 1996-97 WNV outbreak in Romania(WNV-RO97-50-1996 similar to WNV-ISR-hISR2000, geno-type lineage I subtype 2b; WNV-RO96-1030-1996 and WNV-ROM96(0334)-1996, belonging to a different subtype, subtype1 [Figure]) (20). The fact that no such divergence of genotypesof WNV isolates was observed during the 1999 New York epi-demic (Figure) was interpreted as being compatible with a sin-gle, new introduction of this virus to the Western Hemisphere.While this manuscript was under review, another groupreported WNV sequences from four patients of the 2000 Israeloutbreak: two isolates most closely related to WNV-R097-50-1996 and two identical to the WNV-NY1999 isolates (21).Analysis of additional isolates from the Israel 2000 and otheroutbreaks, including isolates obtained in 2000, 2001, and sub-sequent years in the USA, will be required to establish theextent to which avian migration and viral mutation contributeto the epidemiology of WNV-related disease.This study was supported by the National Institutes of Health(NS-29425).Dr. Briese is associate director of the Emerging Diseases Labora-tory and assistant professor at the University of California, Irvine.  Insummer 2002, he will join the faculty in the Mailman School of Pub-lic Health at Columbia University as an associate professor of Epide-miology.References  1. Goldblum N, Sterk VV, Paderski B. West Nile fever. The clinical featuresof the disease and the isolation of West Nile virus from the blood of ninehuman cases. Am J Hyg 1954;59:89-103.  2. Marberg K, Goldblum N, Sterk VV, Jasinska-Klingberg W, KlingbergMA. The natural history of West Nile fever. I. Clinical observations dur-ing an epidemic in Israel. Am J Hyg 1956;64:259-65.  3. Taylor RM, Work TH, Hurlbut HS, Rizk F. A study of the ecology ofWest Nile virus in Egypt. Am J Trop Med Hyg 1956;5:579-620.  4. Monath TP, Heinz FX. Flaviviruses. In: Fields BN, Knipe DM,  HowleyPM, editors. Virology. Philadelphia: Lippincott-Raven; 1996. p.961-1034.  5. Hubálek Z, Halouzka J. West Nile fever—a reemerging mosquito-borneviral disease in Europe. Emerg Infect Dis 1999;5:643-50.  6. Heinz FX, Collett MS, Purcell RH, Gould EA, Howard CR, Houghton M,et al. Family Flaviviridae. In: Van Regenmortel MH, Fauquet CM, BishopDHL, Carstens E, Estes MK, Lemon S, et al., editors. Virus Taxonomy.7th report of the International Committee for the Taxonomy of Viruses.San Diego: Academic Press; 2000. p.859-78.  7. Calisher CH. Antigenic classification and taxonomy of flaviviruses (fam-ily Flaviviridae) emphasizing a universal system for the taxonomy ofviruses causing tick-borne encephalitis [see comments]. Acta Virol1988;32:469-78.  8. Berthet FX, Zeller HG, Drouet MT, Rauzier J, Digoutte JP, Deubel V.Extensive nucleotide changes and deletions within the envelope glyco-protein gene of Euro-African West Nile viruses. J Gen Virol1997;78:2293-7.es  •  Vol. 8, No. 5, May 2002DISPATCHES  9. McIntosh BM, Jupp PG, Dos Santos I, Meenehan GM. Epidemics of WestNile and Sindbis viruses in South Africa with Culex (Culex) univittatusTheobald as vector. S Afr J Sci 1976;72:295-300.10. 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Entomologic and avian investigations of an epi-demic of West Nile fever in Romania in 1996, with serologic and molecu-lar characterization of a virus isolate from mosquitoes. Am J Trop MedHyg 1999;61:600-11.21. Hindiyeh M, Shulman LM, Mendelson E, Weiss L, Grossman Z, Bin H.Isolation and characterization of West Nile virus from the blood ofviremic patients during the 2000 outbreak in Israel. Emerg Infect Dis2001;7:748–50.Address for correspondence: T. Briese, Emerging Diseases Laboratory,Departments of Neurology, Microbiology and Molecular Genetics, 3107Gillespie Neuroscience Building, University of California at Irvine, Irvine,California 92697-4292, USA; fax: 949-824-1229; e-mail: tbriese@uci.edues  •  Vol. 8, No. 5, May 2002 531

Phylogenetic analysis of a human isolate from the 2000 Israel West Nile virus epidemic

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Phylogenetic Analysis of a Human Isolate from the 2000 Israel<i>West Nile virus</i>Epidemic

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Extensive nucleotide changes and deletions within the envelope glycoprotein gene of Euro-African West Nile viruses.

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Phylogenetic Analysis of a Human Isolate from the 2000 Israel West Nile virus Epidemic

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