Phylogenetic analysis of a human isolate from the 2000 Israel West Nile virus epidemic. (Dispatches).Specimens from a patient of the 2000 Israel West Nile virus West Nile virus, microorganism and the infection resulting from it, which typically produces no symptoms or a flulike condition. The virus is a flavivirus and is related to a number of viruses that cause encephalitis. 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. ********** West Nile fever West Nile fever West Nile meningoencephalitis Infectious disease An acute, mosquito-borne flaviviral infection endemic–rarely, epidemic–in the Near East, Africa, former Soviet Union, India Clinical After a 3-6 day incubation, children present with a is typically a mild febrile illness characterized by headache, myalgias, lymphadenopathy lymphadenopathy /lym·phad·e·nop·a·thy/ (-op´ah-the) disease of the lymph nodes. angioimmunoblastic lymphadenopathy , angioimmunoblastic lymphadenopathy with dysproteinemia , and a maculopapular rash; West Nile fever occurs sporadically throughout endemic areas of northeastern Africa and tropical Asia (1-5). The causative agent, West Nile virus (WNV), is a member of the genus Flavivirus (family Flaviviridae), which is grouped together with Alfuy virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Kunjin virus, Murray Valley encephalitis virus Murray Valley encephalitis virus (MVEV) is a zoonotic flavivirus endemic to northern Australia and Papua New Guinea. It is the causal agent of Murray Valley encephalitis (previously known as Australian encephalitis) and in humans can cause permanent neurological disease or death. , St. Louis encephalitis St. Louis encephalitis see St. Louis encephalitis. virus, Usutu virus, and Yaounde virus in the Japanese encephalitis virus antigenic complex (6,7). Two different lineages (I and II) of WNV are characterized genetically (8). Whereas lineage II viruses are isolated in endemic areas, lineage l viruses are isolated during epidemic outbreaks of WNV infection and may cause severe encephalitis in the elderly or immunosuppressed persons. Epidemics of West Nile fever were first reported in the 1950s in Israel (2) and the 1970s in South Africa (9). Sites of notable recent outbreaks include Romania (1996, 1997), Italy (1998), Russia (1999), France (2000), United States (1999, 2000) and Israel (2000) (5,10). Sequence analysis of the WNV responsible for the United States outbreak in 1999 (WNV-NY 1999) showed a close phylogenetic relationship to a WNV isolated from a goose in Israel in 1998 (WNV-ISR1998) (11,12). WNV is transmitted mainly by mosquito vectors, although it has also been isolated in several tick species (3,5,13). Birds are an important WNV reservoir. In several avian species, virus replication generates serum titers sufficient to sustain arthropod arthropod Any member of the largest phylum, Arthropoda, in the animal kingdom. Arthropoda consists of more than one million known invertebrate species in four subphyla: Uniramia (five classes, including insects), Chelicerata (three classes, including arachnids and horseshoe transmission (4,5). Birds, during seasonal migrations, are also believed to be instrumental in the geographic spread of WNV (3-5,14). The virus is only occasionally transmitted to humans or other mammals. Viremia viremia /vi·re·mia/ (vi-re´me-ah) the presence of viruses in the blood. vi·re·mi·a n. The presence of viruses in the bloodstream. in mammals is low level; thus, mammals are considered to be dead-end hosts. The Study From July through November 2000, a WNV epidemic occurred in central and northern Israel. More than 430 people were diagnosed with WNV infection; 29 of these patients had fatal encephalitis. We report phylogenetic analysis of WNV sequences isolated from the brain of an encephalitis patient from the 2000 Israel epidemic. A 72-year-old woman with a history of recurrent meningioma meningioma /me·nin·gi·o·ma/ (me-nin?je-o´mah) a benign, slow-growing tumor of the meninges, usually next to the dura mater, which may invade the skull or cause hyperostosis, and often causes increased intracranial pressure; it is usually of the sphenoidal sphenoidal pertaining to the sphenoid bone. ridge, dementia, and depression was hospitalized because of fever and general deterioration of 5 days' duration. On admission, the patient was responsive only to painful stimuli and had generalized muscle stiffness and limb tremors. Clinical and laboratory values were consistent with viral encephalitis; thus, the patient was initially treated with intravenous acyclovir for presumptive herpes simplex encephalitis. When polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is (PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) ) analysis of cerebrospinal fluid (CSF Cerebrospinal Fluid (CSF) Analysis Definition Cerebrospinal fluid (CSF) analysis is a laboratory test to examine a sample of the fluid surrounding the brain and spinal cord. ) showed no evidence of herpes simplex virus Herpes simplex virus A virus that can cause fever and blistering on the skin, mucous membranes, or genitalia. Mentioned in: Conjunctivitis herpes simplex virus infection, and WNV antibodies were detected in serum and CSF, acyclovir was discontinued and ribavirin was initiated at an oral dosage of 2.4 g per day. The patient's clinical status continued to deteriorate with aspiration pneumonia and intermittent generalized seizures. Intravenous immunoglobulin was added (35 g/d for 2 days) without improvement. Approximately one month after onset of illness, the patient died of respiratory failure. Postmortem examination of the brain showed multiple meningiomas, generalized atrophy, and surgical resection of the right parietal lobe. Histology was remarkable for neurofibrillary plaques consistent with Alzheimer's disease, and scattered microglial nodules Nodules A small mass of tissue in the form of a protuberance or a knot that is solid and can be detected by touch. Mentioned in: Leprosy and perivascular perivascular /peri·vas·cu·lar/ (-vas´ku-lar) near or around a vessel. perivascular around a vessel. perivascular cellulitis lymphocytic inflammation in the medulla medulla: see brain stem. , pons, and midbrain midbrain: see brain. were consistent with viral encephalitis. RNA RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic was extracted from frontal cortex and cerebellum with TRI-Reagent (Molecular Research Center, Cincinnati, OH). Four micrograms of total RNA from each brain region were used as a template for reverse transcription-polymerase chain reaction (RT-PCR RT-PCR reverse transcriptase-polymerase chain reaction. See PCR1. ) with primer sets representing three regions of sequence conservation in flavivirus genomes: NS3-1 (EDL/Fla-U5004, 5'--GGA ACD (Automatic Call Distributor) A computerized phone system that responds to the caller with a voice menu and connects the call to the appropriate agent. It can also distribute calls equally to agents. TCM (1) (Trellis-Coded Modulation/Viterbi Decoding) A technique that adds forward error correction to a modulation scheme by adding an additional bit to each baud. TCM is used with QAM modulation, for example. GGH TCN CCH AT and EDL/Fla-L5457, 5'--GTG AAR TGD GCY TCR TCR T cell receptor. TCC AT), NS5-1.1 (EDL/Fla-U9093, 5'--AGY MGR GCH ATH TGG TWY ATG ATG antithymocyte globulin. lymphocyte immune globulin (antithymocyte globulin equine, ATG, ATG equine, LIG) Atgam Pharmacologic class: Immunoglobulin Therapeutic class: Immunosuppressant TGG and EDL/Fla-L9279, 5'--TCC CAV CCD CCD in full charge-coupled device Semiconductor device in which the individual semiconductor components are connected so that the electrical charge at the output of one device provides the input to the next device. GCK GTR TCA TCA 1. trichloroacetic acid. 2. tricarboxylic acid cycle (Krebs cycle). TCA Tricyclic antidepressant, see there TC), and NS5-2 (EDL/Fla-U9954, 5'--GSS AAA AAA: see American Automobile Association. (Triple A) A common single-cell battery used in a myriad of electronic devices of all variety. Like its double A (AA) cousin, it provides 1.5 volts of DC power. When used in series, the voltage is multiplied. KCH TAY GCN CAV ATG TGG and EDL/Fla-L10098 5'--AGC ATR ATR Achilles tendon reflex, see Ankle reflex TCT TCH GTN GTN gestational trophoblastic neoplasia. GTC GTC See: Good 'til cancelled order GTC See good-till-canceled order (GTC). ATC CA) (15,16). Amplification products were obtained with RNA derived from the cerebellum in reactions with all three primer sets; no amplification products were obtained with RNA from the cortex. These amplification products were cloned into the pGEM-Teasy vector (Promega, Madison, WI) and subjected to automated dideoxy sequencing (ABI Abi (ā`bī) [short for Abijah], in the Bible, King Hezekiah's mother. (Application Binary Interface) A specification for a specific hardware platform combined with the operating system. Prism Model 377, Foster City, CA). Sequences were submitted to GenBank (NS3, GenBank accession no. AF394218; NS5, GenBank accession nos. AF394219 and AF394220). Signal of cerebellar amplification products in ethidium bromide-stained gels was reduced in comparison with similar studies performed with brain materials from four patients of the 1999 New York City New York City: see New York, city. New York City City (pop., 2000: 8,008,278), southeastern New York, at the mouth of the Hudson River. The largest city in the U.S. outbreak (data not shown; New York patients were 75 years to 80 years of age, 3 male, 1 female, who died of severe WNV encephalitis during the 1999 outbreak [16]). The 5'-nuclease real-time RT-PCR analysis (17) indicated a relative virus load of 140 copies/200 ng RNA in the Israeli sample, compared with a range of 20 to 7000 copies/200 ng RNA in specimens analyzed from the New York City outbreak (Table). However, since the virus load of the sample from Israel was within the range of virus loads observed with the New York samples, this result for a single Israeli sample may not indicate a strain difference. Quantitative analysis was restricted to the NS5 target because no signal was obtained with primer/probe set prNS3 (fwd, 5 f- GCa CTG AGA GGA CTG CCc AT; probe, 5'-6FAM-TAc CAG CAG 1 Chronic atrophic gastritis 2 Coronary angiography, see there ACA ACA - Application Control Architecture TCc GCA GTG cCC AGA-T-TAMRA; rev, 5'--TGg GTG AGG GTa GCA TGa CA), because of point mutations in WNV-ISR2000 sequence that prevented efficient hybridization hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun) 1. crossbreeding; the act or process of producing hybrids. 2. molecular hybridization 3. with the primer and probe oligonucleotides (fwd--2 mismatches, probe--3 mismatches, rev--3 mismatches; given above in lower case). Sensitivity was not substantially reduced in assays with primer/probe set prNS5, which had one mismatch in the 3'-terminal sequence of the probe oligonucleotide (Table). Sequence analysis of the cloned NS3 and NS5 gene fragments indicated similarity to completely sequenced Romanian and Russian WNV isolates WNV-RO97-50-1996 and WNV-RUS-VLG4-1999, respectively; thus, to facilitate detailed phylogenetic analysis, E gene sequence from the Israel human brain sample was amplified. An E gene sequence of 1,509 nucleotides (GenBank accession number AF394217) was amplified from total RNA by using GeneChoice UNIPOL polymerase (PGC Scientific, Gaithersburg, MD) and primers EDL/E-U1006 (5'--GGA GTG TCT GGA GCA ACA TGG GT) or EDL/E-U1476 (5'--TCC TGC GGC GCC TTC AT) and EDL/E-L2244 (5'--CCC CTC CAA Caa See CCC. CTG ATC CAA AGT AGT antiglobulin test. CC) or EDL/E-L2538 (5'--TCC ATC CAA GCC TCC ACA TCA), respectively. Sequence analysis of this fragment confirmed data from NS3 and NS5 sequence analyses, indicating a closer relationship of WNV-ISR-hISR2000 sequence to Romanian and Russian isolates than to the 1997/98/99 Israeli and the WNV-NY 1999 isolates (Figure). [FIGURE OMITTED] The extent to which this WNV genotype contributed to human disease in the 2000 epidemic remains to be determined. WNV-ISR-hISR2000 may have been carried into Israel by migrating birds from reservoirs in southeastern Europe or reservoirs in northeastern Africa, where a highly related virus was isolated in 1998 (WNV-KEN-KN3829-1998) (18). The 2000 Israel isolates in birds (and pigs, strains ISR-00GooMaS and ISR-00PigC) were different from the previous Israeli isolates (1997/98/99; strains ISR-97Gool, ISR-98Gool, ISR-IS98Stl, ISR-99Goo, and ISR-99Gull [Figure]), but similar to the human isolate. Nonetheless, precedent exists for implicating more than one genotypic variant in a WNV outbreak. During the 1999 outbreak in Volgograd, Russia, two different genotypes were isolated: WNV-RUS-ASTR986-1999 (similar to 1997/98/99 Israeli and the WNV-NY1999 isolates, genotype 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 were identified during the 1996-97 WNV outbreak in Romania (WNV-RO97-50-1996 similar to WNV-ISR-hlSR2000, genotype lineage I subtype 2b; WNV-RO96-1030-1996 and WNV-RoM96(0334)-1996, belonging to a different subtype, subtype 1 [Figure]) (20). The fact that no such divergence of genotypes of WNV isolates was observed during the 1999 New York epidemic (Figure) was interpreted as being compatible with a single, new introduction of this virus to the Western Hemisphere. While this manuscript was under review, another group reported WNV sequences from four patients of the 2000 Israel outbreak: two isolates most closely related to WNV-R097-501996 and two identical to the WNV-NY1999 isolates (21). Analysis of additional isolates from the Israel 2000 and other outbreaks, including isolates obtained in 2000, 2001, and subsequent years in the USA, will be required to establish the extent to which avian migration and viral mutation contribute to the epidemiology of WNV-related disease.
Table. Real Time reverse transcription-polymerase chain reaction
(RT-PCR) analysis of RNA extracts from 2000 Israel West Nile patient
specimens using primer set NY1999-NS5
NS5 standard
NY1999 Armored RNA
Amount (a) [C.sub.T] (b) Dil. (c) Amount (d)
2.5x[10.sup.6] 16.4 1:[10.sup.1] 4.5x[10.sup.6]
2.5x[10.sup.5] 20.0 1:[10.sup.2] 3.4x[10.sup.5]
2.5x[10.sup.4] 23.6 1:[10.sup.3] 3.1x[10.sup.4]
2.5x[10.sup.3] 27.2 1:[10.sup.4] 3.1x[10.sup.3]
2.5x[10.sup.2] 31.1 1:[10.sup.5] 3.4x[10.sup.2]
2.5x[10.sup.1] 34.9 1:[10.sup.6] 3.1x[10.sup.1]
2.5x[10.sup.0] 36.8 (h) 1:[10.sup.7] 3.0x[10.sup.0] (h)
0 >45 0 0
Armored RNA
extract NY 1999 specimens
Patient
Dil. (e) Amount (d) no. [C.sub.T] Amount (d)
1:[10.sup.1] 4.4x[10.sup.5] 1 29.3 6.9x[10.sup.2]
1:[10.sup.2] 4.0x[10.sup.4] 2 25.6 7.3x[10.sup.3]
1:[10.sup.3] 4.3x[10.sup.3] 3 30.0 4.6x[10.sup.2]
1:[10.sup.4] 6.0x[10.sup.2] 5 34.8 2.3x[10.sup.1]
1:[10.sup.5] 9.8x[10.sup.1]
1:10 2.1x[10.sup.0]
1:[10.sup.7] n.d (i)
0 0
NS5 standard
ISR2000 ISR2000 specimens
Amount (f) [C.sub.T] Sample [C.sub.T] Amount (g)
2.5x[10.sup.6] 16.5 cereb. 31.1 1.4x[10.sup.2]
2.5x[10.sup.5] 20.0 cortex 36.4 (h) 1.9x[10.sup.0]
2.5x[10.sup.4] 23.1
2.5x[10.sup.3] 26.7
2.5x[10.sup.2] 30.3
2.5x[10.sup.1] 34.1
2.5x[10.sup.0] 37.3 (h)
0 >45
(a) Plasmid DNA p88-D-21 was quantitated spectrophotometrically, and
dilutions containing the indicated copy number of target sequence were
added to each polymerase chain reaction (PCR) assay.
(b) [C.sub.T], Cycle number at which signal crosses threshold.
(c) Armored RNA West Nile virus (HNY 1999) standard (Ambion, Austin,
TX) was diluted 1:10, boiled, reverse transcribed, and then diluted to
result in amounts per PCR assay equivalent to the indicated dilution of
the stock (5 [micro]L).
(d) Amount calculated based on calibration curve obtained with NS5
Standard NY 1999 (column 1).
(e) Dilutions of Armored RNA West Nile virus (HNY1999) standard
(Ambion) were extracted with TRI-Reagent (Molecular Research Center,
Cincinnati, OH) and then subjected to RT-PCR to result in amounts per
assay equivalent to the indicated dilution of the stock (5 [micro]L).
(f) Plasmid DNA pISR-Dfrag-D6 was quantitated spectrophotometrically,
and dilutions containing the indicated copy number of target sequence
were added to each PCR assay.
(g) Amount calculated based on calibration curve obtained with NS5
Standard ISR2000 (column 5).
(h) Poisson effects take place at low template concentration; duplicate
assay deviations: 36.2 / 37.4, NY1999; 6.0 x [10.sup.0] / 0, armored RNA;
37.4 / 37.1, ISR2000; and 36.4 / >45, cortex.
(i) n.d., not determined.
This study was supported by the National Institutes of Health (NS-29425). References (1.) Goldblum N, Sterk VV, Paderski B. West Nile fever. The clinical features of the disease and the isolation of West Nile virus from the blood of nine human cases. Am J Hyg 1954;59:89-103. (2.) Marberg K, Goldblum N, Sterk VV, Jasinska-Klingberg W, Klingberg MA. The natural history of West Nile fever. I. Clinical observations during 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 of West Nile virus in Egypt. Am J Trop Med Hyg 1956;5:579-620. (4.) Monath TP, Heinz FX. Flaviviruses. In: Fields BN, Knipe DM, Howley PM, editors. Virology. Philadelphia: Lippincott-Raven; 1996. p.961-1034. (5.) Hubalek Z, Halouzka J. West Nile fever--a reemerging mosquito-borne viral 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, Bishop DHL, 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 (family Flaviviridae) emphasizing a universal system for the taxonomy of viruses causing tick-borne encephalitis [see comments]. Acta Virol 1988;32:469-78. (8.) Berthet FX, Zeller HG, Drouet MT, Rauzier J, Digoutte JP, Deubel V. Extensive nucleotide changes and deletions within the envelope glycoprotein gene of Euro-African West Nile viruses. J Gen Virol 1997;78:2293-7. (9.) McIntosh BM, Jupp PG, Dos Santos I, Meenehan GM. Epidemics of West Nile and Sindbis viruses in South Africa with Culex Culex /Cu·lex/ (ku´leks) a genus of mosquitoes found throughout the world, many species of which are vectors of disease-producing organisms. Cu·lex n. (Culex) univittatus Theobald as vector. S Aft J Sci 1976;72:295-300. (10.) Jordan I, Briese T, Fischer N, Lau JY-N, Lipkin WI. Ribavirin inhibits West Nile virus replication and cytopathic effect in neural cells. J Infect Dis 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. Lancet 1999;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 encephalitis in the northeastern United States. Science 1999;286:2333-7. (13.) Hurlbut HS. West Nile virus infection in arthropods. Am J Trop Med Hyg 1956;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. Bull World 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 Kunjin/West Nile-like flavivirus in brains of patients with New York encephalitis. Lancet 1999;354:1261-2. (17.) Briese T, Glass WG, Lipkin WI. Detection of West Nile virus sequences in cerebrospinal fluid. Lancet 2000;355:1614-5. (18.) Miller BR, Nasci RS, Godsey MS, Savage HM, Lutwama JJ, Lanciotti RS, et al. First field evidence for natural vertical transmission of West Nile virus in Culex univittatus complex mosquitoes from Rift Valley province Rift Valley Province of Kenya, bordering Uganda, is one of Kenya's seven administrative provinces outside Nairobi. Rift Valley Province is the largest and one of the most economically vibrant provinces in Kenya. , Kenya. Am J Trop Med Hyg 2000;62:240-6. (19.) Lvov DK, Butenko AM, Gromashevsky VL, Larichev VP, Gaidamovich SY, Vyshemirsky OI, et al. Isolation of two strains of West Nile virus during 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 epidemic of West Nile fever in Romania in 1996, with serologic and molecular characterization of a virus isolate from mosquitoes. Am J Trop Med Hyg 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 of viremic patients during the 2000 outbreak in Israel. Emerg Infect Dis 2001;7:748-50. Address for correspondence: T. Briese, Emerging Diseases Laboratory, Departments of Neurology, Microbiology and Molecular Genetics, 3107 Gillespie Neuroscience Building, University of California The University of California has a combined student body of more than 191,000 students, over 1,340,000 living alumni, and a combined systemwide and campus endowment of just over $7.3 billion (8th largest in the United States). at Irvine, Irvine, California 92697-4292, USA; fax: 949-824-1229; e-mail: tbriese@uci.edu Thomas Briese, * Andrew Rambaut, ([dagger]) Melissa Pathmajeyan, * Jihad Bishara, ([double dagger]) Miriam Weinberger, ([double dagger]) Silvio Pitlik, ([double dagger]) and W. Ian Lipkin * * University of California at Irvine, Irvine, California, USA; ([dagger])-University of Oxford, Oxford, UK; and ([double dagger])-Rabin Medical Center-Beilinson Campus, Petah Tikva, Israel |
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