Origin of the West Nile virus responsible for an outbreak of encephalitis in the Northeastern United States

Science

Volumn 286, Issue 5448, 1999, Pages 2333-2337

  Lanciotti, R S ^a   Roehrig, J T ^a   Deubel, V ^a   Smith, J ^a   Parker, M ^a   Steele, K ^a   Crise, B ^a   Volpe, K E ^a   Crabtree, M B ^a   Scherret, J H ^a   Hall, R A ^a   MacKenzie, J S ^a   Cropp, C B ^a   Panigrahy, B ^a   Ostlund, E ^a   Schmitt, B ^a   Malkinson, M ^a   Banet, C ^a   Weissman, J ^a   Komar, N ^a   Savage, H M ^a   Stone, W ^a   McNamara, T ^a   Gubler, D J ^a   more..

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

GLYCOPROTEIN;

AMINO ACID SEQUENCE; ANIMAL TISSUE; ARTICLE; BIRD; ENCEPHALITIS; EPIDEMIC; EPITOPE MAPPING; HUMAN; HUMAN TISSUE; MOSQUITO; NONHUMAN; NUCLEOTIDE SEQUENCE; PHYLOGENY; PRIORITY JOURNAL; UNITED STATES; WEST NILE FLAVIVIRUS;

AMINO ACID SEQUENCE; ANIMALS; ANTIBODIES, MONOCLONAL; ANTIBODIES, VIRAL; BASE SEQUENCE; BIRD DISEASES; BIRDS; DISEASE OUTBREAKS; ENCEPHALITIS VIRUSES, JAPANESE; FLUORESCENT ANTIBODY TECHNIQUE, INDIRECT; GENOME, VIRAL; HUMANS; MOLECULAR SEQUENCE DATA; NEW ENGLAND; NEW YORK CITY; PHYLOGENY; SONGBIRDS; VIRAL ENVELOPE PROTEINS; WEST NILE FEVER; WEST NILE VIRUS;

ANIMALIA; ANSER SP.; AVES; CORVIDAE; CORVUS; FLAVIVIRUS; PHOENICOPTERUS CHILENSIS; WEST NILE VIRUS;

EID: 0033579282     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5448.2333     Document Type: Article

References (24)

1. The following RT-PCR and sequencing protocol was used. RNA was extracted from chicken embryo allantoic fluid with the QIAamp Viral RNA kit (Qiagen); 140 μl of starting material was extracted, according to the manufacturer's protocol, and the RNA was resuspended in a final volume of 100 μl of ribonuclease-free water. DNA templates for sequencing were then generated as follows: The entire RNA genome of WN-NY99 (flamingo 382-99) was converted/copied into six overlapping double-stranded DNA fragments in multiple RT-PCR reactions using the WN-specific primer pairs 109/1442c, 1248/2737c, 2414/5237c, 5119/7990c, 7336/9794c, and 9661/10,489c (where c = complementary). The 3′ end of WN-NY99 virus was amplified by addition of a polyadenylate [poly(A)] tail onto the genome with poly(A) polymerase, followed by a RT-PCR reaction with the WN-specific primer 10,141 in combination with an oligo(dT) anchor primer. The 5′ end of WN-NY99 was amplified with the 5′ RACE System kit (Life Technologies, Gaithersburg, MD) and WN-specific primers 619c and 348c. Sequencing of additional WN isolates in the prM-E region (see text) was accomplished with the use of the primer pairs in the corresponding region of the genome, as described above. All WN-specific RT-PCR and sequencing primers were designed with the use of OLIGO (Molecular Biology Insights Inc., Cascade, CO) and the published sequences of WN (GenBank accession numbers M12294 and M10103) and Kunjin (GenBank accession number D00246). RT-PCR reactions were performed with the TITAN One Tube RT-PCR kit (Boehringer Mannheim) following the manufacturer's protocol. The resulting DNA fragments were purified by electrophoresis on 1% agarose gels; the DNA bands were excised, then isolated using the QIAquick gel extraction kit (Qiagen). Both strands of the purified DNAs were sequenced with the use of the Taq DyeDeoxy Terminator Cycle sequencing kit (Perkin-Elmer/Applied Biosystems) and a total of 51 WN-specific primers spaced about 400 bases apart on the genome (primer sequences are available upon request). Cycle sequencing was performed by combining ∼400 ng of gel-purified DNA (0.2 pmol) with 30 pmol of WN-specific primer and following the manufacturer's protocol.
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24. We thank K. Tsuchiya, A. Kerst, B. Holloway, K. Gottfried, M. Godsey, N. Panella, B. R. Miller, A. H. Hunt, R. S. Nasci, B. J. Biggerstaff, and C. J. Mitchell (CDC) for technical assistance and valuable scientific discussions; S. Murri and M.-T. Drouet (Institut Pasteur), G. Gustafson, J. Pedersen, D. Petersen, K. Moser, and D. Johnson (NVSL), K. Hynes and R. Diana (New York State Department of Environmental Conservation), L. Sileo and K. Converse (National Wildlife Center), A. Ngbokoli and R. Manduca (Wildlife Conservation Society), and D. J. White (New York State Department of Health) for technical assistance; E. Gould (Institute of Virology and Environmental Microbiology, Natural Environment Research Council, UK) for providing WN-specific mAb H5.46; M. Layton, A. Fine, J. Miller, D. Nash, A. Ramon, and I. Poshni (New York City Department of Health) for providing human specimens for testing; K. Reibner (New York City Office of the Chief Medical Examiner) for performing the autopsies; and R. Deavours and A. Mather (CDC) for secretarial and editorial assistance.