Origin of the West Nile Virus Responsible for an Outbreak of Encephalitis in the Northeastern United States

Author:

Lanciotti R. S.1,Roehrig J. T.1,Deubel V.2,Smith J.3,Parker M.3,Steele K.3,Crise B.3,Volpe K. E.1,Crabtree M. B.1,Scherret J. H.4,Hall R. A.4,MacKenzie J. S.4,Cropp C. B.1,Panigrahy B.5,Ostlund E.5,Schmitt B.5,Malkinson M.6,Banet C.6,Weissman J.6,Komar N.1,Savage H. M.1,Stone W.7,McNamara T.8,Gubler D. J.1

Affiliation:

1. Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80522, USA.

2. Unité des Arbovirus et Virus des Fièvres Hémorragiques, Institut Pasteur–Paris, 75724 Paris Cedex 15, France.

3. Divisions of Virology and Pathology, Virology Division, U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, MD 21701, USA.

4. Department of Microbiology and Parasitology, University of Queensland, Brisbane, Queensland 4072, Australia.

5. National Veterinary Services Laboratories (NVSL), Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Ames, IA 50010, USA.

6. Division of Avian Diseases, Kimron Veterinary Institute, Ministry of Agriculture and Rural Development, Beit-Dagon 50250, Israel.

7. Wildlife Pathology Unit, Division of Fish, Wildlife and Marine Resources, New York State Department of Environmental Conservation, Delmar, NY 12054, USA.

8. Wildlife Conservation Society, Bronx, NY 10460, USA.

Abstract

In late summer 1999, an outbreak of human encephalitis occurred in the northeastern United States that was concurrent with extensive mortality in crows ( Corvus species) as well as the deaths of several exotic birds at a zoological park in the same area. Complete genome sequencing of a flavivirus isolated from the brain of a dead Chilean flamingo ( Phoenicopterus chilensis ), together with partial sequence analysis of envelope glycoprotein (E-glycoprotein) genes amplified from several other species including mosquitoes and two fatal human cases, revealed that West Nile (WN) virus circulated in natural transmission cycles and was responsible for the human disease. Antigenic mapping with E-glycoprotein–specific monoclonal antibodies and E-glycoprotein phylogenetic analysis confirmed these viruses as WN. This North American WN virus was most closely related to a WN virus isolated from a dead goose in Israel in 1998.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

Reference24 articles.

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 and ) and Kunjin (GenBank accession number ). 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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