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.

Abstract. West Nile fever caused fatal disease in humans, horses, and birds in the northeastern United States during 1999. We studied birds from two wildlife facilities in New York City, New York, that died or were euthanatized and were suspected to have West Nile virus infections. Using standard histologic and ultrastructural methods, virus isolation, immunohistochemistry, in situ hybridization and reverse-transcriptase polymerase chain reaction, we identified West Nile virus as the cause of clinical disease, severe pathologic changes, and death in 27 birds representing eight orders and 14 species. Virus was detected in 23/26 brains (88%), 24/ 25 hearts (96%), 15/18 spleens (83%), 14/20 livers (70%), 20/20 kidneys (100%), 10/13 adrenals (77%), 13/ 14 intestines (93%), 10/12 pancreata (83%), 5/12 lungs (42%), and 4/8 ovaries (50%) by one or more methods. Cellular targets included neurons and glial cells in the brain, spinal cord, and peripheral ganglia; myocardial fibers; macrophages and blood monocytes; renal tubular epithelium; adrenal cortical cells; pancreatic acinar cells and islet cells; intestinal crypt epithelium; oocytes; and fibroblasts and smooth muscle cells. Purkinje cells were especially targeted, except in crows and magpies. Gross hemorrhage of the brain, splenomegaly, meningoencephalitis, and myocarditis were the most prominent lesions. Immunohistochemistry was an efficient and reliable method for identifying infected cases, but the polyclonal antibody cross-reacted with St. Louis encephalitis virus and other flaviviruses. In contrast, the in situ hybridization probe pWNV-E (WN-USAMRIID99) reacted only with West Nile virus. These methods should aid diagnosticians faced with the emergence of West Nile virus in the United States.

The ecology of the strain of West Nile virus (WNV) introduced into the United States in 1999 has similarities to the native flavivirus, St. Louis encephalitis (SLE) virus, but has unique features not observed with SLE virus or with WNV in the old world. The primary route of transmission for most of the arboviruses in North America is by mosquito, and infected native birds usually do not suffer morbidity or mortality. An exception to this pattern is eastern equine encephalitis virus, which has an alternate direct route of transmission among nonnative birds, and some mortality of native bird species occurs. The strain of WNV circulating in the northeastern United States is unique in that it causes significant mortality in exotic and native bird species, especially in the American crow (Corvus brachyrhynchos). Because of the lack of information on the susceptibility and pathogenesis of WNV for this species, experimental studies were conducted at the USGS National Wildlife Health Center. In two separate studies, crows were inoculated with a 1999 New York strain of WNV, and all experimentally infected crows died. In one of the studies, control crows in regular contact with experimentally inoculated crows in the same room but not inoculated with WNV succumbed to infection. The direct transmission between crows was most likely by the oral route. Inoculated crows were viremic before death, and high titers of virus were isolated from a variety of tissues. The significance of the experimental direct transmission among captive crows is unknown.

Abstract. An outbreak of West Nile virus (WNV) in and around New York City during the late summer of 1999 was the cause of extensive mortality among free-ranging birds. Within the Bronx Zoo/Wildlife Conservation Park, viral activity was also observed and produced some morbidity and mortality among specimens in the zoo's bird collection and probably caused morbidity in at least one specimen from the zoo's mammal collection. To determine the extent of the outbreak and attempt to ascertain the temporal appearance of virus within the park, a serologic survey of birds and mammals was performed. The survey showed that 34% of tested birds (125 of 368; 124 species) were positive for antibody to WNV. The virus caused a disease to infection ratio of 22% (27 of 125) among birds with a 70% (19 of 27) case fatality rate. In contrast, only 8% of the mammals (9 of 117; 35 species) possessed antibody to WNV and there was no virus-associated mortality. Testing of banked and fresh sera obtained from both birds and mammals revealed that there was no evidence of WNV circulation before the 1999 outbreak and that birds introduced into the park were not the source of the New York outbreak. West Nile virus RNA was detected in tissues from one bird that died in February 2000, long after the end of the mosquito transmission season. The potential importance of zoologic parks as possible sentinels for emerging diseases is discussed.