Although 34 of the isolates showed sequences identical to the WN-NY99 isolate, we were able to show geographical-based clusters of mutations. In particular, 26 isolates were characterized by mutation of C to T at position 858. This group apparently originated in Stamford, CT and disseminated to sites located as far as 54 miles from Stamford. Sequences of WN virus isolated from both brain and heart tissues from the same avian host were identical in all 14 tested individual birds, suggesting that the mutations we have documented are real and not caused by culture, RNA extraction, or PCR procedures. We conclude that this portion of the viral genome will enable us to follow the geographical and temporal movement of variant WN virus strains as they adapt to North America. viral evolution ͉ epidemiology

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Inactivation of West Nile virus (WNV) in enzyme-linked immunosorbent assay (ELISA) wash buffer at 37°C was studied, as well as inactivation of WNV in cell culture medium over several days at an ambient temperature (28°C). Aliquots of WNV were removed from the 37°C ELISA wash buffer at 5, 15, 30, and 60 min for the former experiment, while daily aliquots of medium were sampled for the latter experiment. No virus was detected in the wash buffer at 30 and 60 min, while virus was readily detected from cell culture medium over this time. In addition, titers of WNV consistently dropped over a 7-day period at 28°C compared to control suspensions of virus held at 4°C. These observations indicate that WNV is readily inactivated in the presence of detergentcontaining buffers. Furthermore, the viability loss at ambient temperature suggests that WNV is easily inactivated during routine transportation and testing of human body fluids such as serum and cerebrospinal fluid.West Nile virus (WNV), like all flaviviruses, is an enveloped virus with a single-stranded RNA genome. WNV is classified as a biosafety level 3 (BSL3) agent, which, by definition, requires special precautions and physical structures for the containment of the virus in a laboratory setting. The possible presence of WNV in any human serum or cerebrospinal fluid sample raises the issue of inadvertent exposure to this virus during routine laboratory work. Particularly, it has been recommended (2) that during the testing of sera for WNV-specific antibodies, aerosol-producing procedures (e.g., enzyme-linked immunosorbent assay [ELISA] plate rinsing) should be performed in a class 2 biological safety cabinet (BSC2). Although these procedures can also be performed safely inside instruments which provide their own degree of aerosol protection, the operation of an ELISA plate washer in a BSC2 raises serious airflow concerns that may compromise the intended BSL3 safety conditions provided by the BSC2.To determine the safety of serologic testing for WNV antibodies, we tested the effect of the specimen diluent buffer on WNV viability under normal testing conditions (37°C). The specimen diluent buffer (herein referred to as WNV wash buffer) is a phosphate-buffered saline (PBS) solution containing 0.05% Tween 20. The role of Tween detergents in viral inactivation is well documented for other enveloped viruses, and we sought to determine the deleterious effect of Tween 20 on WNV viability. As reference laboratories often receive specimens shipped unrefrigerated or without cold packs, we also conducted a series of experiments to determine the effect of ambient temperatures (28°C) on WNV viability in the absence of detergent.For both experiments, a cell-free supernatant from a WNVinfected Vero cell culture was prepared via centrifugation at 8,000 ϫ g for 5 min at an ambient temperature (28°C). The culture was seeded with a WNV isolate obtained from a crow brain in August 2000. The WNV isolate has been verified by reverse transcriptase PCR and immunofluorescent assay procedure...