Inactivation of West Nile Virus during Serologic Testing and Transport

Abstract

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...

West Nile virus in plasma is highly sensitive to methylene blue–light treatment

“…The recent spread of WNV is a case in point. A number of investigations have shown that WNV is rather labile and can be inactivated by simple methods (e.g., by lowering the pH and by detergent, pepsin, and heat treatment, respectively 28‐30 ). It was also described that WNV is sensitive to the alkylating agent PEN 110, which is used to inactivate pathogens in RBCs 31 .…”

Inactivation of Hantaan Virus-Containing Samples for Subsequent Investigations outside Biosafety Level 3 Facilities

“…Recently, inactivation of West Nile virus in a detergent-containing buffer has been demonstrated [16] . In our studies, we could verify a reduction in viral infectivity of at least 2 log scales after lysing infected cells with Nonidet P-40™-containing lysis buffer.…”

Experimental Transmission of Mayaro Virus by Aedes aegypti

“…The remaining sera yielded no plaques by 92 hours post-inoculation compared with plaques that developed ~65 hours post-inoculation from stock virus. Because RNA has been shown to be more stable than infectious virus through storage and shipping processes with variable temperature conditions or multiple freeze-thaw cycles, 51,52 we also tested samples by qRT-PCR. To obtain a result relevant to this study, we created a standard curve from plaque assay-quantified viremic mouse serum and expressed quantities of MAYV in test samples in terms of PFU equivalents based on the amount of RNA detected.…”