Inhibition of West Nile Virus Multiplication in Cell Culture by Anti-Parkinsonian Drugs

The diverse physiological actions of dopamine are mediated by at least five distinct G protein-coupled receptor subtypes. Two D1-like receptor subtypes (D1 and D5) couple to the G protein Gs and activate adenylyl cyclase. The other receptor subtypes belong to the D2-like subfamily (D2, D3, and D4) and are prototypic of G protein-coupled receptors that inhibit adenylyl cyclase and activate K+ channels. The genes for the D1 and D5 receptors are intronless, but pseudogenes of the D5 exist. The D2 and D3 receptors vary in certain tissues and species as a result of alternative splicing, and the human D4 receptor gene exhibits extensive polymorphic variation. In the central nervous system, dopamine receptors are widely expressed because they are involved in the control of locomotion, cognition, emotion, and affect as well as neuroendocrine secretion. In the periphery, dopamine receptors are present more prominently in kidney, vasculature, and pituitary, where they affect mainly sodium homeostasis, vascular tone, and hormone secretion. Numerous genetic linkage analysis studies have failed so far to reveal unequivocal evidence for the involvement of one of these receptors in the etiology of various central nervous system disorders. However, targeted deletion of several of these dopamine receptor genes in mice should provide valuable information about their physiological functions.

West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem. However, our understanding of the molecular interaction of such flaviviruses with mammalian host cells is limited. WNV encodes only 10 proteins, implying that it may use many cellular proteins for infection. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway. RNA interference (RNAi) presents a powerful forward genetics approach to dissect virus-host cell interactions. Here we report the identification of 305 host proteins that affect WNV infection, using a human-genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic-reticulum-associated degradation pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides a comprehensive molecular portrait of WNV-human cell interactions that forms a model for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets.

The authors report on the development and application of a rapid TaqMan assay for the detection of West Nile (WN) virus in a variety of human clinical specimens and field-collected specimens. Oligonucleotide primers and FAM- and TAMRA-labeled WN virus-specific probes were designed by using the nucleotide sequence of the New York 1999 WN virus isolate. The TaqMan assay was compared to a traditional reverse transcriptase (RT)-PCR assay and to virus isolation in Vero cells with a large number ( approximately 500) of specimens obtained from humans (serum, cerebrospinal fluid, and brain tissue), field-collected mosquitoes, and avian tissue samples. The TaqMan assay was specific for WN virus and demonstrated a greater sensitivity than the traditional RT-PCR method and correctly identified WN virus in 100% of the culture-positive mosquito pools and 98% of the culture-positive avian tissue samples. The assay should be of utility in the diagnostic laboratory to complement existing human diagnostic testing and as a tool to conduct WN virus surveillance in the United States.