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You are here: Home / Publications / Bibliographies and Resource Guides / West Nile Virus Bibliography, 2004 -2007 / Research  Printer Friendly Page
West Nile Virus Bibliography, 2004-2007
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Bai, F., T. Wang, U. Pal, F. Bao, L.H. Gould, and E. Fikrig (2005). Use of RNA interference to prevent lethal murine West Nile virus infection. Journal of Infectious Diseases 191(7): 1148-1154. ISSN: 0022-1899.
Descriptors: mice, molecular genetics, West Nile virus, encephalitis, genetic techniques, virus replication.

Benzaghou, I., I. Bougie, F. Picard Jean, and M. Bisaillon (2006). Energetics of RNA binding by the West Nile virus RNA triphosphatase. FEBS Letters 580(3): 867-877.
Abstract: The West Nile virus (WNV) RNA genome harbors the characteristic methylated cap structure present at the 5' end of eukaryotic mRNAs. In the present study, we report a detailed study of the binding energetics and thermodynamic parameters involved in the interaction between RNA and the WNV RNA triphosphatase, an enzyme involved in the synthesis of the RNA cap structure. Fluorescence spectroscopy assays revealed that the initial interaction between RNA and the enzyme is characterized by a high enthalpy of association and that the minimal RNA binding site of NS3 is 13 nucleotides. In order to provide insight into the relationship between the enzyme structure and RNA binding, we also correlated the effect of RNA binding on protein structure using both circular dichroism and denaturation studies as structural indicators. Our data indicate that the protein undergoes structural modifications upon RNA binding, although the interaction does not significantly modify the stability of the protein.
Descriptors: acid anhydride hydrolases, viral RNA, West Nile virus genetics.

Bogachek, M.V., E.V. Protopopova, and V.A. Ternovoi (2005). Immunochemical Properties of Recombinant Polypeptides Mimicking Domains I and II of West Nile Virus Glycoprotein E. Molecular Biology P.: 710-18.
Descriptors: West Nile virus, immunochemical properties, recombinant polypeptides, glycoprotein E, domains I and II.

Chappell, K.J., M.J. Stoermer, D.P. Fairlie, and P.R. Young (2006). Insights to Substrate Binding and Processing by West Nile Virus NS3 Protease through Combined Modeling, Protease Mutagenesis, and Kinetic Studies. Journal of Biological Chemistry 281(50): 38448-58.
Abstract: West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3, site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection.
Descriptors: West Nile virus, substrate binding, protease, combined modeling, kinetic studies, treatment, infections, pathology, enxyme.

Chappell, K.J., T.A. Nall, M.J. Stoermer, N.X. Fang, J.D.A. Tyndall, D.P. Fairlie, and P.R. Young (2005). Site-directed Mutagenesis and Kinetic Studies of the West Nile Virus NS3 Protease Identify Key Enzyme-Substrate Interactions. Journal of Biological Chemistry. 280(4): 2896-2903. ISSN: 0021-9258.
Abstract: The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ac-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in WNV NS3 protease at S1 (V115A/F, D129A/E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases.
Descriptors: West Nile virus, studies, mutagenesis, kinetic, NS3 protease, enzyme substrate, replication, therapeutic target, protease inhibitors.

Cheeran, M.C., S. Hu, W.S. Sheng, A. Rashid, P.K. Peterson, and J.R. Lokensgard (2005). Differential responses of human brain cells to West Nile virus infection. Journal of Neurovirology 11(6): 512-24.
Abstract: In recent years, West Nile virus (WNV) has emerged as a major cause of encephalitis in the United States. However, the neuropathogenesis of this flavivirus is poorly understood. In the present study, the authors used primary human brain cell cultures to investigate two neuropathogenic features: viral replication and induction of cytokines. Although neurons and astrocytes were found to support productive WNV infection, viral growth was poorly permissive in microglial cells. Compared to neuronal cultures that sustained viral growth for at least 2 weeks, replication peaked in astrocytes by 72 h post infection. In response to viral infection, astrocytes produced chemokines (CXCL10 and CCL5), but none of the cytokines (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, IL-6, interferon alpha or gamma) tested could be detected. Although microglial cells failed to support viral replication, WNV induced production of the proinflammatory cytokines IL-6 and TNF-alpha. Microglial cells also released robust amounts of the chemokines CXCL10 and CCL2, as well as lower levels of CCL5, in response to WNV infection. WNV-induced chemokine and cytokine production by microglia was coupled with activation of mitogen-activated protein kinase (MAPK) intracellular signaling pathways. Inhibition of p38 MAPK decreased chemokine production in response to WNV. Taken together, these findings suggest that microglial cell responses may influence the neuropathogenesis of WNV infection.
Descriptors: brain pathology, cytokines immunology, neurons pathology, West Nile virus, astrocytes pathology, astrocytes virology, brain virology, cells, cultured, Cercopithecus aethiops, chemokines, cxc biosynthesis, chemotaxis immunology, cytokines biosynthesis, neurons virology, vero cells.

Chen, Y.P., Y.J. Yang, X.D. Wu, and Z.L. Wang (2006). [Preparation of monoclonal antibodies against West Nile virus envelope protein domain.]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi; Zhonghua Shiyan He Linchuang Bingduxue Zazhi; Chinese Journal of Experimental and Clinical Virology 20(3): 213-5.
Abstract: BACKGROUND: To prepare monoclonal antibodies against West Nile virus (WNV) envelope protein domain. METHODS: BALB/c mice were immunized with recombinant antigen of West Nile virus envelope protein domain, and the spleen cells of the mice were used to prepare the monoclonal antibodies (McAb) by hybridoma technique. RESULTS: Three hybridoma cell strains secreting McAbs against WNV envelope protein domain, designated as 4F7, 6H3 and 8E4, respectively, were obtained and were identified by indirect enzyme linked immunosorbent assay (ELISA), they belonged to IgG1, IgG1 and Ig2a, respectively. Two epitopes of envelope protein domain were determined, among them, 4F7 and 6H3 were against the same epitope and 8E4 to another one. CONCLUSIONS: The results of indirect ELISA, Western blot and indirect immunofluorescence experiment indicated that these three McAbs were specific for West Nile virus envelope protein domain and did not cross-react with Japanese encephalitis virus and other viruses, so they can be used for specific detection of West Nile virus.
Descriptors: West Nile virus, envelope protein, spleen cells, mice, monoclonal antibodies, western blot, enzyme linked immunosorbent assay, Japanese encephalitis.
Language of Text: Chinese.

Chu, J.J., P.W. Leong, and M.L. Ng (2005). Characterization of plasma membrane-associated proteins from Aedes albopictus mosquito (C6/36) cells that mediate West Nile virus binding and infection. Virology 339(2): 249-60.
Abstract: This study isolated and characterized the West Nile virus (WNV) putative receptor molecule(s) from Aedes albopictus mosquito (C6/36) cells. The binding of WNV to C6/36 cells was saturated with 5000 particles per cell. The entry of WNV into C6/36 cells was strongly inhibited when pretreated with proteinase K and to a lesser extent with sodium periodate. However, pretreatment of C6/36 cells with phospholipases, glycosidases, heparinases and neurimidase had no effect on virus entry. By using virus overlay protein blot assay, WNV was observed to bind to the 140-kDa, 95-kDa, 70-kDa and 55-kDa plasma membrane-associated molecules isolated from C6/36 cells. Murine antibodies generated against the 95-kDa and 70-kDa membrane proteins effectively blocked WNV, Japanese encephalitis virus (JEV) and Dengue virus (DV) serotype 2 infection in C6/36 cells. In addition, the binding of the recombinant-WNV envelope domain III protein to C6/36 cells can be inhibited by the anti-95-kDa and anti-70-kDa membrane protein antibodies. These data strongly supported the possibility that the 95-kDa and 70-kDa plasma membrane-associated proteins are part of a receptor complex for mosquito-borne flaviviruses (WNV, JEV and DV) on mosquito cells.
Descriptors: aedes metabolism, membrane proteins metabolism, West Nile fever metabolism, West Nile virus, aedes cytology, aedes virology, cell line, cell membrane metabolism, culicidae, gene expression, protein binding.

Chu, J.J., R. Rajamanonmani, J. Li, R. Bhuvanakantham, J. Lescar, and M.L. Ng (2005). Inhibition of West Nile virus entry by using a recombinant domain III from the envelope glycoprotein. Journal of General Virology 86(Pt 2): 405-12.
Abstract: The envelope glycoprotein located at the outermost surface of the flavivirus particle mediates entry of virus into host cells. In this study, the involvement of domain III of West Nile virus (WNV-DIII) envelope protein in binding to host cell surface was investigated. WNV-DIII was first expressed as a recombinant protein and purified after a solubilization and refolding procedure. The refolded WNV-DIII protein displays a content of beta-sheets consistent with known homologous structures of other flavivirus envelope DIII, shown by using circular dichroism analysis. Purified recombinant WNV-DIII protein was able to inhibit WNV entry into Vero cells and C6/36 mosquito cells. Recombinant WNV-DIII only partially blocked the entry of dengue-2 (Den 2) virus into Vero cells. However, entry of Den 2 virus into C6/36 was blocked effectively by recombinant WNV-DIII. Murine polyclonal serum produced against recombinant WNV-DIII protein inhibited infection with WNV and to a much lesser extent with Den 2 virus, as demonstrated by plaque neutralization assays. Together these results provided strong evidence that immunoglobulin-like DIII of WNV envelope protein is responsible for binding to receptor on the surface of host cells. The data also suggest that similar attachment molecule(s) or receptor(s) were used by WNV and Den 2 virus for entry into C6/36 mosquito cells.
Descriptors: antigens, viral pharmacology, viral envelope proteins pharmacology, virus replication drug effects, West Nile virus drug effects, amino acid sequence, viral biosynthesis, cell line, Cercopithecus aethiops, culicidae, dengue virus drug effects, immune sera immunology, mice, molecular sequence data, neutralization tests, protein structure, tertiary, recombinant proteins pharmacology, sequence alignment, solubility, vero cells, viral envelope proteins biosynthesis, viral envelope proteins chemistry, West Nile virus.

Chung, K.M., M.K. Liszewski, G. Nybakken, A.E. Davis, R.R. Townsend, D.H. Fremont, J.P. Atkinson, and M.S. Diamond (2006). West Nile virus nonstructural protein NS1 inhibits complement activation by binding the regulatory protein factor H. Proceedings of the National Academy of Sciences of the United States of America 103(50): 19111-6.
Abstract: The complement system, by virtue of its dual effector and priming functions, is a major host defense against pathogens. Flavivirus nonstructural protein (NS)-1 has been speculated to have immune evasion activity, because it is a secreted glycoprotein, binds back to cell surfaces, and accumulates to high levels in the serum of infected patients. Herein, we demonstrate an immunomodulatory function of West Nile virus NS1. Soluble and cell-surface-associated NS1 binds to and recruits the complement regulatory protein factor H, resulting in decreased complement activation in solution and attenuated deposition of C3 fragments and C5b-9 membrane attack complexes on cell surfaces. Accordingly, extracellular NS1 may function to minimize immune system targeting of West Nile virus by decreasing complement recognition of infected cells.
Descriptors: West Nile virus, nonstructural protein, complement activation, protein factor H, host defense, immune system.

Chung, K.M., G.E. Nybakken, and B.S. Thompson (2006). Antibodies against West Nile Virus Nonstructural Protein NS1 Prevent Lethal Infection through Fc g Receptor-Dependent and -Independent Mechanisms. Journal of Virology P.: 1340-51.
Descriptors: West Nile virus, antibodies, nonstructural protein, lethal infection, Fc g receptor, NS1.

Cinatl, J.J., M. Michaelis, C. Fleckenstein, G. Bauer, H. Kabickova, M. Scholz, H.F. Rabenau, and H.W. Doerr (2006). West Nile virus infection induces interferon signalling in human retinal pigment epithelial cells. Investigative Ophthalmology and Visual Science 47(2): 645-51.
Abstract: PURPOSE: In addition to neuroinvasive disease, West Nile virus (WNV) infection is frequently associated with self-limiting chorioretinitis and vitritis. However, the mechanisms of ophthalmic WNV infection are rarely investigated, in part because of the lack of reliable in vitro models. The authors therefore established the first model of ocular WNV infection and investigated interaction of WNV with IFN signal-transduction mechanisms. METHODS: Human retinal pigment epithelial (RPE) cells were infected with WNV strain NY385-99 at a multiplicity of infection of 5. Virus replication was evaluated by virus titers at different times after infection. The susceptibility of RPE cells to WNV infection was confirmed by transmission electron microscopy. IFN-beta expression was assessed by quantitative real-time PCR and by measurements of antiviral activity in cell culture supernatants. IFN signaling was evaluated by phosphorylation of transducer and activator of transcription 1 and 2 (STAT1/2) proteins, with immunoblot analysis. RESULTS: RPE cells appeared to be highly sensitive to WNV infection. Maximum viral titers were found 24 hours after infection, followed by a continuous decline during the course of infection. WNV infection of RPE cells was followed by increased IFN-beta expression associated with IFN signaling and subsequent inhibition of WNV replication. CONCLUSIONS: In this study, the first cell culture model of ophthalmic WNV infection was developed and characterized in RPE cells, and the molecular mechanisms of WNV infection were studied. The data suggest that WNV induces a general antiviral state in RPE cells. This general antiviral state correlates with WNV-induced IFN signaling in retinal cells.
Descriptors: interferon beta biosynthesis, pigment epithelium of eye virology, signal transduction physiology, West Nile virus, blotting, western, cell culture techniques, Cercopithecus aethiops, interferon beta genetics, microscopy, electron, transmission, models, biological, phosphorylation, pigment epithelium of eye metabolism, pigment epithelium of eye ultrastructure, reverse transcriptase polymerase chain reaction, stat1 transcription factor metabolism, stat2 transcription factor metabolism, vero cells, virus replication physiology, West Nile virus ultrastructure.

Cosnier, S., R.E. Ionescu, S. Herrmann, L. Bouffier, M. Demeunynck, and R.S. Marks (2006). Electroenzymatic polypyrrole-intercalator sensor for the determination of West Nile virus cDNA. Analytical Chemistry 78(19): 7054-7.
Abstract: The chemical binding of a redox acridone derivative onto a polypyrrole film functionalized by N-hydroxysuccinimide groups provided an electrode capable of anchoring DNA duplex by simple insertion of the grafted acridone intercalator into the dsDNA solution. This electrode was applied for the detection of a ssDNA derived from a West Nile virus sequence. The latter was thus amperometrically detected after its hybridization in solution with a biotinylated complementary oligonucleotide followed by its anchoring and labeling by a glucose oxidase at 1 pg/mL.
Descriptors: West Nile virus, DNA, determination, virus sequence, hybridization, chemical binding, redox acridone derivative, electrode.

Cruz Pacheco, G., L. Esteva, J.A. Montano Hirose, and C. Vargas (2005). Modelling the dynamics of West Nile Virus. Bulletin of Mathematical Biology 67(6): 1157-72.
Abstract: In this work we formulate and analyze a mathematical model for the transmission of West Nile Virus (WNV) infection between vector (mosquito) and avian population. We find the Basic Reproductive Number R0 in terms of measurable epidemiological and demographic parameters. R0 is the threshold condition that determines the dynamics of WNV infection: if R0< or =1 the disease fades out, and for R0 >1 the disease remains endemic. Using experimental and field data we estimate R0 for several species of birds. Numerical simulations of the temporal course of the infected bird proportion show damped oscillations approaching the endemic value.
Descriptors: bird diseases transmission, bird diseases virology, culex virology, insect vectors virology, models, biological, West Nile fever transmission, West Nile fever, West Nile virus growth and development, bird diseases epidemiology, birds, computer simulation, United States epidemiology, West Nile fever epidemiology, West Nile fever, zoonoses epidemiology, zoonoses virology.

Davis, A., M. Bunning, P. Gordy, N. Panella, B. Blitvich, and R. Bowen (2005). Experimental and natural infection of North American bats with West Nile virus. American Journal of Tropical Medicine and Hygiene 73(2): 467-469. ISSN: 0002-9637.
Abstract: Big brown (Eptesicus fuscus) and Mexican free-tailed (Tadarida brasiliensis) bats were inoculated with the New York 99 strain of West Nile virus to assess their potential to serve as amplifying hosts and determine the clinical effect of infection. Groups of three or four bats were bled at daily intervals between 1 and 6 days after inoculation to determine the pattern of viremia. Beginning 2 days after inoculation, virus was isolated each day from one or more E. fuscus bats, in titers ranging from 10 to 180 plaque-forming units per milliliter of serum. Virus was not isolated from any of the sera collected from T brasiliensis bats. None of the bats from either species showed clinical signs associated with exposure to virus. Sera from an additional 149 bats collected in Louisiana in 2002 during an epizootic of West Nile fever were tested for antibodies to virus, and two were found to be positive. These data suggest that bats from these two widely distributed species are unlikely to serve as amplifying hosts for West Nile virus.
Descriptors: Eptesicus fuscus, Tadarida brasiliensis, transmission of viruses, West Nile virus, experimental and natural infection, USA.

Davis, C.W., H.Y. Nguyen, S.L. Hanna, M.D. Sanchez, R.W. Doms, and T.C. Pierson (2006). West Nile virus discriminates between DC-SIGN and DC-SIGNR for cellular attachment and infection. Journal of Virology 80(3): 1290-1301.
Abstract: The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, cells expressing these attachment factors efficiently capture, and are infected by, a diverse array of appropriately glycosylated pathogens, including dengue virus. In this study, we investigated whether these lectins could enhance cellular infection by West Nile virus (WNV), a mosquito-borne flavivirus related to dengue virus. We discovered that DC-SIGNR promoted WNV infection much more efficiently than did DC-SIGN, particularly when the virus was grown in human cell types. The presence of a single N-linked glycosylation site on either the prM or E glycoprotein of WNV was sufficient to allow DC-SIGNR-mediated infection, demonstrating that uncleaved prM protein present on a flavivirus virion can influence viral tropism under certain circumstances. Preferential utilization of DC-SIGNR was a specific property conferred by the WNV envelope glycoproteins. Chimeras between DC-SIGN and DC-SIGNR demonstrated that the ability of DC-SIGNR to promote WNV infection maps to its carbohydrate recognition domain. WNV virions and subviral particles bound to DC-SIGNR with much greater affinity than DC-SIGN. We believe this is the first report of a pathogen interacting more efficiently with DC-SIGNR than with DC-SIGN. Our results should lead to the discovery of new mechanisms by which these well-studied lectins discriminate among ligands.
Descriptors: cell adhesion molecules, C-type lectins, cell surface receptors, West Nile virus, cell adhesion molecules.

Dhingra, V., Q. Li, A.B. Allison, D.E. Stallknecht, and Z.F. Fu (2005). Proteomic profiling and neurodegeneration in West Nile virus-infected neurons. Journal of Biomedicine and Biotechnology 2005(3): 271-279.
Abstract: West Nile virus, a mosquito-borne flavivirus, is a human, equine, and avian pathogen. High-resolution two-dimensional differential-gel electrophoresis (2D-DIGE) was used to characterize protein expression in primary rat neurons and to examine the proteomic profiling to understand the pathogenesis of West-Nile-associated meningoencephalitis. Three pH ranges, 3-10, 4-7, and 5-6, were used to analyze the protein spots. The proteins are labeled with fluorescent dyes Cy3 and Cy5 before being separated on the basis of charge and size respectively on a two-dimensional platform. About 55 proteins showed altered expression levels. These were then subsequently digested and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis using peptide mass fingerprinting and database searching. These cellular proteins could represent distinct roles during infection related to apoptosis. Our findings show that two-dimensional differential gel electrophoresis combined with mass spectrometry is a powerful approach that permits the identification of proteins whose expression was altered due to West Nile virus infection.
Descriptors: West Nile virus, neurodegeneration, infected neurons, differential gel electrophoresis, pathogenesis, cellular proteins.

Diamond, M.S. (2005). Development of effective therapies against West Nile virus infection. Expert Review of Anti Infective Therapy 3(6): 931-944.
Abstract: Since its entry into North America in 1999, West Nile virus has spread throughout the USA and Canada, and now annually causes a clinical spectrum of human disease ranging from a self-limiting acute febrile illness to potentially lethal encephalitis. Although no therapy is currently approved for use in humans, several strategies are being pursued to develop effective prophylaxis and treatments. This review describes the epidemiology, clinical presentation and pathogenesis of West Nile virus infection, and highlights recent progress towards an effective therapy.
Descriptors: antiviral agents, pharmaceutical trends, West Nile fever, West Nile virus drug effects, literature review.

Diamond, M.S. and R.S. Klein (2006). A genetic basis for human susceptibility to West Nile virus. Trends in Microbiology 14(7): 287-289.
Abstract: West Nile virus (WNV) infects thousands of humans annually and causes a spectrum of disease ranging from an acute febrile illness to lethal encephalitis. A new study suggests a link between CCR5Delta32 (a common mutant allele of the chemokine and HIV receptor CCR5) and fatal WNV infection. The study highlights a possible risk in targeting this receptor for the prevention and/or treatment of infectious diseases.
Descriptors: genetic predisposition to disease, CCR5 receptors genetics, West Nile fever, mice, West Nile virus, risk factors.

Ding, X., X. Wu, T. Duan, M. Siirin, H. Guzman, Z. Yang, R.B. Tesh, and S.Y. Xiao (2005). Nucleotide and amino acid changes in West Nile virus strains exhibiting renal tropism in hamsters. American Journal of Tropical Medicine and Hygiene 73(4): 803-807.
Abstract: Recent studies have shown that West Nile virus (WNV) can induce an asymptomatic persistent infection in the kidneys of experimentally infected hamsters. The chronically infected rodents shed virus in their urine for up to 8 months, despite the disappearance of viremia and the development of high levels of neutralizing antibodies. WNV, like most members of the Japanese encephalitis virus complex (Flavivirus; Flaviviridae), is assumed to be mainly neurotropic; little is known about the genetic basis for its renal tropism. In this study, complete sequence analyses were done to compare four WNV isolates from the urines of persistently infected hamsters with the wild-type parent virus (NY 385-99). Nucleotide changes, ranging from 0.05% to 0.09%, were identified in all of the WNV isolates from urine; most of the changes were in coding regions, causing amino acid substitutions in the E, NS1, NS2B, and NS5 proteins. The genetic changes associated with renal tropism were also accompanied by a loss of virulence for hamsters and a change in plaque morphology.
Descriptors: amino acid substitution, kidney, West Nile fever, West Nile virus, chronic disease, animal disease models, kidney pathology, molecular sequence data, urine virology, virulence.

Diniz, J.A., A.P. Da Rosa, H. Guzman, F. Xu, S.Y. Xiao, V.L. Popov, P.F. Vasconcelos, and R.B. Tesh (2006). West Nile virus infection of primary mouse neuronal and neuroglial cells: The role of astrocytes in chronic infection. American Journal of Tropical Medicine and Hygiene 75(4): 691-696.
Abstract: Primary cultures of embryonic murine neurons and newborn mouse astrocytes were inoculated with West Nile virus (WNV) strain NY385-99 to compare the pathogenesis of WNV infection in these types of CNS cells. Two different outcomes were observed. WNV infection in the neurons was rapidly progressive and destructive; within 5 days, all of the neurons were destroyed through apoptosis. WNV infection in the astrocytes evolved more slowly and did not seem to be highly lethal to the cells. The infected astrocytes continued to produce infectious virus (10(4.6)-10(6.5) PFU/mL) for 114 days, in a permissive, persistent infection. During this period, WNV antigen could be shown in the cytoplasm of the infected astrocytes by immunocytochemical assay, transmission electron microscopy of ultrathin sections, and in the cell culture medium by complement fixation test. Our results with this in vitro experimental murine cell model indicate that astrocytes can develop chronic or persistent infection with WNV, suggesting that these cells may play a role in the maintenance of WNV in the CNS.
Descriptors: newborn mouse astrocytes, embryonic murine neurons, West Nile virus, complement fixation tests, cytopathogenic effect, viral hemagglutination tests, in situ nick end labeling, mice, virus replication.

Elghonemy, S., W.G. Davis, and M.A. Brinton (2005). The majority of the nucleotides in the top loop of the genomic 3 terminal stem loop structure are cis-acting in a West Nile virus infectious clone. Virology P.: 238-46.
Descriptors: West Nile virus, infectious clone, nucleotides, genomic 3, terminal loop stem structure.

Faraj, C., M. Elkohli, and M. Lyagoubi (2006). The gonotrophic cycle of Culex pipiens (Diptera: Culicidae), West Nile virus potential vector, in Morocco: Evaluation of its duration in laboratory. [Cycle gonotrophique de Culex pipiens (Diptera: culicidae), vecteur potentiel du virus West Nile, au Maroc: Estimation de la duree en laboratoire]. Bulletin De La Societe De Pathologie Exotique 99(2): 119-121.
Abstract: Culex pipiens gonotrophic cycle has been studied, for the first time in Morocco, under experimental conditions. Its average duration was estimated at 5.54 +/- 1.73 days.
Descriptors: culex growth and development, insect vectors, West Nile virus, Morocco.
Language of Text: French.

Fayzulin, R., F. Scholle, O. Petrakova, I. Frolov, and P.W. Mason (2006). Evaluation of replicative capacity and genetic stability of West Nile virus replicons using highly efficient packaging cell lines. Virology 351(1): 196-209. ISSN: 0042-6822.
Abstract: A stable cell system for high-efficiency packaging of West Nile virus (WNV) subgenomic replicons into virus-like particles (VLPs) was developed. VLPs could be propagated on these packaging cells and produced infectious foci similar to foci produced by WNV. Focus size correlated with the replicative capacity of WNV replicons, indicating that genome copy number, rather than amount of trans-complementing structural proteins, was rate-limiting in packaging of VLPs. Comparison of VLP production from replicon genomes encoding partial or complete C genes indicated that portions of C downstream of the cyclization sequence could improve genome replication or that cis expression of C could enhance packaging. Interestingly, a rapid loss of replicon-encoded reporter gene activity was detected within two serial passages of reporter gene-containing VLPs. The loss of reporter activity correlated with gene deletion and better VLP growth, indicating a powerful selection pressure for WNV genomes lacking reporter genes.
Descriptors: variation genetics, virus assembly, virus replication physiology, West Nile virus, Cercopithecus aethiops, cricetinae, vero cells.

Formosinho, P. and M.M. Santos Silva (2006). Experimental infection of Hyalomma marginatum ticks with West Nile virus. Acta Virologica 50(3): 175-180.
Abstract: To define the possible role of Hyalomma marginatum ticks in the transmission of West Nile virus (WNV) in Portugal an experimental infection was established. Ticks were fed on viremic rabbits previously infected with WNV. In different developmental stage of H. marginatum virus isolation and detection of viral antigen and viral RNA were attempted. The oral infection rates were 3%, 33% and 75% for engorged larvae, nymphs and females after oviposition, respectively. Transstadial transmission rates for nymphs exposed to virus as larvae, for adults exposed as larvae, and for adults exposed as nymphs were 33%, 11% and 46%, respectively. No evidence of transovarial transmission was obtained. Ticks in the stages of nymphs and adults were able to transmit the infection to uninfected hosts. This study demonstrated that H. marginatum could be involved in the natural circulation of WNV in Portugal.
Descriptors: West Nile virus, ticks, experimental infection, vector, Hyalomma marginatum, viremic rabbits, infection rates, transviral transmission.

Fredericksen, B.L. and M.J. Gale (2006). West Nile virus evades activation of interferon regulatory factor 3 through RIG-I-dependent and -independent pathways without antagonizing host defense signaling. Journal of Virology 80(6): 2913-2923. ISSN: 0022-538X.
Abstract: The ability of viruses to control and/or evade the host antiviral response is critical to the establishment of a productive infection. We have previously shown that West Nile virus NY (WNV-NY) delays activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical to the initiation of the antiviral response. Here we demonstrate that the delayed activation of IRF-3 is essential for WNV-NY to achieve maximum virus production. Furthermore, WNV-NY utilizes a unique mechanism to control activation of IRF-3. In contrast to many other viruses that impose a nonspecific block to the IRF-3 pathway, WNV-NY eludes detection by the host cell at early times postinfection. To better understand this process, we assessed the role of the pathogen recognition receptor (PRR) retinoic acid-inducible gene I (RIG-I) in sensing WNV-NY infection. RIG-I null mouse embryo fibroblasts (MEFs) retained the ability to respond to WNV-NY infection; however, the onset of the host response was delayed compared to wild-type (WT) MEFs. This suggests that RIG-I is involved in initially sensing WNV-NY infection, while other PRRs sustain and/or amplify the host response later in infection. The delayed initiation of the host response correlated with an increase in WNV-NY replication in RIG-I null MEFs compared to WT MEFs. Our data suggest that activation of the host response by RIG-I early in infection is important for controlling replication of WNV-NY. Furthermore, pathogenic strains of WNV may have evolved to circumvent stimulation of the host response until after replication is well under way.
Descriptors: interferon regulatory factor 3 antagonists and inhibitors, trans activation genetics, trans activators metabolism, West Nile fever immunology, West Nile virus, cell line, interferon regulatory factor 3 metabolism, mice, plaque assay, virus replication.

Garcia Tapia, D., C.M. Loiacono, and S.B. Kleiboeker (2006). Replication of West Nile virus in equine peripheral blood mononuclear cells. Veterinary Immunology and Immunopathology 110(3-4): 229-244.
Abstract: A cell model of primary monocytes and other mononuclear cells isolated from equine blood was used to study the kinetics of West Nile virus (WNV) replication in a natural host. West Nile virus has emerged on the North American continent as a significant cause of morbidity and mortality in a wide range of avian and mammalian species. While other flaviviruses are known to infect monocytes and lymphocytes, the ability of WNV to productively replicate in specific immune cells of peripheral blood has not been assessed. In this study, enriched populations of monocytes and lymphocytes as well as purified monocytes, CD4+, CD8+ and B lymphocytes were obtained from equine blood. Productive WNV replication was demonstrated by viral growth curves, quantitative RT-PCR for WNV RNA, and indirect immunofluorescence detection of a non-structural WNV protein. Enriched and purified monocytes consistently supported productive viral replication in blood from nine of nine horses tested while a minor subset of CD4+ lymphocytes supported productive replication in cells from three of the nine horses tested. Peak viral titers of 3.2-6.6 log10 PFU/ml were reached at 6 days post-inoculation (p.i.) and titers were maintained through 10-15 days p.i. Activation of monocytes with bacterial lipopolysaccharide, which resulted in activation of nuclear transcription factor kappaB (NF-kappaB) plus elevation of nitric oxide and type I interferon levels, reduced or eliminated WNV replication. These results suggest that immune cells of the peripheral blood may serve as target cells for initial replication of WNV and may play a role in subsequent viral dissemination. Furthermore, primary equine immune cell cultures represent a potentially useful model of a natural WNV host when testing compounds such as antivirals for use in WNV treatment.
Descriptors: horses, mononuclear leukocytes, virus replication, West Nile virus, antigens, CD metabolism, cell adhesion, interferon type I metabolism, mononuclear metabolism, lipopolysaccharides, primary equine immune cell cultures, model of natural WNV host.

Glass, W.G., J.K. Lim, R. Cholera, A.G. Pletnev, J.L. Gao, and P.M. Murphy (2005). Chemokine receptor CCR5 promotes leukocyte trafficking to the brain and survival in West Nile virus infection. Journal of Experimental Medicine 202(8): 1087-1098.
Abstract: The molecular immunopathogenesis of West Nile virus (WNV) infection is poorly understood. Here, we characterize a mouse model for WNV using a subcutaneous route of infection and delineate leukocyte subsets and immunoregulatory factors present in the brains of infected mice. Central nervous system (CNS) expression of the chemokine receptor CCR5 and its ligand CCL5 was prominently up-regulated by WNV, and this was associated with CNS infiltration of CD4+ and CD8+ T cells, NK1.1+ cells and macrophages expressing the receptor. The significance of CCR5 in pathogenesis was established by mortality studies in which infection of CCR5-/- mice was rapidly and uniformly fatal. In the brain, WNV-infected CCR5-/- mice had increased viral burden but markedly reduced NK1.1+ cells, macrophages, and CD4+ and CD8+ T cells compared with WNV-infected CCR5+/+ mice. Adoptive transfer of splenocytes from WNV-infected CCR5+/+ mice into infected CCR5-/- mice increased leukocyte accumulation in the CNS compared with transfer of splenocytes from infected CCR5-/- mice into infected CCR5-/- mice, and increased survival to 60%, the same as in infected CCR5+/+ control mice. We conclude that CCR5 is a critical antiviral and survival determinant in WNV infection of mice that acts by regulating trafficking of leukocytes to the infected brain.
Descriptors: brain metabolism, cell movement, mononuclear leukocytes, CCR5 metabolism, West Nile fever, West Nile virus, adoptive transfer, brain immunology and pathology, cytokines, DNA primers, enzyme linked immunosorbent assay (ELISA), flow cytometry, knockout mice, reverse transcriptase polymerase chain reaction.

Glass, W.G., J.K. Lim, D.H. Mcdermott, A. Pletnev, R. Cholera, J. Gao, S. Lekhong, S.F. Yu, W.A. Frank, J. Pape, R.C. Cheshier, and P.M. Murphy (2006). CCR5 saves lives: The protective role of CCR5 during West Nile virus infection. Journal of Neurochemistry 96(Suppl. 1): 94. ISSN: 0022-3042.
Descriptors: West Nile virus infection, CCR5 protective role, immune system, blood lymphatics, susceptibility, immunopathogenic mechanism.
Notes: Meeting Information: Annual Meeting of the American Society for Neurochemistry, Portland, Oregon, USA; March 11 -15, 2006.

Glass, W.G., D.H. McDermott, J.K. Lim, S. Lekhong, S.F. Yu, W.A. Frank, J. Pape, R.C. Cheshier, and P.M. Murphy (2006). CCR5 deficiency increases risk of symptomatic West Nile virus infection. Journal of Experimental Medicine 203(1): 35-40.
Abstract: West Nile virus (WNV) is a reemerging pathogen that causes fatal encephalitis in several species, including mouse and human. Recently, we showed that the chemokine receptor CCR5 is critical for survival of mice infected with WNV, acting at the level of leukocyte trafficking to the brain. To test whether this receptor is also protective in man, we determined the frequency of CCR5Delta32, a defective CCR5 allele found predominantly in Caucasians, in two independent cohorts of patients, one from Arizona and the other from Colorado, who had laboratory-confirmed, symptomatic WNV infection. The distribution of CCR5Delta32 in a control population of healthy United States Caucasian random blood donors was in Hardy-Weinberg equilibrium and CCR5Delta32 homozygotes represented 1.0% of the total group (n = 1,318). In contrast, CCR5Delta32 homozygotes represented 4.2% of Caucasians in the Arizona cohort (odds ratios [OR] = 4.4 [95% confidence interval [CI], 1.6-11.8], P = 0.0013) and 8.3% of Caucasians in the Colorado cohort (OR = 9.1 [95% CI, 3.4-24.8], P < 0.0001). CCR5Delta32 homozygosity was significantly associated with fatal outcome in the Arizona cohort (OR = 13.2 [95% CI, 1.9-89.9], P = 0.03). We conclude that CCR5 mediates resistance to symptomatic WNV infection. Because CCR5 is also the major HIV coreceptor, these findings have important implications for the safety of CCR5-blocking agents under development for HIV/AIDS.
Descriptors: genetic predisposition to disease, CCR5 receptors deficiency, West Nile fever, homozygote, odds ratio.

Goodell, J.R., F. Puig Basagoiti, B.M. Forshey, P.Y. Shi, and D.M. Ferguson (2006). Identification of compounds with anti-West Nile Virus activity. Journal of Medicinal Chemistry 49(6): 2127-2137. ISSN: 0022-2623.
Abstract: The lack of antiviral compounds targeting flaviviruses represents a significant problem in the development of strategies for treating West Nile Virus (WNV), Dengue, and Yellow Fever infections. Using WNV high-throughput screening techniques developed in our laboratories, we report the identification of several small molecule anti-WNV compounds belonging to four different structural classes including pyrazolines, xanthanes, acridines, and quinolines. The initial set of "hits" was further refined using cell viability-cytotoxicity assays to two 1,3,5-triaryl pyrazoline compounds: 1-(4-chlorophenylacetyl)-5-(4-nitrophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole and 1-benzoyl-5-(4-chlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole. On the basis of their activity and favorable therapeutic indexes, these compounds were identified as viable leads and subjected to additional evaluation using an authentic viral titer reduction assay employing an epidemic strain of WNV. The compounds were further evaluated in a transient replicon reporting system to gain insight into the mechanism of action by identifying the step at which inhibition takes place during viral replication. The results indicate the pyrazolines inhibit RNA synthesis, pointing to viral RNA polymerase, RNA helicase, or other viral replication enzymes as potential targets. Progress was also made in understanding the structural requirements for activity by synthesizing a focused chemical library of substituted pyrazolines. Preliminary SAR data are presented that show the aryl-rings are required for activity against WNV. More importantly, the results indicate WNV activity is tolerant to aryl-substitutions paving the way for the design and development of much larger combinatorial libraries with varied physicochemical properties.
Descriptors: acridines, chemical synthesis, antiviral agents, pyrazoles, quinolines, West Nile virus, xanthenes, virus replication.

Gould, L.H., J. Sui, H. Foellmer, T. Oliphant, T. Wang, M. Ledizet, A. Murakami, K. Noonan, C. Lambeth, K. Kar, J.F. Anderson, A.M. de Silva, M.S. Diamond, R.A. Koski, W.A. Marasco, and E. Fikrig (2005). Protective and therapeutic capacity of human single-chain Fv-Fc fusion proteins against West Nile virus. Journal of Virology 79(23): 14606-14613. ISSN: 0022-538X.
Abstract: West Nile virus has spread rapidly across the United States, and there is currently no approved human vaccine or therapy to prevent or treat disease. Passive immunization with antibodies against the envelope protein represents a promising means to provide short-term prophylaxis and treatment for West Nile virus infection. In this study, we identified a panel of 11 unique human single-chain variable region antibody fragments (scFvs) that bind the envelope protein of West Nile virus. Selected scFvs were converted to Fc fusion proteins (scFv-Fcs) and were tested in mice for their ability to prevent lethal West Nile virus infection. Five of these scFv-Fcs, 11, 15, 71, 85, and 95, protected 100% of mice from death when given prior to infection with virus. Two of them, 11 and 15, protected 80% of mice when given at days 1 and 4 after infection. In addition, four of the scFv-Fcs cross-neutralized dengue virus, serotype 2. Binding assays using yeast surface display demonstrated that all of our scFvs bind to sites within domains I and II of West Nile virus envelope protein. These recombinant human scFvs are potential candidates for immunoprophylaxis and therapy of flavivirus infections.
Descriptors: viral antibodies, viral vaccines , West Nile fever prevention and control, West Nile virus, antibody specificity, immunoglobulin fragments, recombinant fusion proteins.

Gu, B., S. Ouzunov, L. Wang, P. Mason, N. Bourne, A. Cuconati, and T.M. Block (2006). Discovery of small molecule inhibitors of West Nile virus using a high-throughput sub-genomic replicon screen. Antiviral Research 70(2): 39-50.
Abstract: West Nile virus (WNV) is a positive-sense, single-stranded RNA virus of the family Flaviviridae. WNV persistently infects insect cells, but can causes acute cytopathic infection of mammalian cells and is an etiologic agent of viral encephalitis in humans. By using a cell line expressing a WNV subgenomic replicon [Rossi, S.L., Zhao, Q., O'Donnell, V.K., Mason, P.W., 2005. Adaptation of West Nile virus replicons to cells in culture and use of replicon-bearing cells to probe antiviral action. Virology 331 (2), 457-470], we developed a high-throughput assay and used it to screen a library of small molecule compounds for inhibitors of WNV replication in the absence of live virus. Here we report the identification of novel small molecule inhibitors for WNV replicon replication. We demonstrate that the compounds inhibited WNV replication-dependent luciferase expression in the replicon cells and reduced WNV viral protein accumulation and viral RNA copy number in the replicon cells. Two classes of compounds with multiple hits, parazolotrahydrothophenes and pyrozolopyrimidines, showed preliminary structure-activity relationships. In WNV infection assays, one pyrozolopyrimidine compound was confirmed to have antiviral activity. These compounds should be valuable for developing anti-WNV therapeutic drugs as well as research tools to study the mechanism of WNV replication.
Descriptors: antiviral agents, West Nile virus, WNV replication, preclinical methods, viral genetics, luciferases, replicon drug effects.

Gu, B., P. Mason, L. Wang, N. Bourne, S. Rossi, S. Ouzounov, A. Cuconati, A. Mehta, and T. Block (2005). Discovery of West Nile virus inhibitors. Antiviral Research 65(3): A68. ISSN: 0166-3542.
Descriptors: West Nile virus, viral disease inhibitors, drug therapy, cell based replicon assay.
Notes: Meeting Information: 18th International Conference on Antiviral Research, Barcelona, Spain; April 11 -14, 2005.

Guo, J.T., J. Hayashi, and C. Seeger (2005). West Nile Virus Inhibits the Signal Transduction Pathway of Alpha Interferon. Journal of Virology P.: 1343-50.
Descriptors: West Nile virus, signal transduction pathway, alpha interferon, inhibits.

Hahn, D.C., N.M. Nemeth, E. Edwards, P.R. Bright, and N. Komar (2006). Passive West Nile virus antibody transfer from maternal Eastern screech-owls (Megascops asio) to progeny. Avian Diseases 50(3): 454-455. ISSN: 0005-2086.
Abstract: Transovarial antibody transfer in owls has not been demonstrated for West Nile virus (WNV). We sampled chicks from captive adult WNV-antibody-positive Eastern Screech-Owls (Megascops asio) to evaluate the prevalence of transovarial maternal antibody transfer, as well as titers and duration of maternal antibodies. Twenty-four owlets aged 1 to 27 days old circulated detectable antibodies with neutralizing antibody titers ranging from 20 to 1600 (median 1:40). Demonstrating that WNV antibodies are passively transferred transovarially is important for accurate interpretation of serologic data from young birds.
Descriptors: viral blood antibodies, bird diseases, maternally acquired immunity, West Nile virus, screech owls.

Higgs, S., B. Schneider, D. Vanlandingham, K. Klingler, and E. Gould (2005). Nonviremic transmission of West Nile virus. Proceedings of the National Academy of Sciences of the United States of America. 102(25): 8871-8874. ISSN: 0027-8424.
Abstract: West Nile virus (WNV) is now the predominant circulating arthropod-borne virus in the United States with >15,000 human cases and >600 fatalities since 1999. Conventionally, mosquitoes become infected when feeding on viremic birds and subsequently transmit the virus to susceptible hosts. Here, we demonstrate nonviremic transmission of WNV between cofeeding mosquitoes. Donor, Culex pipiens quinquefasciatus mosquitoes infected with WNV were fed simultaneously with uninfected "recipient" mosquitoes on naive mice. At all times, donor and recipient mosquitoes were housed in separate sealed containers, precluding the possibility of mixing. Recipients became infected in all five trials, with infection rates as high as 5.8% and no detectable viremia in the hosts. Remarkably, a 2.3% infection rate was observed when 87 uninfected mosquitoes fed adjacent to a single infected mosquito. This phenomenon could potentially enhance virus survival, transmission, and dispersion and obviate the requirement for viremia. All vertebrates, including immune and insusceptible animals, might therefore facilitate mosquito infection. Our findings question the status of dead-end hosts in the WNV transmission cycle and may partly explain the success with which WNV established and rapidly dispersed throughout North America.
Descriptors: West Nile virus, disease transmission, Culex quinquefasciatus, Culex pipiens, mosquitoes, insect vectors, hematophagy, mice, Culex-pipiens-quinquefasciatus.

Hunsperger, E.A. and J.T. Roehrig (2006). Temporal analyses of the neuropathogenesis of a West Nile virus infection in mice. Journal of Neurovirology 12(2): 129-139.
Abstract: A West Nile virus (WNV) infection in humans can produce neurological symptoms including acute flaccid paralysis, encephalitis, meningitis and myelitis. To investigate the pathogenesis of WNV in the peripheral and the central nervous system (PNS and CNS), the authors used a murine footpad inoculation model of WNV infection. Survival curves of virus-infected animals of ages 4- to 6-weeks-old demonstrated age-dependent mortality where older animals (6-weeks-old) had a higher mortality rate compared to younger animals (4- and 5-weeks-old). The mice that survived the virus infection formed WNV-reactive antibodies, confirming viral infection and clearance. The localization of viral RNA (vRNA) and antigen in infected murine tissues was investigated using TaqMan and immunohistochemistry (IHC) respectively. During a nine day infection, vRNA levels in the spinal cord and brainstem fluctuated, suggesting early viral clearance from these tissues by days 3-4 p.i. with later re-introduction. Viral antigens detected using IHC were primarily observed in three main regions of the brain: cortex, hippocampus and brainstem. Additionally, the dorsal root ganglion neurons of the PNS stained positive for viral antigens. These data are consistent with multiple routes of neuroinvasion following a peripheral inoculation of virus and do not preclude the previous observation that virus-infected peripheral neurons can introduce virus into the CNS by a retrograde transport mechanism.
Descriptors: West Nile fever, West Nile virus isolation and purification, disease models, disease progression, inbred C57BL mice, viral RNA analysis, viral genetics, reverse transcriptase polymerase chain reaction, time factors.

Iglesias, M.C., M.P. Frenkiel, K. Mollier, P. Souque, P. Despres, and P. Charneau (2006). A single immunization with a minute dose of a lentiviral vector-based vaccine is highly effective at eliciting protective humoral immunity against West Nile virus. Journal of Gene Medicine 8(3): 265-274.
Abstract: BACKGROUND: Lentiviral vectors, due to their capacity to transduce non-dividing cells, have become precious and worldwide used gene transfer systems. Their ability to efficiently and stably transduce dendritic cells (DCs) has led to their successful use as vaccination vectors for eliciting strong, specific and protective cellular immune responses mostly in anti-tumoral but also in anti-viral applications. However, the ability of lentiviral vectors to elicit an antibody-based protective immunity has, to date, not been evaluated. In the present study, we evaluated the potential of a lentiviral vector-based vaccine to elicit humoral immunity against West Nile virus (WNV). WNV is a mosquito-borne flavivirus that emerged in North America and causes encephalitis in humans, birds and horses. Neutralizing anti-WNV antibodies have been shown to be crucial for protection against WNV encephalitis. METHODS: The ability of lentiviral vector TRIP/sE(WNV), expressing the secreted soluble form of the envelope E-glycoprotein (sE(WNV)) from the highly virulent IS-98-ST1 strain of WNV, to induce a specific humoral response and protection against WNV infection was assessed in a mouse model of WNV encephalitis. RESULTS: Remarkably, a single immunization with a minute dose of TRIP/sE(WNV) was efficient at eliciting a long-lasting, protective and sterilizing humoral immunity, only 1 week after priming. CONCLUSIONS: This study broadens the applicability of lentiviral vectors as efficient non-replicating vaccines against pathogens for which a neutralizing humoral response is one active arm of the protective immunity. The TRIP/sE(WNV) lentiviral vector appears to be a promising tool for veterinary vaccination against zoonotic WNV.
Descriptors: antibody formation, lentivirus, viral vaccines, West Nile fever, West Nile virus, genetic vectors, immunization schedule, mice, transduction, viral envelope proteins.

Johnson, B.W., O. Kosoy, D.A. Martin, A.J. Noga, B.J. Russell, A.A. Johnson, and L.R. Petersen (2005). West Nile virus infection and serologic response among persons previously vaccinated against yellow fever and Japanese encephalitis viruses. Vector Borne and Zoonotic Diseases 5(2): 137-145. ISSN: 1530-3667.
Abstract: It is hypothesized that previous heterologous flaviviral exposure may modulate clinical illness among persons infected with West Nile virus (WNV). Little is known about the serological response in such persons. In summer 2003, a WNV outbreak occurred in Colorado, the location of the Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases (DVBID). DVBID employees, most previously vaccinated with yellow fever virus (YFV) or Japanese encephalitis virus (JEV) vaccines, were studied to determine whether previous vaccination affected symptom development among those subsequently infected with WNV during the outbreak, as well as their serological response. Serum samples collected in December 2003 and previously banked samples were tested using the plaque reduction neutralization test (PRNT) against WNV, Saint Louis encephalitis virus, dengue- 4 virus, JEV, and YFV. Specimens shown to have WNV antibody by PRNT were tested by IgM and IgG enzymelinked immunosorbent assays (ELISAs). Ten (9%) of 113 serosurvey participants had WNV neutralizing antibody titers in December 2003. PRNT titers from previous specimens showed that one of the ten had seroconverted to WNV before 2003. Of the remaining nine participants, seven reported illness in the summer of 2003, two of which were unvaccinated and five previously vaccinated. In the December 2003 specimens, five persons previously unvaccinated or vaccinated only against YFV had a fourfold or greater neutralizing titer with WNV than with other flaviviruses, whereas no persons previously vaccinated against JEV or JEV and YFV showed a similar difference in neutralizing titers. Eight of nine persons infected in 2003 had negative or indeterminate WNV MAC-ELISA results in the December 2003 sample; the ninth person was vaccinated against YFV one month previously, and was also YFV positive by MAC-ELISA. We conclude that previous flaviviral vaccination does not markedly affect the development of WNV fever and that the IgM antibody response in patients without neuroinvasive WNV disease is transient.
Descriptors: Japanese encephalitis vaccines, adverse effects, West Nile fever immunology, West Nile virus, yellow fever vaccine, viral biosynthesis, Colorado, enzyme linked immunosorbent assay.

Julander, J.G., Q.A. Winger, A.L. Olsen, C.W. Day, R.W. Sidwell, and J.D. Morrey (2005). Treatment of West Nile virus-infected mice with reactive immunoglobulin reduces fetal titers and increases dam survival. Antiviral Research 65(2): 79-85. ISSN: 0166-3542.
Abstract: The objectives of this study were to determine if injection of West Nile virus (WNV) into timed-pregnant mice would result in fetal infection and if administration of WNV-reactive immunoglobulin would increase dam survival and reduce fetal viral titers. Dams injected on 7.5 days post-coitus (dpc) had detectable viral titers in the placenta 10.5dpc with a mean titer of 10(4.9) 50% cell-culture infectious doses per gram of tissue (CCID(50)/g tissue). The mean placental titer increased to 10(8.6)CCID(50)/g tissue at 12.5dpc. Infectious virus was detectable 12.5dpc in 10 of 10 fetuses with a mean titer of 10(7.5)CCID(50)/g tissue. Treatment of dams (challenged with WNV on 7.5dpc) with WNV-reactive human immunoglobulin (Ig) on 8.5 and 9.5dpc resulted in a significant reduction of virus in fetuses as compared with non-reactive human Ig-treated females on 12.5dpc (P< or =0.001). Treatment also resulted in survival of dams to term. Treatment of dams with WNV-reactive human Ig on 12.5 and 13.5dpc also resulted in reduction of viral titer on 14.5dpc, indicating that later treatment may also be efficacious. This suggests that Ig treatment may be useful in treating fetal WNV infection in women.
Descriptors: viral antibodies, immunoglobulins, pregnancy complications, West Nile fever , West Nile virus, disease transmission, mice, infectious immunology.

Kajaste Rudnitski, A., T. Mashimo, M.P. Frenkiel, J.L. Guenet, M. Lucas, and P. Despres (2006). The 2',5'-Oligoadenylate Synthetase 1b Is a Potent Inhibitor of West Nile Virus Replication Inside Infected Cells. Journal of Biological Chemistry. 281(8): 4624-4637. ISSN: 0021-9258.
Abstract: The 2',5'-oligoadenylate synthetase (OAS) proteins associated with endoribonuclease RNase L are components of the interferon-regulated OAS/RNase L system, which is an RNA decay pathway known to play an important role in the innate antiviral immunity. A large body of evidence suggests a critical role for the 1b isoform of the mouse Oas gene (Oas1b) in resistance to West Nile virus (WNV) infection in vivo. WNV is a positive, single-stranded RNA virus responsible for severe encephalitis in a large range of animal species and humans. To investigate the molecular basis for the sensitivity of WNV to the Oas1b antiviral pathway, we established a stable mouse fibroblastic cell clone that up-regulates Oas1b protein expression under the control of the Tet-Off expression system. We showed that murine cells respond to Oas1b expression by efficiently inhibiting WNV replication. The antiviral action of Oas1b was essentially restricted to the early stages in virus life cycle. We found that the inability of WNV to productively infect the Oas1b-expressing cells was attributable to a dramatic reduction in positive-stranded viral RNA level. Thus, Oas1b represents an antiviral pathway that exerts its inhibitory effect on WNV replication by preventing viral RNA accumulation inside infected cells.
Descriptors: Internet resource, West Nile virus, oligoadenylate synthase, OAS, RNaseL, proteins, antiviral immunity.

Kalitzky, M., H. Rohde and P. Borowski (2005). West Nile virus: New targets for potential antivirals. In: P.F. Torrence Antiviral Drug Discovery for Emerging Diseases and Bioterrorism Threats, John Wiley & Sons Ltd: Chichester, UK, p. 263-278. ISBN: 0471668273.
Descriptors: West Nile virus, anti-viral drug discovery, emerging diseases, protein synthesis, enzyme inhibition, virus replication.

Kanai, R., K. Kar, K. Anthony, L.H. Gould, M. Ledizet, E. Fikrig, W.A. Marasco, R.A. Koski, and Y. Modis (2006). Crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes. Journal of Virology 80(22): 11000-11008. ISSN: 0022-538X.
Abstract: West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.
Descriptors: epitopes, viral envelope proteins chemistry, West Nile virus, amino acid sequence, crystallography, x ray, dengue virus, encephalitis viruses, tick borne, epitope mapping, molecular sequence data, protein binding, tertiary protein structure, sequence alignment.

Karaca, K., R. Bowen, L.E. Austgen, M. Teehee, L. Siger, D. Grosenbaugh, L. Loosemore, J.C. Audonnet, R. Nordgren, and J.M. Minke (2005). Recombinant canarypox vectored West Nile virus (WNV) vaccine protects dogs and cats against a mosquito WNV challenge. Vaccine 23(29): 3808-3813.
Abstract: The safety and efficacy of a canarypox vector expressing PrM and E genes of West Nile virus (WNV) (ALVAC-WNV) was evaluated in dogs and cats. One group of 17 dogs (vaccinated with 10(5.6) TCID(50)) and two groups of cats (groups 1 [n=14] vaccinated with 10(7.5) TCID(50) and 2 [n=8] 10(5.6) TCID(50)) were vaccinated twice at 28-day intervals. Fifteen dogs and eleven cats served as negative controls. The cats and dogs were challenged 120 and 135 days after the second immunization, respectively via the bites of Aedes albopictus mosquitoes infected with WNV. The first dose of vaccine induced a detectable antibody response in four dogs and five cats (one immunized with low and four with high doses). After the second dose, all the vaccinated dogs and all of the cats, immunized with high dose had detectable antibody titers, whereas only four of eight cats in the low dose group were seropositive. None of the vaccinated dogs and one vaccinated cat developed viremia following the WNV mosquito-challenge. In contrast, 14 of the 15 control dogs and 9 of the 11 control cats developed viremia. The experimental vaccine described in this study may be of value in the prevention of WNV infection in dogs and cats.
Descriptors: canarypox vector, cat diseases, dog diseases, viral vaccines, West Nile fever, West Nile virus, viral blood antibodies, synthetic genetics, viral envelope proteins, viral vaccines, West Nile fever prevention and control.

Kaufmann, B., G.E. Nybakken, P.R. Chipman, W. Zhang, M.S. Diamond, D.H. Fremont, R.J. Kuhn, and M.G. Rossmann (2006). West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody. Proceedings of the National Academy of Sciences of the United States of America 103(33): 12400-12404.
Abstract: Flaviviruses, such as West Nile virus (WNV), are significant human pathogens. The humoral immune response plays an important role in the control of flavivirus infection and disease. The structure of WNV complexed with the Fab fragment of the strongly neutralizing mAb E16 was determined to 14.5-Angstrom resolution with cryo-electron microscopy. E16, an antibody with therapeutic potential, binds to domain III of the WNV envelope glycoprotein. Because of steric hindrance, Fab E16 binds to only 120 of the 180 possible binding sites on the viral surface. Fitting of the previously determined x-ray structure of the Fab-domain III complex into the cryo-electron microscopy density required a change of the elbow angle between the variable and constant domains of the Fab. The structure suggests that the E16 antibody neutralizes WNV by blocking the initial rearrangement of the E glycoprotein before fusion with a cellular membrane.
Descriptors: monoclonal antibodies, immunoglobulin Fab fragments, West Nile virus chemistry, binding sites, cell line, cricetinae, cryoelectron microscopy, crystallography, x ray, hydrogen ion concentration, models, protein conformation, viral fusion proteins.

Keller, B.C., B.L. Fredericksen, M.A. Samuel, R.E. Mock, P.W. Mason, M.S. Diamond, and M.J. Gale (2006). Resistance to alpha/beta interferon is a determinant of West Nile virus replication fitness and virulence. Journal of Virology 80(19): 9424-9434. ISSN: 0022-538X.
Abstract: The emergence of West Nile virus (WNV) in the Western Hemisphere is marked by the spread of pathogenic lineage I strains, which differ from typically avirulent lineage II strains. To begin to understand the virus-host interactions that may influence the phenotypic properties of divergent lineage I and II viruses, we compared the genetic, pathogenic, and alpha/beta interferon (IFN-alpha/beta)-regulatory properties of a lineage II isolate from Madagascar (MAD78) with those of a new lineage I isolate from Texas (TX02). Full genome sequence analysis revealed that MAD78 clustered, albeit distantly, with other lineage II strains, while TX02 clustered with emergent North American isolates, more specifically with other Texas strains. Compared to TX02, MAD78 replicated at low levels in cultured human cells, was highly sensitive to the antiviral actions of IFN in vitro, and demonstrated a completely avirulent phenotype in wild-type mice. In contrast to TX02 and other pathogenic forms of WNV, MAD78 was defective in its ability to disrupt IFN-induced JAK-STAT signaling, including the activation of Tyk2 and downstream phosphorylation and nuclear translocation of STAT1 and STAT2. However, replication of MAD78 was rescued in cells with a nonfunctional IFN-alpha/beta receptor (IFNAR). Consistent with this finding, the virulence of MAD78 was unmasked upon infection of mice lacking IFNAR. Thus, control of the innate host response and IFN actions is a key feature of WNV pathogenesis and replication fitness.
Descriptors: interferon alpha immunology, interferon beta immunology, virus replication, West Nile fever, mice, molecular sequence data, phenotype, phylogeny, protein tyrosine kinase metabolism, stat transcription factors metabolism, signal transduction, drug effects, West Nile virus isolation and purification.

Khairallah, M., S. Ben Yahia, S. Attia, S. Zaouali, B. Jelliti, S. Jenzri, A. Ladjimi, and R. Messaoud (2006). Indocyanine green angiographic features in multifocal chorioretinitis associated with West Nile virus infection. Retina 26(3): 358-359.
Descriptors: chorioretinitis, diagnostic use of coloring agents, eye infections, indocyanine green, West Nile fever diagnosis, viral blood antibodies, chorioretinitis, viral virology, fluorescein angiography, immunoglobulin M blood.

Kilpatrick, A.M., L.D. Kramer, S.R. Campbell, E.O. Alleyne, A.P. Dobson, and P. Daszak (2005). West Nile virus risk assessment and the bridge vector paradigm. Emerging Infectious Diseases 11(3): 425-429. ISSN: 1080-6040.
Abstract: In the northeast United States, control of West Nile virus (WNV) vectors has been unfocused because of a lack of accurate knowledge about the roles different mosquitoes play in WNV transmission. We analyzed the risk posed by 10 species of mosquitoes for transmitting WNV to humans by using a novel risk-assessment measure that combines information on the abundance, infection prevalence, vector competence, and biting behavior of vectors. This analysis suggests that 2 species (Culex pipiens L. and Cx. restuans Theobald [Diptera: Cilicidae]) not previously considered important in transmitting WNV to humans may be responsible for up to 80% of human WNV infections in this region. This finding suggests that control efforts should be focused on these species which may reduce effects on nontarget wetland organisms. Our risk measure has broad applicability to other regions and diseases and can be adapted for use as a predictive tool of future human WNV infections.
Descriptors: culex mosquitoes physiology, insect vectors, West Nile fever transmission, New England, risk, West Nile virus.

Kinney, R.M., C.Y. Huang, M.C. Whiteman, R.A. Bowen, S.A. Langevin, B.R. Miller, and A.C. Brault (2006). Avian virulence and thermostable replication of the North American strain of West Nile virus. Journal of General Virology 87(Pt 12): 3611-3622.
Abstract: The NY99 genotype of West Nile virus (WNV) introduced into North America has demonstrated high virulence for American crows (AMCRs), whilst a closely related WNV strain (KEN-3829) from Kenya exhibits substantially reduced virulence in AMCRs [Brault, A. C., Langevin, S. A., Bowen, R. A., Panella, N. A., Biggerstaff, B. J., Miller, B. R. & Nicholas, K. (2004). Emerg Infect Dis 10, 2161-2168]. Viruses rescued from infectious cDNA clones of both the NY99 and KEN-3829 strains demonstrated virulence comparable to that of their parental strains in AMCRs. To begin to define parameters that might explain the different virulence phenotypes between these two viruses, temperature-sensitivity assays were performed for both viruses at the high temperatures experienced in viraemic AMCRs. Growth curves of the two WNV strains were performed in African green monkey kidney (Vero; 37-42 degrees C) and duck embryonic fibroblast (DEF; 37-45 degrees C) cells cultured at temperatures that were tolerated by the cell line. Unlike the NY99 virus, marked decreases in KEN-3829 viral titres were detected between 36 and 120 h post-infection (p.i.) at temperatures above 43 degrees C. Replication of KEN-3829 viral RNA was reduced 6500-fold at 72 h p.i. in DEF cells incubated at 44 degrees C relative to levels of intracellular virus-specific RNA measured at 37 degrees C. In contrast, replication of virus derived from the NY99 infectious cDNA at 44 degrees C demonstrated only a 17-fold reduction in RNA level. These results indicated that the ability of WNV NY99 to replicate at the high temperatures measured in infected AMCRs could be an important factor leading to the increased avian virulence and emergence of this strain of WNV.
Descriptors: bird diseases, crows, virus replication, West Nile fever, West Nile virus, body temperature, cell line, Cercopithecus aethiops, ducks, plaque assay, viral RNA biosynthesis, survival analysis, temperature, vero cells.

Koh, W.L. and M.L. Ng (2005). Molecular mechanisms of West Nile virus pathogenesis in brain cell. Emerging Infectious Diseases 11(4): 629-362. ISSN: viral blood.
Abstract: We analyzed the response of human glioma cells to West Nile virus infection by investigating host transcriptional changes. Changes in expression of 23 genes showed similarities to those in other neurodegenerative diseases. These changes may be useful as potential biomarkers and elucidate novel mechanisms behind the neuropathology of infection with this virus.
Descriptors: brain virology, West Nile virus, biological markers, tumor cell line, cytopathogenic effect, viral gene expression, glioblastoma, microarray analysis, reproducibility of results, reverse transcriptase polymerase chain reaction, West Nile fever physiopathology.

Lee, J.W.M., J.J.H. Chu, and M.L. Ng (2006). Quantifying the Specific Binding between West Nile Virus Envelope Domain III Protein and the Cellular Receptor [alpha]V[beta]3 Integrin. Journal of Biological Chemistry. 281(3): 1352-1360. ISSN: 0021-9258.
Abstract: A previous study has illustrated that the [alpha]V[beta]3 integrin served as the functional receptor for West Nile virus (WNV) entry into cells. Domain III (DIII) of WNV envelope protein (E) was postulated to mediate virus binding to the cellular receptor. In this study, the specificity and affinity binding of WNV E DIII protein to [alpha]V[beta]3 integrin was confirmed with co-immunoprecipitation and receptor competition assay. Binding of WNV E DIII protein to [alpha]V[beta]3 integrin induced the phosphorylation of focal adhesion kinase that is required to mediate ligand-receptor internalization into cells. A novel platform was then developed using the atomic force microscopy to measure this specific binding force between WNV E DIII protein and the cellular receptor, [alpha]V[beta]3 integrin. The single protein pair-interacting force measured was in the range of 45 +/- 5 piconewtons. This interacting force was highly specific as minimal force was measured in the WNV E DIII protein interaction with [alpha]V[beta]5 integrin molecules and heparan sulfate. These experiments provided an insight to quantitate virus-receptor interaction. Force measurement using atomic force microscopy can serve to quantitatively analyze the effect of candidate drugs that modulate virus-host receptor affinity.
Descriptors: Internet-resource, West Nile virus, Domain III protein, binding, cellular receptor, virus binding.

Lewis, M., J. Renclawowicz, and P. van den Driessche (2006). Traveling waves and spread rates for a West Nile virus model. Bulletin of Mathematical Biology 68(1): 3-23.
Abstract: A reaction-diffusion model for the spatial spread of West Nile virus is developed and analysed. Infection dynamics are based on a modified version of a model for cross infection between birds and mosquitoes (Wonham et al., 2004, An epidemiological model for West-Nile virus: Invasion analysis and control application. Proc. R. Soc. Lond. B 271), and diffusion terms describe movement of birds and mosquitoes. Working with a simplified version of the model, the cooperative nature of cross-infection dynamics is utilized to prove the existence of traveling waves and to calculate the spatial spread rate of infection. Comparison theorem results are used to show that the spread rate of the simplified model may provide an upper bound for the spread rate of a more realistic and complex version of the model.
Descriptors: biological models, West Nile fever, epidemiology algorithms, animal migration, birds, culicidae virology, North America.

Lewis, M.A., J. Renclawowicz, P. van den Driessche, and M. Wonham (2006). A comparison of continuous and discrete-time West Nile virus models. Bulletin of Mathematical Biology 68(3): 491-509.
Abstract: The first recorded North American epidemic of West Nile virus was detected in New York state in 1999, and since then the virus has spread and become established in much of North America. Mathematical models for this vector-transmitted disease with cross-infection between mosquitoes and birds have recently been formulated with the aim of predicting disease dynamics and evaluating possible control methods. We consider discrete and continuous time versions of the West Nile virus models proposed by Wonham et al. [Proc. R. Soc. Lond. B 271:501-507, 2004] and by Thomas and Urena [Math. Comput. Modell. 34:771-781, 2001], and evaluate the basic reproduction number as the spectral radius of the next-generation matrix in each case. The assumptions on mosquito-feeding efficiency are crucial for the basic reproduction number calculation. Differing assumptions lead to the conclusion from one model [Wonham, M.J. et al., [Proc. R. Soc. Lond. B] 271:501-507, 2004] that a reduction in bird density would exacerbate the epidemic, while the other model [Thomas, D.M., Urena, B., Math. Comput. Modell. 34:771-781, 2001] predicts the opposite: a reduction in bird density would help control the epidemic.
Descriptors: biological models, West Nile virus, birds, culicidae virology, feeding behavior, North America, population density.

Li, J., R. Bhuvanakantham, J. Howe, and M.L. Ng (2006). The glycosylation site in the envelope protein of West Nile virus (Sarafend) plays an important role in replication and maturation processes. Journal of General Virology 87(Pt 3): 613-622.
Abstract: The complete genome of West Nile (Sarafend) virus [WN(S)V] was sequenced. Phylogenetic trees utilizing the complete genomic sequence, capsid gene, envelope gene and NS5 gene/3' untranslated region of WN(S)V classified WN(S)V as a lineage II virus. A full-length infectious clone of WN(S)V with a point mutation in the glycosylation site of the envelope protein (pWNS-S154A) was constructed. Both growth kinetics and the mode of maturation were affected by this mutation. The titre of the pWNS-S154A virus was lower than the wild-type virus. This defect was corrected by the expression of wild-type envelope protein in trans. The pWNS-S154A virus matured intracellularly instead of at the plasma membrane as shown for the parental WN(S)V.
Descriptors: viral genome, viral envelope proteins, West Nile virus, amino acid sequence, cell line, Cercopithecus aethiops, glycosylation, molecular sequence data, sequence alignment, species specificity, vero cells, viral envelope proteins.

Li, L., A. Barrett, and D. Beasley (2005). Differential expression of domain III neutralizing epitopes on the envelope proteins of West Nile virus strains. Virology 335(1): 99-105. ISSN: 0042-6822.
Descriptors: West Nile virus, viral proteins, viral antigens, epitopes, neutralization, amino acid sequences, antigenic variation, nucleotide sequences, structure activity relationships, viral-envelope-protein, viral-antibodies, molecular-sequence-data, Internet-resource.

Lim, J.K., W.G. Glass, D.H. McDermott, and P.M. Murphy (2006). CCR5: No longer a "good for nothing" gene--chemokine control of West Nile virus infection. Trends in Immunology 27(7): 308-312.
Abstract: The chemokine receptor CCR5 was identified in 1996 as a crucial host factor exploited by HIV for cell entry. CCR5 presumably functions normally in antimicrobial host defense because it generally mediates leukocyte chemotactic responses; however, evidence of antimicrobial functions for CCR5 in humans has been elusive. Recently, genetic analyses in mice and humans have provided strong evidence for the CCR5 control of infection by West Nile virus (WNV), a re-emerging pathogen capable of causing fatal encephalitis. Thus, the same receptor can benefit or harm the host, depending on the virus. Although CCR5 might be a logical target for new drug development in HIV/AIDS, the benefits of blocking CCR5 could carry the cost of an increased risk of WNV disease in co-infected patients.
Descriptors: CCR5 receptor, West Nile fever, anti-HIV agents, drug therapy, drug development, chemokine receptor.

Liu, R., J. Shuai, J. Wu, and H. Zhu (2006). Modeling spatial spread of West Nile virus and impact of directional dispersal of birds. Mathematical Biosciences and Engineering 3(1): 145-160. ISSN: 1547-1063.
Descriptors: West Nile virus spread, modeling, bird dispersal, disease transmission, spatial spread model, long range dispersal patterns.

Liu, W.J., X.J. Wang, D.C. Clark, M. Lobigs, R.A. Hall, and A.A. Khromykh (2006). A single amino acid substitution in the West Nile virus nonstructural protein NS2A disables its ability to inhibit alpha/beta interferon induction and attenuates virus virulence in mice. Journal of Virology 80(5): 2396-2404. ISSN: 0022-538X.
Abstract: Alpha/beta interferons (IFN-alpha/beta) are key mediators of the innate immune response against viral infection. The ability of viruses to circumvent IFN-alpha/beta responses plays a crucial role in determining the outcome of infection. In a previous study using subgenomic replicons of the Kunjin subtype of West Nile virus (WNV(KUN)), we demonstrated that the nonstructural protein NS2A is a major inhibitor of IFN-beta promoter-driven transcription and that a single amino acid substitution in NS2A (Ala30 to Pro [A30P]) dramatically reduced its inhibitory effect (W. J. Liu, H. B. Chen, X. J. Wang, H. Huang, and A. A. Khromykh, J. Virol. 78:12225-12235). Here we show that incorporation of the A30P mutation into the WNV(KUN) genome results in a mutant virus which elicits more rapid induction and higher levels of synthesis of IFN-alpha/beta in infected human A549 cells than that detected following wild-type WNV(KUN) infection. Consequently, replication of the WNV(KUN)NS2A/A30P mutant virus in these cells known to be high producers of IFN-alpha/beta was abortive. In contrast, both the mutant and the wild-type WNV(KUN) produced similar-size plaques and replicated with similar efficiency in BHK cells which are known to be deficient in IFN-alpha/beta production. The mutant virus was highly attenuated in neuroinvasiveness and also attenuated in neurovirulence in 3-week-old mice. Surprisingly, the mutant virus was also partially attenuated in IFN-alpha/betagamma receptor knockout mice, suggesting that the A30P mutation may also play a role in more efficient activation of other antiviral pathways in addition to the IFN response. Immunization of wild-type mice with the mutant virus resulted in induction of an antibody response of similar magnitude to that observed in mice immunized with wild-type WNV(KUN) and gave complete protection against challenge with a lethal dose of the highly virulent New York 99 strain of WNV. The results confirm and extend our previous original findings on the role of the flavivirus NS2A protein in inhibition of a host antiviral response and demonstrate that the targeted disabling of a viral mechanism for evading the IFN response can be applied to the development of live attenuated flavivirus vaccine candidates.
Descriptors: interferon alpha biosynthesis, interferon beta biosynthesis, viral nonstructural proteins genetics, amino acid substitution, antibodies, viral blood, cell line, cricetinae, animal disease models, knockout mice, plaque assay, survival analysis, virulence genetics, virus replication, West Nile fever, West Nile virus.

Long, M.T., W. Jeter, J. Hernandez, D.C. Sellon, D. Gosche, K. Gillis, E. Bille, and E.P. Gibbs (2006). Diagnostic performance of the equine IgM capture ELISA for serodiagnosis of West Nile virus infection. Journal of Veterinary Internal Medicine 20(3): 608-613.
Abstract: The objectives of these studies were to assess the diagnostic performance (sensitivity and specificity) of the IgM capture enzyme-linked immunosorbent assay (ELISA; MAC) for diagnosis of West Nile (WN) virus in horses and to examine the performance of this test by using different criteria for seropositivity. A total of 36 horses classified as WN virus infected (group 1) and 383 horses from 4 subpopulations of hoses classified as noninfected (groups 2, 3, 4, and 5) were used in the study. The sensitivity (proportion of infected horses that tested positive for WN virus IgM antibodies) and specificity (proportion of noninfected horses that tested negative) were calculated at different cutoff points by using receiver operating curve (ROC) analysis. Using a selected cutoff point = 2.0, the sensitivity and specificity of the MAC were 91.7 and 99.2%, respectively. The area under the ROC curve = 0.95 (95% confidence interval [CI], 0.89 to 1.0), suggesting that the MAC is a useful tool for diagnosis of recent WN virus exposure in horses. In fulfillment of the 2nd objective, 2 other indices were developed and these indices approached 1.0 for the AUC with smaller 95% CIs. These indices were then used to test 602 additional diagnostic samples submitted from suspect horses between 2002 and 2004. Using the standard cutoff, 194 (32%) of the horses were interpreted as positive. Utilizing newly predicted cutoff criteria from each index, additional horses were identified as positive. In conclusion, the MAC as used for identification of WN virus-diseased horses undergoing recent exposure performs reliably at the standard cutoff for seropositivity. A negative test might not completely rule out WN virus disease, but horses that test negative were most likely not exposed to WNV. Performance of the test can be further improved by investigation of other indexes of seropositivity.
Descriptors: sensitivity and specificity of the IgM capture enzyme-linked immunosorbent assay (ELISA), diagnosis of West Nile virus, horses, seropositivity, predictive value of tests, serologic tests, West Nile fever.

Macdonald, J., J. Tonry, R.A. Hall, B. Williams, G. Palacios, M.S. Ashok, O. Jabado, D. Clark, R.B. Tesh, T. Briese, and W.I. Lipkin (2005). NS1 protein secretion during the acute phase of West Nile virus infection. Journal of Virology 79(22): 13924-13933. ISSN: 0022-538X.
Abstract: The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNV NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.
Descriptors: viral nonstructural proteins, West Nile fever, West Nile virus, cell line, Cercopithecus aethiops, cricetinae, animal disease models, gene expression regulation, Sprague Dawley rats.

Mateo, R., S.Y. Xiao, H. Guzman, H. Lei, A.P. Da Rosa, and R.B. Tesh (2006). Effects of immunosuppression on West Nile virus infection in hamsters. American Journal of Tropical Medicine and Hygiene 75(2): 356-362. ISSN: 0002-9637.
Abstract: A research study, comparing the pathogenesis of experimental West Nile virus (WNV) infection in immunocompetent and immunosuppressed golden hamsters, is described. Cyclophosphamide was used to immunosuppress the animals. The immunosuppressed hamsters had a prolonged period of viremia, depressed humoral immune response, more extensive and severe pathology, and higher fatality rate than the untreated immunocompetent animals. Histopathological and immunohistochemical studies of tissues from the two groups showed that pathologic changes in the untreated infected animals were confined to the brain and spinal cord, whereas the histopathological changes and WNV antigen distribution in the immunosuppressed animals were much more extensive and diffuse, involving the adrenal, kidney, heart and lung, and brain and spinal cord. Results of this study in the hamster model provide insight into the increased severity of WNV infection observed in immunosuppressed people.
Descriptors: central nervous system, West Nile fever, West Nile virus, antibody formation, viral isolation and purification, Cercopithecus aethiops, cyclophosphamide, disease models, animal immunocompetence, immunocompromised host, immunosuppressive agents.

Mattheus, A., L. Coleman, J.-S. Temple, and M.-B. Doughty (2006). Purification and enzymatic characterization of West Nile virus RNA dependent RNA polymerase. FASEB Journal 20(5, Part 2): A903. ISSN: 0892-6638.
Descriptors: West Nile virus, purification and enzymatic characterization, RNA, polymerase chain reaction, laboratory techniques, gel filtration chromatography.
Notes: Meeting Information: Experimental Biology 2006 Meeting, San Francisco, California, USA; April 01 -05, 2006.

Mcmurtrey, C., W. Bardet, A. Fleshman, and W. Hildebrand (2006). Direct HLA class I epitope discovery in West Nile virus infection. Journal of Immunology 176(Suppl. S): S211. ISSN: 0022-1767.
Descriptors: West Nile virus infection, epitope discovery, western blot, electrophoretic techniques, immunologic techniques, mass spectrometry, quantitative polymerase chain reaction.
Notes: Meeting Information: Annual Meeting of the American-Association-of-Immunologists, Boston, Massachusettes, USA; May 12 -16, 2006.

Mehlhop, E. and M.S. Diamond (2006). Protective immune responses against West Nile virus are primed by distinct complement activation pathways. Journal of Experimental Medicine 203(5): 1371-1381.
Abstract: West Nile virus (WNV) causes a severe infection of the central nervous system in several vertebrate animals including humans. Prior studies have shown that complement plays a critical role in controlling WNV infection in complement (C) 3(-/-) and complement receptor 1/2(-/-) mice. Here, we dissect the contributions of the individual complement activation pathways to the protection from WNV disease. Genetic deficiencies in C1q, C4, factor B, or factor D all resulted in increased mortality in mice, suggesting that all activation pathways function together to limit WNV spread. In the absence of alternative pathway complement activation, WNV disseminated into the central nervous system at earlier times and was associated with reduced CD8+ T cell responses yet near normal anti-WNV antibody profiles. Animals lacking the classical and lectin pathways had deficits in both B and T cell responses to WNV. Finally, and somewhat surprisingly, C1q was required for productive infection in the spleen but not for development of adaptive immune responses after WNV infection. Our results suggest that individual pathways of complement activation control WNV infection by priming adaptive immune responses through distinct mechanisms.
Descriptors: CD8 positive T lymphocytes, West Nile fever, West Nile virus antibodies, B lymphocytes immunology, central nervous system, knockout mice, spleen.

Mehlhop, E., K. Whitby, T. Oliphant, A. Marri, M. Engle, and M.S. Diamond (2005). Complement activation is required for induction of a protective antibody response against West Nile virus infection. Journal of Virology 79(12): 7466-7477. ISSN: 0022-538X.
Abstract: Infection with West Nile virus (WNV) causes a severe infection of the central nervous system (CNS) with higher levels of morbidity and mortality in the elderly and the immunocompromised. Experiments with mice have begun to define how the innate and adaptive immune responses function to limit infection. Here, we demonstrate that the complement system, a major component of innate immunity, controls WNV infection in vitro primarily in an antibody-dependent manner by neutralizing virus particles in solution and lysing WNV-infected cells. More decisively, mice that genetically lack the third component of complement or complement receptor 1 (CR1) and CR2 developed increased CNS virus burdens and were vulnerable to lethal infection at a low dose of WNV. Both C3-deficient and CR1- and CR2-deficient mice also had significant deficits in their humoral responses after infection with markedly reduced levels of specific anti-WNV immunoglobulin M (IgM) and IgG. Overall, these results suggest that complement controls WNV infection, in part through its ability to induce a protective antibody response.
Descriptors: viral blood antibodies, complement activation, West Nile fever, West Nile virus, complement c3 genetics, cricetinae, mice, neutralization tests, complement genetics, West Nile fever.

Morrey, J.D., V. Siddharthan, A.L. Olsen, G.Y. Roper, H. Wang, T.J. Baldwin, S. Koenig, S. Johnson, J.L. Nordstrom, and M.S. Diamond (2006). Humanized monoclonal antibody against West Nile virus envelope protein administered after neuronal infection protects against lethal encephalitis in hamsters. Journal of Infectious Diseases 194(9): 1300-1308.
Abstract: Humans infected with West Nile virus (WNV) may clinically present with symptoms that are suggestive of neurological infection. Nearly all treatments of WNV disease have been effective in animal models only if administered before or soon after viral challenge. Here, we evaluated whether a potent neutralizing anti-WNV humanized monoclonal antibody (MAb), hE16, could improve the course of disease in a hamster model when administered after the virus had infected neurons in the brain. Five days after viral injection, WNV was detected in the brains of hamsters by cytopathic assay, quantitative reverse-transcription polymerase chain reaction, and immunohistochemical staining of WNV envelope in neurons. Notably, 80%-90% of the hamsters treated 5 days after viral injection by intraperitoneal injection with hE16 survived the disease, compared with 37% of the placebo-treated hamsters (P< or =.001). The hamsters that received hE16 directly in the brain also exhibited markedly improved survival rates, compared with those in the placebo-treated hamsters. In prospective experiments, hamsters with high levels of infectious WNV in their cerebrospinal fluid were also protected by hE16 when administered 5 days after viral injection. These experiments suggest that humanized MAbs with potent neutralizing activity are a possible treatment for human patients after WNV has infected neurons in the central nervous system.
Descriptors: administration of monoclonal antibodies, West Nile fever, drug therapy, West Nile virus, antiviral agents, therapeutic use, cricetinae, dose-response relationship.

Murgai, M., M. Mayda, M. Rios, R. Hammamieh, and M. Jett (2006). Gene expression analysis of the West Nile virus and the Dengue serotypes. FASEB Journal 20(4, Part 1): A62. ISSN: 0892-6638.
Descriptors: West Nile virus, Dengue fever, vector borne diseases, meningitis, encephalitis, microarray gene expression analysis, genetic technique, cDNA microarrays.
Notes: Meeting Information: Experimental Biology 2006 Meeting, San Francisco, California, USA; April 01 -05, 2006.

Nemeth, N., D. Hahn, D. Gould, and R. Bowen (2006). Experimental West Nile Virus Infection in Eastern Screech Owls (Megascops asio). Avian Diseases. 50(2): 252-258. ISSN: 0005-2086.
Abstract: Eastern Screech Owls (EASOs) were experimentally infected with the pathogenic New York 1999 strain of West Nile virus (WNV) by subcutaneous injection or per os. Two of nine subcutaneously inoculated birds died or were euthanatized on 8 or 9 days postinfection (DPI) after <24 hr of lethargy and recumbency. All subcutaneously inoculated birds developed levels of viremia that are likely infectious to mosquitoes, with peak viremia levels ranging from 105.0 to 109.6 plaque-forming units/ml. Despite the viremia, the remaining seven birds did not display signs of illness. All birds alive beyond 5 DPI seroconverted, although the morbid birds demonstrated significantly lower antibody titers than the clinically normal birds. Cagemates of infected birds did not become infected. One of five orally exposed EASOs became viremic and seroconverted, whereas WNV infection in the remaining four birds was not evident. All infected birds shed virus via the oral and cloacal route. Early during infection, WNV targeted skin, spleen, esophagus, and skeletal muscle. The two morbid owls had myocardial and skeletal muscle necrosis and mild encephalitis and nephritis, whereas some of the clinically healthy birds that were sacrificed on 14 DPI had myocardial arteritis and renal phlebitis. WNV is a significant pathogen of EASOs, causing pathologic lesions with varying clinical outcomes.
Descriptors: Strigiformes, wild birds, West Nile virus, pathogenicity, vertebrate viruses, subcutaneous injection, drug delivery systems, viremia, disease transmission, insect vectors, symptoms, seroconversion, morbidity, oral administration, disease course, disease severity, hosts, new host records, pathogen-shedding, Internet-resource.
Language of Text: Summary in Spanish.

Nybakken, G.E., C.A. Nelson, B.R. Chen, M.S. Diamond, and D.H. Fremont (2006). Crystal structure of the West Nile virus envelope glycoprotein. Journal of Virology 80(23): 11467-11474. ISSN: 0022-538X.
Abstract: The envelope glycoprotein (E) of West Nile virus (WNV) undergoes a conformational rearrangement triggered by low pH that results in a class II fusion event required for viral entry. Herein we present the 3.0-A crystal structure of the ectodomain of WNV E, which reveals insights into the flavivirus life cycle. We found that WNV E adopts a three-domain architecture that is shared by the E proteins from dengue and tick-borne encephalitis viruses and forms a rod-shaped configuration similar to that observed in immature flavivirus particles. Interestingly, the single N-linked glycosylation site on WNV E is displaced by a novel alpha-helix, which could potentially alter lectin-mediated attachment. The localization of histidines within the hinge regions of E implicates these residues in pH-induced conformational transitions. Most strikingly, the WNV E ectodomain crystallized as a monomer, in contrast to other flavivirus E proteins, which have crystallized as antiparallel dimers. WNV E assembles in a crystalline lattice of perpendicular molecules, with the fusion loop of one E protein buried in a hydrophobic pocket at the DI-DIII interface of another. Dimeric E proteins pack their fusion loops into analogous pockets at the dimer interface. We speculate that E proteins could pivot around the fusion loop-pocket junction, allowing virion conformational transitions while minimizing fusion loop exposure.
Descriptors: West Nile cirus, crystal structure, envelope glycoprotein, low pH, conformational rearrangement.

Nybakken, G.E., T. Oliphant, S. Johnson, S. Burke, M.S. Diamond, and D.H. Fremont (2005). Structural basis of West Nile virus neutralization by a therapeutic antibody. Nature 437(7059): 764-768. ISSN: 0028-0836.
Descriptors: West Nile virus neutralization, therapeutic antibody, monoclonal antibody, E16 DIII epitope, immune response.

O'Leary, D.R., S. Kuhn, K.L. Kniss, A.F. Hinckley, S.A. Rasmussen, W.J. Pape, L.K. Kightlinger, B.D. Beecham, T.K. Miller, D.F. Neitzel, S.R. Michaels, G.L. Campbell, R.S. Lanciotti, and E.B. Hayes (2006). Birth outcomes following West Nile Virus infection of pregnant women in the United States: 2003-2004. Pediatrics 117(3): E537-E545.
Abstract: BACKGROUND: Congenital West Nile virus (WNV) infection was first described in a single case in 2002. The proportion of maternal WNV infections resulting in congenital infection and clinical consequences of such infections are unknown. METHODS: In 2003 and 2004, women in the United States who acquired WNV infection during pregnancy were reported to the Centers for Disease Control and Prevention by state health departments. Data on pregnancy outcomes were collected. One of the maternal WNV infections was identified retrospectively after the infant was born. Maternal sera, placenta, umbilical cord tissue, and cord serum were tested for WNV infection by using serologic assays and reverse-transcription polymerase chain reaction. Infant health was assessed at delivery and through 12 months of age. RESULTS: Seventy-seven women infected with WNV during pregnancy were clinically followed in 16 states. A total of 71 women delivered 72 live infants; 4 women had miscarriages, and 2 had elective abortions. Of the 72 live infants, 67 were born at term, and 4 were preterm; gestational age was unknown for 1. Of 55 live infants from whom cord serum was available, 54 tested negative for anti-WNV IgM. One infant born with umbilical hernia and skin tags had anti-WNV IgM in cord serum but not in peripheral serum at age 1 month. An infant who had no anti-WNV IgM in cord blood, but whose mother had WNV illness 6 days prepartum, developed WNV meningitis at age 10 days. Another infant, whose mother had acute WNV illness at delivery, was born with a rash and coarctation of the aorta and had anti-WNV IgM in serum at 1 month of age; cord serum was not available. A fourth infant, whose mother had onset of WNV illness 3 weeks prepartum that was not diagnosed until after delivery, had WNV encephalitis and underlying lissencephaly detected at age 17 days and subsequently died; cord serum was not available. The following major malformations were noted among live-born infants: aortic coarctation (n = 1); cleft palate (n = 1); Down syndrome (n = 1); lissencephaly (n = 1); microcephaly (n = 2); and polydactyly (n = 1). One infant had glycogen storage disease type 1. Abnormal growth was noted in 8 infants. CONCLUSIONS: Of 72 infants followed to date in 2003 and 2004, almost all seemed normal, and none had conclusive laboratory evidence of congenital WNV infection. Three infants had WNV infection that could have been congenitally acquired. Seven infants had major malformations, but only 3 of these had defects that could have been caused by maternal WNV infection based on the timing of the infections and the sensitive developmental period for the specific malformations, and none had any conclusive evidence of WNV etiology. However, the sensitivity and specificity of IgM testing of cord blood to detect congenital WNV infection are currently unknown, and congenital WNV infection among newborns with IgM-negative serology cannot be ruled out. Prospective studies comparing pregnancy outcomes of WNV-infected and -uninfected women are needed to better define the outcomes of WNV infection during pregnancy.
Descriptors: pregnancy complications, West Nile fever, children, vertical disease transmission, fetal blood, immunoglobulin M analysis, human milk, viral RNA analysis, West Nile virus isolation and purification.
Notes: Comment In: Pediatrics. 2006 Mar;117(3):936-9.

Oh, W., M.R. Yang, E.W. Lee, K.M. Park, S. Pyo, J.S. Yang, H.W. Lee, and J. Song (2006). Jab1 mediates cytoplasmic localization and degradation of West Nile virus capsid protein. Journal of Biological Chemistry 281(40): 30166-30174.
Abstract: The clinical manifestations of West Nile virus (WNV), a member of the Flavivirus family, include febrile illness, sporadic encephalitis, and paralysis. The capsid (Cp) of WNV is thought to participate in these processes by inducing apoptosis through mitochondrial dysfunction and activation of caspase-9 and caspase-3. To further identify the molecular mechanism of the WNV capsid protein (WNVCp), yeast two-hybrid assays were employed using WNV-Cp as bait. Jab1, the fifth subunit of the COP9 signalosome, was subsequently identified as a molecule that interacts with WNVCp. Immunoprecipitation and glutathione S-transferase pulldown assays confirmed that direct interaction could occur between WNVCp and Jab1. Immunofluorescence microscopy demonstrated that the overexpressed WNVCp, which localized to the nucleolus, was translocated to the cytoplasm upon its co-expression with Jab1. When treated with leptomycin B, Jab1-facilitated nuclear exclusion of WNVCp was prevented, which indicated that the CRM1 complex is required for Jab1-facilitated nuclear export of WNVCp. Moreover, Jab1 promoted the degradation of WNVCp in a proteasome-dependent way. Consistent with this, WNVCp-mediated cell cycle arrest at the G(2) phase in H1299 was prevented by exogenous Jab1. Finally, an analysis of WNVCp deletion mutants indicated that the first 15 amino acids were required for interaction with Jab1. Furthermore, the double-point mutant of the WNVCp, P5A/P8A, was incapable of binding to Jab1. These results indicate that Jab1 has a potential protective effect against pathogenic WNVCp and might provide a novel target site for the treatment of disease caused by WNV.
Descriptors: capsid proteins, cytoplasm metabolism, intracellular signaling peptides and proteins, peptide hydrolases, West Nile virus, active transport, cell nucleus genetics, amino acid sequence, cell line, tumor, cell nucleus, enzymology, gene expression regulation, protein genetics, molecular sequence data, signal transduction genetics, two hybrid system techniques.

Oh, W.K. and J. Song (2006). Hsp70 functions as a negative regulator of West Nile virus capsid protein through direct interaction. Biochemical and Biophysical Research Communications 347(4): 994-1000. ISSN: 0006-291X.
Abstract: West Nile virus (WNV) is a member of the Flavivirus family and induces febrile illness, sporadic encephalitis, and paralysis. The capsid (Cp) of WNV is thought to play a role in inducing these symptoms through caspase-3- and caspase-9-dependent apoptosis. Using WNVCp as bait for a yeast two-hybrid assay, we identified that Hsp70 interacted with WNVCp. The interaction between Hsp70 and WNVCp was further substantiated using purified proteins. Deletion analysis of Hsp70 indicated that WNVCp could bind to the substrate binding domain of Hsp70. The presence of WNVCp in the Hsp70-dependent folding system inhibited the refolding of beta-galactosidase (beta-gal), which showed that WNVCp might function as a negative regulator of Hsp70. Finally, the cytotoxic effect of WNVCp in 293T cells was prevented by ectopic Hsp70, suggesting a negative regulatory role of Hsp70 on WNVCp. Our findings suggest a possible negative regulatory role of Hps70 in the pathway of WNV infection.
Descriptors: capsid protein metabolism, Hsp70 heat shock proteins, West Nile virus, apoptosis, tumor cell line, protein binding, two hybrid system techniques.

Oliphant, T., M. Engle, G.E. Nybakken, C. Doane, S. Johnson, L. Huang, S. Gorlatov, E. Mehlhop, A. Marri, K.M. Chung, G.D. Ebel, L.D. Kramer, D.H. Fremont, and M.S. Diamond (2005). Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nature Medicine 11(5): 522-530.
Abstract: Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans.
Descriptors: monoclonal antibodies, viral envelope (E) protein, neutralization of West Nile virus in vivo, mice, treatment option.

Oliphant, T., G.E. Nybakken, M. Engle, Q. Xu, C.A. Nelson, S. Sukupolvi Petty, A. Marri, B.E. Lachmi, U. Olshevsky, D.H. Fremont, T.C. Pierson, and M.S. Diamond (2006). Antibody recognition and neutralization determinants on domains I and II of west Nile Virus envelope protein. Journal of Virology 80(24): 12149-12159. ISSN: 0022-538X.
Abstract: Previous studies have demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope (E) strongly protect against infection in animals. Herein, we observed significantly less efficient neutralization by 89 MAbs that recognized domain I (DI) or II (DII) of WNV E protein. Moreover, in cells expressing Fc gamma receptors, many of the DI- and DII-specific MAbs enhanced infection over a broad range of concentrations. Using yeast surface display of E protein variants, we identified 25 E protein residues to be critical for recognition by DI- or DII-specific neutralizing MAbs. These residues cluster into six novel and one previously characterized epitope located on the lateral ridge of DI, the linker region between DI and DIII, the hinge interface between DI and DII, and the lateral ridge, central interface, dimer interface, and fusion loop of DII. Approximately 45% of DI-DII-specific MAbs showed reduced binding with mutations in the highly conserved fusion loop in DII: 85% of these (34 of 40) cross-reacted with the distantly related dengue virus (DENV). In contrast, MAbs that bound the other neutralizing epitopes in DI and DII showed no apparent cross-reactivity with DENV E protein. Surprisingly, several of the neutralizing epitopes were located in solvent-inaccessible positions in the context of the available pseudoatomic model of WNV. Nonetheless, DI and DII MAbs protect against WNV infection in mice, albeit with lower efficiency than DIII-specific neutralizing MAbs.
Descriptors: West Nile virus, monoclonal antibodies, epitope, envelope, MAbs, neutralization, gamma receptors, mice.

Olsen, A., V. Siddharthan, and J. Morrey (2006). Transforming growth factor-beta 1 improves blood-brain barrier properties in mice infected with West Nile virus. Antiviral Research 70(1): A78. ISSN: 0166-3542.
Descriptors: mice, West Nile virus, growth factor beta 1, blood brain barrier, neural coordination, drug therapy.
Notes: Meeting Information: 19th International Conference on Antiviral Research, San Juan, Puerto Rico; May 7 -11, 2006.

Olsen, A.L., D. Chen, and J.D. Morrey (2005). Presumptive identification of a protein associated with West Nile virus encephalitis in CSF of hamsters. Antiviral Research 65(3): A39. ISSN: 0166-3542.
Descriptors: West Nile virus encephalitis, hamsters, presumptive identification, nervous system disease, proteomics approach, mass spectrophotometer analysis, cerebrospinal fluid.
Notes: Meeting Information: 18th International Conference on Antiviral Research, Barcelona, Spain; April 11 -14, 2005.

Ong, S.P., B.G. Choo, J.J. Chu, and M.L. Ng (2006). Expression of vector-based small interfering RNA against West Nile virus effectively inhibits virus replication. Antiviral Research 72(3): 216-223. ISSN: 0166-3542.
Abstract: RNA interference is one of the effective emerging anti-viral strategies to inhibit virus infection in cells. In this study, a small interfering RNA expressing vector (pSilencer-NS5) targeting the NS5 gene of West Nile virus (WNV) was employed to target and destroy WNV transcripts. Real-time PCR revealed drastic reduction in WNV RNA transcripts in pSilencer-NS5-transfected Vero cells. The virus infectious titre was also significantly reduced by 90% as determined by plaque assays. The resulting decrease in virus replication was shown to be specific since both scrambled and nucleotide(s) mismatch siRNA against WNV NS5 gene did not have any effect on WNV productive yields. Furthermore, Western immunoblot analysis on the expression of viral NS5 and envelope (E) proteins showed significant down-regulation on the expression of viral NS5 and envelope (E) proteins in virus-infected cells that were pre-transfected with pSilencer-NS5. These data clearly supported the notion that the expression of vector-based siRNA against WNV NS5 gene is able to exert its silencing effect on WNV-infected cells without inducing cytotoxicity, hence holding promise in therapeutic treatment of this important emerging infectious disease.
Descriptors: West Nile virus, virus replication, small interfering RNA, anti viral strategies, western immunoblot analysis.

Owen, J., F. Moore, N. Panella, E. Edwards, R. Bru, M. Hughes, and N. Komar (2006). Migrating birds as dispersal vehicles for West Nile virus. EcoHealth 3(2): 79-85. ISSN: 1612-92021612-9210.
Descriptors: West Nile virus, migrating birds, dispersal vehicles, locomotor activity, virus spread.

Paisley, J.E., A.F. Hinckley, D.R. O'Leary, W.C. Kramer, R.S. Lanciotti, G.L. Campbell, and E.B. Hayes (2006). West Nile virus infection among pregnant women in a northern Colorado community, 2003 to 2004. Pediatrics 117(3): 814-820.
Abstract: OBJECTIVE: Since West Nile virus (WNV) was first detected in New York in 1999, it has spread across North America and become a major public health concern. In 2002, the first documented case of intrauterine WNV infection was reported, involving an infant with severe brain abnormalities. To determine the frequencies of WNV infections during pregnancy and of intrauterine WNV infections, we measured WNV-specific antibodies in cord blood from infant deliveries after a community-wide epidemic of WNV disease. METHODS: Five hundred sixty-six pregnant women who presented to Poudre Valley Hospital (Fort Collins, CO) for delivery between September 2003 and May 2004 provided demographic and health history data through self-administered questionnaires and hospital admission records. Umbilical cord blood was collected from 549 infants and screened for WNV-specific IgM and IgG antibodies with enzyme-linked immunosorbent assays, with confirmation by plaque-reduction neutralization tests. Newborn growth parameters, Apgar scores, and hearing test results were recorded. RESULTS: Four percent (95% confidence interval: 2.4-5.7%) of cord blood samples tested positive for WNV-specific IgG antibodies. No cord blood samples were positive for WNV-specific IgM antibodies. There were no significant differences between infants of seropositive and seronegative mothers with respect to any of the growth parameters or outcomes measured. CONCLUSIONS: Intrauterine WNV infections seemed to be infrequent. In our study, WNV infection during pregnancy did not seem to affect adversely infant health at birth. Larger prospective studies are necessary to measure more completely the effects of maternal WNV infection on pregnancy and infant health outcomes.
Descriptors: pregnancy complications, infectious epidemiology, West Nile fever, Colorado, fetal blood, immunoglobulin A blood, immunoglobulin G blood, children, seroepidemiologic studies, West Nile virus isolation and purification.

Papin, J.F., R.A. Floyd, and D.P. Dittmer (2005). Methylene blue photoinactivation abolishes West Nile virus infectivity in vivo. Antiviral Research 68(2): 84-87. ISSN: 0166-3542.
Abstract: The prevalence of West Nile virus (WNV) infections and associated morbidity has accelerated in recent years. Of particular concern is the recent demonstration that this virus can be transmitted by blood products and can cause severe illness and mortality in transfusion recipients. We have evaluated methylene blue (MB)+light as a safe and cost-effective means to inactivate WNV in vitro. This regimen inactivated WNV with an IC50 of 0.10 microM. Up to 10(7)pfu/ml of WNV could be inactivated by MB+light with no residual infectivity. MB+light inactivated three primary WNV isolates from the years 1999, 2002 and 2003 and prevented mortality in a murine model for WNV infection. Since MB is already approved for human use at a dose of 100mg/kg/day, we conjecture that MB+light treatment of blood products for high-risk patients will be efficacious and suitable for use in resource-limited settings.
Descriptors: methylene blue, West Nile virus, blood transfusion, light, inbred BalbC mice, plaque assay, sterilization, West Nile fever.

Paterson, R. (2005). How West Nile virus crosses the blood-brain barrier. Lancet. Neurology 4(1): 18.
Descriptors: membrane glycoproteins, adverse effects, cell surface receptors biosynthesis, viral load, West Nile fever, West Nile virus isolation and purification, blood brain barrier, toll like receptors.

Pierson, T.C., M.S. Diamond, A.A. Ahmed, L.E. Valentine, C.W. Davis, M.A. Samuel, S.L. Hanna, B.A. Puffer, and R.W. Doms (2005). An infectious West Nile virus that expresses a GFP reporter gene. Virology 334(1): 28-40. ISSN: 0042-6822.
Descriptors: West Nile virus, GFP reporter gene, cell transfection, plasmid encoding, DNA, viral replication.

Pierson, T.C., M.D. Sanchez, B.A. Puffer, A.A. Ahmed, B.J. Geiss, L.E. Valentine, L.A. Altamura, M.S. Diamond, and R.W. Doms (2006). A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection. Virology 346(1): 53-65. ISSN: 0042-6822.
Abstract: West Nile virus (WNV) is a neurotropic flavivirus within the Japanese encephalitis antigenic complex that is responsible for causing West Nile encephalitis in humans. The surface of WNV virions is covered by a highly ordered icosahedral array of envelope proteins that is responsible for mediating attachment and fusion with target cells. These envelope proteins are also primary targets for the generation of neutralizing antibodies in vivo. In this study, we describe a novel approach for measuring antibody-mediated neutralization of WNV infection using virus-like particles that measure infection as a function of reporter gene expression. These reporter virus particles (RVPs) are produced by complementation of a sub-genomic replicon with WNV structural proteins provided in trans using conventional DNA expression vectors. The precision and accuracy of this approach stem from an ability to measure the outcome of the interaction between antibody and viral antigens under conditions that satisfy the assumptions of the law of mass action as applied to virus neutralization. In addition to its quantitative strengths, this approach allows the production of WNV RVPs bearing the prM-E proteins of different WNV strains and mutants, offering considerable flexibility for the study of the humoral immune response to WNV in vitro. WNV RVPs are capable of only a single round of infection, can be used under BSL-2 conditions, and offer a rapid and quantitative approach for detecting virus entry and its inhibition by neutralizing antibody.
Descriptors: viral antibodies immunology, West Nile virus, cell line, Cercopithecus aethiops, cricetinae, reporter genes, neutralization tests, time factors, viral envelope proteins, West Nile fever immunology.

Pierson, T., M. Diamond, A. Ahmed, L. Valentine, C. Davis, M. Samuel, S. Hanna, B. Puffer, and R. Doms (2005). An infectious West Nile Virus that expresses a GFP reporter gene. Virology 334(1): 28-40. ISSN: 0042-6822.
Descriptors: West Nile virus, genetic vectors, green fluorescent protein, reporter genes, gene expression regulation, neutralization, virus replication, humans, cultured cells, nucleotide sequences, viral-antibodies, molecular-sequence-data, Internet-resource.

Prince, H.E., M. Lape Nixon, M.P. Busch, L.H. Tobler, G.A. Foster, and S.L. Stramer (2005). Utilization of follow-up specimens from viremic blood donors to assess the value of west nile virus immunoglobulin G avidity as an indicator of recent infection. Clinical and Diagnostic Laboratory Immunology 12(9): 1123-1126.
Abstract: The value of West Nile virus immunoglobulin G avidity for distinguishing recent from past infection was investigated using 348 follow-up specimens from 170 viremic blood donors. Low avidity accurately indicated infection within the previous 4 months. However, due to rapid avidity maturation in some individuals, high avidity did not accurately indicate past infection.
Descriptors: blood donors, West Nile fever, West Nile virus antibodies, antibody affinity, follow-up studies, blood immunoglobulin G.

Prince, H.E., L.H. Tobler, M. Lape Nixon, G.A. Foster, S.L. Stramer, and M.P. Busch (2005). Development and Persistence of West Nile Virus-Specific Immunoglobulin M (IgM), IgA, and IgG in Viremic Blood Donors. Journal of Clinical Microbiology. 43(9): 4316-20. ISSN: 0095-1137.
Abstract: West Nile Virus (WNV) antibody development and persistence were investigated in blood donors who made WNV RNA-positive (viremic) donations in 2003. Plasma samples from the index donations and follow-up serum or plasma samples were tested for WNV immunoglobulin M (IgM), IgA, and IgG by using enzyme-linked immunosorbent assays. Antibody development was investigated with 154 samples collected from 84 donors 1 to 21 days after their RNA-positive, antibody-negative, index donation. WNV IgM and IgA were first detected on day 3, and all samples collected after day 9 were WNV IgM and IgA positive; WNV IgG was first detected on day 4, and all samples collected after day 16 were positive. Antibody persistence in this donor group (index donations antibody negative) was evaluated by using 128 samples collected from 89 donors on days 22 to 440 of follow-up; 88% of samples were WNV IgM positive, 86% were WNV IgA positive, and 100% were WNV IgG positive. In linear regression analysis, trendlines for WNV IgM and IgA reached the value discriminating positive from negative results at 218 days and 232 days of follow-up, respectively. Similar WNV IgM and IgA persistence trends characterized 27 donors whose index samples were positive for WNV IgM and IgA, as well as 14 donors whose index samples were positive for WNV IgG but negative for WNV IgM. These findings show that WNV IgG emerges after WNV IgM and IgA and that both WNV IgM and IgA typically persist for at least 6 months after infection. Thus, unlike some other flavivirus infections, WNV infection is not characterized by a relatively rapid disappearance of virus-specific IgA.
Descriptors: West Nile virus, viremic blood donors, specific immunoglobulin M, IgM, persistence, plasma samples, enzyme linked immunosorbent assays.

Puig Basagoiti, F., T.S. Deas, P. Ren, M. Tilgner, D.M. Ferguson, and P.Y. Shi (2005). High-throughput assays using a luciferase-expressing replicon, virus-like particles, and full-length virus for West Nile virus drug discovery. Antimicrobial Agents and Chemotherapy 49(12): 4980-4988. ISSN: 0066-4804.
Descriptors: West Nile virus, high throughput assays, lucifrase, drug discovery, genetic systems.

Ramanathan, M.P., J.A. Chambers, P. Pankhong, M. Chattergoon, W. Attatippaholkun, K. Dang, N. Shah, and D.B. Weiner (2006). Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway. Virology 345(1): 56-72. ISSN: 0042-6822.
Abstract: The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV.
Descriptors: apoptosis, caspases, viral nonstructural proteins, West Nile virus, amino acid sequence, cell line, crow, DNA helicases, enzyme inhibitors, gene silencing, molecular sequence data, peptide hydrolases, protein structure, tertiary genetics, vacuoles, West Nile fever.

Ramanathan, M.P., J.A. Chambers, J. Taylor, B.T. Korber, M.D. Lee, A. Nalca, K. Dang, P. Pankhong, W. Attatippaholkun, and D.B. Weiner (2005). Expression and evolutionary analysis of West Nile virus (Merion strain). Journal of Neurovirology 11(6): 544-556.
Abstract: The authors report a new strain of West Nile virus (WNV) with the expression analysis of its individual open reading frames. Since its sudden appearance in the summer of 1999 in New York City, the virus has spread rapidly across the continental United States into Canada and Mexico. Besides, its rapid transmission by various vectors, the spread of this virus through organ transplantation, blood transfusion, and mother-child transmission through breast milk is of concern. In order to understand molecular variations of WNV in North America and to generate new tools for understanding WNV biology, a complete clone of WNV has been constructed. Investigations so far have focused only on half of its genes products and a detailed molecular and cell biological aspects on all of WNV gene have yet to be clearly established. The open reading frames of WNV were recovered through an reverse transcriptase-polymerase chain reaction (RT-PCR)-PCR using brain tissue from a dead crow collected in Merion, PA, and cloned into a mammalian expression vector. The deduced amino acid sequences of individual open reading frames were analyzed to determine various structural motifs and functional domains. Expression analysis shows that in neuronal cells, C, NS1, and NS5 proteins are nuclear localized whereas the rest of the antigens are confined to the cytoplasm when they are expressed in the absence of other viral antigens. This is the first report that provides an expression analysis as well as intracellular distribution pattern for all of WNV gene products, cloned from an infected bird. Evolutionary analysis of Merion strain sequences indicates that this strain is distinct phylogenetically from the previously reported WNV strains.
Descriptors: viral gene expression regulation, West Nile fever, West Nile virus, amino acid sequence, bird diseases, birds, molecular sequence data, phylogeny, reverse transcriptase polymerase chain reaction.

Ramanathan, M.P., P. Pankhong, M.A. Kutzler, C.A. Chambers, J.J. Kim, G. Gunasekaran, T.C. Pierson, and D.B. Weiner (2006). Protein kinase CK2: A potential target in the development of novel therapeutics against West Nile virus. Journal of Neurovirology 12(Suppl. 1): 69. ISSN: 1355-0284.
Descriptors: West Nile virus, protein kinase CK2, potential target in therapeutics developmen.
Notes: Meeting Information: 7th International Symposium on NeuroVirology, Philadelphia, Pennsylvania, USA; May 31 -June 03, 2006.

Ramanathan, M., J. Chambers, P. Pankhong, M. Chattergoon, W. Attatippaholkun, K. Dang, N. Shah, and D. Weiner (2006). Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway. Virology 345(1): 56-72. ISSN: 0042-6822.
Descriptors: West Nile virus, viral proteins, proteolysis, cysteine proteinases, Corvidae, crows, cloning (DNA), complementary DNA, gene expression regulation, apoptosis, amino acid sequences, enzyme activation, nucleotide sequences, viral-nonstructural-proteins, caspase- molecular-sequence-data, Internet-resource.

RASGON, J.L., M. VENKATESAN, C.J. WESTBROOK, and M.C. HAUER (2006). Polymorphic microsatellite loci from the West Nile virus vector Culex tarsalis. Molecular Ecology Notes. 6(3): 680-682. ISSN: 1471-8278.
Abstract: Since its introduction in 1999, West Nile virus (WNV) has spread across North America. Culex tarsalis is a highly efficient WNV vector species. Many traits such as virus susceptibility, autogeny and host preference vary geographically and temporally in C. tarsalis. Culex tarsalis genomic libraries were developed and were highly enriched for microsatellite inserts (42-96%). We identified 12 loci that were polymorphic in wild C. tarsalis populations. These microsatellites are the first DNA-based genetic markers developed for C. tarsalis and will be useful for investigating population structure and constructing genetic maps in this mosquito.
Descriptors: Internet-resource, West Nile virus, vectors, C. tarsalis, genomic libraries, loci, microsatellites, genetic markers.

Rawal, A., P.J. Gavin, and C.D. Sturgis (2006). Cerebrospinal fluid cytology in seasonal epidemic West Nile virus meningo-encephalitis. Diagnostic Cytopathology 34(2): 127-129.
Abstract: The incidence of West Nile Virus (WNV) infection has progressively increased in North America since the first epidemic in 1999. Formal scholarly documentation of cerebrospinal fluid (CSF) cytology changes in patients with WNV infection is limited. We report our experience with CSF cytospins from a population of consecutive patients with documented CSF WNV-specific IgM. Thirty-two patients (12 male, 20 female) with a median age of 52 yr (range, 19-88) diagnosed with WNV meningo-encephalitis were studied. Symptoms were present for a mean of 5 days (range, 1-14) prior to lumbar puncture. CSF proteins were elevated in 94% of patients (30/32) with a mean value of 79 mg/dl (range, 36-185). CSF glucose was normal to elevated in all cases. All cytomorphologically adequate samples demonstrated a pleocytosis with a mean of 156 cells/mm3 (range, 13-683). Nearly, all (26/28) patients showed increased CSF neutrophils--mean 43% (range, 1-83). Mean lymphocyte and monocyte fractions were 44% (range, 8-85) and 14% (range, 2-27), respectively. Three cases showed 1-4% plasma cells. Mean total leukocyte counts (TLC) (197 cells/mm3) and mean neutrophil fractions (50%) were greater in patients sampled within the first 3 days of symptoms than in those sampled beyond day 3 (mean TLC, 126 cells/mm3; mean neutrophil fraction, 37%). Relative lymphocyte proportions increased from a mean of 39 to 48% after 3 days of illness. WNV should be considered as a potential etiology of infectious CSF pleocytosis in the North American late summer and early fall seasons. 2006 Wiley-Liss, Inc.
Descriptors: cerebrospinal fluid, meningoencephalitis, West Nile virus, immunoglobulin M metabolism, seasons, West Nile virus.

Rios, M., M.J. Zhang, A. Grinev, K. Srinivasan, S. Daniel, O. Wood, I.K. Hewlett, and A.I. Dayton (2006). Monocytes-macrophages are a potential target in human infection with West Nile virus through blood transfusion. Transfusion 46(4): 659-667.
Abstract: BACKGROUND: West Nile virus (WNV) transmission by transfusion was documented in 2002. Approximately 80 percent of WNV infections are asymptomatic and 1 percent develop severe neurological illness. In animals, Langerhans-dendritic cells support initial viral replication, followed by replication in lymphoid tissues and dissemination to organs and possibly to the CNS. The cellular tropism of WNV infection after transfusion and the particular human blood cells that sustain viral replication remain largely unknown. Whether primary monocyte-derived macrophages (MDMs) support WNV infection-replication and produce infectious virions, with an in vitro system, was investigated. STUDY DESIGN AND METHODS: Elutriated monocytes (CD33+/CD14+) from suitable blood donors were cultured in the presence of macrophage-colony-stimulating factor, infected with WNV-NY99 at different time points, washed, and cultivated for up to 47 days. Supernatants were tested for WNV replication by TaqMan reverse transcription-polymerase chain reaction (RT-PCR), with primers for the envelope and/or 3'NC regions, and by cDNA-PCR to detect WNV minus-strand RNA and for the presence of functional virions by infectivity assays in Vero cells. RESULTS: RT-PCR TaqMan of supernatants demonstrated productive infection of MDMs. Viral load reached 2 to 5 log above baseline in 3 to 6 days and then declined, with detectable viral replication persisting for up to 47 days. WNV minus-strand RNA was detected in Day 4 cultures, indicating active viral replication. Infected MDM cultures showed no cytopathic changes. Supernatants that were TaqMan-positive for the presence of WNV-infected Vero cells and produced cytopathic effects within 3 to 5 days of culture. CONCLUSION: The susceptibility of monocytes-macrophages to productive infection in vitro is compatible with a potential role in initial WNV replication and propagation after transmission by transfusion.
Descriptors: blood transfusion, macrophages, monocytes, West Nile fever prevention and control, West Nile virus isolation and purification, cultured cells, reverse transcriptase polymerase chain reaction.

Roosendaal, J., E.G. Westaway, A. Khromykh, and J.M. Mackenzie (2006). Regulated cleavages at the West Nile virus NS4A-2K-NS4B junctions play a major role in rearranging cytoplasmic membranes and Golgi trafficking of the NS4A protein. Journal of Virology 80(9): 4623-4632. ISSN: 0022-538X.
Abstract: A common feature associated with the replication of most RNA viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviviruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile virus, Kunjin virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS2B-3-4A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.
Descriptors: cell membrane, golgi apparatus, viral nonstructural proteins, West Nile virus metabolism, cell line, cell membrane ultrastructure, Cercopithecus aethiops, genetic vectors, electron microscopy, protein binding, protein transport, replicon genetics.

Rossi, S.L., Q. Zhao, V.K. O' Donnell, and P.W. Mason (2005). Adaptation of West Nile virus replicons to cells in culture and use of replicon-bearing cells to probe antiviral action. Virology 331(2): 457-470. ISSN: 0042-6822.
Descriptors: West Nile virus, virus replicons, cell culture, hamsters, humans, monkeys, WNV genomes.

Saldanha, J., S. Shead, A. Heath, and M. Drebot (2005). Collaborative study to evaluate a working reagent for West Nile virus RNA detection by nucleic acid testing. Transfusion 45(1): 97-102. ISSN: 0041-1132.
Descriptors: West Nile virus, RNA detection, nucleic acid testing, assays.

Samuel, M.A. and M.S. Diamond (2005). Alpha/beta interferon protects against lethal West Nile virus infection by restricting cellular tropism and enhancing neuronal survival. Journal of Virology 79(21): 13350-13361. ISSN: 0022-538X.
Abstract: West Nile virus (WNV) is a mosquito-borne flavivirus that is neurotropic in humans, birds, and other animals. While adaptive immunity plays an important role in preventing WNV spread to the central nervous system (CNS), little is known about how alpha/beta interferon (IFN-alpha/beta) protects against peripheral and CNS infection. In this study, we examine the virulence and tropism of WNV in IFN-alpha/beta receptor-deficient (IFN- alpha/betaR-/-) mice and primary neuronal cultures. IFN-alpha/betaR-/- mice were acutely susceptible to WNV infection through subcutaneous inoculation, with 100% mortality and a mean time to death (MTD) of 4.6 +/- 0.7 and 3.8+/- 0.5 days after infection with 10(0) and 10(2) PFU, respectively. In contrast, congenic wild-type 129Sv/Ev mice infected with 10(2) PFU showed 62% mortality and a MTD of 11.9 +/- 1.9 days. IFN-alpha/betaR-/- mice developed high viral loads by day 3 after infection in nearly all tissues assayed, including many that were not infected in wild-type mice. IFN-alpha/betaR-/- mice also demonstrated altered cellular tropism, with increased infection in macrophages, B cells, and T cells in the spleen. Additionally, treatment of primary wild-type neurons in vitro with IFN-beta either before or after infection increased neuronal survival independent of its effect on WNV replication. Collectively, our data suggest that IFN-alpha/beta controls WNV infection by restricting tropism and viral burden and by preventing death of infected neurons.
Descriptors: interferon alpha analysis, interferon beta analysis, West Nile fever , West Nile virus, antiviral agents, brain immunology, cultured cells, cultured, inbred mice, skeletal muscle, organ specificity, messenger RNA genetics, reverse transcriptase polymerase chain reaction, spinal cord , spleen, virus replication.

Samuel, M.A., K. Whitby, B.C. Keller, A. Marri, W. Barchet, B.R. Williams, R.H. Silverman, M.J. Gale, and M.S. Diamond (2006). PKR and RNase L contribute to protection against lethal West Nile Virus infection by controlling early viral spread in the periphery and replication in neurons. Journal of Virology 80(14): 7009-7019. ISSN: 0022-538X.
Abstract: West Nile virus (WNV) is a neurotropic, mosquito-borne flavivirus that can cause lethal meningoencephalitis. Type I interferon (IFN) plays a critical role in controlling WNV replication, spread, and tropism. In this study, we begin to examine the effector mechanisms by which type I IFN inhibits WNV infection. Mice lacking both the interferon-induced, double-stranded-RNA-activated protein kinase (PKR) and the endoribonuclease of the 2',5'-oligoadenylate synthetase-RNase L system (PKR(-/-) x RL(-/-)) were highly susceptible to subcutaneous WNV infection, with a 90% mortality rate compared to the 30% mortality rate observed in congenic wild-type mice. PKR(-/-) x RL(-/-) mice had increased viral loads in their draining lymph nodes, sera, and spleens, which led to early viral entry into the central nervous system (CNS) and higher viral burden in neuronal tissues. Although mice lacking RNase L showed a higher CNS viral burden and an increased mortality, they were less susceptible than the PKR(-/-) x RL(-/-) mice; thus, we also infer an antiviral role for PKR in the control of WNV infection. Notably, a deficiency in both PKR and RNase L resulted in a decreased ability of type I IFN to inhibit WNV in primary macrophages and cortical neurons. In contrast, the peripheral neurons of the superior cervical ganglia of PKR(-/-) x RL(-/-) mice showed no deficiency in the IFN-mediated inhibition of WNV. Our data suggest that PKR and RNase L contribute to IFN-mediated protection in a cell-restricted manner and control WNV infection in peripheral tissues and some neuronal subtypes.
Descriptors: endoribonucleases, meningoencephalitis, neurons, virus replication genetics, West Nile fever, West Nile virus metabolism, cerebellar cortex, endoribonucleases deficiency, type II interferon metabolism, macrophages, meningoencephalitis, knockout mice, organ specificity, superior cervical ganglion.

Sanchez, M.D., T.C. Pierson, D. McAllister, S.L. Hanna, B.A. Puffer, L.E. Valentine, M.M. Murtadha, J.A. Hoxie, and R.W. Doms (2005). Characterization of neutralizing antibodies to West Nile virus. Virology 336(1): 70-82. ISSN: 0042-6822.
Abstract: We produced nine monoclonal antibodies (MAbs) directed against the West Nile virus E glycoprotein using three different immunization strategies: inactivated virus, naked DNA, and recombinant protein. Most of the MAbs bound to conformation dependent epitopes in domain III of the E protein. Four of the MAbs neutralized WNV infection and bound to the same region of domain III with high affinity. The neutralizing MAbs were obtained from mice immunized with inactivated virus alone or in combination with a DNA plasmid. In contrast, MAbs obtained by immunization with a soluble version of the E glycoprotein did not exhibit neutralizing activity. These non-neutralizing antibodies were cross-reactive with several other flaviviruses, including Saint Louis encephalitis, Japanese encephalitis, Yellow Fever and Powassan viruses. Interestingly, some non-neutralizing MAbs bound with high affinity to domains I or III, indicating that both affinity and the precise epitope recognized by an antibody are important determinants of WNV neutralization.
Descriptors: monoclonal antibodies, viral immunology, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick borne diseases, enzyme linked immunosorbent assay, epitope mapping, glycoproteins, mice, tertiary protein structure, yellow fever virus.

Savage, H.M., M. Anderson, E. Gordon, L. McMillen, L. Colton, D. Charnetzky, M. Delorey, S. Aspen, K. Burkhalter, B.J. Biggerstaff, and M. Godsey (2006). Oviposition activity patterns and West Nile virus infection rates for members of the Culex pipiens complex at different habitat types within the hybrid zone, Shelby County, TN, 2002 (Diptera: Culicidae). Journal of Medical Entomology 43(6): 1227-1238. ISSN: 0022-2585.
Abstract: Oviposition activity and West Nile virus (family Flaviviridae, genus Flavivirus, WNV) infection rates were assessed for members of the Culex pipiens complex from July through December 2002 by using gravid traps placed at four ecologically different sites in the southern portion of the hybrid zone in Shelby County, TN. Molecular assays identified three members of the Cx. pipiens complex: Cx. pipiens pipiens L., Cx. p. quinquefasciatus Say, and Cx. p. pipiens-Cx. p. quinquefasciatus hybrids (hybrids). The Cx. pipiens complex accounted for 90% of mosquitoes collected in gravid traps. All 285 WNV-positive mosquitoes were Culex mosquitoes, and 277 (97%) were Cx. pipiens complex mosquitoes. Infection rates among members of the Cx. pipiens complex were not significantly different. Infection rates were significantly higher at two urban sites than at a rural site, and WNV was not detected at a forested site. At urban sites, abundances of members of the Cx. pipiens complex corresponded to a simple latitude model of the hybrid zone. Cx. p. quinquefasciatus was most abundant (46.4%), followed by hybrids (34.1%) and Cx. p. pipiens (19.5%). The relative abundances at a rural site were reversed with Cx. p. pipiens (48.4%) being most abundant. This demonstrates that spatial habitat variation may profoundly influence the distribution of members of the Cx. pipiens complex within the hybrid zone. Members of the Cx. pipiens complex did not display different oviposition patterns. However, oviposition patterns assessed hourly at urban and rural sites were significantly different. At urban sites, oviposition activity of Cx. pipiens complex mosquitoes was bimodal with an evening peak associated with sunset and a morning peak associated with sunrise. At the rural site, the evening peak was pronounced and the morning peak weak and similar to nighttime activity.
Descriptors: West Nile virus, Culex mosquitoes, traps, habitat variation, infection rates, oviposition activity, evening peak, morning peak.

Sbrana, E., J.H. Tonry, S.Y. Xiao, A.P. da Rosa, S. Higgs, and R.B. Tesh (2005). Oral transmission of West Nile virus in a hamster model. American Journal of Tropical Medicine and Hygiene 72(3): 325-329. ISSN: 0002-9637.
Abstract: The results of experiments comparing the pathogenesis of West Nile virus (WNV) following infection by mosquito bite, needle inoculation, and ingestion are reported. Adult hamsters were readily infected by all three routes. The level and duration of viremia, clinical manifestations, pathology, and antibody response in the hamsters following mosquito infection and needle inoculation were similar; after oral infection, the onset of viremia was delayed and the mortality was lower, but the level and duration of viremia, histopathology, and antibody response were similar to the other routes. The results from this and previously published studies indicate that a wide variety of animal species are susceptible to oral infection with WNV and that orally infected animals develop a viremia and illness similar to that following the bite of infected mosquitoes. Oral infection appears to be an alternative transmission mechanism used by a number of different flaviviruses; its potential role in the natural history of WNV is discussed.
Descriptors: West Nile fever transmission, West Nile virus isolation and purification, viral blood antibodies, Cercopithecus aethiops, cricetinae, culex mosquitoes, disease models, animal, liver virology, mesocricetus, vero cells, viremia physiopathology, hamsters.

Scherbik, S.V., J.M. Paranjape, B.M. Stockman, R.H. Silverman, and M.A. Brinton (2006). RNase L plays a role in the antiviral response to West Nile virus. Journal of Virology 80(6): 2987-2999. ISSN: 0022-538X.
Abstract: Alleles at the Flv locus determine disease outcome after a flavivirus infection in mice. Although comparable numbers of congenic resistant and susceptible mouse embryo fibroblasts (MEFs) are infected by the flavivirus West Nile virus (WNV), resistant MEFs produce approximately 100- to 150-fold lower titers than susceptible ones and flavivirus titers in the brains of resistant and susceptible animals can differ by >10,000-fold. The Flv locus was previously identified as the 2'-5' oligoadenylate synthetase 1b (Oas1b) gene. Oas gene expression is up-regulated by interferon (IFN), and after activation by double-stranded RNA, some mouse synthetases produce 2-5A, which activates latent RNase L to degrade viral and cellular RNAs. To determine whether the lower levels of intracellular flavivirus genomic RNA from resistant mice detected in cells at all times after infection were mediated by RNase L, RNase L activity levels in congenic resistant and susceptible cells were compared. Similar moderate levels of RNase L activation by transfected 2-5A were observed in both types of uninfected cells. After WNV infection, the mRNAs of IFN-beta and three Oas genes were up-regulated to similar levels in both types of cells. However, significant levels of RNase L activity were not detected until 72 h after WNV infection and the patterns of viral RNA cleavage products generated were similar in both types of cells. When RNase L activity was down-regulated in resistant cells via stable expression of a dominant negative RNase L mutant, approximately 5- to 10-times-higher yields of WNV were produced. Similarly, about approximately 5- to 10-times-higher virus yields were produced by susceptible C57BL/6 RNase L-/- cells compared to RNase L+/+ cells that were either left untreated or pretreated with IFN and/or poly(I) . poly(C). The data indicate that WNV genomic RNA is susceptible to RNase L cleavage and that RNase L plays a role in the cellular antiviral response to flaviviruses. The results suggest that RNase L activation is not a major component of the Oas1b-mediated flavivirus resistance phenotype.
Descriptors: endoribonucleases, West Nile virus, 2',5' oligoadenylate synthetase, adenine nucleotides, cell line, cricetinae, enzyme activation, gene expression regulation, interferons pharmacology, mice, mice congenic, mice, virus replication, West Nile virus.

Scholle, F. and P.W. Mason (2005). West Nile virus replication interferes with both poly(I:C)-induced interferon gene transcription and response to interferon treatment. Virology 342(1): 77-87. ISSN: 0042-6822.
Abstract: West Nile virus (WNV), the leading cause of viral encephalitis in the United States, is an arthropod-transmitted member of the family Flaviviridae. We have explored the interaction of this positive-strand RNA virus with signaling pathways involved in induction of the host's innate immune response. Phosphorylation of STAT-1 in response to interferon (IFN) treatment and the ability of IFN to establish an antiviral state were reduced in WNV replicon-bearing cell lines. Similarly, the activation of IRF3 and stimulation of IFN-beta transcription in response to the double-stranded RNA (dsRNA) mimetic poly(I:C) were inhibited in replicon-bearing and WNV-infected HeLa cells. In contrast, WNV replicons did not affect IRF3 activation by Sendai virus infection, suggesting that not all IRF3 activating pathways are inhibited by WNV. Taken together, these findings demonstrate that WNV replication in cultured cells interferes with both the response to IFN and synthesis of IFN-beta in response to dsRNA.
Descriptors: interferon beta genetics, West Nile virus, cell nucleus active transport, dimerization, hela cells, natural immunity, interferon regulatory factor 3, viral genetics, signal transduction, transcription, genetic drug effects, virus replication.

Seidah, N.G. (2006). Unexpected similarity between the cytosolic West Nile virus NS3 and the secretory furin-like serine proteinases. Biochemical Journal 393(Pt 2): E1-E3. ISSN: 0264-6021.
Abstract: Many viral proteins undergo proteolytic processing events that are required for virus infection and virion assembly. In this issue of Biochemical Journal, Strongin and co-workers report that the NS3 protease from West Nile virus unexpectedly cleaves certain substrates at pairs of basic residues, a specificity that resembles that of the furin-like PCs (proprotein convertases). This led to the demonstration that furin/PC inhibitors containing poly(D-arginine) are also potent inhibitors of NS3, and that anthrax toxin protective antigen and myelin basic protein are potential NS3 substrates. Structural modelling based on Dengue virus NS3 provided a possible rationale for the observed cleavage specificity of West Nile virus NS3.
Descriptors: cytosol, furin, viral nonstructural proteins, West Nile virus, RNA helicases antagonists and inhibitors, amino acid sequence, serine endopeptidases, substrate specificity.
Notes: Comment On: Biochem J. 2006 Jan 15;393(Pt 2):503-11.

Seregin, A., R. Nistler, V. Borisevich, G. Yamshchikov, E. Chaporgina, C.W. Kwok, and V. Yamshchikov (2006). Immunogenicity of West Nile virus infectious DNA and its noninfectious derivatives. Virology 356(1-2): 115-125. ISSN: 0042-6822.
Abstract: The exceptionally high virulence of the West Nile NY99 strain makes its suitability in the development of a live WN vaccine uncertain. The aim of this study is to investigate the immunogenicity of noninfectious virus derivatives carrying pseudolethal mutations, which preclude virion formation without affecting preceding steps of the viral infectious cycle. When administered using DNA immunization, such constructs initiate an infectious cycle but cannot lead to a viremia. While the magnitude of the immune response to a noninfectious replication-competent construct was lower than that of virus or infectious DNA, its overall quality and the protective effect were similar. In contrast, a nonreplicating construct of similar length induced only a marginally detectable immune response in the dose range used. Thus, replication-competent noninfectious constructs derived from infectious DNA may offer an advantageous combination of the safety of noninfectious formulations with the quality of the immune response characteristic of infectious vaccines.
Descriptors: West Nile virus, immunogenicity, DNA, infectious, non infectious, virulence, immune response, NY99 strain.

Sheets, R.L., J. Stein, T.S. Manetz, C. Andrews, R. Bailer, J. Rathmann, and P.L. Gomez (2006). Toxicological safety evaluation of DNA plasmid vaccines against HIV-1, Ebola, Severe Acute Respiratory Syndrome, or West Nile virus is similar despite differing plasmid backbones or gene-inserts. Toxicological Sciences 91(2): 620-630.
Abstract: The Vaccine Research Center has developed a number of vaccine candidates for different diseases/infectious agents (HIV-1, Severe Acute Respiratory Syndrome virus, West Nile virus, and Ebola virus, plus a plasmid cytokine adjuvant-IL-2/Ig) based on a DNA plasmid vaccine platform. To support the clinical development of each of these vaccine candidates, preclinical studies were performed to screen for potential toxicities (intrinsic and immunotoxicities). All treatment-related toxicities identified in these repeated-dose toxicology studies have been confined primarily to the sites of injection and seem to be the result of both the delivery method (as they are seen in both control and treated animals) and the intended immune response to the vaccine (as they occur with greater frequency and severity in treated animals). Reactogenicity at the site of injection is generally seen to be reversible as the frequency and severity diminished between doses and between the immediate and recovery termination time points. This observation also correlated with the biodistribution data reported in the companion article (Sheets et al., 2006), in which DNA plasmid vaccine was shown to remain at the site of injection, rather than biodistributing widely, and to clear over time. The results of these safety studies have been submitted to the Food and Drug Administration to support the safety of initiating clinical studies with these and related DNA plasmid vaccines. Thus far, standard repeated-dose toxicology studies have not identified any target organs for toxicity (other than the injection site) for our DNA plasmid vaccines at doses up to 8 mg per immunization, regardless of disease indication (i.e., expressed gene-insert) and despite differences (strengths) in the promoters used to drive this expression. As clinical data accumulate with these products, it will be possible to retrospectively compare the safety profiles of the products in the clinic to the results of the repeated-dose toxicology studies, in order to determine the utility of such toxicology studies for signaling potential immunotoxicities or intrinsic toxicities from DNA vaccines. These data build on the biodistribution studies performed (see companion article, Sheets et al., 2006) to demonstrate the safety and suitability for investigational human use of DNA plasmid vaccine candidates for a variety of infectious disease prevention indications.
Descriptors: DNA vaccines, viral vaccines, acquired immunodeficiency syndrome, ebola like viruses, HIV-1 genetics, hemorrhagic fever, rabbits, severe acute respiratory syndrome, tissue distribution, West Nile fever genetics, West Nile virus.

Sheets, R.L., J. Stein, T.S. Manetz, C. Duffy, M. Nason, C. Andrews, W.P. Kong, G.J. Nabel, and P.L. Gomez (2006). Biodistribution of DNA plasmid vaccines against HIV-1, Ebola, Severe Acute Respiratory Syndrome, or West Nile virus is similar, without integration, despite differing plasmid backbones or gene inserts. Toxicological Sciences 91(2): 610-619.
Abstract: The Vaccine Research Center has developed a number of vaccine candidates for different diseases/infectious agents (HIV-1, Severe Acute Respiratory Syndrome virus, West Nile virus, and Ebola virus, plus a plasmid cytokine adjuvant-IL-2/Ig) based on a DNA plasmid vaccine platform. To support the clinical development of each of these vaccine candidates, preclinical studies have been performed in mice or rabbits to determine where in the body these plasmid vaccines would biodistribute and how rapidly they would clear. In the course of these studies, it has been observed that regardless of the gene insert (expressing the vaccine immunogen or cytokine adjuvant) and regardless of the promoter used to drive expression of the gene insert in the plasmid backbone, the plasmid vaccines do not biodistribute widely and remain essentially in the site of injection, in the muscle and overlying subcutis. Even though approximately 10(14) molecules are inoculated in the studies in rabbits, by day 8 or 9 ( approximately 1 week postinoculation), already all but on the order of 10(4)-10(6) molecules per microgram of DNA extracted from tissue have been cleared at the injection site. Over the course of 2 months, the plasmid clears from the site of injection with only a small percentage of animals (generally 10-20%) retaining a small number of copies (generally around 100 copies) in the muscle at the injection site. This pattern of biodistribution (confined to the injection site) and clearance (within 2 months) is consistent regardless of differences in the promoter in the plasmid backbone or differences in the gene insert being expressed by the plasmid vaccine. In addition, integration has not been observed with plasmid vaccine candidates inoculated i.m. by Biojector 2000 or by needle and syringe. These data build on the repeated-dose toxicology studies performed (see companion article, Sheets et al., 2006) to demonstrate the safety and suitability for investigational human use of DNA plasmid vaccine candidates for a variety of infectious disease prevention indications.
Descriptors: vaccines, viral vaccines, acquired immunodeficiency syndrome, ebola-like viruses, HIV-1, hemorrhagic fever, inbred strains of mice, plasmids, promoter regions, severe acute respiratory syndrome, DNA administration, West Nile virus.

Shirato, K., H. Miyoshi, H. Kariwa, and I. Takashima (2006). The kinetics of proinflammatory cytokines in murine peritoneal macrophages infected with envelope protein-glycosylated or non-glycosylated West Nile virus. Virus Research 121(1): 11-16. ISSN: 0168-1702.
Abstract: The envelope (E) protein glycosylation status of the New York strain of West Nile (WN) virus is an important determinant of virus neuroinvasiveness. To elucidate the determinant of the difference between E protein-glycosylated and non-glycosylated WN virus infections, the cytokine expression of murine peritoneal macrophages infected with each virus was examined. Tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta were up-regulated with replication of the E protein-glycosylated virus. Interferon (IFN) beta and IL-6 were up-regulated with the clearance of both viruses. These results suggest that TNFalpha and IL-1beta expression are related to the virulence of E protein-glycosylated WN virus.
Descriptors: interleukin 1 metabolism, peritoneal macrophages, tumor necrosis factor, alpha metabolism, viral envelope proteins, West Nile fever, cultured cells, glycosylation, inbred Balb C mice, up-regulation.

Shiryaev, S.A., B.I. Ratnikov, A.V. Chekanov, S. Sikora, D.V. Rozanov, A. Godzik, J. Wang, J.W. Smith, Z. Huang, I. Lindberg, M.A. Samuel, M.S. Diamond, and A.Y. Strongin (2006). Cleavage targets and the D-arginine-based inhibitors of the West Nile virus NS3 processing proteinase. Biochemical Journal 393(Pt 2): 503-511. ISSN: 0264-6021.
Abstract: Mosquito-borne WNV (West Nile virus) is an emerging global threat. The NS3 proteinase, which is essential for the proteolytic processing of the viral polyprotein precursor, is a promising drug target. We have isolated and biochemically characterized the recombinant, highly active NS3 proteinase. We have determined that the NS3 proteinase functions in a manner that is distantly similar to furin in cleaving the peptide and protein substrates. We determined that aprotinin and D-arginine-based 9-12-mer peptides are potent inhibitors of WNV NS3 with K(i) values of 26 nM and 1 nM respectively. Consistent with the essential role of NS3 activity in the life cycle of WNV and with the sensitivity of NS3 activity to the D-arginine-based peptides, we showed that nona-D-Arg-NH2 reduced WNV infection in primary neurons. We have also shown that myelin basic protein, a deficiency of which is linked to neurological abnormalities of the brain, is sensitive to NS3 proteolysis in vitro and therefore this protein represents a convenient test substrate for the studies of NS3. A three-dimensional model of WNV NS3 that we created may provide a structural guidance and a rationale for the subsequent design of fine-tuned inhibitors. Overall, our findings represent a foundation for in-depth mechanistic and structural studies as well as for the design of novel and efficient inhibitors of WNV NS3.
Descriptors: arginine analysis, protease inhibitors, viral nonstructural proteins, West Nile virus, amino acid sequence, cultured cells, furin, mice, molecular sequence data, myelin sheath, inhibitors of WNV NS3 proteinase.
Notes: Comment In: Biochem J. 2006 Jan 15;393(Pt 2):e1-3.

Shrestha, B., M.A. Samuel, and M.S. Diamond (2006). CD8+ T cells require perforin to clear West Nile virus from infected neurons. Journal of Virology 80(1): 119-129. ISSN: 0022-538X.
Abstract: Injury to neurons after West Nile virus (WNV) infection is believed to occur because of viral and host immune-mediated effects. Previously, we demonstrated that CD8+ T cells are required for the resolution of WNV infection in the central nervous system (CNS). CD8+ T cells can control infection by producing antiviral cytokines (e.g., gamma interferon or tumor necrosis factor alpha) or by triggering death of infected cells through perforin- or Fas ligand-dependent pathways. Here, we directly evaluated the role of perforin in controlling infection of a lineage I New York isolate of WNV in mice. A genetic deficiency of perforin molecules resulted in higher viral burden in the CNS and increased mortality after WNV infection. In the few perforin-deficient mice that survived initial challenge, viral persistence was observed in the CNS for several weeks. CD8+ T cells required perforin to control WNV infection as adoptive transfer of WNV-primed wild-type but not perforin-deficient CD8+ T cells greatly reduced infection in the brain and spinal cord and enhanced survival of CD8-deficient mice. Analogous results were obtained when wild-type or perforin-deficient CD8+ T cells were added to congenic primary cortical neuron cultures. Taken together, our data suggest that despite the risk of immunopathogenesis, CD8+ T cells use a perforin-dependent mechanism to clear WNV from infected neurons.
Descriptors: West Nile virus, infected neurons, perforin, CD8+T cells, host immune mediated effects, central nervous system, genetic deficiency.

Shrestha, B., T. Wang, M.A. Samuel, K. Whitby, J. Craft, E. Fikrig, and M.S. Diamond (2006). Gamma interferon plays a crucial early antiviral role in protection against West Nile virus infection. Journal of Virology 80(11): 5338-5348. ISSN: 0022-538X.
Abstract: West Nile virus (WNV) causes a severe central nervous system (CNS) infection in humans, primarily in the elderly and immunocompromised. Prior studies have established an essential protective role of several innate immune response elements, including alpha/beta interferon (IFN-alpha/beta), immunoglobulin M, gammadelta T cells, and complement against WNV infection. In this study, we demonstrate that a lack of IFN-gamma production or signaling results in increased vulnerability to lethal WNV infection by a subcutaneous route in mice, with a rise in mortality from 30% (wild-type mice) to 90% (IFN-gamma(-/-) or IFN-gammaR(-/-) mice) and a decrease in the average survival time. This survival pattern in IFN-gamma(-/-) and IFN-gammaR(-/-) mice correlated with higher viremia and greater viral replication in lymphoid tissues. The increase in peripheral infection led to early CNS seeding since infectious WNV was detected several days earlier in the brains and spinal cords of IFN-gamma(-/-) or IFN-gammaR(-/-) mice. Bone marrow reconstitution experiments showed that gammadelta T cells require IFN-gamma to limit dissemination by WNV. Moreover, treatment of primary dendritic cells with IFN-gamma reduced WNV production by 130-fold. Collectively, our experiments suggest that the dominant protective role of IFN-gamma against WNV is antiviral in nature, occurs in peripheral lymphoid tissues, and prevents viral dissemination to the CNS.
Descriptors: West Nile virus, gamma interferon, infection, antiviral, CNS, mice, alpha-beta interferon, virus replication, dendritic cells.

Siddharthan, V., G.J. Roper, H. Wang, A.L. Olsen, L. Preece, C. Andy, B. Taro, and J.D. Morrey (2006). Identification of West Nile virus-infected cells in the central nervous system of rodents early in infection: Implications for treatment. Antiviral Research 70(1): A79. ISSN: 0166-3542.
Descriptors: West Nile virus infected cells, central nervous system, rodents, treatment, confocal microscopy, immunofluorescence.
Notes: Meeting Information: 19th International Conference on Antiviral Research, San Juan, Puerto Rico; May 7 -11, 2006.

Siger, L., R. Bowen, K. Karaca, M. Murray, S. Jagannatha, B. Echols, R. Nordgren, and J.M. Minke (2006). Evaluation of the efficacy provided by a Recombinant Canarypox-Vectored Equine West Nile Virus vaccine against an experimental West Nile Virus intrathecal challenge in horses. Veterinary Therapeutics Research in Applied Veterinary Medicine 7(3): 249-256.
Abstract: Efficacy of the Recombitek Equine West Nile Virus (WNV) vaccine was evaluated against a WNV intrathecal challenge model that results in WNV-induced clinical disease. Ten vaccinated (twice at days 0 and 35) and 10 control horses were challenged 2 weeks after administration of the second vaccine with a virulent WNV by intrathecal administration. After the challenge, eight of 10 controls developed clinical signs of encephalomyelitis whereas one vaccinate exhibited muscle fasciculation only once. Nine controls and one vaccinate developed a fever. Histopathology revealed mild to moderate nonsuppurative encephalitis in eight controls and one vaccinate. None of the vaccinates and all of the controls developed WNV viremia after challenge. All vaccinated horses developed antibodies to WNV after vaccination. These and results of previous studies demonstrate efficacy of the Recombitek WNV vaccine against WNV-induced clinical disease and natural challenge with WNV-infected mosquitoes.
Descriptors: Recombitek Equine West Nile Virus vaccine, WNV intrathecal challenge model, clinical studies, horses, development of encephalomyelitis, histopathology, antibodies to WNV, mosquito vectors.

Sitati, E.M. and M.S. Diamond (2006). CD4+ T-cell responses are required for clearance of West Nile virus from the central nervous system. Journal of Virology 80(24): 12060-12069. ISSN: 0022-538X.
Abstract: Although studies have established that innate and adaptive immune responses are important in controlling West Nile virus (WNV) infection, the function of CD4(+) T lymphocytes in modulating viral pathogenesis is less well characterized. Using a mouse model, we examined the role of CD4(+) T cells in coordinating protection against WNV infection. A genetic or acquired deficiency of CD4(+) T cells resulted in a protracted WNV infection in the central nervous system (CNS) that culminated in uniform lethality by 50 days after infection. Mice surviving past day 10 had high-level persistent WNV infection in the CNS compared to wild-type mice, even 45 days following infection. The absence of CD4(+) T-cell help did not affect the kinetics of WNV infection in the spleen and serum, suggesting a role for CD4-independent clearance mechanisms in peripheral tissues. WNV-specific immunoglobulin M (IgM) levels were similar to those of wild-type mice in CD4-deficient mice early during infection but dropped approximately 20-fold at day 15 postinfection, whereas IgG levels in CD4-deficient mice were approximately 100- to 1,000-fold lower than in wild-type mice throughout the course of infection. WNV-specific CD8(+) T-cell activation and trafficking to the CNS were unaffected by the absence of CD4(+) T cells at day 9 postinfection but were markedly compromised at day 15. Our experiments suggest that the dominant protective role of CD4(+) T cells during primary WNV infection is to provide help for antibody responses and sustain WNV-specific CD8(+) T-cell responses in the CNS that enable viral clearance.
Descriptors: West Nile virus, CNS, clearance, CD4+T cell responses, immune responses, spleen, serum, viral clearance.

Steinman, A., C. Banet Noach, L. Simanov, N. Grinfeld, Z. Aizenberg, O. Levi, D. Lahav, M. Malkinson, S. Perk, and N.Y. Shpigel (2006). Experimental Infection of Common Garter Snakes (Thamnophis sirtalis) with West Nile Virus. Vector Borne and Zoonotic Diseases 6(4): 361-368. ISSN: 1530-3667.
Abstract: The role of various reptilian species in the infectious cycle of several arboviruses is documented, but their role in that of West Nile virus (WNV) is uncertain. Common garter snakes (Thamnophis sirtalis) were infected subcutaneously with 10(5) plaque forming units (PFU) WNV-Isr 98, five of nine snakes became viremic, and five exhibited persistent low levels of neutralizing antibodies. Four of the parentally infected snakes died and high titers of virus were found in multiple organ samples. In contrast, orally infected garter snakes did not become viremic, but viral RNA was detected in cloacal swabs. Since oral infection of predator birds by WNV is known, their ingestion of infected snakes may also result in their becoming infected.
Descriptors: reptilian species, West Nile virus, garter snakes, subcutaneous infection, oral infection, viremic, RNA, predator birds.

Styer, L.M., K.A. Bernard, and L.D. Kramer (2006). Enhanced early West Nile virus infection in young chickens infected by mosquito bite: Effect of viral dose. American Journal of Tropical Medicine and Hygiene 75(2): 337-345. ISSN: 0002-9637.
Abstract: Mosquito transmission of arboviruses potentially affects the course of viral infection in the vertebrate host. Studies were performed to determine if viral infection differed in chickens infected with West Nile virus (WNV) by mosquito bite or needle inoculation. Mosquito-infected chickens exhibited levels of viremia and viral shedding that were up to 1,000 times higher at 6, 12, and 24 hours post-feeding (PF) compared with those inoculated with 10(3) PFU by needle. Follow-up studies were conducted to determine if enhanced early infection was due to a higher viral dose inoculated by mosquitoes. Needle inoculation with successively higher doses of WNV led to higher early viremia and viral shedding; a dose >or= 10(4) PFU by needle was required to attain the high early viremia observed in mosquito-infected chickens. Mosquitoes inoculated WNV at this level as estimated by feeding on a hanging drop of blood (mean: 10(2.5), range: 10(0.7)-10(4.6) PFU). These results indicate that enhanced early infection in mosquito-infected chickens may be explained by higher viral dose delivered by mosquitoes. On the other hand, chickens infected by multiple mosquitoes (N = 3-11) had viremic titers that were 25-50 times higher at 6 and 12 hours PF than in chickens infected by a single mosquito, suggesting that viral dose is not the only factor involved in enhanced early infection. The likelihood that enhanced early infection in mosquito-infected chickens is due to a higher viral dose inoculated by mosquitoes and/or other factors (saliva, inoculation location, or viral source) is discussed.
Descriptors: chickens, culex mosquitos, insect vectors, poultry diseases, West Nile fever, West Nile virus, viral blood, Cercopithecus aethiops, cloaca, subcutaneous injections, insect bites and stings, poultry diseases , specific pathogen free organisms, vero cells.

Teehee, M.L., M.L. Bunning, S. Stevens, and R.A. Bowen (2005). Experimental infection of pigs with West Nile virus. Archives of Virology 150(6): 1249-12. ISSN: 0304-8608.
Abstract: Young adult and weanling pigs were challenged with the New York 99 strain of West Nile virus through the bite of infected mosquitoes. Each of six adult pigs seroconverted, but virus was isolated from serum of only one pig following challenge. Three of five weanling pigs developed viremia, with peak titers of 10(1.9) and 10(3.1) PFU/mL. Clinical signs attributable to West Nile virus infection were not observed in any of these animals. An additional four pigs were challenged by feeding West Nile virus-infected mice, and none of the four developed a detectable viremia or seroconverted. These results suggest that pigs are unlikely to play a significant role as amplifying hosts of West Nile virus.
Descriptors: West Nile fever transmission, West Nile virus, infected mosquitoes, viral blood antibodies, mice, neutralization tests, swine, viremia.

Temple, J.S., L. Coleman, and M.B. Doughty (2005). Cloning and expression of West Nile virus RNA polymerase. Abstracts of Papers American Chemical Society 230: U512. ISSN: 0065-7727.
Descriptors: West Nile virus, cloning and expression, RNA polymerase, molecular genetics, polymerase chain reaction techniques.
Notes: Meeting Information: 230th National Meeting of the American Chemical Society, Washington, DC, USA; August 28 -September 1, 2005.

Tesh, R.B., M. Siirin, H. Guzman, A.P. Travassos da Rosa, X. Wu, T. Duan, H. Lei, M.R. Nunes, and S.Y. Xiao (2005). Persistent West Nile virus infection in the golden hamster: studies on its mechanism and possible implications for other flavivirus infections. Journal of Infectious Diseases 192(2): 287-295.
Abstract: Golden hamsters (Mesocricetus auratus) experimentally infected with West Nile virus (WNV) developed chronic renal infection and persistent shedding of virus in urine for up to 8 months, despite initial rapid clearance of virus from blood and the timely appearance of high levels of specific neutralizing antibodies. Infectious WNV could be recovered by direct culture of their urine and by cocultivation of kidney tissue for up to 247 days after initial infection. Only moderate histopathologic changes were observed in the kidneys or brain of the chronically infected hamsters, although WNV antigen was readily detected by immunohistochemistry within epithelium, interstitial cells, and macrophages in the distal renal tubules. Comparison of WNV isolates from serial urine samples from individual hamsters over several months indicated that the virus underwent both genetic and phenotypic changes during persistent infection. These findings are similar to previous reports of persistent infection with tickborne encephalitis and Modoc viruses.
Descriptors: flavivirus infections, West Nile fever, viral blood antibodies, antibody formation, animal disease models, enzyme linked immunosorbent assay, kidney, Mesocricetus auratus, golden hamster, viremia immunology, West Nile virus.

Throsby, M., C. Geuijen, J. Goudsmit, A.Q. Bakker, J. Korimbocus, R.A. Kramer, M. Clijsters van der Horst, M. de Jong, M. Jongeneelen, S. Thijsse, R. Smit, T.J. Visser, N. Bijl, W.E. Marissen, M. Loeb, D.J. Kelvin, W. Preiser, J. ter Meulen, and J. de Kruif (2006). Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile Virus. Journal of Virology 80(14): 6982-6992. ISSN: 0022-538X.
Abstract: Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 mug/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.
Descriptors: monoclonal antibodies immunology, viral immunology, viral envelope proteins, West Nile fever, West Nile virus, monoclonal genetics, viral genetics, antibody specificity, molecular cloning, mice, protein structure.

Tiawsirisup, S., K.B. Platt, R.B. Evans, and W.A. Rowley (2005). A comparision of West Nile Virus transmission by Ochlerotatus trivittatus (COQ.), Culex pipiens (L.), and Aedes albopictus (Skuse). Vector Borne and Zoonotic Diseases 5(1): 40-47. ISSN: 1530-3667.
Abstract: Transmission of West Nile virus (WNV) by Ochlerotatus trivittatus, Culex pipiens, and Aedes albopictus were compared 14 days after taking blood meals from viremic chickens with titers ranging from 10(2.5) to 10(9.5) cell infective dose (50)s (CID50s)/mL serum. Transmission occurred in one of four (25%) Oc. trivittatus and one of 25 (4%) Cx. pipiens that fed on chickens with titers of 10(5.5) CID50s/mL. No transmission occurred among two of 16 (13%) Oc. trivittatus or one of 25 (4%) Cx. pipiens that became infected after blood meals with titers of 10(5.0) and 10(4.5) CID50s/mL, the next lowest blood meal titers evaluated. Seventeen of 28 (61%) Ae. albopictus transmitted WNV after blood meals with titers of 10(7.0) CID50s/mL, but no infection or transmission was observed among 21 Ae. albopictus that fed on chickens with titers of 10(5.0) CID50s/mL, the next lowest titer evaluated. Transmission by all three species increased dramatically after blood meals with WNV titers of > or = 10(5.5) CID50s/mL. No significant differences occurred in dissemination and transmission rates of the three species after taking blood meals with titers of > 10(7.0) CID50s/mL. The cumulative mean +/- SE transmission rates of Oc. trivittatus, Cx. pipiens, and Ae. albopictus after blood meals with titers of > or = 10(7.0) CID50s/mL were 45.5 +/- 4.1%, 46.8 +/- 4.5%, and 72.4 +/- 5.5%. The cumulative mean dissemination rates of the three species were 78.3 +/- 6.7%, 74.8 +/- 2.6%, and 88.6 +/- 2.1%. The rates of transmission by the three species that developed disseminated infections after blood meals with titers of > or = 10(7.0) CID50s/mL were 58.8 +/- 4.4%, 62.6 +/- 5.8%, and 81.6 +/- 5.4%, respectively. In a previous study, we found that susceptibility of the three species to WNV was essentially the same when fed on chickens with WNV titers of > 10(7.0) CID50s/mL, but Oc. trivittatus and Cx. pipiens were more susceptible than Ae. albopictus to WNV at lower virus titers. The current study strongly suggests that Ae. albopictus is a more efficient vector than Oc. trivittatus and Cx. pipiens when fed blood meals with titers of > 10(7.0) CID50s/mL. However, Oc. trivittatus and Cx. pipiens might be more efficient as vectors when infected by blood meals with titers of < 10(7.0) CID50s/mL.
Descriptors: insect vectors, West Nile fever transmission, West Nile virus isolation and purification, chickens, culex mosquitoes, disease susceptibility, disease vectors, ochlerotatus, species specificity, viral load.

Tiawsirisup, S., K.B. Platt, F. Fabiosa, and W.A. Rowley (2006). West Nile virus titers in Aedes vexans (Meigen) and Culex pipiens (L.) saliva. Thai Journal of Veterinary Medicine 36(1): 87. ISSN: 0125-6491.
Descriptors: West Nile virus titers, mosquito saliva, blood meals, virus isolation, titration plaque assay, Aedes vexans, Culex pipiens, birds.
Notes: Meeting Information: Proceedings of the Annual Conference of the Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand; April 27-28 , 2006.

Tiawsirisup, S., K.B. Platt, B.J. Tucker, and W.A. Rowley (2005). Eastern cottontail rabbits (Sylvilagus floridanus) develop West Nile virus viremias sufficient for infecting select mosquito species. Vector Borne and Zoonotic Diseases 5(4): 342-350. ISSN: 1530-3667.
Abstract: The potential of the eastern cottontail rabbit (CTR; Sylvilagus floridanus) to contribute to an enzootic West Nile virus (WNV) cycle was demonstrated by characterizing the WNV viremia profile of 15 CTRs and demonstrating that mosquitoes could become infected by feeding on these CTRs. Eight CTRs were infected with a titer of 10(5.0) cell-infectious dose 50% endpoints (CID50s) of WNV (NY99-Crow) by needle and seven CTRs by bite of one or more WNV-infected mosquitoes. There were no marked differences between the WNV viremia profiles of CTRs infected by either method. West Nile virus was detected in serums of all CTRs by 24 h p.i. The daily mean titers of all 15 CTRs on days 1-4 p.i. were 10(4.1+/-0.4), 10(4.7+/-0.3), 10(4.1+/-0.6), and 10(3.7+/-0.6) respectively, declining to 10(1.2+/-0.1) CID50s/ml of serum by day 6 p.i. No virus was detected in the blood of any CTR on day 7 p.i. The average duration of WNV titers of >or=10(4.3) and <10(5.0) CID50s/mL for all CTRs was 2.2 +/- 0.6 and 1.0 +/- 0.1 days, respectively. The minimum estimated infection rates (MEIRs) of Culex pipiens (L.) and Culex salinarius (Coq.) that fed on CTRs with titers of >or=10(4.3) and >10(5.0) were 11.5 +/- 5.5 and 21 +/- 6.0%, respectively. These rates increased to 20.5 +/- 6.4% and 25.0 +/- 3.0% when CTR serum titers were >10(5.0) CID50s/mL. Neither Aedes aegypti (L.) nor Aedes albopictus (Skuse) were infected by feeding on CTRs with titers of <10(5.0) CID50s/mL. The MEIRs of these two species were 11.5 +/- 3.5% and 1.5 +/- 0.5% after feeding on CTRs with titers of >10(5.0) CID50s/ml. None of the CTRs infected by mosquito bite or by needle showed any symptoms of WNV disease.
Descriptors: cottontail rabbit, West Nile virus, viremia, Culex mosquitos, infection rates.

Tilley, P.A., J.D. Fox, G.C. Jayaraman, and J.K. Preiksaitis (2006). Nucleic acid testing for west nile virus RNA in plasma enhances rapid diagnosis of acute infection in symptomatic patients. Journal of Infectious Diseases 193(10): 1361-1364.
Abstract: Although nucleic acid amplification testing (NAAT) for West Nile virus (WNV) is useful in screening blood donors, such methods have not been studied in symptomatic patients. For diagnosis of WNV infection, 1.0 mL of plasma was tested by NAAT, and WNV-specific immunoglobulin M was assayed. Of 276 WNV cases, 191 were tested by both serology and NAAT. Of these, 86 (45.0%), 111 (58.1%), and 180 (94.2%) were detected by NAAT, serology, and combined NAAT and serology, respectively. NAAT-based screening was most useful within 8 days of the onset of symptoms. Viremia is common in early symptomatic WNV infection, and NAAT enhances diagnostic yield.
Descriptors: nucleic acid amplification techniques, viral RNA analysis, West Nile fever, West Nile virus isolation and purification, Alberta, routine diagnostic tests, immunoglobulin M, predictive value of tests, viral blood RNA, reverse transcriptase polymerase chain reaction, specimen handling, viremia diagnosis.

Tobler, L.H., C. Bianco, S.A. Glynn, G.B. Schreiber, B.J. Dille, H.E. Prince, R.S. Lanciotti, J.M. Linnen, J. Gallarda, V. Shyamala, D. Smith, S.H. Kleinman, and M.P. Busch (2005). Detection of West Nile virus RNA and antibody in frozen plasma components from a voluntary market withdrawal during the 2002 peak epidemic. Transfusion 45(4): 480-486.
Abstract: BACKGROUND: The US West Nile virus (WNV) epidemic in the summer and fall of 2002 included the first documented cases of transfusion-transmitted WNV infection. In December 2002, the FDA supported a voluntary market withdrawal by the blood banking community of frozen blood components collected in WNV high-activity areas. At the time, the prevalence of viremia and serologic markers for WNV in the blood supply was undefined. STUDY DESIGN AND METHODS: In collaboration with America's Blood Centers, 1468 frozen plasma components (of approx. 60,000 frozen units voluntarily withdrawn from the market) were selectively retrieved from the peak epidemic regions and season (June 23, 2002-September 28, 2002). These units were unlinked, subaliquoted, and tested by WNV enzyme immunoassays (EIAs; Focus Technologies and Abbott Laboratories) and nucleic acid amplification tests (NATs; Gen-Probe Inc. and Roche Molecular Systems). RESULTS: Of the 1468 EIA results from Abbott and Focus, 7 were anti-immunoglobulin M (IgM)- and anti-immunoglobulin G (IgG)-reactive by both assays, 8 and 1 were IgM-only-reactive, and 8 and 23 were IgG-only-reactive, respectively. NAT by Gen-Probe and Roche Molecular Systems yielded one RNA-positive, antibody-negative unit containing approximately 440 RNA copies per mL. An additional 10-fold replicate NAT testing by Gen-Probe on 14 of 15 IgM-reactive specimens yielded 2 additional IgM- and IgG-reactive units with low-level viremia (i.e., 7/10 and 2/10 replicates tested reactive). CONCLUSION: The prevalence of acute (RNA-positive) and recent (IgM-seroreactive) WNV infections indicates that transfusion risk in high-risk areas could have been considerable and that voluntary market withdrawal of frozen components likely averted some WNV transfusion transmissions. The existence of very-low-level viremic units raises concerns, because WNV minipool NAT screening will miss such units and individual NAT may not completely correct this situation.
Descriptors: blood banks, plasma virology, West Nile fever epidemiology, West Nile virus isolation and purification, viral blood antibodies, consumer product safety, disease outbreaks, viral analysis, risk factors, seroepidemiologic studies.
Notes: Comment In: Transfusion. 2005 Apr;45(4):460-2.

Tonry, J.H., S.Y. Xiao, M. Siirin, H. Chen, A.P. da Rosa, and R.B. Tesh (2005). Persistent shedding of West Nile virus in urine of experimentally infected hamsters. American Journal of Tropical Medicine and Hygiene 72(3): 320-324. ISSN: 0002-9637.
Abstract: Adult hamsters that survived experimental West Nile virus (WNV) infection developed persistent viruria. Infectious WNV could be cultured from their urine for up to 52 days. Immunohistochemical examination of kidneys of viruric animals showed foci of WNV antigen in renal tubular epithelial and vascular endothelial cells. These findings are compatible with virus replication and persistent infection of renal epithelial cells. The potential clinical and virologic significance of these findings as well as their possible epidemiologic importance are discussed.
Descriptors: virus shedding, West Nile fever transmission, antigens, viral analysis, cricetinae, disease models, animal, kidney pathology, liver virology, mesocricetus, West Nile virus isolation and purification.

Torrence, P.F., N. Gupta, C. Whitney, and J.D. Morrey (2006). Evaluation of synthetic oligonucleotides as inhibitors of West Nile virus replication. Antiviral Research 70(2): 60-65.
Abstract: A series of synthetic oligonucleotide phosphorothioate 15-mers were generated against specific sequences in the West Nile virus RNA genome. These antisense oligonucleotides targeted (1) conserved features of the West Nile virus RNA genome that may be expected to lead to inhibition of virus replication since such features play essential roles in the virus lifecycle; (2) G-quartet oligonucleotides with potential facilitated uptake properties and that also targeted conserved sequences among a range of West Nile virus strains. Several formulations with significant in vitro antiviral activity were found. Among the active oligonucleotides were examples that targeted both C-rich RNA sequences of the West Nile RNA genome as well as recognized conserved sequences key to West Nile virus replication. Since the antiviral activity of the latter oligonucleotides diminished upon 2'-O-methyl substitution, it is likely that their activity involves RNase H-catalyzed RNA degradation. One G-rich oligonucleotide that did not target a West Nile virus RNA sequence also was found. These results suggest the potential of antisense strategies for the control of West Nile virus replication if the attendant problem of oligonucleotide delivery can be adequately addressed.
Descriptors: oligonucleotides, antisense genetics, virus replication, West Nile virus, Cercopithecus aethiops, vero cells.

Tyler, K.L., J. Pape, R.J. Goody, M. Corkill, and B.K. Kleinschmidt DeMasters (2006). CSF findings in 250 patients with serologically confirmed West Nile virus meningitis and encephalitis. Neurology 66(3): 361-365.
Abstract: OBJECTIVE: To provide a large, comprehensive evaluation of the CSF findings in patients with serologically confirmed West Nile virus (WNV), CNS disease, and their correlation with outcome. METHODS: CSF samples from 334 WNV-infected hospitalized patients were analyzed. Information was available and extracted from the medical records of 250 of these patients, and CSF parameters correlated with clinical and epidemiologic features of disease (e.g., patient age, sex, outcome). RESULTS: Patients with meningitis had a mean of 226 cells/mm3, and those with encephalitis had a mean of 227 cells/mm3. Three percent of meningitis patients and 5% of encephalitis patients had fewer than 5 cells/mm3, and approximately 8% of both groups had more than 500 cells/mm3. Patients with meningitis had a mean of 41% neutrophils, and those with encephalitis had 45%. Forty-five percent of meningitis patients and 37% of encephalitis patients had at least 50% neutrophils in their initial CSF specimen. Neither the mean percent neutrophils nor their distribution differed significantly between groups. Forty-seven percent of encephalitis patients and 16% of meningitis patients had CSF protein of 100 mg/dL or greater (p < 0.01). Although specific CSF parameters, including nucleated cell count and protein concentration, correlated significantly with outcome, multivariate analysis suggested that their total predictive value was modest. Age was an additional predictor of outcome independent of CSF variables in all patients. CONCLUSIONS: Serologically confirmed West Nile virus meningitis and encephalitis produce similar degrees of CSF pleocytosis and are frequently associated with substantial CSF neutrophilia. Patients with encephalitis have higher CSF protein concentrations and are more likely to have adverse outcomes, including admission to long-term care facilities or even death after their acute illness. CSF findings were only a modest predictor of disease outcome, with patient age adding important independent prognostic information.
Descriptors: encephalitis, cerebrospinal fluid, viral meningitis, West Nile fever, age factors, cell count, leukocytosis, multivariate analysis, osmolar concentration, predictive value of tests, serologic tests, treatment outcome.

Ujjinamatada, R.K., Y.S. Agasimundin, P. Zhang, R.S. Hosmane, R. Schuessler, P. Borowski, K. Kalicharran, and A. Fattom (2005). A novel imidazole nucleoside containing a diaminodihydro-S-triazine as a substituent: Inhibitory activity against the West Nile virus NTPase/helicase. Nucleosides, Nucleotides and Nucleic Acids 24(10-12): 1775-1788.
Abstract: The attempted synthesis of a ring-expanded guanosine (1) containing the imidazo[4,5-e][1,3]diazepine ring system by condensation of 1-(2'-deoxy-beta-D-erythropentofuranosyl)-4-ethoxycarbonylimidazole-5-carbaldehyde (2) with guanidine resulted in the formation of an unexpected product, 1-(2'-deoxy-beta-D-erythropentofuranosyl)-5-(2, 4-diamino-3, 6-dihydro-1,3, 5-triazin-6-yl)imidazole-4-carboxamide (7). The structure as well as the pathway of formation of 7 was corroborated by isolation of the intermediate, followed by its conversion to the product. Nucleoside 7 showed promising in vitro anti-helicase activity against the West Nile virus NTPase/helicase with an IC50 of 3-10 microg/mL.
Descriptors: antiviral agents, imidazoles, RNA helicases, viral proteins, West Nile virus, dose-response relationship, guanosine, nucleoside triphosphatases.

Vaidyanathan, R. and T.W. Scott (2006). Apoptosis in mosquito midgut epithelia associated with West Nile virus infection. Apoptosis 11(9): 1643-1651. ISSN: 1360-8185.
Abstract: The mosquito Culex pipiens pipiens is a documented vector of West Nile virus (WNV, Flaviviridae, Flavivirus). Our laboratory colony of C. p. pipiens, however, was repeatedly refractory to experimental transmission of WNV. Our goal was to identify if a cellular process was inhibiting virus infection of the midgut. We examined midguts of mosquitoes fed control and WNV-infected blood meals. Three days after feeding, epithelial cells from abdominal midguts of mosquitoes fed on WNV fluoresced under an FITC filter following Acridine Orange staining, indicating apoptosis in this region. Epithelial cells from experimental samples examined by TEM exhibited ultrastructural changes consistent with apoptosis, including shrinkage and detachment from neighbors, heterochromatin condensation, nuclear degranulation, and engulfment of apoptotic bodies by adjacent cells. Virions were present in cytoplasm and within cytoplasmic vacuoles of apoptotic cells. No apoptosis was detected by TEM in control samples. In parallel, we used Vero cell plaque assays to quantify infection after 7 and 10 day extrinsic incubation periods and found that none of the mosquitoes (0/55 and 0/10) which imbibed infective blood were infected. We propose that programmed cell death limits the number of WNV-infected epithelial cells and inhibits disseminated viral infections from the mosquito midgut.
Descriptors: apoptosis, culex mosquitoes, intestinal mucosa, West Nile virus, Cercopithecus aethiops, insect vectors, electron transmission microscopy.

Venter, M., T.G. Myers, M.A. Wilson, T.J. Kindt, J.T. Paweska, F.J. Burt, P.A. Leman, and R. Swanepoel (2005). Gene expression in mice infected with West Nile virus strains of different neurovirulence. Virology 342(1): 119-140. ISSN: 0042-6822.
Abstract: West Nile virus causes febrile illness in humans with a proportion of cases progressing to meningoencephalitis, encephalitis, hepatitis, and death. Isolates of the virus fall into two genetic lineages, with differences in neuroinvasiveness for mice occurring between strains within both lineages. We used DNA microarrays to compare gene expression in mice infected peripherally with seven lineage 1 and 2 strains confirmed to be of either high or low neuroinvasiveness in mice and associated with severe or benign infection in humans and birds. The 4 strains with highest neuroinvasiveness induced increased expression of 47 genes in the brain, 111 genes in the liver, and 70 genes in the spleen, relative to the 3 least neuroinvasive strains. Genes involved in interferon signaling pathways, protein degradation, T-cell recruitment, MHC class I and II antigen presentation, and apoptosis were identified that may have both pathogenic and protective effects, but increased expression of certain acute proteins, central nervous system specific proteins and proteins associated with T-cell hepatitis, implicate mechanisms related to exalted virulence.
Descriptors: gene expression, West Nile fever genetics, West Nile virus, brain metabolism, gene expression profiling, liver metabolism, mice, oligonucleotide array sequence analysis, reverse transcriptase polymerase chain reaction, spleen metabolism.

Vijayasri, S. and S. Agrawal (2005). Domain-based homology modeling and mapping of the conformational epitopes of envelope glycoprotein of West Nile virus. Journal of Molecular Modeling Online 11(3): 248-255.
Abstract: Knowledge-based modeling has proved significantly accurate for generating the quality models for proteins whose sequence identity with the structurally known targets is greater than or equal to 40%. On the other hand, models obtained for low sequence identities are not reliable. Hence, a reliable and alternative strategy that uses knowledge of domains in the protein can be used to improve the quality of the model generated by the homology method. Here, we report a method for developing a 3D-model for the envelope glycoprotein (Egp) of west nile virus (WNV), using knowledge of structurally conserved functional domains amongst the target sequence (Egp of WNV) and its homologous templates belonging to the same protein family, flaviviridae. This strategy is found to be highly effective in reducing the root mean square deviation (RMSD) value at the Calpha positions of the target and its experimental homologues. The 3D structure of a protein is a prerequisite for structure-based drug design as well as for identifying the conformational epitopes that are essential for the designing vaccines. The conformational epitopes are mapped from the 3D structure of Egp of WNV modeled using the concept of an antigenic domain. A total of five such epitope regions/sites have been identified. They have been found distributed in the loop regions (surface) of the whole protein model composed of dimerization, central and immunological domains. These sites are proposed as the binding sites for HLA proteins/B-cell receptors. Binding is required to activate the immune response against WNV.
Descriptors: epitope mapping, gene products, 3D models, envelope glycoprotein (Egp), West Nile virus .

Vinayagamoorthy, T., K. Mulatz, M. Drebot, and R. Hodkinson (2005). Molecular typing of West Nile Virus, Dengue, and St. Louis encephalitis using multiplex sequencing. Journal of Molecular Diagnostics 7(2): 152-159.
Abstract: We report the development of an assay to simultaneously identify three of the clinically important flaviviruses (West Nile Virus, Dengue, and St. Louis encephalitis). This assay is based on the nucleotide sequence variations within a 266-bp region of the non-structural protein 5. Further, based on the nucleotide variations in the same region of the non-structural protein 5, four of the present Dengue serotypes were identified. To identify some of the subtypes of WNV we have developed a second assay using multiplex sequencing technology. The format of the result of this assay is an electropherogram of two genomic segments of the WNV genome: a 48-nucleotide sequence from the anchored core protein C and a 45-nucleotide sequence coding for the non-structural proteins (proteinase and putative helicase genes).
Descriptors: dengue virus, St. Louis encephalitis virus, viral genome, DNA sequence analysis, West Nile virus, DNA base sequence, viral genetics, molecular sequence data, variation genetics, West Nile virus classification.

Wang, T., Y. Gao, E. Scully, C.T. Davis, J.F. Anderson, T. Welte, M. Ledizet, R. Koski, J.A. Madri, A. Barrett, Z. Yin, J. Craft, and E. Fikrig (2006). Gamma delta T cells facilitate adaptive immunity against West Nile virus infection in mice. Journal of Immunology 177(3): 1825-1832.
Abstract: West Nile (WN) virus causes fatal meningoencephalitis in laboratory mice, and gammadelta T cells are involved in the protective immune response against viral challenge. We have now examined whether gammadelta T cells contribute to the development of adaptive immune responses that help control WN virus infection. Approximately 15% of TCRdelta(-/-) mice survived primary infection with WN virus compared with 80-85% of the wild-type mice. These mice were more susceptible to secondary challenge with WN virus than the wild-type mice that survived primary challenge with the virus. Depletion of gammadelta T cells in wild-type mice that survived the primary infection, however, does not affect host susceptibility during secondary challenge with WN virus. Furthermore, gammadelta T cells do not influence the development of Ab responses during primary and at the early stages of secondary infection with WN virus. Adoptive transfer of CD8(+) T cells from wild-type mice that survived primary infection with WN virus to naive mice afforded partial protection from lethal infection. In contrast, transfer of CD8(+) T cells from TCRdelta(-/-) mice that survived primary challenge with WN virus failed to alter infection in naive mice. This difference in survival correlated with the numeric and functional reduction of CD8 memory T cells in these mice. These data demonstrate that gammadelta T cells directly link innate and adaptive immunity during WN virus infection.
Descriptors: gamma delta T cells, T lymphocyte subsets, West Nile fever, West Nile virus, adoptive transfer, CD8 positive T lymphocytes, genetic predisposition to disease genetics, cellular genetics, immunoglobulin G biosynthesis, immunoglobulin M biosynthesis, immunologic memory genetics, lymphocyte depletion, knockout inbred c57bl, mice, receptors.

Ward, M.P. (2006). Spread of equine West Nile virus encephalomyelitis during the 2002 Texas epidemic. American Journal of Tropical Medicine and Hygiene 74(6): 1090-1095. ISSN: 0002-9637.
Abstract: Using reports of clinical West Nile virus (WNV) encephalomyelitis in Texas equids during 2002, the distribution of disease was analyzed using cluster statistics and spatial modeling to develop hypotheses of disease spread during the first year of its detection. Significant (P < 0.05) clusters of cases reported early during the outbreak were identified in east, northcentral, and north Texas, and significant (P < 0.05) clusters late during the outbreak were detected in central, south, and west Texas. Two counties on the south Texas coast first reported disease significantly (P < 0.05) earlier than their 10 nearest neighboring counties. The estimated incidence of disease was greatest in the high plains of north Texas and in northcentral Texas. Higher rates were also estimated in eastern and southern areas of the Gulf Coast. The spatial and temporal distribution observed indicates that the equine WNV epidemic began in two parts of Texas and spread elsewhere throughout the state. The mechanism of introduction and spread remains speculative.
Descriptors: disease outbreaks, horse diseases, West Nile fever, cluster analysis, geography, statistical models, Texas, zoonoses.

Ward, M.P. (2005). Epidemic West Nile virus encephalomyelitis: A temperature-dependent, spatial model of disease dynamics. Preventive Veterinary Medicine 71(3-4): 253-264.
Abstract: Since first being detected in New York in 1999, West Nile virus (WNV) has spread throughout the United States and more than 20,000 cases of equine WNV encephalomyelitis have been reported. A spatial model of disease occurrence was developed, using data from an outbreak of serologically confirmed disease in an unvaccinated population of horses at 108 locations in northern Indiana between 3 August and 17 October 2002. Daily maximum temperature data were recorded at meteorological stations surrounding the study area. The distribution of the total number of degree-days elapsing between July 4 and the date of diagnosis of each case was best described by a normal distribution (mean=5243 degrees F, S.D.=1047). The days on which the average risk was >25, >50 and >75% were predicted (versus observed) to occur on August 23 (August 9), August 31 (September 2) and September 9 (September 9). The epidemic was predicted to occur 3 days earlier, or 4 days later, than observed if temperatures in the study area were uniformly increased, or decreased, by 5 degrees F, respectively. Maps indicated that WNV encephalomyelitis risk always remained greater in the northwest quadrant of the study area. Since WNV might exist at a hypoendemic level of infection, and occasionally re-emerge as a cause of epidemics in equine populations, by identifying factors that contributed to this epidemic, the potential impact of future epidemics can be reduced. Such studies rely on a GIS framework, availability of meteorological and possibly remotely sensed data and information on host and landscape factors. An early-warning system for WNV transmission in equine populations could be developed.
Descriptors: animal disease outbreaks, horse diseases, West Nile fever, horses, Indiana, statistical models, space-time clustering, West Nile virus.

Warner, R.D., R.C. Kimbrough, J.L. Alexander, J.J. Rush Pierce, T. Ward, and L.P. Martinelli (2006). Human west nile virus neuroinvasive disease in Texas, 2003 epidemic: Regional differences. Annals of Epidemiology 16(10): 749-755. ISSN: 1047-2797.
Abstract: PURPOSE: Arboviral diseases, such as West Nile virus (WNV) epizootics, tend to be geographically unique because of the biomes that support the vector(s) and reservoir host(s). Understanding such details aids in preventive efforts. We studied the 2003 epidemic of human West Nile neuroinvasive disease (WNND) in Texas because it initially appeared that incidence was not uniform across regions of the state. METHODS: The epidemic was described by age, sex, and region of residence. These variables were used to compare age-specific incidence, standardized cumulative incidence, and age-adjusted relative risk (RR). We verified case data and used routine software, with population estimates from the US Census Bureau. RESULTS: Regardless of sex, risk increased with age. Males had the greater risk (RR, 1.69); however, males aged 5 to 17 years had the greatest RR. Of the five regions compared, two posed more (RRs, 7.98 and 2.14) and one posed less (RR, 0.40) risk than the remainder of the state. Proportions of Culex vector species differed significantly between regions. CONCLUSIONS: During 2003, the risk for WNND varied considerably across Texas. This suggests that various risks for WNV infection deserve additional research for preventive interventions to be regionally appropriate and effective.
Descriptors: West Nile virus, epizootics, reservoir hosts, preventive efforts, epidemic, variables, risk, Culex vector, Texas.

Wicker, J.A., M.C. Whiteman, D.W. Beasley, C.T. Davis, S. Zhang, B.S. Schneider, S. Higgs, R.M. Kinney, and A.D. Barrett (2006). A single amino acid substitution in the central portion of the West Nile virus NS4B protein confers a highly attenuated phenotype in mice. Virology 349(2): 245-253. ISSN: 0042-6822.
Abstract: West Nile virus (WNV) NS4B is a small hydrophobic nonstructural protein that is hypothesized to participate both in viral replication and evasion of host innate immune defenses. The protein has four cysteine residues (residues 102, 120, 227, and 237). Since cysteines are often critical for the function of proteins, each of the four cysteine residues found in WNV NS4B was mutated to serine by site-directed mutagenesis. While three of these substitutions had little effect on replication or mouse virulence phenotypes, the C102S mutation was associated with a temperature-sensitive phenotype at 41 degrees C as well as attenuation of the neuroinvasive and neurovirulence phenotypes in mice.
Descriptors: amino acid substitution, viral nonstructural proteins, West Nile virus, amino acid sequence, Cercopithecus aethiops, cysteine genetics, animal disease models, heat, lethal dose (LD) 50, mice, West Nile fever, cysteine residues, C102S mutation.

Wolf, R.F., J.F. Papin, R. Hines Boykin, M. Chavez Suarez, G.L. White, M. Sakalian, and D.P. Dittmer (2006). Baboon model for West Nile virus infection and vaccine evaluation. Virology 355(1): 44-51. ISSN: 0042-6822.
Abstract: Animal models that closely mimic the human condition are of paramount significance to study pathogenic mechanisms, vaccine and therapy scenarios. This is particularly true for investigations that involve emerging infectious diseases. Nonhuman primate species represent an alternative to the more intensively investigated rodent animal models and in a number of instances have been shown to represent a more reliable predictor of the human response to infection. West Nile virus (WNV) has emerged as a new pathogen in the Americas. It has a 5% fatality rate, predominantly in the elderly and immune compromised. Typically, infections are cleared by neutralizing antibodies, which suggests that a vaccine would be efficacious. Previously, only macaques had been evaluated as a primate model for WNV vaccine design. The macaques did not develop WNV disease nor express the full complement of IgG subclasses that is found in humans. We therefore explored baboons, which exhibit the similar four IgG subclasses observed in humans as a new model for WNV infection and vaccine evaluation. In this present report, we describe the experimental infection of baboons with WNV and test the efficacy of an inactivated WNV vaccination strategy. All experimentally infected animals developed transient viremia and subsequent neutralizing antibodies. Anti-WNV IgM antibodies peaked at 20 days post-infection. Anti-WNV IgG antibodies appeared later and persisted past 60 days. Prior vaccination with chemically inactivated virus induced neutralizing titers and a fast, high titer IgG recall response, which resulted in lower viremia upon challenge. This report is the first to describe the development of the baboon model for WNV experimental infection and the utility of this model to characterize the immunologic response against WNV and a candidate WNV vaccine.
Descriptors: animal disease models, Papio, West Nile fever, West Nile virus vaccines, immunoglobulin G, immunoglobulin M, neutralization tests, viremia.

Yiannakoulias, N.W., D.P. Schopflocher, and L.W. Svenson (2006). Modelling geographic variations in West Nile virus. Canadian Journal of Public Health 97(5): 374-378.
Abstract: BACKGROUND: This paper applies a method for modelling the spatial variation of West Nile virus (WNv) in humans using bird, environmental and human testing data. METHODS: We used data collected from 503 Alberta municipalities. In order to manage the effects of residual spatial autocorrelation, we used generalized linear mixed models (GLMM) to model the incidence of infection. RESULTS: There were 275 confirmed cases of WNv in the 2003 calendar year in Alberta. Our spatial model indicates that living in the grasslands natural region and levels of human testing are significant positive predictors of WNv; living in an urban area is a significant negative predictor. CONCLUSION: Infected bird data contribute little to our model. The variability of West Nile virus incidence in Alberta may be partly confounded by the variations in the rate of testing in different parts of the province. However, variation in infection is also associated with known environmental risk factors. Our findings are consistent with existing knowledge of WNv in North America.
Descriptors: ecology, linear models, rural health, urban health, West Nile fever, Alberta, birds, seroepidemiologic studies.

Yuan, F., Z. Lou, X. Li, Y.W. Chen, J.I. Bell, Z. Rao, and G.F. Gao (2005). Refolding, crystallization and preliminary X-ray structural studies of the West Nile virus envelope (E) protein domain III. Acta Crystallographica. Section F, Structural Biology and Crystallization Communications 61(Pt 4): 421-423. ISSN: 1744-3091.
Abstract: Domain III of the West Nile virus envelope protein, the putative receptor-binding domain, is a major virion-surface determinant for virulence. This protein was reported to be intrinsically unstable and has defied previous crystallization attempts. It has now been purified from inclusion bodies by protein refolding and was crystallized using the hanging-drop vapour-diffusion method at 291 K. The crystals belong to space group P222(1), with unit-cell parameters a = 52.6, b = 59.7, c = 95.0 A. A complete data set was collected to 2.8 A at 100 K with Cu Kalpha X-rays from a rotating-anode generator.
Descriptors: viral envelope proteins, West Nile virus, binding sites, crystallization methods, inclusion bodies, viral protein folding, x ray diffraction.

Zhang, J.S., P.H. Zhang, B.Y. Si, H. Yang, and W.C. Cao (2005). [Comparison and discrimination of the biological characteristics between West Nile virus and Japanese encephalitis virus]. Chinese Journal of Experimental and Clinical Virology 19(4): 340-343.
Abstract: BACKGROUND: To compare the biological characteristics of West Nile virus (WNV) and Japanese encephalitis virus (JEV), including cells sensitivity, pathogenicity, viral morphology, as well as the results of immunological and molecular biological detection. METHODS: Cytopathic effect (CPE) and pathogenicity were observed in C6/36 cells and in suckling mice inoculated intracerebrally with the WNV or JEV, respectively. The sliced tissue samples for electron microscopic examination were prepared for the morphologic observation of the viruses. Serum antibody to WNV or JEV was detected using indirect immunofluorescence assay (IFA), and the viral RNA was analyzed by RT-PCR method. RESULTS: WNV or JEV-caused CPE was characterized by cell fusion and cell shedding, respectively. There was no significant difference in the pathogenicity to suckling mice between WNV and JEV. The morphologic observation showed that the shape and size of the two virions were similar. WNV and JEV were found to have antigenic cross-reactivity. The viral RNA could be detected from both WNV and JEV samples with universal primer set, but only nucleoside fragments of corresponding virus could be amplified when specific primers were used. CONCLUSION: CPE in C6/36 cell and detection of the viral RNA should be useful in discrimination of WNV and JEV, and simultaneously examining the titers of serum antibodies against WNV and JEV may be helpful to diagnosis of infection with these agents.
Descriptors: West Nile virus, Japanese encephalitis virus, biological characteristics, pathogenicity, morphology, viral RNA, diagnosis.
Language of Text: Chinese.

Zhang, S., L. Li, S.E. Woodson, C.Y. Huang, R.M. Kinney, A.D. Barrett, and D.W. Beasley (2006). A mutation in the envelope protein fusion loop attenuates mouse neuroinvasiveness of the NY99 strain of West Nile virus. Virology 353(1): 35-40. ISSN: 0042-6822.
Abstract: Substitutions were engineered individually and in combinations at the fusion loop, receptor-binding domain and a stem-helix structure of the envelope protein of a West Nile virus strain, NY99, and their effects on mouse virulence and presentation of epitopes recognized by monoclonal antibodies (MAbs) were assessed. A single substitution within the fusion loop (L107F) attenuated mouse neuroinvasiveness of NY99. No substitutions attenuated NY99 neurovirulence. The L107F mutation also abolished binding of a non-neutralizing MAb, 3D9, whose epitope had not been previously identified. MAb 3D9 was subsequently shown to be broadly cross-reactive with other flaviviruses, consistent with binding near the highly conserved fusion loop.
Descriptors: mutation, viral envelope proteins, West Nile fever, West Nile virus, monoclonal antibodies, mice, neutralization tests, virulence.

Zhou, H., N.J. Singh, and K.S. Kim (2006). Homology modeling and molecular dynamics study of West Nile virus NS3 protease: A molecular basis for the catalytic activity increased by the NS2B cofactor. Proteins 65(3): 692-701.
Abstract: The West Nile virus (WNV) NS3 serine protease, which plays an important role in assembly of infective virion, is an attractive target for anti-WNV drug development. Cofactors NS2B and NS4A increase the catalytic activity of NS3 in dengue virus and Hepatitis C virus, respectively. Recent studies on the WNV-NS3 characterize the catalytically active form of NS3 by tethering the 40-residue cofactor NS2B. It is suggested that NS2B is essential for the NS3 activity in WNV, while there is no information of the WNV-NS3-related crystal structure. To understand the role of NS2B/substrate in the NS3 catalytic activity, we built a series of models: WNV-NS3 and WNV-NS3-NS2B and WNV-NS3-NS2B-substrate using homology modeling and molecular modeling techniques. Molecular dynamics (MD) simulations were performed for 2.75 ns on each model, to investigate the structural stabilization and catalytic triad motion of the WNV NS3 protease with and without NS2B/substrate. The simulations show that the NS3 rearrangement occurs upon the NS2B binding, resulting in the stable D75-OD1...H51-NH hydrogen bonding. After the substrate binds to the NS3-NS2B active site, the NS3 protease becomes more stable, and the catalytic triad is formed. These results provide a structural basis for the activation and stabilization of the enzyme by its cofactor and substrate.
Descriptors: West Nile virus, serine protease, infective virion, drug development, dengue virus, Hepatitis C, modeling techniques.


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