| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 South Grand Ave, St Louis, MO 63104, USA
Correspondence
Thomas J. Chambers
thomas_chambers2{at}merck.com
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
The GenBank/EMBL/DDBJ accession numbers for the brain-associated viral variants are EU391638–EU391642.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). WNV causes zoonotic infections involving mainly wild avian hosts and is transmitted primarily by Culex sp. mosquitoes, but also incidentally infects humans and horses, as well as other vertebrate species. WNV was originally described in 1937 from an infected human in Uganda (Smithburn et al., 1940), but is now widespread in several continents (Africa, Europe, Asia and North America), mostly as a result of migration of viraemic birds (Rappole et al., 2000).
WNVs can be subdivided into two distinct lineages on the basis of serological data and genetic characterization (Price & O'Leary, 1967; Berthet et al., 1997). Lineage 1 viruses, which include the highly virulent NY99 strain, are the most widespread and typically associated with epidemics or epizootics (Petersen & Roehrig, 2001; Roehrig et al., 2002). Lineage 1 viruses can be further differentiated into at least three clades: clade 1a, which is found worldwide; clade 1b, which includes the Australian Kunjin virus (Lanciotti et al., 1999; Scherret et al., 2002); and clade 1c containing isolates from India (Bondre et al., 2007). Lineage 2 comprises virus isolates from sub-Saharan Africa and Madagascar (Scherret et al., 2001; Burt et al., 2002). Viruses of lineage 2 have not been associated with large disease outbreaks of this type (Lanciotti et al., 1999).
WN25 and WN25A are variants of a lineage 1 strain (WN-Israel) isolated in 1951 in association with outbreaks of West Nile fever and some cases of central nervous system disease (Goldblum et al., 1954). WN25 was derived by passage of its parental WN-Israel strain in Aedes aegypti cultures (25 passages) followed by plaque purification and growth in C6/36 cells, as described previously (Lustig et al., 1992). WN25A was derived from WN25 by selection for monoclonal antibody neutralization resistance (Halevy et al., 1994). Both WN25 and WN25A viruses are attenuated for neuroinvasiveness in normal mice and have been tested as experimental vaccines against WNV in geese (Lustig et al., 2000). The genetic basis for the attenuation has not been defined. For both viruses, loss of the neuroinvasive phenotype in normal mice is associated with acquisition of an N-linked glycosylation site at the conserved position in the envelope (E) protein. However, WN25A contains an additional substitution at position 307 in the E protein, at the site of neutralization resistance (Halevy et al., 1994; Chambers et al., 1998).
WN25 and WN25A viruses are highly pathogenic for severe combined immunodeficiency (SCID) mice, causing systemic infection and uniform death within several days of infection (Halevy et al. 1994). However, they differ in their capacity for virulence reversion during infection in this model. WN25 virus recovered from the brains of SCID mice exhibited increased neuroinvasiveness when tested in normal mice, whereas WN25A did not. This indicates a differential propensity of the two viruses to undergo neuroadaptation in the mouse model.
The objective of the current study was to obtain additional characterization of the virulence properties of WN25A virus in the mouse model. Data have not been reported on the properties of this attenuated strain in B-cell-deficient mice. Another objective was to determine whether there was any evidence for genetic or phenotypic instability of the virus in cases where virus replication and dissemination occurred in such mice. Such data are relevant for the further assessment of WN25A virus as a candidate live-attenuated viral vaccine.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Chambers et al., 1998). A working virus stock for the current experiments was prepared by passage of the original WN25A stock in Vero cells. Plaque assays were carried out in confluent Vero cell monolayers by infecting with 10-fold dilutions of virus in alpha-MEM plus 10 % FBS for 1 h at 37 °C, followed by removal of the medium and replacement with an overlay solution of 1 % ME agarose in alpha-MEM plus 5 % FBS. Plaques of 4 mm in size were visible by 3 days post-infection and were stained with 0.05 % neutral red in PBS, followed by fixation in 7 % formalin and counterstaining with 1 % crystal violet solution.
Tissue titrations.
Infectious WN25A virus in mouse tissues was detected by plaque assay of tissue homogenates on Vero cells. Tissues were harvested from mice that exhibited signs of encephalitis after perfusion with cold (4 °C) PBS and dissection of the organs and tissues and immediate storage on dry ice. Samples were thawed on ice and 10 % tissue homogenates were prepared in PBS plus 10 % FBS using a ground glass dounce homogenizer. Tissue homogenates were diluted serially and plated on 60 mm dishes of confluent Vero cells, followed by incubation for 1 h at 37 °C. Plates were then washed two to three times with 5–8 ml alpha-MEM plus 5 % FBS to remove any particulate material. Ten millilitres of the same overlay medium used for the plaque assay (described above) was added to the plates, followed by incubation for 3–6 days at 37 °C to detect plaques. Plates were then stained as described above and the total p.f.u. for each organ or tissue tested was calculated. For measurement of virus in sera, blood was obtained by cardiac puncture of moribund mice prior to perfusion and stored on ice. After 30 min, the serum was centrifuged at 4 °C at 1500 r.p.m. in a Sorvall RT 6000D centrifuge, and the sera were removed and stored at –70 °C until plaque titration. Sera were then tested in 10-fold dilutions using the plaque assay conditions described above.
Animal experiments.
Normal C57BL/6 and B-cell-deficient (congenic µMT mice, strain B6-Igh6-6tm1Cgn) were used (Jackson Laboratories). Viruses were diluted in PBS plus 10 % FBS and inoculated by the intracerebral (i.c.), intraperitoneal (i.p.) or subcutaneous (s.c.) route. Neurovirulence was assessed by i.c. inoculation of 30 µl virus into the left cerebral hemisphere of mice after anaesthetization. Neuroinvasiveness was tested by inoculation of virus in volumes of approximately 0.25 ml by the i.p. or s.c. route. Mice were observed until found dead or until exhibiting signs of encephalitis and then euthanized. Reinoculation experiments with virus recovered from the brain of mice inoculated with WN25A were carried out by injection of µMT mice by the i.p. route. The brain-associated virus was passaged once in Vero cells to prepare a working virus stock for these experiments.
Nucleotide sequencing of brain-associated virus.
Brains were obtained from mice exhibiting signs of encephalitis, after perfusion to remove contaminating blood. Brains were processed by preparation of 10 % (w/v) extracts as described above, and a proportion of the extract was used for isolation of RNA. Brain homogenate was mixed with Trizol (Gibco-BRL) and the RNA was purified using the recommendations of the manufacturer. RNA pellets were resuspended in 10 mM Tris/HCl (pH 8.0) and stored at –70 °C. RNA was used for cDNA synthesis by reverse transcriptase (Superscript II) in the presence of an antisense primer corresponding to WNV nt 2656–2669 of the reported sequence of the Nigerian strain of WNV (Wengler et al., 1985), as described previously (Chambers et al., 1998). PCR amplification was carried out using DeepVent DNA polymerase (New England Biolabs) in the presence of the same 3' primer and a 5' primer corresponding to WNV nt 881–898. PCR products were isolated from low-melting-temperature agarose gels (BioWhittaker Molecular Applications) and purified using a Wizard PCR prep kit (Promega). Purified PCR products were then cloned using the ZeroBLUNT-TOPO procedure (Invitrogen). Colonies of bacterial cells containing the cloned PCR fragments were identified by screening miniprep DNA with restriction enzyme digestions and then nucleotide sequencing using an ABI sequencer.
Molecular modelling.
A model of the WNV25A virus E protein was derived from the structure of the WNV NY99 E protein (PDB accession no. 2HG0; Nybakken et al., 2006), using modelling tools available in SWISS-MODEL. The sequence for the WN25A E protein was imported into the model and the predicted structure was found to resemble that of the reference protein, based on comparison of the three-dimensional images generated by the viewer. The model was used to analyse effects of mutations at the N-linked glycosylation site in domain I where mutations in the WN25A viral variants recovered from B-cell-deficient mice were observed. Rotamer positions for the amino acid substitutions tyrosine, asparagine and serine at position 155 were selected on the basis of the most favourable energy parameters calculated by the modelling tools. The predicted effects of these configurations in the context of the E0–F0 loop encompassing residues 139–160 were analysed with respect to space filling and hydrogen bonding to determine whether differences in the local structure could be detected. Electrostatic potentials were calculated and mapped on to the surface of the space-filling models using programs on the SWISS-MODEL platform.
Statistical analysis.
Differences in mortality end points were analysed using Fisher's exact test. Differences in mean survival times were measured using log-rank testing.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
illustrates the survival data based on results of several experiments done in mice ranging from 6 to 8 weeks of age. Most mice inoculated by the i.p. route developed fatal disease, with an overall mortality rate of 92 %. The majority of mice succumbed to infection within 21 days, with a mean survival time of 18 days; however, the range was large (9–72 days) (see also Table 1). Mice that survived the infection beyond 72 days did not exhibit any characteristic signs of encephalitis and were sacrificed after a few additional months of observation. In contrast to these results, normal 6–8-week-old C57BL/6 mice did not exhibit mortality or any signs of illness when given 1x107 p.f.u. WN25A by the i.p. route (Fig. 1; Table 1).
|
|
also shows that the majority of mice (80 %) inoculated with a dose of 1x106 p.f.u. also succumbed to this dose within 21 days. The range of survival times was also less variable (11–29 days, mean 15.6 days) than at the 1x107 dose, and late mortality was not observed as frequently as with the 1x107 dose. Doses below 1x106 p.f.u. were tested in separate experiments (see below).
After s.c. inoculation, only 68 % of mice succumbed to infection at the 1x107 p.f.u. dose (Fig. 1; Table 1). The mean survival time was 28.5 days, and the range was 10–70 days. A lower percentage of mice developed fatal infection within 21 days compared with the group infected by the i.p. route (9/19 vs 18/22, respectively). Mice inoculated with 1x106 p.f.u. by the s.c. route were also susceptible to fatal disease. Although only a small number was tested, the mortality rate was 57 % and the mean survival time was 38 days (range 23–53 days), which was not very different from those for the 1x107 dose. Differences between the i.p. and s.c. routes were compared for statistical significance. At the 1x107 dose, mortality rates and mean survival times were both significantly different (P=0.046 and 0.001, respectively). At the 1x106 dose, neither the mortality rates nor the average survival times were significantly different (P=0.593 and 0.082, respectively).
B-cell-deficient mice of 6–8 weeks of age were also tested for susceptibility to i.c. inoculation with WN25A virus to confirm that the virus exhibited a neurovirulent phenotype in these strains. Inoculation with a dose of 370 p.f.u. caused uniform mortality. Survival time was limited to 7–8 days. Similar results were obtained in normal C57BL/6 mice of the same age. Dose ranging was not done in these experiments.
Virus burdens in tissues of B-cell-deficient mice
Mice that succumbed to infection with WN25A virus exhibited typical signs of flavivirus encephalitis (ruffling, hunching and hind-limb paralysis). To determine the pattern of virus distribution in tissues of these mice, various organs and tissues were recovered and tested for virus burden using plaque assays of tissue homogenates. Table 2 shows the results of these experiments. Viraemia was detected in virtually all mice that succumbed to encephalitis, with levels of virus in the sera ranging from 2.6 to 3.8 log p.f.u. ml–1 (mean of 3.4). The highest viral titres were measured in brain (8.8 log p.f.u. g–1). Low to moderate levels of infectious virus in the range of 0.38–1.5 log p.f.u. g–1 were detected in most peripheral tissues tested (lung, liver, heart, peritoneum, muscle and kidney). However, neither the adrenal gland nor the spleen yielded infectious virus. Mice that did not exhibit signs of illness were not tested for viraemia or systemic infection, so it is not known whether viraemia occurred in the absence of encephalitis.
|
). These differences include acquisition of an N-linked oligosaccharide acceptor site at the conserved flavivirus glycosylation site in the E protein (aa 155) and substitution of glutamic acid for lysine at aa 307 at the presumed site of monoclonal antibody neutralization resistance.
To determine whether neuroinvasion of WN25A virus in B-cell-deficient mice was associated with any new mutations in the E protein that might affect its attenuated phenotype, viruses were isolated from the brains of five mice and the sequences of the E proteins of these viruses were determined (Table 3). Sequence data were compared with that of the parental WN25A virus used for inoculation of these mice, as well as the sequences of the virulent WN-Israel virus parent of WN25A and that of the WN25A virus originally reported (Chambers et al., 1998). As indicated in Methods, the current WN25A stock differs from the WN25A by a passage in Vero cells.
|
Comparison of the sequence of the WN25A virus used in the current experiments with the previously determined sequence for WN25A (Chambers et al., 1998) revealed substitutions at positions 249 (arginine for lysine) and 253 (methionine for isoleucine). These presumably arose during the Vero cell passage used to prepare the WN25A stock. These substitutions were also present in E proteins of the five brain-associated variants from the B-cell-deficient mice.
Neuroinvasiveness of brain-associated WN25A virus
Viruses from the brains of B-cell-deficient mice that developed encephalitis were further investigated to determine whether loss of the N-linked glycosylation site was associated with reversion to the virulence phenotype of the WN-Israel virus. One of the brain-associated viruses (WN25A.9, Table 3), from a mouse that succumbed at 8 weeks post-inoculation, was tested in B-cell-deficient and normal C57BL/6 mice at various doses in comparison with the parental WN25A virus stock.
B-cell-deficient mice (6–8 weeks of age) were inoculated with WN25A.9 virus by the i.p. route at doses ranging from 2x104 to 2x107 p.f.u. The mortality rate at the highest dose was approximately 82 % (9/11) but decreased to 36 % (5/14) at the lowest dose (Table 4). The mortality rates for mice receiving the same doses of the parental WN25A virus were not significantly different (non-significant for comparison at each dose). Survival curves for the two viruses resembled those for mice described earlier for i.p. inoculation of WN25A virus, with most of the mice succumbing within the initial 21 days (data not shown). Mean survival times for the mice in the two groups were not significantly different. The longest observed time to death occurred in one mouse inoculated with WN25A.9 virus (124 days). Thus, WN25A.9 caused lower mortality than WN25A at all but the lowest dose tested, but the differences from WN25A were not significant at any dose.
|
). In addition, mice of different ages were tested to assess whether there was an age-related susceptibility of normal mice to these viruses and whether the two viruses differed in this respect.
|
). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Lustig et al., 2000). Mortality rates in B-cell-deficient mice are lower than those observed in SCID mice, which exhibited 100 % mortality and shorter survival times, reflecting the profound immunodeficiency of SCID mice (Halevy et al., 1994). B-cell-deficient mice are also highly vulnerable to the WNV NY99 strain, with very low doses causing high mortality rates, incubation periods for fatal disease shorter than observed here for WN25A, virus burdens in the periphery considerably higher and kinetics of virus accumulation more rapid than for WN25A (Diamond et al., 2003). These differences are presumably governed by molecular determinants that differentiate the pathogenicity of different WNVs in the mouse model (Beasley et al., 2002).
The factors governing the incomplete mortality of B-cell-deficient mice from WN25A virus infection in this model are not known. Clearance of a large burden of WNV by non-specific immune responses such as macrophages contributes to resistance against early death from WNV encephalitis in immunodeficient mice (Ben-Nathan et al., 1996). Cell-mediated immune responses also play an important role in protection against WNV disease and virus clearance (Diamond et al., 2003; Shrestha & Diamond, 2004) and may contribute to the variable survival observed in the case of B-cell-deficient mice that do not rapidly succumb to infection with WN25A virus. A variety of physical stresses that are believed to have effects on the blood–brain barrier (Ben-Nathan et al., 1989; Kobiler et al., 1989; Katz et al., 2002) may also account for some of the variability in susceptibility observed here. In any case, the susceptibility of B-cell-deficient mice to WN25A virus is consistent with many previous reports, demonstrating a requirement for virus-specific antibodies to control flavivirus infection in various mouse challenge models (Mathews & Roehrig, 1984; Schlesinger et al., 1985; Brandriss et al., 1986; Phillpotts et al., 1987; Kimura-Kuroda & Yasui, 1988; Broom et al., 2000; Roehrig et al., 2001; Ben-Nathan et al., 2003).
Lineage 1 strains of WNV, including the WN25 and WN25A viruses, as well as a highly attenuated WNV infectious clone (Yamshchikov et al., 2001, 2004), have been evaluated as live-attenuated viral vaccines. Both WN25 and WN25A viruses induced protective immunity in geese against a virulent WNV field isolate (Lustig et al., 2000). Despite the efficacy of these experimental vaccines, stability of the vaccine phenotype remains an important issue, because of the propensity of WNVs to exhibit genetic heterogeneity both in cell substrates in vitro (Lee et al., 2004) and in the natural environment (Beasley et al., 2001, 2002; Davis et al., 2004). Any phenotypic instability resulting from genetic heterogeneity could present difficulties in ensuring the safety of a live-attenuated vaccine against WNV due to potential loss of its attenuated properties. In this regard, WN25A viruses recovered from the brains of B-cell-deficient mice that developed fatal encephalitis exhibited genetic instability within the E protein, involving loss of the N-linked glycosylation site at position 155. This was of interest due to the possibility that this represented a reversion to the virulence phenotype of WN-Israel virus. To evaluate the significance of this mutation, one brain-associated variant (WN25A.9) that lacked the N-linked site was tested for virulence properties. WN25A.9 did not display consistently increased neuroinvasive properties for either B-cell-deficient or normal mice, although there was evidence of attenuation of neuroinvasiveness in normal mice below 5 weeks of age. These observations indicated a lack of neuroadaptation or reversion of WN25A virus during replication in immunodeficient mice. This contrasts with what has been observed for the WN25 virus (from which WN25A was derived), where partial reversion was observed after passage in SCID mice (Halevy et al., 1994), involving restoration of the parental WN-Israel sequence (Chambers et al., 1998). Thus, the loss of the N-linked site in WN25A.9 represents a pseudoreversion to the phenotype of the parental WN-Israel virus. Brain-associated viruses were not recovered from any normal mice with encephalitis, so it is not currently known whether loss of the N-linked site is unique to B-cell-deficient mice or occurs in the context of neuroinvasion by WN25A in general.
The effect of N-linked glycosylation of WN25A on mouse neuroinvasiveness contrasts with results in studies of other WNV strains, where it has been observed to be a determinant of virulence for the NY99 strain (Shirato et al., 2004; Beasley et al., 2005). However, available data suggest that WNV neuroinvasiveness is influenced by multiple genetic determinants, and strain variation in non-structural regions may affect properties conferred by the E protein in the context of glycosylation (Beasley et al., 2005). Furthermore, for WN25A, it is also notable that glycosylation of the N-linked acceptor site occurs at residue 155 rather than residue 154, which may alter the local structure of the protein in a manner that destabilizes its function, as suggested from the model of the WNV NY99 E protein (Nybakken et al., 2006; discussed below).
The basis for lack of reversion of WN25A virus in this model is not currently known. The glutamic acid residue at position 307 in domain III, the site of monoclonal antibody resistance involved in the derivation of WN25A from WN25, may be a contributing factor. A strong attenuating effect of this mutation may reduce the capacity for rapid reversion. However, it must also be acknowledged that other mutations may be present in the genome of WN25A or have accumulated in the genomes of the brain-associated variants such as WN25A.9 that could affect virulence properties independent of the effects of residue 307. Complete genomic sequencing of these various strains is needed for a better understanding of their properties.
Another explanation for the lack of reversion is that substitution of the serine residue instead of tyrosine at position 155 might affect the local structure of domain I in the absence of the N-linked glycan that could independently reduce virulence properties by altering the function of the E protein. To gain insight into this question, a model for the WN25A E protein based on the structure of the WNV NY99 E protein was examined (Figs 2 and 3). These two proteins share 97.5 % amino acid sequence identity through the 400 aa of the crystallized WNV NY99 protein. The ribbon model, with its side-chain orientations and relevant predicted hydrogen bonds, and the space-filling model together with electrostatic potentials generated by surface amino acids of WN25A E protein resembled those of WNV NY99. The glycosylation site for WN25A occurs within the unique 
A' helix between the E0 and F0 loops of domain I, where residues 156–160 are conserved between WN25A and WNV NY99, except for an isoleucine instead of valine at position 159 in WNV NY99. However, the residue at 155 in WN25A is more C-terminal in the helix than residue 154 in the WNV NY99 structure and is predicted to project towards the β-strands of the E0–F0 loop instead of outward above the surface of the helix (Fig. 2b). In the space-filling model, there is also slight displacement of the asparagine residue relative to tyrosine (Fig. 2a).
|
|
), using optimal orientations for the side-chain substitutions as determined using the SWISS-MODEL database viewer. The ribbon depiction of the region around residue 155 was used to display a local structure, involving 4 aa that form a hydrophobic tetrad: residue 155, residue 1 (phenylalanine), residue 159 (isoleucine) and residue 164 (alanine).
Modelling of a tyrosine at position 155 (Fig. 3, left panel) indicates the side chain being directed into the space formed between the E0 and F0 strands. This orientation is predicted to be stabilized by the formation of two hydrogen bonds within the tetrad, and between the arginine residue of the F0 loop and possibly by interactions with the adjacent phenylalanine ring from residue 1. Modelling of an asparagine residue at position 155 (Fig. 3, middle panel) indicates a predicted orientation of the side chain above the surface of the A' helix where it is accessible for attachment of an N-linked glycan at this site. There are no predicted interactions with residues within the F0 loop. Modelling of a serine residue at position 155 (Fig. 3, right panel) indicates that its side chain projects above the A' helix as for asparagine, with no predicted interactions of its side-chain hydroxyl with adjacent residues. These predicted structures support a hypothesis that the asparagine and serine residues at position 155 contribute to a difference in local structure involving the A' helix and the E0–F0 loop, which may contribute to a relative destabilization of the domain I/II interface compared with the case of tyrosine. In addition, the shift in the N-linked acceptor site to position 155 in WN25A virus rather than 154 as found in other WNV strains may be an important factor in explaining the differential effect of glycosylation on virulence properties.
The findings of the current study provide some additional information on the attenuation phenotype of the WN25A virus, which has previously undergone characterization of its virulence properties only in normal and SCID mice. Further studies of the genetic and phenotypic stability of WN25A virus in this model or in other immunodeficient mouse strains may be relevant for consideration of this virus as a live-attenuated vaccine against WNV.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Beasley, D. W., Li, L., Suderman, M. T. & Barrett, A. D. (2002). Mouse neuroinvasive phenotype of West Nile virus strains varies depending upon virus genotype. Virology 296, 17–23.[CrossRef][Medline]
Beasley, D. W. C., Whiteman, M. C., Zhang, S., Huang, C. Y.-H., Schneider, B. S., Smith, D. R., Gromowski, G. D., Higgs, S., Kinney, R. M. & Barrett, A. D. T. (2005). Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains. J Virol 79, 8339–8347.
Ben-Nathan, D., Lustig, S. & Feuerstein, G. (1989). The influence of cold or isolation stress on neuroinvasiveness and virulence of an attenuated variant of West Nile virus. Arch Virol 109, 1–10.[CrossRef][Medline]
Ben-Nathan, D., Huitinga, I., Lustig, S., van Rooijen, N. & Kobiler, D. (1996). West Nile virus neuroinvasion and encephalitis induced by macrophage depletion in mice. Arch Virol 141, 459–469.[CrossRef][Medline]
Ben-Nathan, D., Lustig, S., Tam, G., Robinzon, S., Segal, S. & Rager-Zisman, B. (2003). Prophylactic and therapeutic efficacy of human intravenous immunoglobulin in treating West Nile virus infection in mice. J Infect Dis 188, 5–12.[CrossRef][Medline]
Berthet, F. X., Zeller, H. G., Drouet, M. T., Rauzier, J., Digoutte, J. P. & Deubel, V. (1997). Extensive nucleotide changes and deletions within the envelope glycoprotein gene of Euro-African West Nile viruses. J Gen Virol 78, 2293–2297.[Abstract]
Bondre, V. P., Jadi, R. S., Mishra, A. C., Yergolkar, P. N. & Arankalle, V. A. (2007). West Nile virus isolates from India: evidence for a distinct genetic lineage. J Gen Virol 88, 875–884.
Brandriss, M. W., Schlesinger, J. J., Walsh, E. E. & Briselli, M. (1986). Lethal 17D yellow fever encephalitis in mice. I. Passive protection by monoclonal antibodies to the envelope proteins of 17D yellow fever and dengue 2 viruses. J Gen Virol 67, 229–234.
Broom, A. K., Wallace, M. J., Mackenzie, J. S., Smith, D. W. & Hall, R. A. (2000). Immunisation with gamma globulin to Murray Valley encephalitis virus and with an inactivated Japanese encephalitis virus vaccine as prophylaxis against Australian encephalitis: evaluation in a mouse model. J Med Virol 61, 259–265.[CrossRef][Medline]
Burke, S. D. & Monath, T. P. (2001). Flaviviruses. In Fields Virology, vol. 1, 4th edn, pp. 1043–125. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.
Burt, F. J., Grobbelaar, A. A., Leman, P. A., Anthony, F. S., Gibson, G. V. & Swanepoel, R. (2002). Phylogenetic relationships of Southern African West Nile virus isolates. Emerg Infect Dis 8, 820–826.[Medline]
Chambers, T. J., Halevy, M., Nestorowicz, A., Rice, C. M. & Lustig, S. (1998). West Nile virus envelope proteins: nucleotide sequence analysis of strains differing in mouse neuroinvasiveness. J Gen Virol 79, 2375–2380.[Abstract]
Davis, C. T., Beasley, D. W., Guzman, H., Siirin, M., Parsons, R. E., Tesh, R. B. & Barrett, A. D. (2004). Emergence of attenuated West Nile virus variants in Texas, 2003. Virology 330, 342–350.[CrossRef][Medline]
Diamond, M. S., Shrestha, B., Marri, A., Mahan, D. & Engle, M. (2003). B cells and antibody play critical roles in the immediate defense of disseminated infection by West Nile encephalitis virus. J Virol 77, 2578–2586.
Goldblum, N., Strek, V. V. & Padersky, B. (1954). West Nile fever: the clinical features of the disease and isolation of West Nile from the blood of nine human cases. Am J Hyg 59, 89–103.[Medline]
Halevy, M., Akov, Y., Ben-Nathan, B., Kobiler, D., Lachmi, B. & Lustig, S. (1994). Loss of active neuroinvasiveness in attenuated strains of West Nile virus: pathogenicity in immunocompetent and SCID mice. Arch Virol 137, 355–370.[CrossRef][Medline]
Katz, Y., Lustig, S., Ben-Shlomo, I., Kobiler, D. & Ben-Nathan, D. (2002). Inhalation anesthetic-induced neuroinvasion by an attenuated strain of West Nile virus in mice. J Med Virol 66, 576–580.[CrossRef][Medline]
Kimura-Kuroda, J. & Yasui, K. (1988). Protection of mice against Japanese encephalitis virus by passive administration with monoclonal antibodies. J Immunol 141, 3606–3610.[Abstract]
Kobiler, D., Lustig, S., Gozes, Y., Ben-Nathan, D. & Akov, Y. (1989). Sodium dodecylsulphate induces a breach in the blood–brain barrier and enables a West Nile virus variant to penetrate into mouse brain. Brain Res 496, 314–316.[CrossRef][Medline]
Lanciotti, R. S., Roehrig, J. T., Deubel, V., Smith, J., Parker, M., Steele, K., Crise, B., Volpe, K. E., Crabtree, M. B. & other authors (1999). Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 286, 2333–2337.
Lee, E., Hall, R. A. & Lobigs, M. (2004). Common E protein determinants for attenuation of glycosaminoglycan-binding variants of Japanese encephalitis and West Nile viruses. J Virol 78, 8271–8280.
Lustig, S., Danenberg, H. D., Kafri, Y., Kobiler, D. & Ben-Nathan, D. (1992). Viral neuroinvasion and encephalitis induced by lipopolysaccharide and its mediators. J Exp Med 176, 707–712.
Lustig, S., Olshevsky, U., Ben-Nathan, D., Lachmi, B. E., Malkinson, M., Kobiler, D. & Halevy, M. (2000). A live attenuated West Nile virus strain as a potential veterinary vaccine. Viral Immunol 13, 401–410.[Medline]
Mathews, J. H. & Roehrig, J. T. (1984). Elucidation of the topography and determination of the protective epitopes on the E glycoprotein of Saint Louis encephalitis virus by passive transfer with monoclonal antibodies. J Immunol 132, 1533–1537.[Abstract]
Nybakken, G. E., Nelson, C. A., Chen, B. R., Diamond, M. S. & Fremont, D. H. (2006). Crystal structure of the West Nile virus envelope glycoprotein. J Virol 80, 11467–11474.
Petersen, L. R. & Roehrig, J. T. (2001). West Nile virus: a reemerging global pathogen. Emerg Infect Dis 7, 611–614.[Medline]
Phillpotts, R. J., Stephenson, J. R. & Porterfield, J. S. (1987). Passive immunization of mice with monoclonal antibodies raised against tick-borne encephalitis virus: brief report. Arch Virol 93, 295–301.[CrossRef][Medline]
Price, W. H. & O'Leary, W. (1967). Geographic variation in the antigenic character of West Nile virus. Am J Epidemiol 85, 84–86.
Rappole, J. H., Derrickson, S. R. & Hubalek, Z. (2000). Migratory birds and spread of West Nile virus in the western hemisphere. Emerg Infect Dis 6, 319–328.[Medline]
Roehrig, J. T., Staudinger, L. A., Hunt, A. R., Mathews, J. H. & Blair, C. D. (2001). Antibody prophylaxis and therapy for flavivirus encephalitis infections. Ann N Y Acad Sci 951, 286–297.[Medline]
Roehrig, J. T., Layton, M., Smith, P., Campbell, G. L., Nasci, R. & Lanciotti, R. S. (2002). The emergence of West Nile virus in North America: ecology, epidemiology, and surveillance. Curr Top Microbiol Immunol 267, 223–240.[Medline]
Scherret, J. H., Poidinger, M., Mackenzie, J. S., Broom, A. K., Deubel, V., Lipkin, W. I., Briese, T., Gould, E. A. & Hall, R. A. (2001). The relationships between West Nile and Kunjin viruses. Emerg Infect Dis 7, 697–705.[Medline]
Scherret, J. H., Mackenzie, J. S., Hall, R. A., Deubel, V. & Gould, E. A. (2002). Phylogeny and molecular epidemiology of West Nile and Kunjin viruses. Curr Top Microbiol Immunol 267, 373–390.[Medline]
Schlesinger, J. J., Brandriss, M. W. & Walsh, E. E. (1985). Protection against 17D yellow fever encephalitis in mice by passive transfer of monoclonal antibodies to the nonstructural glycoprotein gp48 and by active immunization with gp48. J Immunol 135, 2805–2809.[Abstract]
Shirato, K., Miyoshi, H., Goto, A., Ako, Y., Ueki, T., Kariwa, H. & Takashima, I. (2004). Viral envelope protein glycosylation is a molecular determinant of the neuroinvasiveness of the New York strain of West Nile virus. J Gen Virol 85, 3637–3645.
Shrestha, B. & Diamond, M. S. (2004). Role of CD8+ T cells in control of West Nile virus infection. J Virol 78, 8312–8321.
Smithburn, K. C., Hughes, T. P., Burke, A. W. & Paul, J. H. (1940). A neurotropic virus isolated from the blood of a native of Uganda. Am J Trop Med 20, 471–492.
Wengler, G., Castle, E., Leidner, U., Nowak, T. & Wengler, G. (1985). Sequence analysis of the membrane protein V3 of the flavivirus West Nile virus and of its gene. Virology 147, 264–267.[CrossRef][Medline]
Yamshchikov, V. F., Wengler, G., Perelygin, A. A., Brinton, M. A. & Compans, R. W. (2001). An infectious clone of the West Nile flavivirus. Virology 281, 294–304.[CrossRef][Medline]
Yamshchikov, G., Borisevich, V., Seregin, A., Chaporgina, E., Mishina, M., Mishin, V., Kwok, C. W. & Yamshchikov, V. (2004). An attenuated West Nile prototype virus is highly immunogenic and protects against the deadly NY99 strain: a candidate for live WN vaccine development. Virology 330, 304–312.[CrossRef][Medline]
Received 6 July 2007;
accepted 27 November 2007.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
