Discrimination of West Nile virus and Japanese encephalitis virus strains using RT-PCR RFLP analysis

West Nile (WN) virus is a mosquito-borne flavivirus that induces lethal encephalitis in humans and horses. Since an outbreak of WN encephalitis in humans and horses occurred in New York City in late August 1999, the possibility exists that WN virus will invade regions that have close links with the United States, such as Japan. We developed a genetic diagnostic method that discriminates between strains of WN virus and Japanese encephalitis (JE) virus. The method involves RT-PCR restriction fragment length polymorphism (RFLP) analysis with a RT-PCR primer set, a nested PCR primer set, and a restriction enzyme. We detected WN and JE viruses in experimentally infected animal brain, spleen, and serum samples. Our method is useful in distinguishing WN viruses from the endemic background of JE viruses, and in discriminating the highly virulent WN strain, which was isolated in New York in 1999, from other WN virus strains.     

Subpopulation of peripheral blood lymphocytes in human encephalitis caused by group B arboviruses (Dengue, West Nile and Japanese B encephalitis.

Sera from twenty patients with clinically diagnosed viral encephalitis were studied during the acute and convalescent phases of illness for evidence of group B arboviral infection by demonstrating a rise in the titre of haemagglutination inhibiting (HI) antibodies. The immune status of these individuals was studied by quantitating peripheral T, T and 'null' lymphocytes. The percentage of total T cells in arboviral encephalitic (AE) group decreased significantly while that of B cells and 'active' T cells remained unaltered when compared to that of normal individuals. The null cell percentage, on the other hand increased significantly in the AE group. The absolute number of circulating T and B cells did not alter in the AE group while that of null cells rose significantly. The percentage and concentration of T. B and null cells during the convalescent phase of AE did not differ significantly when compared to those of the acute phase.     

Infection and injury of neurons by West Nile encephalitis virus

West Nile virus (WNV) infects neurons and leads to encephalitis, paralysis, and death in humans, animals, and birds. We investigated the mechanism by which neuronal injury occurs after WNV infection. Neurons in the anterior horn of the spinal cords of paralyzed mice exhibited a high degree of WNV infection, leukocyte infiltration, and degeneration. Because it was difficult to distinguish whether neuronal injury was caused by viral infection or by the immune system response, a novel tissue culture model for WNV infection was established in neurons derived from embryonic stem (ES) cells. Undifferentiated ES cells were relatively resistant to WNV infection. After differentiation, ES cells expressed neural antigens, acquired a neuronal phenotype, and became permissive for WNV infection. Within 48 h of exposure to an exceedingly low multiplicity of infection (5 x 10(-4)), 50% of ES cell-derived neurons became infected, producing nearly 10(7) PFU of infectious virus per ml, and began to die by an apoptotic mechanism. The establishment of a tractable virus infection model in ES cell-derived neurons facilitates the study of the molecular basis of neurotropism and the mechanisms of viral and immune-mediated neuronal injury after infection by WNV or other neurotropic pathogens.     

Immunization of flavivirus West Nile recombinant envelope domain III protein induced specific immune response and protection against West Nile virus infection

The domain III of the West Nile virus (WNV) envelope glycoprotein (E) was shown to serve as virus attachment domain to the cellular receptor, and neutralizing Abs have been mapped to this specific domain. In this study, domain III of the WNV E protein (WNV E DIII) was expressed as a recombinant protein and its potential as a subunit vaccine candidate was evaluated in BALB/C mice. Immunization of WNV E DIII protein with oligodeoxynucleotides (CpG-DNA) adjuvant by i.p. injection was conducted over a period of 3 wk. The immunized mice generated high titer of WNV-neutralizing Abs. Murine Ab against WNV E DIII protein was also capable of neutralizing Japanese encephalitis virus. The IgG isotypes generated were predominantly IgG2a in the murine sera against the recombinant protein. Splenocyte cultures from the mice coadministrated with WNV E DIII protein and CpG secreted large amounts of IFN-gamma and IL-2 and showed proliferation of T cells in the presence of WNV E DIII protein. Overall, this study highlighted that recombinant WNV E DIII protein delivered in combination with CpG adjuvant to mice generated a Th1 immune response type against WNV and can serve as a potential vaccine to prevent WNV infection.     

Drak2 contributes to West Nile virus entry into the brain and lethal encephalitis

Death-associated protein kinase-related apoptosis-inducing kinase-2 (Drak2), a member of the death-associated protein family of serine/threonine kinases, is specifically expressed in T and B cells. In the absence of Drak2, mice are resistant to experimental autoimmune encephalomyelitis due to a decrease in the number of cells infiltrating the CNS. In the present study, we investigated the role of Drak2 in West Nile virus (WNV)-induced encephalitis and found that Drak2(-/-) mice were also more resistant to lethal WNV infection than wild-type mice. Although Drak2(-/-) mice had an increase in the number of IFN-gamma-producing T cells in the spleen after infection, viral levels in the peripheral tissues were not significantly different between these two groups of mice. In contrast, there was a reduced viral load in the brains of Drak2(-/-) mice, which was accompanied by a decrease in the number of Drak2(-/-) CD4(+) and CD8(+) T cells in the brain following WNV infection. Moreover, we detected viral Ags in T cells isolated from the spleen or brain of WNV-infected mice. These results suggest that following a systemic infection, WNV might cross the blood brain barrier and enter the CNS by being carried by infected infiltrating T cells.     

Changing the protease specificity for activation of a flavivirus, tick-borne encephalitis virus

The infectivity of flavivirus particles depends on a maturation process that is triggered by the proteolytic cleavage of the precursor of the M protein (prM). This activation cleavage is naturally performed by ubiquitous cellular proteases of the furin family, which typically recognize the multibasic sequence motif R-X-R/K-R. Previously, we demonstrated that a tick-borne encephalitis virus (TBEV) mutant with an altered cleavage motif, R-X-R, produced immature, noninfectious particles that could be activated by exogenous trypsin, which cleaves after single basic residues. Here, we report the adaptation of this mutant to chymotrypsin, a protease specific for large, hydrophobic amino acid residues. Using selection pressure in cell culture, two different mutations conferring a chymotrypsin-dependent phenotype were identified. Surprisingly, one of these mutations (Ser85Phe) occurred three positions upstream of the natural cleavage site. The other mutation (Arg89His) arose at the natural cleavage position but involved a His residue, which is not a typical chymotrypsin cleavage site. Efficient cleavage of protein prM and activation by the heterologous protease were confirmed using various recombinant TBEV mutants. Mutants with only the originally selected mutations exhibited unimpaired export kinetics and were genotypically stable during at least six cell culture passages. However, in contrast to the wild-type virus or trypsin-dependent mutants, chymotrypsin-dependent mutants were not neurovirulent in suckling mice. Our results demonstrate that flaviviruses with altered protease specificities can be generated and suggest that this approach can be used for the construction of viral mutants or vectors that can be activated on demand and have restricted tissue tropism and virulence.     

Studies on the antigenic structure of Japanese encephalitis virus using monoclonal antibodies

The antigenic relationships among 11 strains of Japanese encephalitis (JE) virus were analyzed by using monoclonal antibodies (NARMA) against the Nakayama-RFVL strain in hemagglutination-inhibition (HI) and neutralization (Nt) tests. Of the 14 JE virus-specific HI antibodies, all except NARMA 5 showed Nt reactivity with the homologous strain. The HI and Nt titers of these antibodies were not parallel. The 14 antibodies included the following characteristic antibodies: NARMA 3 is a species-specific antibody with HI and Nt reactivities against JE virus, NARMA 13 is a species-specific HI antibody, NARMA 6 is a Nakayama strain-specific antibody with HI and Nt reactivities, and NARMA 5 is a Nakayama strain-specific HI antibody. The 11 strains of JE virus were divided into four major antigenic groups. However, slight antigenic differences were found among some strains of the same group. Furthermore, competitive binding assays were performed to determine the distribution of antigenic determinants by enzyme-linked immunosorbent assay. The results suggest the existence of at least five HI sites on the JE virus virion, and indicate that the JE species-specific HI site and the flavivirus genus-specific HI site are topologically distinct.     

Caspase 3-dependent cell death of neurons contributes to the pathogenesis of West Nile virus encephalitis

West Nile virus (WNV) is a neurotropic, arthropod-borne flavivirus that has become a significant global cause of viral encephalitis. To examine the mechanisms of WNV-induced neuronal death and the importance of apoptosis in pathogenesis, we evaluated the role of a key apoptotic regulator, caspase 3. WNV infection induced caspase 3 activation and apoptosis in the brains of wild-type mice. Notably, congenic caspase 3(-/-) mice were more resistant to lethal WNV infection, although there were no significant differences in the tissue viral burdens or the kinetics of viral spread. Instead, decreased neuronal death was observed in the cerebral cortices, brain stems, and cerebella of caspase 3(-/-) mice. Analogously, primary central nervous system (CNS)-derived neurons demonstrated caspase 3 activation and apoptosis after WNV infection, and treatment with caspase inhibitors or a genetic deficiency in caspase 3 significantly decreased virus-induced death. These studies establish that caspase 3-dependent apoptosis contributes to the pathogenesis of lethal WNV encephalitis and suggest possible novel therapeutic targets to restrict CNS injury.     

Characterization of the West Nile virus protease substrate specificity and inhibitors

West Nile virus (WNV), a mosquito-borne member of Flaviviridae, is a human pathogen causing widespread disease for which there is no vaccine or chemotherapy. The two-component viral serine protease consists of a heterodimeric complex between the hydrophilic domain of the cofactor, NS2B (NS2BH) and the protease domain (NS3-pro). The protease is essential for polyprotein processing followed by assembly of viral replicase and genome replication. Therefore, the protease is an excellent target for development of antiviral therapeutics. Here, we report the expression in Escherichia coli, purification, and characterization of biochemical and kinetic properties of the WNV protease. Furthermore, we show that the WNV and the dengue virus type 2 (DENV-2) proteases are inhibited by aprotinin with inhibitor constants of 0.16 and 0.026 microM, respectively. Molecular modeling of the WNV protease/aprotinin complex, based on the known crystal structures of the WNV NS2BH-N3pro and aprotinin, suggest a potentially strong interaction between the P2 Lys and the protease activator peptide, NS2BH. This conclusion based on molecular modeling is in agreement with our data of a higher k(cat)/Km value with the substrate, Boc-Gly-Lys-Arg-MCA than the Boc-Gly-Arg-Arg-MCA and is also consistent with the results of an earlier study that were based on substrate-based inhibitor peptides.     

The Israeli strain IS-98-ST1 of West Nile virus as viral model for West Nile encephalitis in the Old World

West Nile virus (WNV) recently became a major public health concern in North America, the Middle East, and Europe. In contrast with the investigations of the North-American isolates, the neurovirulence properties of Middle-Eastern strains of WNV have not been extensively characterized. Israeli WNV strain IS-98-ST1 that has been isolated from a white stork in 1998, was found to be highly neuroinvasive in adult C57BL/6 mice. Strain IS-98-ST1 infects primary neuronal cells from mouse cortex, causing neuronal death. These results demonstrate that Israeli strain IS-98-ST1 provides a suitable viral model for WNV-induced disease associated with recent WNV outbreaks in the Old World.     

Antibodies to West Nile virus: a double-edged sword

Antibodies, although critical for host defense against West Nile virus (WNV), can both neutralize and enhance viral infection. In this issue of Cell Host & Microbe, Pierson et al. dissect these opposing effects and demonstrate that, when 120 epitopes are available per WNV virion, approximately 25 are occupied by antibody at 50% neutralization. At lower occupancies, enhancement of infection dominates; at higher ones, neutralization ensues. These results are important for WNV vaccine design and for potential therapeutic use of antibodies to WNV.     

Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis

West Nile virus (WNV), a mosquito-borne single-stranded (ss)RNA flavivirus, causes human disease of variable severity. We investigated the involvement of Toll-like receptor (Tlr) 3, which recognizes viral double-stranded (ds)RNA, on WNV infection. Tlr3-deficient (Tlr3(-/-)) mice were more resistant to lethal WNV infection and had impaired cytokine production and enhanced viral load in the periphery, whereas in the brain, viral load, inflammatory responses and neuropathology were reduced compared to wild-type mice. Peripheral WNV infection led to a breakdown of the blood-brain barrier and enhanced brain infection in wild-type but not in Tlr3(-/-) mice, although both groups were equally susceptible upon intracerebroventricular administration of the virus. Tumor necrosis factor-alpha receptor 1 signaling is vital for blood-brain barrier compromise upon Tlr3 stimulation by dsRNA or WNV. Collectively, WNV infection leads to a Tlr3-dependent inflammatory response, which is involved in brain penetration of the virus and neuronal injury.     

Resuscitating mutations in a furin cleavage-deficient mutant of the flavivirus tick-borne encephalitis virus

Cleavage of the viral surface protein prM by the proprotein convertase furin is a key step in the maturation process of flavivirus particles. A mutant of tick-borne encephalitis virus (TBEV) carrying a deletion mutation within the furin recognition motif of protein prM (changing R-T-R-R to R-T-R) was previously shown to be noninfectious in BHK-21 cells. We now demonstrate how natural selection can overcome this lethal defect in two different growth systems by distinct resuscitating mutations. In BHK-21 cells, a spontaneous codon duplication created a minimal furin cleavage motif (R-R-T-R). This mutation restored infectivity by enabling intracellular prM cleavage. A completely different mutation pattern was observed when the mutant virus was passaged in mouse brains. The "pr" part of protein prM, which is removed by cleavage, contains six conserved Cys residues. The mutations selected in mice changed the number of Cys residues to five or seven by substitution mutations near the original cleavage site, probably causing a major perturbation of the structural integrity of protein prM. Although viable in mice, such Cys mutants could not be passaged in BHK-21 cells under normal growth conditions (37 degrees C), but one of the mutants exhibited a low level of infectivity at a reduced incubation temperature (28 degrees C). No evidence for the cleavage of protein prM in BHK-21 cells was obtained. This suggests that under certain growth conditions, the structural perturbation of protein prM can restore the infectivity of TBEV by circumventing the need for intracellular furin-mediated cleavage. This is the first example of a flavivirus using such a molecular mechanism.     

Serological detection of St. Louis encephalitis virus and West Nile virus in equines from Santa Fe, Argentina

St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) present ecological and antigenic similarities and are responsible for serious human diseases. In addition, WNV is a significant pathogen in terms of equine health. The purpose of our study was to analyse the seroprevalence of SLEV and WNV in equine sera collected in Santa Fe Province, Argentina. The seroprevalence determined using the plaque reduction neutralisation test was 12.2% for SLEV, 16.2% for WNV and 48.6% for a combination of both viruses. These results provide evidence of the co-circulation of SLEV and WNV in equines in Santa Fe.     

Persistence of West Nile virus

West Nile virus (WNV) is a widespread global pathogen that results in significant morbidity and mortality. Data from animal models provide evidence of persistent renal and neurological infection from WNV; however, the possibility of persistent infection in humans and long-term neurological and renal outcomes related to viral persistence remain largely unknown. In this paper, we provide a review of the literature related to persistent infection in parallel with the findings from cohorts of patients with a history of WNV infection. The next steps for enhancing our understanding of WNV as a persistent pathogen are discussed.     

Common E protein determinants for attenuation of glycosaminoglycan-binding variants of Japanese encephalitis and West Nile viruses

Natural isolates and laboratory strains of West Nile virus (WNV) and Japanese encephalitis virus (JEV) were attenuated for neuroinvasiveness in mouse models for flavivirus encephalitis by serial passage in human adenocarcinoma (SW13) cells. The passage variants displayed a small-plaque phenotype, augmented affinity for heparin-Sepharose, and a marked increase in specific infectivity for SW13 cells relative to the respective parental viruses, while the specific infectivity for Vero cells was not altered. Therefore, host cell adaptation of passage variants was most likely a consequence of altered receptor usage for virus attachment-entry with the involvement of cell surface glycosaminoglycans (GAG) in this process. In vivo blood clearance kinetics of the passage variants was markedly faster and viremia was reduced relative to the parental viruses, suggesting that affinity for GAG (ubiquitously present on cell surfaces and extracellular matrices) is a key determinant for the neuroinvasiveness of encephalitic flaviviruses. A difference in pathogenesis between WNV and JEV, which was reflected in more efficient growth in the spleen and liver of the WNV parent and passage variants, accounted for a less pronounced loss of neuroinvasiveness of GAG binding variants of WNV than JEV. Single gain-of-net-positive-charge amino acid changes at E protein residue 49, 138, 306, or 389/390, putatively positioned in two clusters on the virion surface, define molecular determinants for GAG binding and concomitant virulence attenuation that are shared by the JEV serotype flaviviruses.     

CD8+ T cells mediate recovery and immunopathology in West Nile virus encephalitis

C57BL/6J mice infected intravenously with the Sarafend strain of West Nile virus (WNV) develop a characteristic central nervous system (CNS) disease, including an acute inflammatory reaction. Dose response studies indicate two distinct kinetics of mortality. At high doses of infection (10(8) PFU), direct infection of the brain occurred within 24 h, resulting in 100% mortality with a 6-day mean survival time (MST), and there was minimal destruction of neural tissue. A low dose (10(3) PFU) of infection resulted in 27% mortality (MST, 11 days), and virus could be detected in the CNS 7 days postinfection (p.i.). Virus was present in the hypogastric lymph nodes and spleens at days 4 to 7 p.i. Histology of the brains revealed neuronal degeneration and inflammation within leptomeninges and brain parenchyma. Inflammatory cell infiltration was detectable in brains from day 4 p.i. onward in the high-dose group and from day 7 p.i. in the low-dose group, with the severity of infiltration increasing over time. The cellular infiltrates in brain consisted predominantly of CD8(+), but not CD4(+), T cells. CD8(+) T cells in the brain and the spleen expressed the activation markers CD69 early and expressed CD25 at later time points. CD8(+) T-cell-deficient mice infected with 10(3) PFU of WNV showed increased mortalities but prolonged MST and early infection of the CNS compared to wild-type mice. Using high doses of virus in CD8-deficient mice leads to increased survival. These results provide evidence that CD8(+) T cells are involved in both recovery and immunopathology in WNV infection.