Flaviviruses have imperfect icosahedral symmetry.

Flaviviruses assemble initially in an immature, noninfectious state and undergo extensive conformational rearrangements to generate mature virus. Previous cryo-electron microscopy (cryo-EM) structural studies of flaviviruses assumed icosahedral symmetry and showed the concentric organization of the external glycoprotein shell, the lipid membrane, and the internal nucleocapsid core. We show here that when icosahedral symmetry constraints were excluded in calculating the cryo-EM reconstruction of an immature flavivirus, the nucleocapsid core was positioned asymmetrically with respect to the glycoprotein shell. The core was positioned closer to the lipid membrane at the proximal pole, and at the distal pole, the outer glycoprotein spikes and inner membrane leaflet were either perturbed or missing. In contrast, in the asymmetric reconstruction of a mature flavivirus, the core was positioned concentric with the glycoprotein shell. The deviations from icosahedral symmetry demonstrated that the core and glycoproteins have varied interactions, which likely promotes viral assembly and budding.

Systematic analysis of protein identity between Zika virus and other arthropod-borne viruses.

OBJECTIVE: To analyse the proportions of protein identity between Zika virus and dengue, Japanese encephalitis, yellow fever, West Nile and chikungunya viruses as well as polymorphism between different Zika virus strains. METHODS: We used published protein sequences for the Zika virus and obtained protein sequences for the other viruses from the National Center for Biotechnology Information (NCBI) protein database or the NCBI virus variation resource. We used BLASTP to find regions of identity between viruses. We quantified the identity between the Zika virus and each of the other viruses, as well as within-Zika virus polymorphism for all amino acid k-mers across the proteome, with k ranging from 6 to 100. We assessed accessibility of protein fragments by calculating the solvent accessible surface area for the envelope and nonstructural-1 (NS1) proteins. FINDINGS: In total, we identified 294 Zika virus protein fragments with both low proportion of identity with other viruses and low levels of polymorphisms among Zika virus strains. The list includes protein fragments from all Zika virus proteins, except NS3. NS4A has the highest number (190 k-mers) of protein fragments on the list. CONCLUSION: We provide a candidate list of protein fragments that could be used when developing a sensitive and specific serological test to detect previous Zika virus infections.

Comparative pathology of neurovirulent lineage 1 (NY99/385) and lineage 2 (SPU93/01) West Nile virus infections in BALBc mice.

The pathology in mice infected with neurovirulent South African lineage 2 West Nile virus (WNV) strains has not previously been described. Three- to 4-month-old male BALBc mice were infected with South African neurovirulent lineage 2 (SPU93/01) or lineage 1 (NY385/99) WNV strains and the gross and microscopic central nervous system (CNS) and extra-CNS pathology of both investigated and compared. Mice infected with both lineages showed similar illness, paralysis, and death from days 7 to 11 postinfection (PI). Two survivors of each lineage were euthanized on day 21 PI. WNV infection was confirmed by nested real-time reverse transcription polymerase chain reaction of tissues, mostly brain, in the majority of mice euthanized sick or that died and in 1 healthy lineage 2 survivor. Gross lesions caused by both lineages were identical and included marked gastric and proximal small intestinal fluid distension as described in a previous mouse study, but intestinal microscopic lesions differed. CNS lesions were subtle. Immunohistochemical (IHC)-positive labeling for WNV E protein was found in neurons multifocally in the brain of 3 lineage 1-infected and 3 lineage 2-infected mice from days 9 to 11 PI, 4 of these including brainstem neurons, and of cecal myenteric ganglion neurons in 1 lineage 2-infected day 8 PI mouse. Findings supported hypotheses in hamsters that gastrointestinal lesions are likely of brainstem origin. Ultrastructurally, virus-associated cytoplasmic vesicular or crystalline structures, or amorphous structures, were found to label IHC positive in control-positive avian cardiomyocytes and mouse thalamic neurons, respectively, and WNV-like 50-nm particles, which were scarce, did not label.

Membrane curvature in flaviviruses.

Coordinated interplay between membrane proteins and the lipid bilayer is required for such processes as transporter function and the entrance of enveloped viruses into host cells. In this study, three-dimensional cryo-electron microscopy density maps of mature and immature flaviviruses were analyzed to assess the curvature of the membrane leaflets and its relation to membrane-bound viral glycoproteins. The overall morphology of the viral membrane is determined by the icosahedral scaffold composed of envelope (E) and membrane (M) proteins through interaction of the proteins' stem-anchor regions with the membrane. In localized regions, small membrane areas exhibit convex, concave, flat or saddle-shaped surfaces that are constrained by the specific protein organization within each membrane leaflet. These results suggest that the organization of membrane proteins in small enveloped viruses mediate the formation of membrane curvature.

Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane.

Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.

Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354.

Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.

The endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex.

The cytoplasmic replication of positive-sense RNA viruses is associated with a dramatic rearrangement of host cellular membranes. These virus-induced changes result in the induction of vesicular structures that envelop the virus replication complex (RC). In this study, we have extended our previous observations on the intracellular location of West Nile virus strain Kunjin virus (WNV(KUN)) to show that the virus-induced recruitment of host proteins and membrane appears to occur at a pre-Golgi step. To visualize the WNV(KUN) replication complex, we performed three-dimensional (3D) modeling on tomograms from WNV(KUN) replicon-transfected cells. These analyses have provided a 3D representation of the replication complex, revealing the open access of the replication complex with the cytoplasm and the fluidity of the complex to the rough endoplasmic reticulum. In addition, we provide data that indicate that a majority of the viral RNA species housed within the RC is in a double-stranded RNA (dsRNA) form.

Crystallization and preliminary X-ray diffraction analysis of West Nile virus.

West Nile virus, a human pathogen, is closely related to other medically important flaviviruses of global impact such as dengue virus. The infectious virus was purified from cell culture using polyethylene glycol (PEG) precipitation and density-gradient centrifugation. Thin amorphously shaped crystals of the lipid-enveloped virus were grown in quartz capillaries equilibrated by vapor diffusion. Crystal diffraction extended at best to a resolution of about 25 A using synchrotron radiation. A preliminary analysis of the diffraction images indicated that the crystals had unit-cell parameters a approximately b approximately 480 A, gamma = 120 degrees , suggesting a tight hexagonal packing of one virus particle per unit cell.

Imported West Nile virus encephalitis in an Israeli tourist.

West Nile virus is an arbovirus that has caused large outbreaks of febrile illness, meningitis and encephalitis in Europe, North America and the Middle East. We describe the first laboratory-confirmed human case of West Nile virus infection in Australia, in a 58-year-old tourist who was almost certainly infected in Israel. The case is a reminder of the need to consider exotic pathogens in travellers and of the risk of introducing new pathogens into Australia.

Role of nonstructural protein NS2A in flavivirus assembly.

Flavivirus nonstructural (NS) proteins are involved in RNA replication and modulation of the host antiviral response; however, evidence is mounting that some NS proteins also have essential roles in virus assembly. Kunjin virus (KUN) NS2A is a small, hydrophobic, transmembrane protein that is part of the replication complex and inhibits interferon induction. Previously, we have shown that an isoleucine (I)-to-asparagine (N) substitution at position 59 of the NS2A protein blocked the production of secreted virus particles in cells electroporated with viral RNA carrying this mutation. We now show that prolonged incubation of mutant KUN NS2A-I59N replicon RNA, in an inducible BHK-derived packaging cell line (expressing KUN structural proteins C, prM, and E), generated escape mutants that rescued the secretion of infectious virus-like particles. Sequencing identified three groups of revertants that included (i) reversions to wild-type, hydrophobic Ile, (ii) pseudorevertants to more hydrophobic residues (Ser, Thr, and Tyr) at codon 59, and (iii) pseudorevertants retaining Asn at NS2A codon 59 but containing a compensatory mutation (Thr-to-Pro) at NS2A codon 149. Engineering hydrophobic residues at NS2A position 59 or the compensatory T149P mutation into NS2A-I59N replicon RNA restored the assembly of secreted virus-like particles in packaging cells. T149P mutation also rescued virus production when introduced into the full-length KUN RNA containing an NS2A-I59N mutation. Immunofluorescence and electron microscopy analyses of NS2A-I59N replicon-expressing cells showed a distinct lack of virus-induced membranes normally present in cells expressing wild-type replicon RNA. The compensatory mutation NS2A-T149P restored the induction of membrane structures to a level similar to those observed during wild-type replication. The results further confirm the role of NS2A in virus assembly, demonstrate the importance of hydrophobic residues at codon 59 in this process, implicate the involvement of NS2A in the biogenesis of virus-induced membranes, and suggest a vital role for the virus-induced membranes in virus assembly.

Axonal transport mediates West Nile virus entry into the central nervous system and induces acute flaccid paralysis.

West Nile virus (WNV) has emerged as a significant cause of epidemic viral encephalitis and flaccid limb paralysis, yet the mechanism by which it enters the CNS remains uncertain. We used compartmentalized neuron cultures to demonstrate that WNV spreads in both retrograde and anterograde directions via axonal transport. Transneuronal spread of WNV required axonal release of viral particles and was blocked by addition of a therapeutic neutralizing antibody. To test the physiologic significance of axonal transport in vivo, we directly inoculated the sciatic nerve of hamsters with WNV. Intrasciatic infection resulted in paralysis of the hind limb ipsilateral but not contralateral to the injection site. Limb paralysis was blocked either by surgical transection of the sciatic nerve or treatment with the therapeutic neutralizing antibody. Collectively, these studies establish that WNV undergoes bidirectional spread in neurons and that axonal transport promotes viral entry into the CNS and acute limb paralysis. Moreover, antibody therapeutics directly inhibit transneuronal spread of WNV infection and prevent the development of paralysis in vivo.

Structure of immature West Nile virus.

The structure of immature West Nile virus particles, propagated in the presence of ammonium chloride to block virus maturation in the low-pH environment of the trans-Golgi network, was determined by cryo-electron microscopy (cryo-EM). The structure of these particles was similar to that of immature West Nile virus particles found as a minor component of mature virus samples (naturally occurring immature particles [NOIPs]). The structures of mature infectious flaviviruses are radically different from those of the immature particles. The similarity of the ammonium chloride-treated particles and NOIPs suggests either that the NOIPs have not undergone any conformational change during maturation or that the conformational change is reversible. Comparison with the cryo-EM reconstruction of immature dengue virus established the locations of the N-linked glycosylation sites of these viruses, verifying the interpretation of the reconstructions of the immature flaviviruses.

West Nile virus infection of primary mouse neuronal and neuroglial cells: the role of astrocytes in chronic infection.

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.

Manifestations of West Nile neuroinvasive disease.

Since its introduction to North America in 1999, West Nile virus, an arthropod-borne flavivirus, has become the most significant cause of epidemic encephalitis in the western hemisphere. While most human infections with the virus are asymptomatic and the majority of symptomatic persons experience febrile illness, severe neurologic manifestations, including meningitis, encephalitis, and poliomyelitis may be seen. This review summarizes the virology, epidemiology and pathogenesis of human infection with West Nile virus, and details recent advances in our understanding of the pathophysiology and various clinical manifestations of infection.

West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody.

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.

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.

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.

West Nile virus infection induces interferon signalling in human retinal pigment epithelial cells.

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.

Identifying the region influencing the cis-mode of maturation of West Nile (Sarafend) virus using chimeric infectious clones.

West Nile (Sarafend) virus [WN(S)V] has been shown to egress by budding at the plasma membrane of infected cells. However, the region influencing this mode of virus release remains to be deciphered. In this study, we have constructed three chimeric clones in which specific regions of West Nile (Wengler) virus [WN(W)V] were replaced for the corresponding regions of WN(S)V in the full-length infectious clone of WN(S)V to define the region responsible for the cis-mode of WN(S)V maturation. The WN(W)V matures by the trans-mode. All of the resulting chimeric viruses were found to be infective. Transmission electron microscopy analyses performed in Vero cells infected with these chimeric viruses disclosed that the 5' end of the WN(S)V genome plays a major role in influencing the process of maturation at the plasma membrane.

Fatal West Nile virus encephalitis in a renal transplant recipient.

West Nile virus (WNV), a mosquito-transmitted single-stranded RNA flavivirus, causes human disease of variable severity. We report clinical and pathologic findings of fatal encephalitis from the transmission of WNV from an organ donor to a kidney transplant recipient. The patient developed a febrile illness 18 days after transplantation, which progressed to encephalitis. Postmortem examination demonstrated extensive viral encephalopathic changes. Immunohistochemical studies highlighted WNV antigens within neurons, especially in the cerebellum and brainstem. Flavivirus virions were detected ultrastructurally within the cerebellum, and WNV was isolated from the brain and the brainstem. Thus, this case demonstrates the first death in the first solid organ transplant-associated transmission of WNV. Immunosuppression of the transplant recipient might have been responsible for the fulminant viral effects. The pathologic diagnosis helped guide subsequent epidemiologic and laboratory studies.