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.

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.

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.

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.

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.

West Nile virus: a primer for the clinician.

This paper provides the clinician with an understanding of the epidemiologic and biological characteristics of West Nile virus in North America, as well as useful information on the diagnosis, reporting, and management of patients with suspected West Nile virus infection and on advising patients about prevention. Information was gathered from the medical literature and from national surveillance data through May 2002. Since the identification of West Nile virus in New York City in 1999, enzootic activity has been documented in 27 states and the District of Columbia. Continued geographic expansion is likely. Overall, one in 150 infections results in severe neurologic illness. Advanced age is by far the most important risk factor for neurologic disease and, once disease develops, for worse clinical outcome. Surveillance has identified 149 persons with West Nile virus-related illness in 10 states. Encephalitis is more commonly reported than meningitis, and concomitant muscle weakness and flaccid paralysis may provide a clinical clue to the presence of West Nile virus infection. Peak incidence occurs in late summer, although onset has occurred from July through December. Immunoglobulin M antibody testing of serum specimens and cerebrospinal fluid is the most efficient method of diagnosis, although cross-reactions are possible in patients recently vaccinated against or recently infected with related flaviviruses. Testing can be arranged through local, state, or provincial (in Canada) health departments. Prevention rests on elimination of mosquito breeding sites; judicious use of pesticides; and avoidance of mosquito bites, including mosquito repellent use.

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.

Variations in biological features of West Nile viruses.

Pathological findings in humans, horses, and birds with West Nile (WN) encephalitis show neuronal degeneration and necrosis in the central nervous system (CNS), with diffuse inflammation. The mechanisms of WN viral penetration of the CNS and pathophysiology of the encephalitis remain largely unknown. Since 1996, several epizootics involving hundreds of humans, horses, and thousands of wild and domestic bird cases of encephalitis and mortality have been reported in Europe, North Africa, the Middle East, Russia, and the USA (see specific chapters in this issue). However, biological and molecular markers of virus virulence should be characterized to assess whether novel strains with increased virulence are responsible for this recent proliferation of outbreaks.

Cryosubstitution technique reveals new morphology of flavivirus-induced structures.

Cryotechniques in combination with electron microscopy were used in an attempt to obtain more precise morphological details of flavivirus-induced structures. From conventional chemical fixation procedure, proliferation of endoplasmic reticulum, formation of microtubule paracrystals and clusters of smooth membrane vesicles (with 'thread-like' enclosures) were observed. These induced changes are typical for flavivirus infections. The images obtained from cryosections were disappointing as the structures were not well preserved. On the other hand, cryosubstituted-infected cells gave revealing images of the virus-induced structures. The most obvious difference between the cryosubstituted and chemical fixed processes was on the morphology of the 'thread-like' structure. The 'thread-like' structures instead appeared as dense cores. The morphology of the virus particles was also better defined. The envelope of the virus appeared clearly differentiated from the nucleocapsid. The most important finding was that the cryosubstituted technique was able to preserve the structures of the flavivirus nucleocapsids which so far has not been convincing reported with chemical processing.

Brefeldin A affects West Nile virus replication in Vero cells but not C6/36 cells.

A fungal metabolite brefeldin A (BFA) was used to study virus-host interaction in glycoprotein processing in West Nile virus-infected Vero and C6/36 cells. The results indicated that as little as 1 microgram/ml of BFA resulted in complete breakdown in the Golgi organelle in infected Vero cells. This led to modifications of the glycoproteins which could not be efficiently used in infectious virion formation. In contrast, as much as 10 micrograms/ml of BFA in culture medium did not affect either glycoprotein formation or production of infectious particles in C6/36 cells. The results showed that in Vero cells, the transport of glycoproteins to the Golgi apparatus is important in West Nile virus infection. It also showed that BFA could be used as a tool to understand further the trafficking of glycoprotein from the ER to Golgi in flavivirus infection in Vero cells.

Spinal cord slices with attached dorsal root ganglia: a culture model for the study of pathogenicity of encephalitic viruses.

We describe here a culture system for long-term growth of organotypic slices of spinal cord, with attached dorsal root ganglia (DRG) derived from 13-14 day mouse fetuses. This is a unique in vitro tool in which both central and peripheral nervous tissue grow and differentiate in culture to become heavily myelinated. During cultivation the slices and the ganglia become flattened so as to allow microscopical and immunocytochemical staining. When both central and peripheral myelin had been formed (usually around the third week of cultivation), cultures were infected with 5 x 10(6) PFU of West Nile Virus (WNV). Progeny virions appeared first in about 10% of the neurons and were subsequently observed between lamellae in the central myelin sheath of several axons. Such viral arrangement in relation to myelin membranes, might provide a novel concept for a possible mechanism underlying slow viral infection.

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.