The Organisation of Ebola Virus Reveals a Capacity for Extensive, Modular Polyploidy

BACKGROUND: Filoviruses, including Ebola virus, are unusual in being filamentous animal viruses. Structural data on the arrangement, stoichiometry and organisation of the component molecules of filoviruses has until now been lacking, partially due to the need to work under level 4 biological containment. The present study provides unique insights into the structure of this deadly pathogen. METHODOLOGY AND PRINCIPAL FINDINGS: We have investigated the structure of Ebola virus using a combination of cryo-electron microscopy, cryo-electron tomography, sub-tomogram averaging, and single particle image processing. Here we report the three-dimensional structure and architecture of Ebola virus and establish that multiple copies of the RNA genome can be packaged to produce polyploid virus particles, through an extreme degree of length polymorphism. We show that the helical Ebola virus inner nucleocapsid containing RNA and nucleoprotein is stabilized by an outer layer of VP24-VP35 bridges. Elucidation of the structure of the membrane-associated glycoprotein in its native state indicates that the putative receptor-binding site is occluded within the molecule, while a major neutralizing epitope is exposed on its surface proximal to the viral envelope. The matrix protein VP40 forms a regular lattice within the envelope, although its contacts with the nucleocapsid are irregular. CONCLUSIONS: The results of this study demonstrate a modular organization in Ebola virus that accommodates a well-ordered, symmetrical nucleocapsid within a flexible, tubular membrane envelope

Identification and characterization of \u3ci\u3eShigella boydii\u3c/i\u3e 20 serovar nov., a new and emerging \u3ci\u3eShigella\u3c/i\u3e serotype

Analysis of 163 putative Shigella isolates from Canada and the USA showed biochemical reactions consistent with Shigella species, although none of the isolates reacted with antiserum raised against any of the well-established or provisional Shigella serotypes. All these isolates, provisionally designated serotype SH108, were positive for the ipaH gene and the invasion-associated locus. All fermented mannitol, were serologically indistinguishable from each other and showed no reaction in antisera prepared against Escherichia coli serotypes O1 to O181. PCR-RFLP analysis of the genes involved in O-antigen synthesis revealed a common pattern among these isolates that was distinct from recognized Shigella serotypes and E. coli. Between 1999 and 2003, isolates from across Canada were submitted to the National Laboratory for Enteric Pathogens for antibiotic susceptibility testing, phage typing and PFGE. These assays revealed heterogeneity among the members of this serotype. Antimicrobial susceptibility testing with seven antibiotics identified six profiles, with 90% (45/50) of the isolates resistant to four or more antibiotics and 72%(36/50) resistant to five or more. All isolates were typable using a panel of 16 phages, with 11 different phage types (PTs) represented. The most common PTs found were PT 3 (64 %), PT 6 (10 %) and PT 16 (6 %). Analysis of Xbal-restricted genomic DNA revealed 16 highly related patterns that were not readily distinguishable from those obtained for some other Shigella serotypes. The World Health Organization Collaborating Center for Shigella has added serotype SH108 to the Shigella scheme as S. boydii serotype 20 (serovar nov.). Strain SH108 (isolate 99-4528) is the reference strain for this serotype

Characterization of Waterborne Outbreak–associated Campylobacter jejuni, Walkerton, Ontario

The Walkerton, Canada, waterborne outbreak of 2000 resulted from entry of Escherichia coli O157:H7 and Campylobacter spp. from neighboring farms into the town water supply. Isolates of Campylobacter jejuni and Campylobacter coli obtained from outbreak investigations were characterized by phenotypic and genotypic methods, including heat-stable and heat-labile serotyping, phage typing, biotyping, fla–restriction fragment length polymorphism (RFLP) typing, and pulsed-field gel electrophoresis. Two main outbreak strains were identified on the basis of heat-stable serotyping and fla-RFLP typing. These strains produced a limited number of types when tested by other methods. Isolates with types indistinguishable from, or similar to, the outbreak types were found only on one farm near the town of Walkerton, whereas cattle from other farms carried a variety of Campylobacter strains with different type characteristics. Results of these analyses confirmed results from epidemiologic studies and the utility of using several different typing and subtyping methods for completely characterizing bacterial populations

Sub-tomogram averaging of Ebola virus.

<p>(A–D) Sections of the density map of the sub-tomogram average are shown from the top sliced just below the envelope (A), the middle of the virus (B), a side view of the virus (C), and an end-on slice (D). Putative locations of several VP40 proteins adjacent to the membrane are circled. (E,F) Images showing just the nucleocapsid. The helix is right handed (arrow in E). No helical symmetry was applied to this data. Color coding as follows; beige, lipid envelope; green, membrane associated proteins (VP40); blue and purple, outer and inner nucleocapsid.</p

3D structure of the Ebola spike.

<p>The density map of the EBOV GP spike viewed from the side, end-on, and side (with envelope) shows the docked GP1–GP2 structure in yellow (PDB entry 3CSY <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029608#pone.0029608-Lee1" target="_blank">[42]</a>), glycosylation sites (green), and receptor binding site (RBS; red, highlighted). (A) The reconstruction showing the spike (orange) and the envelope (beige). (B) Difference map generated by subtracting the docked structure from reconstruction of the entire spike. The color scheme shows the following putative regions; green, mucin domain; pink, deletions 190–213, 279–298; purple-blue, GP2 stalk. The docked KZ52 neutralizing antibody is shown in purple.</p

Quantitation of Ebola virus length.

<p>(A) Histogram of virion length, with cryo-EM images showing single, continuous and linked particles. A total of 2090 virions with continuous nucleocapsids (no obvious segmentations) were measured, showing the relationship between length and genome copy number per virus. Empty and linked EBOV structures were excluded from the histogram data. A single G1-single/comma shaped EBOV is shown (inset on the right, G1 = 1 copy of genome). (B) Low magnification cryo-images showing: G1- single/comma shape, G1- single/linear, G5-continuous (G5 = 5 copies of genome). (C) High magnification of a G1- (single genome) virion with a region filtered to emphasize the nucleocapsid. (D) Low magnification image of a G4-linked EBOV, each genome copy is indicated and numbered, the red arrows show the transition points between nucleocapsids. The circular holes (filled with vitreous ice) appear as lighter regions and the support film (“quantifoil”) appears dark grey. A “linker” region is shown at higher magnification (inset).</p