Heteroaggregation of an enveloped bacteriophage with colloidal sediments and effect on virus viability.

Four sediments in the colloidal size range: goethite, montmorillonite, illite, and kaolinite, were suspended with the bacteriophage φ6, a model enveloped virus, to determine relative rates of heteroaggregation and the effect of aggregation on virus viability. Turbidity was measured on combinations of virus and each sediment type at low concentration to determine aggregation rates. Aggregation of sediment with virus occurred regardless of mineral type, and larger fraction of virus is expected to aggregate with increasing sediment concentration leading to higher deposition rates. The negatively charged sediments, aggregated with φ6 (also negatively charged at neutral pH) at a faster rate than the positively charged sediments, yielding turbidity slopes of 4.94 × 10-3 s-1 and 7.50 × 10-4 s-1 for φ6-montmorillonite and φ6-illite aggregates, respectively, and 2.98 × 10-5 s-1 and 2.84 × 10-5 s-1, for φ6-goethite and φ6-kaolinite, respectively. This indicates that the interaction between sediments and virus is hydrophobic, rather than electrostatic. Large numbers of virions remained viable post-aggregation, despite the fragility of the viral envelope, indicating that small-sized aggregates, which may travel more readily through porous media, may pose an infection risk. The fraction of φ6 that remained viable varied with sediment type, with montmorillonite-φ6 aggregates experiencing the greatest reduction in infectivity at 35%. TEM analyses reveal that in all sediment-φ6 combinations, infectivity loss was likely due to disassembly of the viral envelope as a result of aggregation.

Component tree analysis of cystovirus φ6 nucleocapsid Cryo-EM single particle reconstructions.

The 3-dimensional structure of the nucleocapsid (NC) of bacteriophage φ6 is described utilizing component tree analysis, a topological and geometric image descriptor. The component trees are derived from density maps of cryo-electron microscopy single particle reconstructions. Analysis determines position and occupancy of structure elements responsible for RNA packaging and transcription. Occupancy of the hexameric nucleotide triphosphorylase (P4) and RNA polymerase (P2) are found to be essentially complete in the NC. The P8 protein lattice likely fixes P4 and P2 in place during maturation. We propose that the viral procapsid (PC) is a dynamic structural intermediate where the P4 and P2 can attach and detach until held in place in mature NCs. During packaging, the PC expands to accommodate the RNA, and P2 translates from its original site near the inner 3-fold axis (20 sites) to the inner 5-fold axis (12 sites) with excess P2 positioned inside the central region of the NC.

Zika virus-like particle (VLP) based vaccine.

The newly emerged mosquito-borne Zika virus poses a major public challenge due to its ability to cause significant birth defects and neurological disorders. The impact of sexual transmission is unclear but raises further concerns about virus dissemination. No specific treatment or vaccine is currently available, thus the development of a safe and effective vaccine is paramount. Here we describe a novel strategy to assemble Zika virus-like particles (VLPs) by co-expressing the structural (CprME) and non-structural (NS2B/NS3) proteins, and demonstrate their effectiveness as vaccines. VLPs are produced in a suspension culture of mammalian cells and self-assembled into particles closely resembling Zika viruses as shown by electron microscopy studies. We tested various VLP vaccines and compared them to analogous compositions of an inactivated Zika virus (In-ZIKV) used as a reference. VLP immunizations elicited high titers of antibodies, as did the In-ZIKV controls. However, in mice the VLP vaccine stimulated significantly higher virus neutralizing antibody titers than comparable formulations of the In-ZIKV vaccine. The serum neutralizing activity elicited by the VLP vaccine was enhanced using a higher VLP dose and with the addition of an adjuvant, reaching neutralizing titers greater than those detected in the serum of a patient who recovered from a Zika infection in Brazil in 2015. Discrepancies in neutralization levels between the VLP vaccine and the In-ZIKV suggest that chemical inactivation has deleterious effects on neutralizing epitopes within the E protein. This along with the inability of a VLP vaccine to cause infection makes it a preferable candidate for vaccine development.

Novel Respiratory Syncytial Virus-Like Particle Vaccine Composed of the Postfusion and Prefusion Conformations of the F Glycoprotein.

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants and children and represents an important global health burden for the elderly and the immunocompromised. Despite decades of research efforts, no licensed vaccine for RSV is available. We have developed virus-like particle (VLP)-based RSV vaccines assembled with the human metapneumovirus (hMPV) matrix protein (M) as the structural scaffold and the RSV fusion glycoprotein (F) in either the postfusion or prefusion conformation as its prime surface immunogen. Vaccines were composed of postfusion F, prefusion F, or a combination of the two conformations and formulated with a squalene-based oil emulsion as adjuvant. Immunization with these VLP vaccines afforded full protection against RSV infection and prevented detectable viral replication in the mouse lung after challenge. Analyses of lung cytokines and chemokines showed that VLP vaccination mostly induced the production of gamma interferon (IFN-γ), a marker of the Th1-mediated immune response, which is predominantly required for viral protection. Conversely, immunization with a formalin-inactivated RSV (FI-RSV) vaccine induced high levels of inflammatory chemokines and cytokines of the Th2- and Th17-mediated types of immune responses, as well as severe lung inflammation and histopathology. The VLP vaccines showed restricted production of these immune mediators and did not induce severe bronchiolitis or perivascular infiltration as seen with the FI-RSV vaccine. Remarkably, analysis of the serum from immunized mice showed that the VLP vaccine formulated using a combination of postfusion and prefusion F elicited the highest level of neutralizing antibody and enhanced the Th1-mediated immune response.

Exfoliation and intercalation of montmorillonite by small peptides.

Understanding structural changes in clay minerals induced by complexation with organic matter is relevant to soil science and agricultural applications. In this study, the effect of peptide storage in montmorillonite and the thermal stability of peptide-clay complexes was examined through characterization by X-ray diffraction (XRD), electron microscopy, UV absorption, and thermogravimetric analysis (TGA). XRD analysis of small peptide-montmorillonite clay complexes produced profiles consisting of reflections associated with the smectite 001 reflection and related peaks similar to that produced by a mixed layer clay mineral structure. Shifts in higher order diffraction maxima were attributed to disorder caused by the intercalation with the peptides. Increasing peptide concentrations resulted in greater shifts towards smaller 2θ from 6.37° (1.39 nm) to 5.45° (1.62 nm) as the interlayer space expanded. The expansion was accompanied by broadening of the 001 reflection (FWHM increases from 0.51 to 1.22° 2θ). The XRD line broadening was interpreted as caused by poorer crystallinity resulting from intercalation and tactoid exfoliation. SEM images revealed montmorillonite platelets with upwardly rolled edges that tend toward cylindrical structures with the production of tubules. High-resolution TEM images revealed bending of montmorillonite platelets, confirming exfoliation. The distribution of basal spacings in the micrographs was determined from the spatial frequencies obtained by Fourier analysis of density profiles. The distribution indicated the presence of discrete coherent crystallite domains. XRD and TGA results indicated that higher peptide concentrations resulted in a greater fraction of intercalated peptides and that surface adsorption of peptides mediated intercalation. Therefore, higher peptide concentration led to more stable organoclay complexes. However, UV absorption and TGA found that peptide adsorption onto montmorillonite had a finite limit at approximately 16% by weight.

The ϕ6 cystovirus protein P7 becomes accessible to antibodies in the transcribing nucleocapsid: a probe for viral structural elements.

Protein P7 is a component of the cystovirus viral polymerase complex. In the unpackaged procapsid, the protein is situated in close proximity to the viral directed RNA polymerase, P2. Cryo-electron microscopy difference maps from the species ϕ6 procapsid have demonstrated that P7 and P2 likely interact prior to viral RNA packaging. The location of P7 in the post-packaged nucleocapsid (NC) remains unknown. P7 may translocate closer to the five-fold axis of a filled procapsid but this has not been directly visualized. We propose that monoclonal antibodies (Mabs) can be selected that serve as probe- reagents for viral assembly and structure. A set of Mabs have been isolated that recognize and bind to the ϕ6 P7. The antibody set contains five unique Mabs, four of which recognize a linear epitope and one which recognizes a conformational epitope. The four unique Mabs that recognize a linear epitope display restricted utilization of Vκ and VH genes. The restricted genetic range among 4 of the 5 antibodies implies that the antibody repertoire is limited. The limitation could be the consequence of a paucity of exposed antigenic sites on the ϕ6 P7 surface. It is further demonstrated that within ϕ6 nucleocapsids that are primed for early-phase transcription, P7 is partially accessible to the Mabs, indicating that the nucleocapsid shell (protein P8) has undergone partial disassembly exposing the protein's antigenic sites.

Morphology of influenza B/Lee/40 determined by cryo-electron microscopy.

Cryo-electron microscopy projection image analysis and tomography is used to describe the overall architecture of influenza B/Lee/40. Algebraic reconstruction techniques with utilization of volume elements (blobs) are employed to reconstruct tomograms of this pleomorphic virus and distinguish viral surface spikes. The purpose of this research is to examine the architecture of influenza type B virions by cryo-electron tomography and projection image analysis. The aims are to explore the degree of ribonucleoprotein disorder in irregular shaped virions; and to quantify the number and distribution of glycoprotein surface spikes (hemagglutinin and neuraminidase) on influenza B. Projection image analysis of virion morphology shows that the majority (∼83%) of virions are spherical with an average diameter of 134±19 nm. The aspherical virions are larger (average diameter = 155±47 nm), exhibit disruption of the ribonucleoproteins, and show a partial loss of surface protein spikes. A count of glycoprotein spikes indicates that a typical 130 nm diameter type B virion contains ∼460 surface spikes. Configuration of the ribonucleoproteins and surface glycoprotein spikes are visualized in tomogram reconstructions and EM densities visualize extensions of the spikes into the matrix. The importance of the viral matrix in organization of virus structure through interaction with the ribonucleoproteins and the anchoring of the glycoprotein spikes to the matrix is demonstrated.

Disassembly of the cystovirus ϕ6 envelope by montmorillonite clay.

Prior studies of clay-virus interactions have focused on the stability and infectivity of nonenveloped viruses, yielding contradictory results. We hypothesize that the surface charge distribution of the clay and virus envelope dictates how the components react and affect aggregation, viral stability, and infectivity. The bacteriophage Cystoviridae species φ6 used in this study is a good model for enveloped pathogens. The interaction between φ6 and montmorillonite (MMT) clay (the primary component of bentonite) is explored by transmission electron microscopy. The analyses show that MMT-φ6 mixtures undergo heteroaggregation, forming structures in which virtually all the virions are either sequestered between MMT platelet layers or attached to platelet edges. The virions swell and undergo disassembly resulting in partial or total envelope loss. Edge-attached viral envelopes distort to increase contact area with the positively charged platelet edges indicating that the virion surface is negatively charged. The nucleocapsid (NCs) remaining after envelope removal also exhibit distortion, in contrast to detergent-produced NCs which exhibit no distortion. This visually discernible disassembly is a mechanism for loss of infectivity previously unreported by studies of nonenveloped viruses. The MMT-mediated sequestration and disassembly result in reduced infectivity, suggesting that clays may reduce infectivity of enveloped pathogenic viruses in soils and sediments.

Protein P7 of the cystovirus φ6 is located at the three-fold axis of the unexpanded procapsid.

The objective of this study was to determine the location of protein P7, the RNA packaging factor, in the procapsid of the φ6 cystovirus. A comparison of cryo-electron microscopy high-resolution single particle reconstructions of the φ6 complete unexpanded procapsid, the protein P2-minus procapsid (P2 is the RNA directed RNA-polymerase), and the P7-minus procapsid, show that prior to RNA packaging the P7 protein is located near the three-fold axis of symmetry. Difference maps highlight the precise position of P7 and demonstrate that in P7-minus particles the P2 proteins are less localized with reduced densities at the three-fold axes. We propose that P7 performs the mechanical function of stabilizing P2 on the inner protein P1 shell which ensures that entering viral single-stranded RNA is replicated.

Bacteriophage φ6--structure investigated by fluorescence Stokes shift spectroscopy.

The Stokes shift of tryptophan (Trp) fluorescence from layers of the lipid-containing bacteriophage φ6 is compared to determine the relative effect of the layers on virus hydrophobicity. In the inner most layer, the empty procapsid (PC) which contains 80-90% of the virion Trp residues, λ(max) = 339.8 nm. The PC emission is substantially more redshifted than the other φ6 layers and nearer to that of the Pseudomonad host cell than the other φ6 layers. The Trp emission from the nucleocapsid (NC) with λ(max) = 337.4 nm, is blueshifted by 2.4 nm relative to the PC although the number of Trp in the NC is identical to the PC. This shift represents an increase in Trp hydrophobicity, likely a requirement for the maintenance of A-form doubled-stranded RNA. Fluorescence from the completely assembled virion indicates it is in a considerably more hydrophobic environment with λ(max) = 330.9 nm. Density measurements show that the water content in the NC does not change during envelope assembly, therefore the blueshifted φ6 emission suggests that the envelope changes the PC environment, probably via the P8 layer. This change in hydrophobicity likely arises from charge redistribution or envelope-induced structural changes in the PC proteins.

Toroidal surface complexes of bacteriophage ϕ12 are responsible for host-cell attachment.

Cryo-electron tomography and subtomogram averaging are utilized to determine that the bacteriophage ϕ12, a member of the Cystoviridae family, contains surface complexes that are toroidal in shape, are composed of six globular domains with six-fold symmetry, and have a discrete density connecting them to the virus membrane-envelope surface. The lack of this kind of spike in a reassortant of ϕ12 demonstrates that the gene for the hexameric spike is located in ϕ12's medium length genome segment, likely to the P3 open reading frames which are the proteins involved in viral-host cell attachment. Based on this and on protein mass estimates derived from the obtained averaged structure, it is suggested that each of the globular domains is most likely composed of a total of four copies of P3a and/or P3c proteins. Our findings may have implications in the study of the evolution of the cystovirus species in regard to their host specificity.

Hybrid phosphorescence and fluorescence native spectroscopy for breast cancer detection.

Fluorescence and phosphorescence measurements are performed on normal and malignant ex vivo human breast tissues using UV LED and xenon lamp excitation. Tryptophan (trp) phosphorescence intensity is higher in both normal glandular and adipose tissue when compared to malignant tissue. An algorithm based on the ratio of trp fluorescence intensity at 345 nm to phosphorescence intensity at 500 nm is successfully used to separate normal from malignant tissue types. Normal specimens consistently exhibited a low I(345)I(500) ratio (<10), while for malignant specimens, the I(345)I(500) ratio is consistently high (>15). The ratio analysis correlates well with histopathology. Intensity ratio maps with a spatial resolution of 0.5 mm are generated in which local regions of malignancy could be identified.

Proteins and dipicolinic acid released during heat shock activation of Bacillus subtilis spores probed by optical spectroscopy.

UV fluorescence and absorption spectroscopy from Bacillus subtilis spores detected proteins and dipicolinic acid (DPA) released into the supernatant after heat treatments ranging from 20 degrees to 90 degrees C. The protein and DPA concentration in the supernatant was greater with higher heat treatment temperatures, undergoing a substantial increase for T > or = 60 degrees C, and supporting the theory that spores undergo a phase transition from a glassylike to a rubberylike state at 56 degrees C. Gel electrophoresis detected several small proteins with molecular weights between 6 and 11 kDa. These proteins may be small acid-soluble spore proteins that are present in spores but break down during germination. A 30 kDa protein extracted above 60 degrees C is related to the rubber-glass phase transition.

Virus particles and receptor interaction monitored by fluorescence spectroscopy.

Native fluorescence spectroscopy (NFS), primarily from tryptophan (trp), was used for in situ investigation of the virus-receptor attachment process in phi6, a lipid-containing bacteriophage from the Cystoviridae family. NFS allowed us to monitor the viral attachment directly to its receptor, which was isolated from the pseudomonad host. Immediately upon mixing, an increase in tryptophan emission intensity was observed followed by a subsequent decrease in emission intensity. The initial increase in emission intensity reflects changes in trp quantum efficiency as the phi6 surface proteins change their conformation as a result of virus attachment to the pilus. The cystovirus spike protein P3 is responsible for receptor recognition and the fluorescence changes observed are likely to be the consequence of its conformational transition at this initial infection stage, providing a kinetic view of this process. The subsequent decrease in trp emission intensity could be due to changes in viral proteins as a result of disassembly of the pili. The technique may have important applications for the dynamic monitoring of additional stages of the virus replication cycle such as assembly, interaction with nucleic acids and maturation. This work expands on a previous demonstration that fluorescence offered a novel tool to detect virus particle interaction with its host cell.

In situ determination of refractive index and size of Bacillus spores by light transmission.

Light-extinction measurements in the wavelength range of 400 to 1000 nm are performed in situ on Bacillus subtilis endospores during heat-shock-induced activation. Simultaneous information on particle size and refractive indices during activation is calculated from the transmission spectra by use of the Gaussian ray approximation of anomalous diffraction theory. During activation the refractive index of the core decreases from 1.51 to 1.39, and the size increases from 0.38 to 0.6 microm.

Virus particles monitored by fluorescence spectroscopy: a potential detection assay for macromolecular assembly.

Native fluorescence spectroscopy was used for in situ investigations of two lipid-containing bacteriophages from the cystovirus family as well as their Pseudomonad host cells. Both the viruses phi6 and phi12 and their bacterial host proteins contain the amino acid tryptophan (trp), which is the predominant fluorophore in UV. Within proteins, trp's structural environment differs, and the differences are reflected in their spectroscopic signatures. It was observed that the peak of the trp emission from both viruses was at 330 nm, a significantly shorter wavelength than trp in either the Pseudomonad host cells or the amino acid's chemical form. This allowed us to monitor the viral attachment process and subsequent lytic release of progeny virus particles by measurement of the trp emission spectra during the infection process. This work demonstrates that fluorescence may offer a novel tool to detect viruses and monitor viral infection of cells and may be part of a biodefense application.

Native fluorescence and excitation spectroscopic changes in Bacillus subtilis and Staphylococcus aureus bacteria subjected to conditions of starvation.

Fluorescence emission and excitation spectra were measured over a 7-day period for Bacillus subtilis (Bs), a spore-forming, and Staphylococcus aureus (Sa), a nonspore-forming bacteria subjected to conditions of starvation. Initially, the Bs fluorescence was predominantly due to the amino acid tryptophan. Later, a fluorescence band with an emission peak at 410 nm and excitation peak at 345 m, from dipicolinic acid, appeared. Dipicolinic acid is produced during spore formation and serves as a spectral signature for detection of spores. The intensity of the 410-nm band continued to increase over the next 3 days. The Sa fluorescence was predominantly from tryptophan and did not change over time. In 6 of the 17 Bs specimens studied, an additional band appeared with a weak emission peak at 460 cm and excitation peaks at 250, 270, and 400 nm. The addition of beta-hydroxybutyric acid to the Bs or the Sa cultures resulted in a two-order of magnitude increase in the 460-nm emission. The addition of Fe2+ quenched the 460 emission, indicating that a source of the 460-nm emission was a siderophore produced by the bacteria. We demonstrate that optical spectroscopy-based instrumentation can detect bacterial spores in real time.