A recombinant virus-like particle vaccine against adenovirus-7 induces a potent humoral response.

Adenoviruses (AdVs) cause infections in humans that range from mild to severe, and can cause outbreaks particularly in close contact settings. Several human AdV types have been identified, which can cause a wide array of clinical manifestations. AdV types 4 and 7 (AdV-4 and AdV-7), which are among the most commonly circulating types in the United States, are known to cause acute respiratory disease that can result in hospitalization and rarely, death. Currently, the only vaccines approved for use in humans are live virus vaccines against AdV-4 and AdV-7, though these vaccines are only authorized for use in U.S. military personnel. While they are efficacious, use of these live virus vaccines carries considerable risks of vaccine-associated viral shedding and recombination. Here, we present an alternative vaccination strategy against AdV-7 using the virus-like particle platform (AdVLP-7). We describe the production of stable recombinant AdVLP-7, and demonstrate that AdVLP-7 is structurally analogous to wild-type AdV-7 virions (WT AdV-7). Preclinical immunogenicity studies in mice show that AdVLP-7 elicits a potent humoral immune response, comparable to that observed in mice immunized with WT AdV-7. Specifically, AdVLP-7 induces high titers of antibodies against AdV-7-specific antigens that can effectively neutralize AdV-7.

Discovery and Classification of the φ6 Bacteriophage: An Historical Review.

The year 2023 marks the fiftieth anniversary of the discovery of the bacteriophage φ6. The review provides a look back on the initial discovery and classification of the lipid-containing and segmented double-stranded RNA (dsRNA) genome-containing bacteriophage-the first identified cystovirus. The historical discussion describes, for the most part, the first 10 years of the research employing contemporary mutation techniques, biochemical, and structural analysis to describe the basic outline of the virus replication mechanisms and structure. The physical nature of φ6 was initially controversial as it was the first bacteriophage found that contained segmented dsRNA, resulting in a series of early publications that defined the unusual genomic quality. The technology and methods utilized in the initial research (crude by current standards) meant that the first studies were quite time-consuming, hence the lengthy period covered by this review. Yet when the data were accepted, the relationship to the reoviruses was apparent, launching great interest in cystoviruses, research that continues to this day.

The Establishment of the ϕ6 Genome Packaging Assay.

This editorial describes the efforts to establish a genome packaging assay for the ϕ6 bacteriophage, which were performed in the laboratory of Leonard Mindich, Ph [...].

Immunogenicity and Efficacy of Monovalent and Bivalent Formulations of a Virus-Like Particle Vaccine against SARS-CoV-2.

Virus-like particles (VLPs) offer great potential as a safe and effective vaccine platform against SARS-CoV-2, the causative agent of COVID-19. Here, we show that SARS-CoV-2 VLPs can be generated by expression of the four viral structural proteins in a mammalian expression system. Immunization of mice with a monovalent VLP vaccine elicited a potent humoral response, showing neutralizing activity against multiple variants of SARS-CoV-2. Subsequent immunogenicity and efficacy studies were performed in the Golden Syrian hamster model, which closely resembles the pathology and progression of COVID-19 in humans. Hamsters immunized with a bivalent VLP vaccine were significantly protected from infection with the Beta or Delta variant of SARS-CoV-2. Vaccinated hamsters showed reduced viral load, shedding, replication, and pathology in the respiratory tract. Immunized hamsters also showed variable levels of cross-neutralizing activity against the Omicron variant. Overall, the VLP vaccine elicited robust protective efficacy against SARS-CoV-2. These promising results warrant further study of multivalent VLP vaccines in Phase I clinical trials in humans.

Heterologous RNA Recombination in the Cystoviruses φ6 and φ8: A Mechanism of Viral Variation and Genome Repair.

Recombination and mutation of viral genomes represent major mechanisms for viral evolution and, in many cases, moderate pathogenicity. Segmented genome viruses frequently undergo reassortment of the genome via multiple infection of host organisms, with influenza and reoviruses being well-known examples. Specifically, major genomic shifts mediated by reassortment are responsible for radical changes in the influenza antigenic determinants that can result in pandemics requiring rapid preventative responses by vaccine modifications. In contrast, smaller mutational changes brought about by the error-prone viral RNA polymerases that, for the most part, lack a replication base mispairing editing function produce small mutational changes in the RNA genome during replication. Referring again to the influenza example, the accumulated mutations-known as drift-require yearly vaccine updating and rapid worldwide distribution of each new formulation. Coronaviruses with a large positive-sense RNA genome have long been known to undergo intramolecular recombination likely mediated by copy choice of the RNA template by the viral RNA polymerase in addition to the polymerase-based mutations. The current SARS-CoV-2 origin debate underscores the importance of understanding the plasticity of viral genomes, particularly the mechanisms responsible for intramolecular recombination. This review describes the use of the cystovirus bacteriophage as an experimental model for recombination studies in a controlled manner, resulting in the development of a model for intramolecular RNA genome alterations. The review relates the sequence of experimental studies from the laboratory of Leonard Mindich, PhD at the Public Health Research Institute-then in New York City-and covers a period of approximately 12 years. Hence, this is a historical scientific review of research that has the greatest relevance to current studies of emerging RNA virus pathogens.

RNA Packaging in the Cystovirus Bacteriophages: Dynamic Interactions during Capsid Maturation.

The bacteriophage family Cystoviridae consists of a single genus, Cystovirus, that is lipid-containing with three double-stranded RNA (ds-RNA) genome segments. With regard to the segmented dsRNA genome, they resemble the family Reoviridae. Therefore, the Cystoviruses have long served as a simple model for reovirus assembly. This review focuses on important developments in the study of the RNA packaging and replication mechanisms, emphasizing the structural conformations and dynamic changes during maturation of the five proteins required for viral RNA synthesis, P1, P2, P4, P7, and P8. Together these proteins constitute the procapsid/polymerase complex (PC) and nucleocapsid (NC) of the Cystoviruses. During viral assembly and RNA packaging, the five proteins must function in a coordinated fashion as the PC and NC undergo expansion with significant position translation. The review emphasizes this facet of the viral assembly process and speculates on areas suggestive of additional research efforts.

Structural characterization of the PCV2d virus-like particle at 3.3 Šresolution reveals differences to PCV2a and PCV2b capsids, a tetranucleotide, and an N-terminus near the icosahedral 3-fold axes.

Porcine circovirus 2 (PCV2) has a major impact on the swine industry. Eight PCV2 genotypes (a-h) have been identified using capsid sequence analysis. PCV2d has been designated as the emerging genotype. The cryo-electron microscopy molecular envelope of PCV2d virus-like particles identifies differences between PCV2a, b and d genotypes that accompany the emergence of PCV2b from PCV2a, and PCV2d from PCV2b. These differences indicate that sequence analysis of genotypes is insufficient, and that it is important to determine the PCV2 capsid structure as the virus evolves. Structure-based sequence comparison demonstrate that each genotype possesses a unique combination of amino acids located on the surface of the capsid that undergo substitution. We also demonstrate that the capsid N-terminus moves in response to increasing amount of nucleic acid packaged into the capsid. Furthermore, we model a tetranucleotide between the 5- and 2-fold axes of symmetry that appears to be responsible for capsid stability.

Sonographic appearance of angiomyofibroblastoma-like tumor of the scrotum.

A variety of extratesticular tumors and tumor-like conditions can occur in the scrotum. Angiomyofibroblastoma is a rare, slow-growing, well-circumscribed mesenchymal tumor, found predominantly in women, and is even a more unusual occurrence in the male genital tract. We describe a case of a 64-year-old male presenting with a scrotal painless mass. On sonography, a well-circumscribed extratesticular mass was present. As no specific imaging findings were present, it was a surprise to learn that the subsequently excised mass proved to be an angiomyofibroblastoma-like tumor. To the best of our knowledge, the sonographic appearance of this tumor in the scrotum has been reported previously in only one case.A variety of extratesticular tumors and tumor-like conditions can occur in the scrotum. Angiomyofibroblastoma is a rare, slow-growing, well-circumscribed mesenchymal tumor, found predominantly in women, and is even a more unusual occurrence in the male genital tract. We describe a case of a 64-year-old male presenting with a scrotal painless mass. On sonography, a well-circumscribed extratesticular mass was present. As no specific imaging findings were present, it was a surprise to learn that the subsequently excised mass proved to be an angiomyofibroblastoma-like tumor. To the best of our knowledge, the sonographic appearance of this tumor in the scrotum has been reported previously in only one case.

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.

Ultrasound for the Diagnosis of Carotidynia.

The purpose of this work is to draw attention to the characteristic sonographic features of carotidynia, which, in the context of a typical clinical picture support its diagnosis. Six patients presented with primary symptoms of neck pain and focal tenderness. In all patients, focal wall thickening of the distal end of the common carotid artery was found exactly in the region of tenderness, leading to mild lumen narrowing. Treatment with nonsteroidal anti-inflammatory drugs or glucocorticoids led to full symptom resolution. Carotidynia will often be encountered first on sonography performed for evaluation of neck pain and has a characteristic sonographic appearance.

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.

Should Intravenous Contrast be Administered in MRI Evaluation of Pediatric Outpatients with Chronic Headache?

BACKGROUND: The practice of administering intravenous contrast to children varies by institution depending on their routine. OBJECTIVES: To assess the necessity of routine contrast administration in brain magnetic resonance imaging (MRI) of pediatric outpatients referred for chronic headache workups. METHODS: We conducted a retrospective review of consecutive pediatric brain MRI examinations performed during January and February 2014 in 30 pediatric outpatients referred for evaluation of chronic headache. Independent review was performed by two board-certified neuroradiologists. The raters reviewed each MRI first as a non-contrast examination (without seeing the post-contrast images) and then with post-contrast images. RESULTS: No abnormalities were found in six patients. One patient had an indeterminate finding of a tubular cerebellar lesion requiring follow-up. In the remaining patients (n = 23), the findings were subclinical and included: mucosal thickening in the paranasal sinuses in 9 patients, cystic changes of the pineal gland in 8 (size 2-9 mm), small developmental venous anomalies in 6, non-specific FLAIR hyperintensities in 4, opacification of the mastoids in 2, and telangiectasia in 1 patient. The subclinical cases that were missed on pre-contrast images were: one small developmental venous anomaly, one telangiectasia and one small pineal cyst, none of which hold clinical significance. All kappa inter-rater and intra-rater agreement scores resulted in values above 0.75, excellent agreement according to Fleiss guidelines. CONCLUSIONS: There seems to be little reason to medically justify large-scale use of routine IV contrast administration to evaluate a brain MRI of pediatric patients referred for chronic headache.

Acceptance of forensic imaging in Israel.

BACKGROUND: Forensic imaging was officially introduced in Israel in 2011. Religious and cultural opposition to autopsies prevails in most of the population of Israel. OBJECTIVES: To examine the extent to which forensic imaging has been accepted as an adjuvant or partial replacement of forensic autopsy, particularly among those opposed to forensic autopsy. METHODS: The study was conducted in the pediatric population. Data were collected from the National Center of Forensic Medicine and Assaf Harofeh Medical Center during the 18 month period following the introduction of forensic imaging (group A). The data were compared to those of the previous 18 months (group B). The examined parameters were cases submitted, examined, autopsied or imaged depending on family consent. RESULTS: Consent to autopsy was similar in both groups (A = 56% vs. B = 54%). In group A, consent for imaging was 24% of all cases, and of those imaged 77% underwent autopsy. Of those examined externally only, 16% consented to imaging. For 7% of the total cases in group A, estimation of cause of death was based on virtopsy alone. CONCLUSIONS: In a country with a high level of religious and cultural opposition to autopsy, it is a challenge to add forensic imaging to the pediatric forensic investigation. Those consenting to forensic imaging are more likely to be those consenting to autopsy. Consent for forensic imaging only was given in 7% of cases. Greater efforts should be invested to educate and inform the public regarding the benefits of virtual autopsy and the importance of data acquired from forensic images.

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.

Quantitative evaluation of facial growth in children after unilateral ESS for subperiosteal orbital abscess drainage.

OBJECTIVE: To determine the effects of unilateral endoscopic sinus surgery (ESS) on facial skeletal growth in children. DESIGN: Retrospective controlled study. SETTING: Academic tertiary referral medical center. MATERIALS AND METHODS: Included were children who underwent a unilateral ESS procedure between 1995 and 2006 to evacuate a subperiosteal orbital abscess (SPOA) and several years later went through cephalometric measurements comparing their facial development between the surgical and nonsurgical sides. RESULTS: A total of 6 children were recruited for this study (3 girls and 3 boys), between the ages 3 to 10 at time of surgery, and from 9.5 to 23 years of age today. Four of the children had surgery on the right side and 2 on the left. No statistically significant difference was found when evaluating all planes in the cephalometric radiographs according to age at surgery, age today and years from surgery. CONCLUSION: In our study, no significant differences were found in craniofacial growth between the sides of the face in children who underwent ESS for the same medical indication on one side of the face, suggesting that ESS might be safely performed even in young children. LEVEL OF EVIDENCE: -2c.

Variations in eyeball diameters of the healthy adults.

The purpose of the current research was to reevaluate the normative data on the eyeball diameters. Methods. In a prospective cohort study, the CT data of consecutive 250 adults with healthy eyes were collected and analyzed, and sagittal, transverse, and axial diameters of both eyeballs were measured. The data obtained from the left eye and from the right eye were compared. The correlation analysis was performed with the following variables: orbit size, gender, age, and ethnic background. Results. We did not find statistically significant differences correlated with gender of the patients and their age. The right eyeball was slightly smaller than the left one but this difference was statistically insignificant (P = 0.17). We did not find statistically significant differences of the eyeball sizes among the ethnicities we dealt with. Strong correlation was found between the transverse diameter and the width of the orbit (r = 0.88). Conclusion. The size of a human adult eye is approximately 24.2 mm (transverse) × 23.7 mm (sagittal) × 22.0-24.8 mm (axial) with no significant difference between sexes and age groups. In the transverse diameter, the eyeball size may vary from 21 mm to 27 mm. These data might be useful in ophthalmological, oculoplastic, and neurological practice.

Quantitative relations between the eyeball, the optic nerve, and the optic canal important for intracranial pressure monitoring.

OBJECTIVE: To find correlations between diameters of the optic nerve sheath (ONSD), the eyeball, and the optic canal that might be important for intracranial pressure monitoring. METHODS: In a prospective cohort study, the CT data of consecutive 400 adults (18+) with healthy eyes and optic nerves and absence of neurological diseases were collected and analyzed. When the CT scans were obtained, the diameters of the optic nerve sheath, the eyeball, and the optic canal were measured and statistically analyzed. The data obtained from the left and from the right eyeballs and optic nerves were compared. The correlation analysis was performed within these variables, with the gender, and the age. RESULTS: In healthy persons, the ONSD varies from 3.65 mm to 5.17 mm in different locations within the intraorbital space with no significant difference between sexes and age groups. There is a strong correlation between the eyeball transverse diameter (ETD) and ONSD that can be presented as ONSD/ETD index. In healthy subjects, the ONSD/ETD index equals 0.19. CONCLUSION: The calculation of an index when ONSD is divided by the ETD of the eyeball presents precise normative database for ONSD intracranial pressure measurement technique. When the ONSD is measured for intracranial pressure monitoring, the most stable results can be obtained if the diameter is measured 10 mm from the globe. These data might serve as a normative database at emergency departments and in general neurological practice.