Atomic Resolution Structure of the Oncolytic Parvovirus LuIII by Electron Microscopy and 3D Image Reconstruction.

LuIII, a protoparvovirus pathogenic to rodents, replicates in human mitotic cells, making it applicable for use to kill cancer cells. This virus group includes H-1 parvovirus (H-1PV) and minute virus of mice (MVM). However, LuIII displays enhanced oncolysis compared to H-1PV and MVM, a phenotype mapped to the major capsid viral protein 2 (VP2). This suggests that within LuIII VP2 are determinants for improved tumor lysis. To investigate this, the structure of the LuIII virus-like-particle was determined using single particle cryo-electron microscopy and image reconstruction to 3.17 Å resolution, and compared to the H-1PV and MVM structures. The LuIII VP2 structure, ordered from residue 37 to 587 (C-terminal), had the conserved VP topology and capsid morphology previously reported for other protoparvoviruses. This includes a core β-barrel and α-helix A, a depression at the icosahedral 2-fold and surrounding the 5-fold axes, and a single protrusion at the 3-fold axes. Comparative analysis identified surface loop differences among LuIII, H-1PV, and MVM at or close to the capsid 2- and 5-fold symmetry axes, and the shoulder of the 3-fold protrusions. The 2-fold differences cluster near the previously identified MVM sialic acid receptor binding pocket, and revealed potential determinants of protoparvovirus tumor tropism

Generation and characterization of anti-Adeno-associated virus serotype 8 (AAV8) and anti-AAV9 monoclonal antibodies

Adeno-associated viruses (AAVs) are promising viral vectors for therapeutic gene delivery, and the approval of an AAV1 vector for the treatment of lipoprotein lipase deficiency has heralded a new and exciting era for this system. However, preclinical and clinical studies show that neutralization from pre-existing antibodies is detrimental for medical application and this hurdle must be overcome before full clinical realization can be achieved. Thus the binding sites for capsid antibodies must be identified and eliminated through capsid engineering. Towards this goal and to recapitulate patient polyclonal responses, a panel of eight new mouse monoclonal antibodies (MAbs) has been generated against AAV8 and AAV9 capsids, two vectors in development for therapeutic application. Native (capsid) dot blot assays confirmed the specificity of these antibodies for their parental serotypes, with the exception of one MAb, HL2372, selected to cross-react against both capsids. Furthermore, in vitro assays showed that these MAbs are capable of neutralizing virus infection. These MAbs will be utilized for structural mapping of antigenic footprints on their respective capsids to inform development of the next generation of rAAV vectors capable of evading antibody neutralization while retaining parental tropism

An ancient lineage of highly divergent parvoviruses infects both vertebrate and invertebrate hosts

Chapparvoviruses (ChPVs) comprise a divergent, recently identified group of parvoviruses (family Parvoviridae), associated with nephropathy in immunocompromised laboratory mice and with prevalence in deep sequencing results of livestock showing diarrhea. Here, we investigate the biological and evolutionary characteristics of ChPVs via comparative in silico analyses, incorporating sequences derived from endogenous parvoviral elements (EPVs) as well as exogenous parvoviruses. We show that ChPVs are an ancient lineage within the Parvoviridae, clustering separately from members of both currently established subfamilies. Consistent with this, they exhibit a number of characteristic features, including several putative auxiliary protein-encoding genes, and capsid proteins with no sequence-level homology to those of other parvoviruses. Homology modeling indicates the absence of a β-A strand, normally part of the luminal side of the parvoviral capsid protein core. Our findings demonstrate that the ChPV lineage infects an exceptionally broad range of host species, including both vertebrates and invertebrates. Furthermore, we observe that ChPVs found in fish are more closely related to those from invertebrates than they are to those of amniote vertebrates. This suggests that transmission between distantly related host species may have occurred in the past and that the Parvoviridae family can no longer be divided based on host affiliation

Endogenous amdoparvovirus-related elements reveal insights into the biology and evolution of vertebrate parvoviruses

Amdoparvoviruses (family Parvoviridae: genus Amdoparvovirus) infect carnivores, and are a major cause of morbidity and mortality in farmed animals. In this study, we systematically screened animal genomes to identify endogenous parvoviral elements (EPVs) disclosing a high degree of similarity to amdoparvoviruses, and investigated their genomic, phylogenetic and protein structural features. We report the first examples of full-length, amdoparvovirus-derived EPVs in the genome of the Transcaucasian mole vole (Ellobius lutescens). We also identify four EPVs in mammal and reptile genomes that are intermediate between amdoparvoviruses and their sister genus (Protoparvovirus) in terms of their phylogenetic placement and genomic features. In particular, we identify a genome-length EPV in the genome of a pit viper (Protobothrops mucrosquamatus) that is more similar to a protoparvovirus than an amdoparvovirus in terms of its phylogenetic placement and the structural features of its capsid protein (as revealed by homology modeling), yet exhibits characteristically amdoparvovirus-like genome features including: (1) a putative middle ORF gene; (2) a capsid gene that lacks a phospholipase A2 domain; (3) a genome structure consistent with an amdoparvovirus-like mechanism of capsid gene expression. Our findings indicate that amdoparvovirus host range extends to rodents, and that parvovirus lineages possessing a mixture of proto- and amdoparvovirus-like characteristics have circulated in the past. In addition, we show that EPV sequences in the mole vole and pit viper encode intact, expressible replicase genes that have potentially been co-opted or exapted in these host species

IR Spectrometer Using 90-Degree Off-Axis Parabolic Mirrors

A gated spectrometer has been designed for real-time, pulsed infrared (IR) studies at the National Synchrotron Light Source at the Brookhaven National Laboratory. A pair of 90-degree, off-axis parabolic mirrors are used to relay the light from an entrance slit to an output recording camera. With an initial wavelength range of 1500–4500 nm required, gratings could not be used in the spectrometer because grating orders would overlap. A magnesium oxide prism, placed between these parabolic mirrors, serves as the dispersion element. The spectrometer is doubly telecentric. With proper choice of the air spacing between the prism and the second parabolic mirror, any spectral region of interest within the InSb camera array’s sensitivity region can be recorded. The wavelengths leaving the second parabolic mirror are collimated, thereby relaxing the camera positioning tolerance. To set up the instrument, two different wavelength (visible) lasers are introduced at the entrance slit and made collinear with the optical axis via flip mirrors. After dispersion by the prism, these two laser beams are directed to tick marks located on the outside housing of the gated IR camera. This provides first-order wavelength calibration for the instrument. Light that is reflected off the front prism face is coupled into a high-speed detector to verify steady radiance during the gated spectral imaging. Alignment features include tick marks on the prism and parabolic mirrors. This instrument was designed to complement single-point pyrometry, which provides continuous time histories of a small collection of spots from shock-heated targets

Seagrass mapping synthesis: a resource for coastal management in the Great Barrier Reef World Heritage Area

This project provides an up to date synthesis of the available information on seagrass in the Great Barrier Reef World Heritage Area (GBRWHA). It brings together more than 30 years of spatial information and data collection into easy to use spatial GIS layers that provide key information on species, meadow type and age and reliability of the data. The project provides: Seagrass site and meadow-specific data in Geographic Information System (GIS) layers to provide seagrass data to inform research analysis and management advice. A site layer that includes >66,000 individual survey sites with information including latitude/longitude, Natural Resource Management region, site depth, seagrass presence/absence, dominant seagrass species, presence/absence of individual species, survey date, survey method, and data custodian. A meadow layer that includes 1169 individual and/or composite seagrass meadows with information including individual meadow persistence, meadow location (intertidal/subtidal), meadow density based on mean biomass and/or mean percent cover, meadow area, dominant seagrass species, seagrass species present, range of survey dates, survey method, and data custodian. Metadata to enable interpretation of the information and to identify the original data custodians for assistance with interpretation. Outcomes: This study consolidates all available seagrass spatial data for the GBRWHA collected from 1984 to December 2014 by the TropWATER Seagrass Group and CSIRO in a GIS database. It assembles and documents the state of spatial knowledge of seagrass in the GBRWHA. The spatial data is based on methods developed by TropWATER and CSIRO for seagrass habitat surveys of subtidal meadows, and TropWATER methods for intertidal surveys. Methods include sampling by boat (free divers, underwater video camera, grabs, sled with net sampling), helicopter and walking. 447,530 hectares of seagrasses were mapped (modelled deep water seagrass areas are not included in area figures in this report) within the GBRWHA; much of which provides habitat for commercial and traditional fishery species, and an important food resource for dugong and green turtle populations. Data is included for twelve seagrass species from three families. Seagrass was present at 39% of all sites visited. The study identifies areas where much of the data available for management is more than 20 years old or where there are specific habitats unsurveyed. Large areas of central and northern Queensland require updating. Several key habitat types such as reef platform seagrass meadows are poorly represented in the data