Influenza virus

No abstract available

Purification and proteomics of influenza virions

This chapter describes a basic workflow for analyzing the protein composition of influenza virions. In order to obtain suitable material, the chapter describes how to concentrate influenza virions from the growth media of infected cells and to purify them by ultracentrifugation through a density gradient. This approach is also suitable for purifying influenza virions from the allantoic fluid of embryonated chicken eggs. As a small quantity of microvesicles are co-purified with virions, optional steps are included to increase the stringency of purification by enriching material with viral receptor binding and cleaving activity. Material purified in this way can be used for a variety of downstream applications, including proteomics. As a detailed example of this, the chapter also describes a standard workflow for analyzing the protein composition of concentrated virions by liquid chromatography and tandem mass spectrometry

Single-particle measurements of filamentous influenza virions reveal damage induced by freezing

Clinical isolates of influenza virus produce pleiomorphic virions, ranging from small spheres to elongated filaments. The filaments are seemingly adaptive in natural infections, but their basic functional properties are poorly understood and functional studies of filaments often report contradictory results. This may be due to artefactual damage from routine laboratory handling, an issue which has been noted several times without being explored in detail. To determine whether standard laboratory techniques could damage filaments, we used immunofluorescence microscopy to rapidly and reproducibly quantify and characterize the dimensions of filaments. Most of the techniques we tested had minimal impact on filaments, but freezing to −70 °C, a standard storage step before carrying out functional studies on influenza viruses, severely reduced their concentration, median length and the infectivity of the whole virion population. We noted that damage from freezing is likely to have affected most of the functional studies of filaments performed to date, and to address this we show that it can be mitigated by snap-freezing or incorporating the cryoprotectant DMSO. We recommend that functional studies of filaments characterize virion populations prior to analysis to ensure reproducibility, and that they use unfrozen samples if possible and cryoprotectants if not. These basic measures will support the robust functional characterizations of filaments that are required to understand their roles in natural influenza virus infections

Educational material about influenza viruses

To supplement a special edition of the journal Viruses, entitled “What’s New with Flu?”, influenza virus researchers have worked together to generate simple educational material to communicate their science to school students. Educational materials suitable for a range of ages are included, from coloring exercises for younger students through to explanations of cutting-edge science in straightforward language for older students. This article contains a handout with influenza facts, a coloring page, a glossary and word find and a connect-the-dots exercise explaining the ideas behind recently published scientific papers. Together, these materials are intended to make research on influenza viruses more accessible to students and teachers

Altering the Size Distribution of Influenza Virion Populations [Poster]

Harry Smith Vacation Studentship Laboratory-adapted influenza viruses produce predominantly spherical virions. In contrast, clinical and veterinary isolates produce a mixture of virions of different sizes, from 0.1 µm spheres to filaments which can reach tens of microns in length. Filamentous influenza virions were discovered in 1946, but the bulk of influenza research has analysed only spherical forms of the virus and the role of filaments in influenza infections is unclear. Functional studies of filaments require the development of methods to manipulate the ratio of spherical to filamentous virions, and we reasoned that this could be achieved by filtration. To test this, we infected MDCK cells with the filamentous Udorn strain of influenza A virus. We collected virus-containing growth media and passed this through filters with 5 µm, 0.45 µm and 0.2 µm pores. Filtrates and unfiltered virus were compared, using Western blotting to measure their protein composition, plaque assays to measure their infectivity and negative stain transmission electron microscopy to measure individual particle sizes. We found that filtration through a filter with 5 µm pores had little effect on composition, infectivity and the ratio of spherical to filamentous particles. In contrast, sub-micron filters, particularly those with 0.2 µm pores, caused a general depletion of virions but increased the sphere to filament ratio. We therefore concluded that sub-micron pore sizes can be used to preferentially remove filaments from populations of pleomorphic influenza virions, providing a useful tool for subtractive studies of the contribution filaments make to influenza virus infections

Single-particle Measurements Reveal Damage to Filamentous Influenza Virions During Laboratory Handling [Poster]

Most laboratory strains of influenza virus produce near-spherical virions, but clinical isolates also produce extended filaments whose biophysical properties are understudied. Most functional studies of filamentous influenza viruses do not include data on the concentration or lengths of the virions, making it hard to interpret their sometimes contradictory results. Furthermore, anecdotal reports suggest that filaments are damaged during routine laboratory handling. Therefore, to understand filament function we require a tool to assess the number and dimensions of filaments in a sample and an assessment of how filaments respond to standard handling procedures. We initially sought to analyse filament populations using negative stain particle counting, but found that this was low-throughput and could not detect particles longer than 10 µm. Instead, we used confocal microscopy with semi-automated image analysis. This allowed a high-throughput, quantitative analysis of length distributions in filament populations. Using this, we assessed the effects of pipetting, vortexing, sonicating, clarification and freezing on filaments. Most procedures did not appreciably alter filament dimensions. Pipetting and vortexing both slightly reduced filament numbers, but their effects were only appreciable after extended treatment. In contrast, freezing substantially reduced the number and median length of filaments, as well as creating ‘kinks’ in filaments which suggest damage to the capsid. We conclude that confocal microscopy can provide the basic measurements needed to interpret functional studies of filamentous strains. Using this approach, we found that freezing filaments causes previously unappreciated damage, which should be considered when planning further research

Cypria petenensis, a new name for the Ostracod Cypria pelagica Brehm 1932

Recently during the preparation of a comprehensive account of the lake plankton (Hutchinson, in press) a rather unfortunate case of homonymy was discovered in the genus Cypria

Active appearance pyramids for object parametrisation and fitting

Object class representation is one of the key problems in various medical image analysis tasks. We propose a part-based parametric appearance model we refer to as an Active Appearance Pyramid (AAP). The parts are delineated by multi-scale Local Feature Pyramids (LFPs) for superior spatial specificity and distinctiveness. An AAP models the variability within a population with local translations of multi-scale parts and linear appearance variations of the assembly of the parts. It can fit and represent new instances by adjusting the shape and appearance parameters. The fitting process uses a two-step iterative strategy: local landmark searching followed by shape regularisation. We present a simultaneous local feature searching and appearance fitting algorithm based on the weighted Lucas and Kanade method. A shape regulariser is derived to calculate the maximum likelihood shape with respect to the prior and multiple landmark candidates from multi-scale LFPs, with a compact closed-form solution. We apply the 2D AAP on the modelling of variability in patients with lumbar spinal stenosis (LSS) and validate its performance on 200 studies consisting of routine axial and sagittal MRI scans. Intervertebral sagittal and parasagittal cross-sections are typically used for the diagnosis of LSS, we therefore build three AAPs on L3/4, L4/5 and L5/S1 axial cross-sections and three on parasagittal slices. Experiments show significant improvement in convergence range, robustness to local minima and segmentation precision compared with Constrained Local Models (CLMs), Active Shape Models (ASMs) and Active Appearance Models (AAMs), as well as superior performance in appearance reconstruction compared with AAMs. We also validate the performance on 3D CT volumes of hip joints from 38 studies. Compared to AAMs, AAPs achieve a higher segmentation and reconstruction precision. Moreover, AAPs have a significant improvement in efficiency, consuming about half the memory and less than 10% of the training time and 15% of the testing time

Proteomics as a tool for live attenuated influenza vaccine characterisation

Many viral vaccines, including the majority of influenza vaccines, are grown in embryonated chicken eggs and purified by sucrose gradient ultracentrifugation. For influenza vaccines this process is well established, but the viral strains recommended for use in vaccines are updated frequently. As viral strains can have different growth properties and responses to purification, these updates risk changes in the composition of the vaccine product. Changes of this sort are hard to assess, as influenza virions are complex structures containing variable ratios of both viral and host proteins. To address this, we used liquid chromatography and tandem mass spectrometry (LC-MS/MS), a flexible and sensitive method ideally suited to identifying and quantifying the proteins present in complex mixtures. By applying LC-MS/MS to the pilot scale manufacturing process of the live attenuated influenza vaccine (LAIV) FluMist® Quadrivalent vaccine (AstraZeneca), we were able to obtain a detailed description of how viral and host proteins are removed or retained at each stage of LAIV purification. LC-MS/MS allowed us to quantify the removal of individual host proteins at each stage of the purification process, confirming that LAIV purification efficiently depletes the majority of host proteins and identifying the small subset of host proteins which are associated with intact virions. LC-MS/MS also identified substantial differences in the retention of the immunosuppressive viral protein NS1 in purified virions. Finally, LC-MS/MS allowed us to detect subtle variations in the LAIV production process, both upstream of purification and during downstream purification stages. This demonstrates the potential utility of LC-MS/MS for optimising the purification of complex biological mixtures and shows that it is a promising approach for process optimisation in a wide variety of vaccine manufacturing platforms