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Virus binding serves to define virus tropism and species specificity1. Virus binding proteins are classically considered as facilitators of cell surface attachment prior to receptor engagement and virus internalization. For efficient entry vaccinia virus (VACV) – the prototypic poxvirus - relies on four binding proteins and an eleven-protein entry fusion complex (EFC)2. We recently demonstrated that VACV binding and fusion proteins are organized into distinct functional domains, with localization of EFC proteins to virion tips directly influencing membrane fusion activity3. However, the relationship between virus binding protein distribution, virion binding orientation and subsequent membrane fusion remain unexplored. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards EFC-containing virion tips to facilitate virus fusion. Using a cell-derived membrane-bleb model system together with super-resolution and electron microscopy we found that side-bound VACV virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins revealed that three of four contribute to binding orientation, amongst which the chondroitin sulphate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia virus4,5, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection kinetics. Our results extend the role of viral binding proteins from mere attachment factors to active participants in successful viral membrane fusion and further illustrate the influence of virus protein architecture on successful infection.

All poxviruses contain a set of proteinaceous structures termed lateral bodies (LB) that deliver viral effector proteins into the host cytosol during virus entry. To date, the spatial proteotype of LBs remains unknown. Using the prototypic poxvirus, vaccinia virus (VACV), we employed a quantitative comparative mass spectrometry strategy to determine the poxvirus LB proteome. We identified a large population of candidate cellular proteins, the majority being mitochondrial, and 15 candidate viral LB proteins. Strikingly, one-third of these are VACV redox proteins whose LB residency could be confirmed using super-resolution microscopy. We show that VACV infection exerts an anti-oxidative effect on host cells and that artificial induction of oxidative stress impacts early and late gene expression as well as virion production. Using targeted repression and/or deletion viruses we found that deletion of individual LB-redox proteins was insufficient for host redox modulation suggesting there may be functional redundancy. In addition to defining the spatial proteotype of VACV LBs, these findings implicate poxvirus redox proteins as potential modulators of host oxidative anti-viral responses and provide a solid starting point for future investigations into the role of LB resident proteins in host immunomodulation., PLoS Pathogens, 18 (7), ISSN:1553-7374, ISSN:1553-7366

To achieve efficient binding and subsequent fusion, most enveloped viruses encode between one and five proteins1. For many viruses, the clustering of fusion proteins—and their distribution on virus particles—is crucial for fusion activity2,3. Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion4. However, the spatial organization of these proteins and how this influences fusion activity is unknown. Here, we show that the membrane of vaccinia virus is organized into distinct functional domains that are critical for the efficiency of membrane fusion. Using super-resolution microscopy and single-particle analysis, we found that the fusion machinery of vaccinia virus resides exclusively in clusters at virion tips. Repression of individual components of the fusion complex disrupts fusion-machinery polarization, consistent with the reported loss of fusion activity5. Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts the formation of fusion pores and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to the efficiency of membrane fusion, and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection. Super-resolution microscopy and single-particle analysis reveal that vaccinia virus membrane proteins are organized into functional domains whose polarization on the surface of mature virus particles is required for viral entry and virus–cell fusion.

Modulation of the host cell cycle is a common strategy used by viruses to create a pro-replicative environment. To facilitate viral genome replication, vaccinia virus (VACV) has been reported to alter cell cycle regulation and trigger the host cell DNA damage response. However, the cellular factors and viral effectors that mediate these changes remain unknown. Here, we set out to investigate the effect of VACV infection on cell proliferation and host cell cycle progression. Using a subset of VACV mutants we characterize the stage of infection required for inhibition of cell proliferation and define the viral effectors required to dysregulate the host cell cycle. Consistent with previous studies, we show that VACV inhibits, and subsequently shifts the host cell cycle. We demonstrate that these two phenomena are independent of one another, with viral early genes being responsible for cell cycle inhibition, and post-replicative viral gene(s) responsible for the cell cycle shift. Extending previous findings, we show that the viral kinase F10 is required to activate the DNA damage checkpoint and that the viral B1/B12 (pseudo) kinases mediate degradation of checkpoint effectors p53 and p21 during infection. We conclude that VACV modulates host cell proliferation and host cell cycle progression through temporal expression of multiple VACV effector proteins.

The replication and assembly of vaccinia virus (VACV), the prototypic poxvirus, occurs exclusively in the cytoplasm of host cells. While the role of cellular cytoskeletal components in these processes remains poorly understood, vimentin-a type III intermediate filament-has been shown to associate with viral replication sites and to be incorporated into mature VACV virions. Here, we employed chemical and genetic approaches to further investigate the role of vimentin during the VACV lifecycle. The collapse of vimentin filaments, using acrylamide, was found to inhibit VACV infection at the level of genome replication, intermediate- and late-gene expression. However, we found that CRISPR-mediated knockout of vimentin did not impact VACV replication. Combining these tools, we demonstrate that acrylamide treatment results in the formation of anti-viral granules (AVGs) known to mediate translational inhibition of many viruses. We conclude that vimentin is dispensable for poxvirus replication and assembly and that acrylamide, as a potent inducer of AVGs during VACV infection, serves to bolster cell's anti-viral response to poxvirus infection.

Autophagy is an essential degradation program required to maintain cell homeostasis. Amongst its many functions is the engulfment and destruction of cytosolic pathogens, termed Xenophagy. Not surprisingly, many pathogens have adapted various strategies to circumvent or co-opt autophagic degradation during infection. For poxviruses, it is known that infection activates autophagy, which however is not required for successful replication. Despite the fact that these complex viruses replicate exclusively in the cytoplasm, autophagy-mediated control of poxvirus infection has not been explored. Using the prototypic poxvirus, vaccinia virus (VACV), we show that over-expression of the xenophagy receptors p62, NDP52 and Tax1Bp1 restricts poxvirus infection. While NDP52 and Tax1Bp1 were degraded, p62 was found to initially target cytoplasmic virions before being shunted to the nucleus. Nuclear translocation of p62 during infection was dependent upon p62 NLS2, and VACV kinase mediated phosphorylation of associated p62 residues T269/S272. These results indicate that VACV actively targets the xenophagy receptor p62 during the early stages of infection to avoid destruction, and further suggest that poxviruses exhibit a unique multi-layered control of autophagy in order to facilitate cytoplasmic replication.

Induction of memory CD8 T cells residing in peripheral tissues is of interest for T cell-based vaccines as these cells are located at mucosal and barrier sites and can immediately exert effector functions, thus providing protection in case of local pathogen encounter. Different memory CD8 T cell subsets patrol peripheral tissues, but it is unclear which subset is superior in providing protection upon secondary infections. We used influenza virus to induce predominantly tissue resident memory T cells or cytomegalovirus to elicit a large pool of effector-like memory cells in the lungs and determined their early protective capacity and mechanism of reactivation. Both memory CD8 T cell pools have unique characteristics with respect to their phenotype, localization, and maintenance. However, these distinct features do not translate into different capacities to control a respiratory vaccinia virus challenge in an antigen-specific manner, although differential activation mechanisms are utilized. While influenza-induced memory CD8 T cells respond to antigen by local proliferation, MCMV-induced memory CD8 T cells relocate from the vasculature into the tissue in an antigen-independent and partially chemokine-driven manner. Together these results bear relevance for the development of vaccines aimed at eliciting a protective memory CD8 T cell pool at mucosal sites., Mucosal Immunology, 14 (3), ISSN:1933-0219, ISSN:1935-3456

The avian pathogen fowlpox virus (FWPV) has been successfully used as a vaccine vector in poultry and humans, but relatively little is known about its ability to modulate host antiviral immune responses in these hosts, which are replication-permissive and nonpermissive, respectively. FWPV is highly resistant to avian type I interferon (IFN) and able to completely block the host IFN-response. Microarray screening of host IFN-regulated gene expression in cells infected with 59 different, nonessential FWPV gene knockout mutants revealed that FPV184 confers immunomodulatory capacity. We report that the FPV184-knockout virus (FWPV&Delta, 184) induces the cellular IFN response as early as 2 h postinfection. The wild-type, uninduced phenotype can be rescued by transient expression of FPV184 in FWPV&Delta, 184-infected cells. Ectopic expression of FPV184 inhibited polyI:C activation of the chicken IFN-&beta, promoter and IFN-&alpha, activation of the chicken Mx1 promoter. Confocal and correlative super-resolution light and electron microscopy demonstrated that FPV184 has a functional nuclear localisation signal domain and is packaged in the lateral bodies of the virions. Taken together, these results provide a paradigm for a late poxvirus structural protein packaged in the lateral bodies, capable of suppressing IFN induction early during the next round of infection.

The avian pathogen, fowlpox virus (FWPV) has been successfully used as vaccine vector in poultry and humans but relatively little is known about its ability to modulate host antiviral immune responses in these hosts, which are replication permissive and non-permissive, respectively. FWPV is highly resistant to avian type I interferon (IFN) and able to completely block the host IFN-response. Microarray screening of host IFN-regulated gene expression in cells infected with 59 different, non-essential FWPV gene knock-out mutants revealed that FPV184 confers immunomodulatory capacity. We report that FPV184-knockout virus (FWPVΔ184) induces the cellular IFN response as early as 2 hours post-infection. The wild-type, uninduced phenotype can be rescued by transient expression of FPV184 in FWPVΔ184-infected cells. Ectopic expression of FPV184 inhibited polyI:C activation of the chicken IFN-β promoter and IFN-α activation of the chicken Mx promoter. Confocal and correlative super-resolution light and electron microscopy demonstrated that FPV184 has a functional nuclear localisation signal domain and is packaged in the lateral bodies of the virions. Taken together, these results provide a paradigm for a late poxvirus structural protein packaged in the lateral bodies and capable of supressing IFN induction early during the next round of infection.

The replication and assembly of vaccinia virus (VACV), the prototypic poxvirus, occurs exclusively in the cytoplasm of host cells. While the role of cellular cytoskeletal components in these processes remains poorly understood, vimentin - a type III intermediate filament - has been shown to associate with viral replication sites and to be incorporated into mature VACV virions. Here we employed chemical and genetic approaches to further investigate the role of vimentin during the VACV lifecycle. The collapse of vimentin filaments, using acrylamide, was found to inhibit VACV infection at the level of genome replication, intermediate- and late- gene expression. However, we found that CRISPR-mediated knockout of vimentin did not impact VACV replication. Combining these tools, we demonstrate that acrylamide treatment results in the formation of antiviral granules (AVGs) known to mediate translational inhibition of many viruses. We conclude that vimentin is dispensable for poxvirus replication and assembly and that acrylamide, as a potent inducer of AVGs during VACV infection, serves to bolster cell’s antiviral response to poxvirus infection.Summary StatementAcrylamide inhibits poxvirus replication by inducing anti-viral granules and blocking translation. This inhibition is independent of the effect of acrylamide on vimentin filaments which were found to be dispensable for viral replication and assembly.

Unlike most enveloped viruses, poxvirus egress is a complex process whereby cytoplasmic single membrane-bound virions are wrapped in a cell-derived double membrane. These triple membrane-bound particles, termed intracellular enveloped virions (IEVs), are then released from infected cells by fusion. While the wrapping double membrane is thought to be derived from virus-modified trans-Golgi or early endosomal cisternae, the cellular factors that regulate virus wrapping remain largely undefined. To identify novel cell factors required for this process the prototypic poxvirus, vaccinia virus (VACV), was subjected to a high-throughput RNAi screen directed against cellular membrane trafficking proteins. Focusing on the endosomal sorting complexes required for transport (ESCRT), we demonstrate that ESCRT-III and VPS4 are required for packaging of virus into multivesicular bodies (MVBs). EM-based characterization of these MVB-IEVs showed that they account for half of IEV production indicating that MVBs serve as a second major source of VACV wrapping membrane. These data support a model whereby, in addition to cisternae-based wrapping, VACV hijacks ESCRT-mediated MVB formation to facilitate virus egress and spread.

To orchestrate context-dependent signalling programmes, poxviruses encode two dual-specificity enzymes, the F10 kinase and the H1 phosphatase. These signalling mediators are essential for poxvirus production, yet their substrate profiles and systems-level functions remain enigmatic. Using a phosphoproteomic screen of cells infected with wild-type, F10 and H1 mutant vaccinia viruses, we systematically defined the viral signalling network controlled by these enzymes. Quantitative cross-comparison revealed 33 F10 and/or H1 phosphosites within 17 viral proteins. Using this proteotype dataset to inform genotype-phenotype relationships, we found that H1-deficient virions harbour a hidden hypercleavage phenotype driven by reversible phosphorylation of the virus protease I7 (S134). Quantitative phosphoproteomic profiling further revealed that the phosphorylation-dependent activity of the viral early transcription factor, A7 (Y367), underlies the transcription-deficient phenotype of H1 mutant virions. Together, these results highlight the utility of combining quantitative proteotype screens with mutant viruses to uncover proteotype-phenotype-genotype relationships that are masked by classical genetic studies.

The regulation of size, volume and mass in living cells is physiologically important, and dysregulation of these parameters gives rise to many diseases. Cell mass is largely determined by the amount of water, proteins, lipids, carbohydrates and nucleic acids present in a cell, and is tightly linked to metabolism, proliferation and gene expression. Technologies have emerged in recent years that make it possible to track the masses of single suspended cells and adherent cells. However, it has not been possible to track individual adherent cells in physiological conditions at the mass and time resolutions required to observe fast cellular dynamics. Here we introduce a cell balance (a 'picobalance'), based on an optically excited microresonator, that measures the total mass of single or multiple adherent cells in culture conditions over days with millisecond time resolution and picogram mass sensitivity. Using our technique, we observe that the mass of living mammalian cells fluctuates intrinsically by around one to four per cent over timescales of seconds throughout the cell cycle. Perturbation experiments link these mass fluctuations to the basic cellular processes of ATP synthesis and water transport. Furthermore, we show that growth and cell cycle progression are arrested in cells infected with vaccinia virus, but mass fluctuations continue until cell death. Our measurements suggest that all living cells show fast and subtle mass fluctuations throughout the cell cycle. As our cell balance is easy to handle and compatible with fluorescence microscopy, we anticipate that our approach will contribute to the understanding of cell mass regulation in various cell states and across timescales, which is important in areas including physiology, cancer research, stem-cell differentiation and drug discovery.

In biology, the use of deep neural networks (DNNs) for analysis of pathogen infection is hampered by a lack of large verified data sets needed for rapid network evolution. Artificial neural networks detect handwritten digits with high precision thanks to large data sets, such as MNIST, that allow nearly unlimited training. Here, we developed a novel strategy we call mimicry embedding, which allows artificial intelligence (AI)-based analysis of variable pathogen-host data sets. We show that deep learning can be used to detect and classify single pathogens based on small differences., The use of deep neural networks (DNNs) for analysis of complex biomedical images shows great promise but is hampered by a lack of large verified data sets for rapid network evolution. Here, we present a novel strategy, termed “mimicry embedding,” for rapid application of neural network architecture-based analysis of pathogen imaging data sets. Embedding of a novel host-pathogen data set, such that it mimics a verified data set, enables efficient deep learning using high expressive capacity architectures and seamless architecture switching. We applied this strategy across various microbiological phenotypes, from superresolved viruses to in vitro and in vivo parasitic infections. We demonstrate that mimicry embedding enables efficient and accurate analysis of two- and three-dimensional microscopy data sets. The results suggest that transfer learning from pretrained network data may be a powerful general strategy for analysis of heterogeneous pathogen fluorescence imaging data sets. IMPORTANCE In biology, the use of deep neural networks (DNNs) for analysis of pathogen infection is hampered by a lack of large verified data sets needed for rapid network evolution. Artificial neural networks detect handwritten digits with high precision thanks to large data sets, such as MNIST, that allow nearly unlimited training. Here, we developed a novel strategy we call mimicry embedding, which allows artificial intelligence (AI)-based analysis of variable pathogen-host data sets. We show that deep learning can be used to detect and classify single pathogens based on small differences.

Research on Toxoplasma gondii and its interplay with the host is often performed using fluorescence microscopy-based imaging experiments combined with manual quantification of acquired images. We present here an accurate and unbiased quantification method for host-pathogen interactions. We describe how to plan experiments and prepare, stain and image infected specimens and analyze them with the program HRMAn (Host Response to Microbe Analysis). HRMAn is a high-content image analysis method based on KNIME Analytics Platform. Users of this guide will be able to perform infection studies in high-throughput volume and to a greater level of detail. Relying on cutting edge machine learning algorithms, HRMAn can be trained and tailored to many experimental settings and questions.

Research on Toxoplasma gondii and its interplay with the host is often performed using fluorescence microscopy-based imaging experiments combined with manual quantification of acquired images. We present here an accurate and unbiased quantification method for host-pathogen interactions. We describe how to plan experiments and prepare, stain and image infected specimens and analyze them with the program HRMAn (Host Response to Microbe Analysis). HRMAn is a high-content image analysis method based on KNIME Analytics Platform. Users of this guide will be able to perform infection studies in high-throughput volume and to a greater level of detail. Relying on cutting edge machine learning algorithms, HRMAn can be trained and tailored to many experimental settings and questions.

Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo, and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we develop a zebrafish infection model to study the innate immune response to Toxoplasma in vivo. By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision driven automated image analysis, we reveal that Toxoplasma invades and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are being cleared in vivo, we analyzed Toxoplasma-macrophage interactions using time-lapse and correlative light and electron microscopy. Strikingly, macrophages are recruited to the infection site and play a key role in Toxoplasma control. These results highlight in vivo control of Toxoplasma by macrophages, and illuminate the possibility to exploit zebrafish for discoveries within the field of parasite immunity.

The use of deep neural networks (DNNs) for analysis of complex biomedical images shows great promise but is hampered by a lack of large verified datasets for rapid network evolution. Here we present a novel “mimicry embedding” strategy for rapid application of neural network architecture-based analysis of biomedical imaging datasets. Embedding of a novel biological dataset, such that it mimics a verified dataset, enables efficient deep learning and seamless architecture switching. We apply this strategy across various microbiological phenotypes; from super-resolved viruses toin vivoparasitic infections. We demonstrate that mimicry embedding enables efficient and accurate analysis of three-dimensional microscopy datasets. The results suggest that transfer learning from pre-trained network data may be a powerful general strategy for analysis of heterogeneous biomedical imaging datasets.

Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo, and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we developed a zebrafish infection model to study the innate immune response to Toxoplasma in vivo. By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision-driven automated image analysis, we reveal that Toxoplasma invades brain cells and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We also show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are cleared in vivo, we further analyzed Toxoplasma-macrophage interactions using time-lapse microscopy and three-dimensional correlative light and electron microscopy (3D CLEM). Time-lapse microscopy revealed that macrophages are recruited to the infection site and play a key role in Toxoplasma control. High-resolution 3D CLEM revealed parasitophorous vacuole breakage in brain cells and macrophages in vivo, suggesting that cell-intrinsic mechanisms may be used to destroy the intracellular niche of tachyzoites. Together, our results demonstrate in vivo control of Toxoplasma by macrophages, and highlight the possibility that zebrafish may be further exploited as a novel model system for discoveries within the field of parasite immunity. This article has an associated First Person interview with the first author of the paper., Summary: We have established a novel zebrafish infection model to investigate Toxoplasma replication in vivo, and demonstrate a key role for macrophages in parasite control.

Quantitative PCR-based methods have proven to be easy-to-use, cost-effective procedures for the quantification of viral gene expression and viral genome numbers. Quantitative PCR (qPCR) and quantitative reverse transcriptase-PCR (qRT-PCR) are rapid and sensitive approaches that can be used to pinpoint defects in viral DNA replication and transcriptional activity, respectively. Due to the significant nucleotide overlap between Poxviridae these methods can be employed across a wide range of viruses from this family. Here we provide methods for the quantification of vaccinia DNA replication by qPCR and quantification of the three classes of vaccinia gene transcription by qRT-PCR.

Super-resolution microscopy has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for super-resolution microscopy designed to combine high performance and ease of use. We named it NanoJ - a reference to the popular ImageJ software it was de-veloped for. In this paper, we highlight the current capabilities of NanoJ for several essential processing steps: spatio-temporal alignment of raw data (NanoJ-Core), super-resolution image re-construction (NanoJ-SRRF), image quality assessment (NanoJ-SQUIRREL), structural modelling (NanoJ-VirusMapper) and control of the sample environment (NanoJ-Fluidics). We expect to expand NanoJ in the future through the development of new tools designed to improve quantitative data analysis and measure the reliability of fluorescent microscopy studies.

In order to initiate successful infection, viruses have to transmit and deliver their genome from one host cell or organism to another. To achieve this, enveloped viruses must first fuse their membrane with those of the target host cell. Here, we describe the sequence of events leading to the entry of representative enveloped viruses, highlighting the strategies they use to gain access to the host cell cytosol.

For image-based infection biology, accurate unbiased quantification of host–pathogen interactions is essential, yet often performed manually or using limited enumeration employing simple image analysis algorithms based on image segmentation. Host protein recruitment to pathogens is often refractory to accurate automated assessment due to its heterogeneous nature. An intuitive intelligent image analysis program to assess host protein recruitment within general cellular pathogen defense is lacking. We present HRMAn (Host Response to Microbe Analysis), an open-source image analysis platform based on machine learning algorithms and deep learning. We show that HRMAn has the capacity to learn phenotypes from the data, without relying on researcher-based assumptions. Using Toxoplasma gondii and Salmonella enterica Typhimurium we demonstrate HRMAn’s capacity to recognize, classify and quantify pathogen killing, replication and cellular defense responses. HRMAn thus presents the only intelligent solution operating at human capacity suitable for both single image and high content image analysis.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Vaccinia virus plaque assays are employed for quantification of virus titer through serial dilution of virus on a monolayer of cells. Once the virus titer is diluted enough to allow for only few cells of the monolayer to be infected, clonal spread of infection can be detected by observing the lesion in the cell monolayer or using virus-specific staining methods. Beyond simple titration, plaque formation bares priceless underlying information about subtle virus-host interactions and their impact on virus spread during multiple rounds of infection. These include virus infectivity, mode of virus spread, virus replication rate, and spatiotemporal spread efficacy. How this underlying information can be harnessed using a high-content imaging setup is discussed here.

Introductory paragraph Cell motility is essential for viral dissemination1. Vaccinia virus (VACV), a close relative of smallpox virus, is thought to exploit cell motility as a means to enhance the spread of infection1. A single viral protein, F11L, contributes to this by blocking RhoA signalling to facilitate cell retraction2. However, F11L alone is not sufficient for VACV induced cell motility, indicating that additional viral factors must be involved. Here we show that the VACV epidermal growth factor homolog, VGF, promotes infected cell motility and the spread of viral infection. We found that VGF secreted from early infected cells is cleaved by ADAM10 whereupon it acts largely in a paracrine fashion to direct cell motility at the leading edge of infection. Real-time tracking of cells infected in the presence of EGFR/MAPK/FAK/ADAM10 inhibitors, or with VGF and F11 deleted viruses, revealed defects in radial velocity and directional migration efficiency leading to impaired cell-to-cell spread of infection. Furthermore, intravital imaging showed that virus spread and lesion formation are attenuated in the absence of VGF. Our results demonstrate how poxviruses hijack epidermal growth factor receptor induced cell motility to promote rapid and efficient spread of infection in vitro and in vivo.

Quantitative PCR-based methods have proven to be easy-to-use, cost-effective procedures for the quantification of viral gene expression and viral genome numbers. Quantitative PCR (qPCR) and quantitative reverse transcriptase-PCR (qRT-PCR) are rapid and sensitive approaches that can be used to pinpoint defects in viral DNA replication and transcriptional activity, respectively. Due to the significant nucleotide overlap between Poxviridae these methods can be employed across a wide range of viruses from this family. Here we provide methods for the quantification of vaccinia DNA replication by qPCR and quantification of the three classes of vaccinia gene transcription by qRT-PCR.

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artefact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artefacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy.

For image-based infection biology, accurate unbiased quantification of host-pathogen interactions is essential, yet often performed manually or using limited enumeration employing simple image analysis algorithms based on image segmentation. Host protein recruitment to pathogens is often refractory to accurate automated assessment due to its heterogeneous nature. An intuitive intelligent image analysis program to assess host protein recruitment within general cellular pathogen defense is lacking. We present HRMAn (Host Response to Microbe Analysis), an open-source image analysis platform based on machine learning algorithms and deep learning. We show that HRMAn has the capability to learn phenotypes from the data, without relying on researcher-based assumptions. Using Toxoplasma gondii and Salmonella typhimurium we demonstrate HRMAn’s capacity to recognize, classify and quantify pathogen killing, replication and cellular defense responses.