Beyond the surface: Investigation of tumorsphere morphology using volume electron microscopy.

The advent of volume electron microscopy (vEM) has provided unprecedented insights into cellular and subcellular organization, revolutionizing our understanding of cancer biology. This study presents a previously unexplored comparative analysis of the ultrastructural disparities between cancer cells cultured as monolayers and tumorspheres. By integrating a robust workflow that incorporates high-pressure freezing followed by freeze substitution (HPF/FS), serial block face scanning electron microscopy (SBF-SEM), manual and deep learning-based segmentation, and statistical analysis, we have successfully generated three-dimensional (3D) reconstructions of monolayer and tumorsphere cells, including their subcellular organelles. Our findings reveal a significant degree of variation in cellular morphology in tumorspheres. We observed the increased prevalence of nuclear envelope invaginations in tumorsphere cells compared to monolayers. Furthermore, we detected a diverse range of mitochondrial morphologies exclusively in tumorsphere cells, as well as intricate cellular interconnectivity within the tumorsphere architecture. These remarkable ultrastructural differences emphasize the use of tumorspheres as a superior model for cancer research due to their relevance to in vivo conditions. Our results strongly advocate for the utilization of tumorsphere cells in cancer research studies, enhancing the precision and relevance of experimental outcomes, and ultimately accelerating therapeutic advancements.

Total infectome investigation of diphtheritic stomatitis in yellow-eyed penguins (Megadyptes antipodes) reveals a novel and abundant megrivirus.

First identified in 2002, diphtheritic stomatitis (DS) is a devastating disease affecting yellow-eyed penguins (Megadyptes antipodes, or hoiho in te reo Maori). The disease is associated with oral lesions in chicks and has caused significant morbidity and mortality. DS is widespread among yellow-eyed penguin chicks on mainland New Zealand yet appears to be absent from the subantarctic population. Corynebacterium spp. have previously been suspected as causative agents yet, due to inconsistent cultures and inconclusive pathogenicity, their role in DS is unclear. Herein, we used a metatranscriptomic approach to identify potential causative agents of DS by revealing the presence and abundance of all viruses, bacteria, fungi and protozoa - together, the infectome. Oral and cloacal swab samples were collected from presymptomatic, symptomatic and recovered chicks along with a control group of healthy adults. Two novel viruses from the Picornaviridae were identified, one of which - yellow-eyed penguin megrivirus - was highly abundant in chicks irrespective of health status but not detected in healthy adults. Tissue from biopsied oral lesions also tested positive for the novel megrivirus upon PCR. We found no overall clustering among bacteria, protozoa and fungi communities at the genus level across samples, although Paraclostridium bifermentans was significantly more abundant in oral microbiota of symptomatic chicks compared to other groups. The detection of a novel and highly abundant megrivirus has sparked a new line of inquiry to investigate its potential association with DS.

Directed Evolution of Seneca Valley Virus in Tumorsphere and Monolayer Cell Cultures of a Small-Cell Lung Cancer Model.

The Seneca Valley virus (SVV) is an oncolytic virus from the picornavirus family, characterized by a 7.3-kilobase RNA genome encoding for all the structural and functional viral proteins. Directed evolution by serial passaging has been employed for oncolytic virus adaptation to increase the killing efficacy towards certain types of tumors. We propagated the SVV in a small-cell lung cancer model under two culture conditions: conventional cell monolayer and tumorspheres, with the latter resembling more closely the cellular structure of the tumor of origin. We observed an increase of the virus-killing efficacy after ten passages in the tumorspheres. Deep sequencing analyses showed genomic changes in two SVV populations comprising 150 single nucleotides variants and 72 amino acid substitutions. Major differences observed in the tumorsphere-passaged virus population, compared to the cell monolayer, were identified in the conserved structural protein VP2 and in the highly variable P2 region, suggesting that the increase in the ability of the SVV to kill cells over time in the tumorspheres is acquired by capsid conservation and positively selecting mutations to counter the host innate immune responses.

A novel gyrovirus is abundant in yellow-eyed penguin (Megadyptes antipodes) chicks with a fatal respiratory disease.

Yellow-eyed penguins (Megadyptes antipodes), or hoiho in te reo Maori, are predicted to become extinct on mainland Aotearoa New Zealand in the next few decades, with infectious disease a significant contributor to their decline. A recent disease phenomenon termed respiratory distress syndrome (RDS) causing lung pathology has been identified in very young chicks. To date, no causative pathogens for RDS have been identified. In 2020 and 2021, the number of chick deaths from suspected RDS increased four- and five-fold, respectively, causing mass mortality with an estimated mortality rate of >90%. We aimed to identify possible pathogens responsible for RDS disease impacting these critically endangered yellow-eyed penguins. Total RNA was extracted from tissue samples collected during post-mortem of 43 dead chicks and subject to metatranscriptomic sequencing and histological examination. From these data we identified a novel and highly abundant gyrovirus (Anelloviridae) in 80% of tissue samples. This virus was most closely related to Gyrovirus 8 discovered in a diseased seabird, while other members of the genus Gyrovirus include Chicken anaemia virus, which causes severe disease in juvenile chickens. No other exogenous viral transcripts were identified in these tissues. Due to the high relative abundance of viral reads and its high prevalence in diseased animals, it is likely that this novel gyrovirus is associated with RDS in yellow-eyed penguin chicks.

Characterisation of a Seneca Valley virus thermostable mutant.

Seneca Valley virus (SVV) is a newly discovered picornavirus in the Senecavirus genus. SVV-001 strain has shown promise as an oncolytic virus against tumors with neuroendocrine features. There is a need to use a structure-based approach to develop virus-like particles capable to mimicking the architecture of naturally occurring empty capsids that can be used as vaccines or as carriers for targeted cancer treatment. However, these empty capsids are inherently less stable, and tedious to purify. This warrants investigation into factors which confer the SVV capsid stability and into combining this knowledge to recombinantly express stable SVV VLPs. In this study, we isolated a thermostable mutant of SVV by thermal selection assays and we characterized a single mutation located in a capsid protein. The cryo-EM map of this mutant showed conformational shifts that facilitated the formation of additional hydrogen bonds and aromatic interactions, which could serve as capsid stabilizing factors.

A lipopolysaccharide-dependent phage infects a pseudomonad phytopathogen and can evolve to evade phage resistance.

Bacterial pathogens are major causes of crop diseases, leading to significant production losses. For instance, kiwifruit canker, caused by the phytopathogen Pseudomonas syringae pv. actinidiae (Psa), has posed a global challenge to kiwifruit production. Treatment with copper and antibiotics, whilst initially effective, is leading to the rise of bacterial resistance, requiring new biocontrol approaches. Previously, we isolated a group of closely related Psa phages with biocontrol potential, which represent environmentally sustainable antimicrobials. However, their deployment as antimicrobials requires further insight into their properties and infection strategy. Here, we provide an in-depth examination of the genome of ΦPsa374-like phages and show that they use lipopolysaccharides (LPS) as their main receptor. Through proteomics and cryo-electron microscopy of ΦPsa374, we revealed the structural proteome and that this phage possess a T = 9 capsid triangulation, unusual for myoviruses. Furthermore, we show that ΦPsa374 phage resistance arises in planta through mutations in a glycosyltransferase involved in LPS synthesis. Lastly, through in vitro evolution experiments we showed that phage resistance is overcome by mutations in a tail fibre and structural protein of unknown function in ΦPsa374. This study provides new insight into the properties of ΦPsa374-like phages that informs their use as antimicrobials against Psa.

Cryo-EM Structure of a Possum Enterovirus.

Enteroviruses (EVs) represent a substantial concern to global health. Here, we present the cryo-EM structure of a non-human enterovirus, EV-F4, isolated from the Australian brushtail possum to assess the structural diversity of these picornaviruses. The capsid structure, determined to ~3 Å resolution by single particle analysis, exhibits a largely smooth surface, similar to EV-F3 (formerly BEV-2). Although the cellular receptor is not known, the absence of charged residues on the outer surface of the canyon suggest a different receptor type than for EV-F3. Density for the pocket factor is clear, with the entrance to the pocket being smaller than for other enteroviruses.

Characterization of the First SARS-CoV-2 Isolates from Aotearoa New Zealand as Part of a Rapid Response to the COVID-19 Pandemic.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has wreaked havoc across the globe for the last two years. More than 300 million cases and over 5 million deaths later, we continue battling the first real pandemic of the 21st century. SARS-CoV-2 spread quickly, reaching most countries within the first half of 2020, and New Zealand was not an exception. Here, we describe the first isolation and characterization of SARS-CoV-2 variants during the initial virus outbreak in New Zealand. Patient-derived nasopharyngeal samples were used to inoculate Vero cells and, three to four days later, a cytopathic effect was observed in seven viral cultures. Viral growth kinetics was characterized using Vero and VeroE6/TMPRSS2 cells. The identity of the viruses was verified by RT-qPCR, Western blot, indirect immunofluorescence assays, and electron microscopy. Whole-genome sequences were analyzed using two different yet complementary deep sequencing platforms (MiSeq/Illumina and Ion PGM™/Ion Torrent™), classifying the viruses as SARS-CoV-2 B.55, B.31, B.1, or B.1.369 based on the Pango Lineage nomenclature. All seven SARS-CoV-2 isolates were susceptible to remdesivir (EC50 values from 0.83 to 2.42 µM) and ß-D-N4-hydroxycytidine (molnupiravir, EC50 values from 0.96 to 1.15 µM) but not to favipiravir (>10 µM). Interestingly, four SARS-CoV-2 isolates, carrying the D614G substitution originally associated with increased transmissibility, were more susceptible (2.4-fold) to a commercial monoclonal antibody targeting the spike glycoprotein than the wild-type viruses. Altogether, this seminal work allowed for early access to SARS-CoV-2 isolates in New Zealand, paving the way for numerous clinical and scientific research projects in the country, including the development and validation of diagnostic assays, antiviral strategies, and a national COVID-19 vaccine development program.

Processing hair follicles for transmission electron microscopy.

Transmission electron microscopy (TEM) has greatly advanced our knowledge of hair growth and follicle morphogenesis, but complex preparations such as fixation, dehydration and embedding compromise ultrastructure. While recent developments with cryofixation have been shown to preserve the ultrastructure of biological materials close to native state, they do have limitations. This review will focus on each stage of the TEM sample preparation process and their effects on the structural integrity of follicles.

Monoclonal antibodies constructed from COVID-19 convalescent memory B cells exhibit potent binding activity to MERS-CoV spike S2 subunit and other human coronaviruses.

Introduction: The Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are two highly contagious coronaviruses causing MERS and COVID-19, respectively, without an effective antiviral drug and a long-lasting vaccine. Approaches for diagnosis, therapeutics, prevention, etc., particularly for SARS-CoV-2 that is continually spreading and evolving, are urgently needed. Our previous study discovered that >60% of sera from convalescent COVID-19 individuals, but <8% from general population, showed binding activity against the MERS-CoV spike protein, indicating that SARS-CoV-2 infection boosted antibodies cross-reactive with MERS-CoV. Methods: To generate antibodies specific to both SARS-CoV-2 and MERS-CoV, here we screened 60 COVID-19 convalescent sera against MERS-CoV spike extracellular domain and S1 and S2 subunits. We constructed and characterized monoclonal antibodies (mAbs) from COVID-19 convalescent memory B cells and examined their binding and neutralizing activities against human coronaviruses. Results and Discussion: Of 60 convalescent serum samples, 34 showed binding activity against MERS-CoV S2, with endpoint titers positively correlated with the titers to SARS-CoV-2 S2. By sorting single memory B cells from COVID-19 convalescents, we constructed 38 mAbs and found that 11 mAbs showed binding activity with MERS-CoV S2, of which 9 mAbs showed potent cross-reactivity with all or a proportion of spike proteins of alphacoronaviruses (229E and NL63) and betacoronaviruses (SARS-CoV-1, SARS-CoV-2, OC43, and HKU1). Moreover, 5 mAbs also showed weak neutralization efficiency against MERS-CoV spike pseudovirus. Epitope analysis revealed that 3 and 8 mAbs bound to linear and conformational epitopes in MERS-CoV S2, respectively. In summary, we have constructed a panel of antibodies with broad-spectrum reactivity against all seven human coronaviruses, thus facilitating the development of diagnosis methods and vaccine design for multiple coronaviruses.

N-Linked Glycosylation on Anthrax Toxin Receptor 1 Is Essential for Seneca Valley Virus Infection.

Seneca Valley virus (SVV) is a picornavirus with potency in selectively infecting and lysing cancerous cells. The cellular receptor for SVV mediating the selective tropism for tumors is anthrax toxin receptor 1 (ANTXR1), a type I transmembrane protein expressed in tumors. Similar to other mammalian receptors, ANTXR1 has been shown to harbor N-linked glycosylation sites in its extracellular vWA domain. However, the exact role of ANTXR1 glycosylation on SVV attachment and cellular entry was unknown. Here we show that N-linked glycosylation in the ANTXR1 vWA domain is necessary for SVV attachment and entry. In our study, tandem mass spectrometry analysis of recombinant ANTXR1-Fc revealed the presence of complex glycans at N166, N184 in the vWA domain, and N81 in the Fc domain. Symmetry-expanded cryo-EM reconstruction of SVV-ANTXR1-Fc further validated the presence of N166 and N184 in the vWA domain. Cell blocking, co-immunoprecipitation, and plaque formation assays confirmed that deglycosylation of ANTXR1 prevents SVV attachment and subsequent entry. Overall, our results identified N-glycosylation in ANTXR1 as a necessary post-translational modification for establishing stable interactions with SVV. We anticipate our findings will aid in selecting patients for future cancer therapeutics, where screening for both ANTXR1 and its glycosylation could lead to an improved outcome from SVV therapy.

Visualizing Nudivirus Assembly and Egress.

Enveloped viruses hijack cellular membranes in order to provide the necessary material for virion assembly. In particular, viruses that replicate and assemble inside the nucleus have developed special approaches to modify the nuclear landscape for their advantage. We used electron microscopy to investigate cellular changes occurring during nudivirus infection and we characterized a unique mechanism for assembly, packaging, and transport of new virions across the nuclear membrane and through the cytoplasm. Our three-dimensional reconstructions describe the complex remodeling of the nuclear membrane necessary to release vesicle-associated viruses into the cytoplasm. This is the first report of nuclear morphological reconfigurations that occur during nudiviral infection.IMPORTANCE The dynamics of nuclear envelope has a critical role in multiple cellular processes. However, little is known regarding the structural changes occurring inside the nucleus or at the inner and outer nuclear membranes. For viruses assembling inside the nucleus, remodeling of the intranuclear membrane plays an important role in the process of virion assembly. Here, we monitored the changes associated with viral infection in the case of nudiviruses. Our data revealed dramatic membrane remodeling inside the nuclear compartment during infection with Oryctes rhinoceros nudivirus, an important biocontrol agent against coconut rhinoceros beetle, a devastating pest for coconut and oil palm trees. Based on these findings, we propose a model for nudivirus assembly in which nuclear packaging occurs in fully enveloped virions.

Virus-Receptor Interactions and Virus Neutralization: Insights for Oncolytic Virus Development.

Oncolytic viruses (OVs) are replication competent agents that selectively target cancer cells. After penetrating the tumor cell, viruses replicate and eventually trigger cell lysis, releasing the new viral progeny, which at their turn will attack and kill neighbouring cells. The ability of OVs to self-amplify within the tumor while sparing normal cells can provide several advantages including the capacity to encode and locally produce therapeutic protein payloads, and to prime the host immune system. OVs targeting of cancer cells is mediated by host factors that are differentially expressed between normal tissue and tumors, including viral receptors and internalization factors. In this review article, we will discuss the evolution of oncolytic viruses that have reached the stage of clinical trials, their mechanisms of oncolysis, cellular receptors, strategies for targeting cancers, viral neutralization and developments to bypass virus neutralization.

Correction: McCarthy, C.; et al. Developing Picornaviruses for Cancer Therapy. Cancers 2019, 11, 685.

The authors wish to make the following corrections to this paper [1] [...].

Virus-Receptor Interactions: Structural Insights For Oncolytic Virus Development.

Recent advancements in oncolytic virotherapy commend a special attention to developing new strategies for targeting cancer cells with oncolytic viruses (OVs). Modifications of the viral envelope or coat proteins serve as a logical mean of repurposing viruses for cancer treatment. In this review, we discuss how detailed structural knowledge of the interactions between OVs and their natural receptors provide valuable insights into tumor specificity of some viruses and re-targeting of alternate receptors for broad tumor tropism or improved tumor selectivity.

Viral infections alter antennal epithelium ultrastructure in honey bees.

Varroa destructor and its associated viruses, in particular deformed wing virus (DWV), have been identified as probable causes of honey bee (Apis mellif era L.) colony losses. Evidence suggests that elevated DWV titres in bees could compromise sensory and communication abilities resulting in negative consequences for hygienic behaviour. As antennae play a central role in this behaviour, we compared antennal ultrastructure in DWV-symptomatic and asymptomatic bees. The results show that virus capsids accumulate in the basal regions of the antennal epithelium, close to the haemolymph. No virus particles were detected at the level of sensory sensilla, such as pore plates, nor within the sensory cell dendrites associated with these sensilla. However, membranous structures appeared to be more prevalent in supporting cells surrounding the dendrites of DWV-symptomatic bees. Para-crystalline arrays containing large numbers of virus particles were detected in the antennae of DWV-symptomatic bees but not in asymptomatic bees.

Developing Picornaviruses for Cancer Therapy.

Oncolytic viruses (OVs) form a group of novel anticancer therapeutic agents which selectively infect and lyse cancer cells. Members of several viral families, including Picornaviridae, have been shown to have anticancer activity. Picornaviruses are small icosahedral non-enveloped, positive-sense, single-stranded RNA viruses infecting a wide range of hosts. They possess several advantages for development for cancer therapy: Their genomes do not integrate into host chromosomes, do not encode oncogenes, and are easily manipulated as cDNA. This review focuses on the picornaviruses investigated for anticancer potential and the mechanisms that underpin this specificity.

Helical twist direction in the macrofibrils of keratin fibres is left handed.

Macrofibrils, the main structural features within the cortical cells of mammalian hair shafts, are long composite bundles of keratin intermediate filaments (KIFs) embedded in a matrix of keratin-associated proteins. The KIFs can be helically arranged around the macrofibril central axis, making a cylinder within which KIF helical angle relative to macrofibril axis increases approximately linearly from macrofibril centre to edge. Mesophase-based self-assembly has been implicated in the early formation of macrofibrils, which first appear as liquid-crystal tactoids in the bulb of hair follicles. Formation appears to be driven initially by interactions between pre-keratinized KIFs. Differences in the nature of these KIF-KIF interactions could result in all macrofibrils being internally twisted in a single handedness, or a 50:50 mixture of handedness within each cortical cell. We data-mined 41 electron tomograms containing three-dimensional macrofibril data from previously published studies of hair and wool. In all 644 macrofibrils examined we found that within each tomogram all macrofibrils had the same handedness. We concluded that earlier reports of left- and right-handed macrofibrils were due to artefacts of imaging or data processing. A handedness marker was used to confirm (using re-imaged sections from earlier studies) that, in both human and sheep, all macrofibrils are left-handed around the macrofibril axis. We conclude that this state is universal within mammalian hair. This also supports the conclusion that the origin of macrofibril twist is the expression of chiral twisting forces between adjacent KIFs, rather than mesophase splay and bending forces relaxing to twisting forces acting within a confined space.

Monoclonal antibodies point to Achilles' heel in picornavirus capsid.

Picornaviruses are small, icosahedral, nonenveloped, positive-sense, single-stranded RNA viruses that form one of the largest and most important viral families. Numerous Picornaviridae members pose serious health or agricultural threats, causing diseases such as poliomyelitis, hepatitis A, or foot-and-mouth disease. The antigenic characterization of picornavirus capsids plays an important role in understanding the mechanism of viral neutralization and the conformational changes associated with genome release, and it can point to regions which can be targeted by small-molecule compounds to be developed as antiviral inhibitors. In a recent study, Cao and colleagues applied this strategy to hepatitis A virus (HAV) and used cryo-electron microscopy (cryo-EM) to characterize a well-conserved antigenic site recognized by several monoclonal antibodies. They further used computational approaches to identify a small-molecule drug with a strong inhibitory effect on cell attachment.

Structural basis for anthrax toxin receptor 1 recognition by Seneca Valley Virus.

Recently, the use of oncolytic viruses in cancer therapy has become a realistic therapeutic option. Seneca Valley Virus (SVV) is a newly discovered picornavirus, which has earned a significant reputation as a potent oncolytic agent. Anthrax toxin receptor 1 (ANTXR1), one of the cellular receptors for the protective antigen secreted by Bacillus anthracis, has been identified as the high-affinity cellular receptor for SVV. Here, we report the structure of the SVV-ANTXR1 complex determined by single-particle cryo-electron microscopy analysis at near-atomic resolution. This is an example of a shared receptor structure between a mammalian virus and a bacterial toxin. Our structure shows that ANTXR1 decorates the outer surface of the SVV capsid and interacts with the surface-exposed BC loop and loop II of VP1, "the puff" of VP2 and "the knob" of VP3. Comparison of the receptor-bound capsid structure with the native capsid structure reveals that receptor binding induces minor conformational changes in SVV capsid structure, suggesting the role of ANTXR1 as an attachment receptor. Furthermore, our results demonstrate that the capsid footprint on the receptor is not conserved in anthrax toxin receptor 2 (ANTXR2), thereby providing a molecular mechanism for explaining the exquisite selectivity of SVV for ANTXR1.