A multivalent mRNA monkeypox virus vaccine (BNT166) protects mice and macaques from orthopoxvirus disease.

In response to the 2022 outbreak of mpox driven by unprecedented human-to-human monkeypox virus (MPXV) transmission, we designed BNT166, aiming to create a highly immunogenic, safe, accessible, and scalable next-generation vaccine against MPXV and related orthopoxviruses. To address the multiple viral forms and increase the breadth of immune response, two candidate multivalent mRNA vaccines were evaluated pre-clinically: a quadrivalent vaccine (BNT166a; encoding the MPXV antigens A35, B6, M1, H3) and a trivalent vaccine (BNT166c; without H3). Both candidates induced robust T cell responses and IgG antibodies in mice, including neutralizing antibodies to both MPXV and vaccinia virus. In challenge studies, BNT166a and BNT166c provided complete protection from vaccinia, clade I, and clade IIb MPXV. Furthermore, immunization with BNT166a was 100% effective at preventing death and at suppressing lesions in a lethal clade I MPXV challenge in cynomolgus macaques. These findings support the clinical evaluation of BNT166, now underway (NCT05988203).

Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses.

Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

A selective sweep in the Spike gene has driven SARS-CoV-2 human adaptation.

The coronavirus disease 2019 (COVID-19) pandemic underscores the need to better understand animal-to-human transmission of coronaviruses and adaptive evolution within new hosts. We scanned more than 182,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes for selective sweep signatures and found a distinct footprint of positive selection located around a non-synonymous change (A1114G; T372A) within the spike protein receptor-binding domain (RBD), predicted to remove glycosylation and increase binding to human ACE2 (hACE2), the cellular receptor. This change is present in all human SARS-CoV-2 sequences but not in closely related viruses from bats and pangolins. As predicted, T372A RBD bound hACE2 with higher affinity in experimental binding assays. We engineered the reversion mutant (A372T) and found that A372 (wild-type [WT]-SARS-CoV-2) enhanced replication in human lung cells relative to its putative ancestral variant (T372), an effect that was 20 times greater than the well-known D614G mutation. Our findings suggest that this mutation likely contributed to SARS-CoV-2 emergence from animal reservoirs or enabled sustained human-to-human transmission.

Emerging Pandemic Diseases: How We Got to COVID-19.

Infectious diseases prevalent in humans and animals are caused by pathogens that once emerged from other animal hosts. In addition to these established infections, new infectious diseases periodically emerge. In extreme cases they may cause pandemics such as COVID-19; in other cases, dead-end infections or smaller epidemics result. Established diseases may also re-emerge, for example by extending geographically or by becoming more transmissible or more pathogenic. Disease emergence reflects dynamic balances and imbalances, within complex globally distributed ecosystems comprising humans, animals, pathogens, and the environment. Understanding these variables is a necessary step in controlling future devastating disease emergences.

A Structure-Informed Atlas of Human-Virus Interactions.

While knowledge of protein-protein interactions (PPIs) is critical for understanding virus-host relationships, limitations on the scalability of high-throughput methods have hampered their identification beyond a number of well-studied viruses. Here, we implement an in silico computational framework (pathogen host interactome prediction using structure similarity [P-HIPSTer]) that employs structural information to predict ∼282,000 pan viral-human PPIs with an experimental validation rate of ∼76%. In addition to rediscovering known biology, P-HIPSTer has yielded a series of new findings: the discovery of shared and unique machinery employed across human-infecting viruses, a likely role for ZIKV-ESR1 interactions in modulating viral replication, the identification of PPIs that discriminate between human papilloma viruses (HPVs) with high and low oncogenic potential, and a structure-enabled history of evolutionary selective pressure imposed on the human proteome. Further, P-HIPSTer enables discovery of previously unappreciated cellular circuits that act on human-infecting viruses and provides insight into experimentally intractable viruses.

DNA-Packing Portal and Capsid-Associated Tegument Complexes in the Tumor Herpesvirus KSHV.

Assembly of Kaposi's sarcoma-associated herpesvirus (KSHV) begins at a bacteriophage-like portal complex that nucleates formation of an icosahedral capsid with capsid-associated tegument complexes (CATCs) and facilitates translocation of an ∼150-kb dsDNA genome, followed by acquisition of a pleomorphic tegument and envelope. Because of deviation from icosahedral symmetry, KSHV portal and tegument structures have largely been obscured in previous studies. Using symmetry-relaxed cryo-EM, we determined the in situ structure of the KSHV portal and its interactions with surrounding capsid proteins, CATCs, and the terminal end of KSHV's dsDNA genome. Our atomic models of the portal and capsid/CATC, together with visualization of CATCs' variable occupancy and alternate orientation of CATC-interacting vertex triplexes, suggest a mechanism whereby the portal orchestrates procapsid formation and asymmetric long-range determination of CATC attachment during DNA packaging prior to pleomorphic tegumentation/envelopment. Structure-based mutageneses confirm that a triplex deep binding groove for CATCs is a hotspot that holds promise for antiviral development.

A ~40-kb flavi-like virus does not encode a known error-correcting mechanism.

It is commonly held that there is a fundamental relationship between genome size and error rate, manifest as a notional "error threshold" that sets an upper limit on genome sizes. The genome sizes of RNA viruses, which have intrinsically high mutation rates due to a lack of mechanisms for error correction, must therefore be small to avoid accumulating an excessive number of deleterious mutations that will ultimately lead to population extinction. The proposed exceptions to this evolutionary rule are RNA viruses from the order Nidovirales (such as coronaviruses) that encode error-correcting exonucleases, enabling them to reach genome lengths greater than 40 kb. The recent discovery of large-genome flavi-like viruses (Flaviviridae), which comprise genomes up to 27 kb in length yet seemingly do not encode exonuclease domains, has led to the proposal that a proofreading mechanism is required to facilitate the expansion of nonsegmented RNA virus genomes above 30 kb. Herein, we describe a ~40 kb flavi-like virus identified in a Haliclona sponge metatranscriptome that does not encode a known exonuclease. Structural analysis revealed that this virus may have instead captured cellular domains associated with nucleic acid metabolism that have not been previously found in RNA viruses. Phylogenetic inference placed this virus as a divergent pesti-like lineage, such that we have provisionally termed it "Maximus pesti-like virus." This virus represents an instance of a flavi-like virus achieving a genome size comparable to that of the Nidovirales and demonstrates that RNA viruses have evolved multiple solutions to overcome the error threshold.

The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by an RNA-Mediated Dimerization Mechanism.

Human APOBEC3H and homologous single-stranded DNA cytosine deaminases are unique to mammals. These DNA-editing enzymes function in innate immunity by restricting the replication of viruses and transposons. APOBEC3H also contributes to cancer mutagenesis. Here, we address the fundamental nature of RNA in regulating human APOBEC3H activities. APOBEC3H co-purifies with RNA as an inactive protein, and RNase A treatment enables strong DNA deaminase activity. RNA-binding-defective mutants demonstrate clear separation of function by becoming DNA hypermutators. Biochemical and crystallographic data demonstrate a mechanism in which double-stranded RNA mediates enzyme dimerization. Additionally, APOBEC3H separation-of-function mutants show that RNA binding is required for cytoplasmic localization, packaging into HIV-1 particles, and antiviral activity. Overall, these results support a model in which structured RNA negatively regulates the potentially harmful DNA deamination activity of APOBEC3H while, at the same time, positively regulating its antiviral activity.

Structural basis and dynamics of Chikungunya alphavirus RNA capping by nsP1 capping pores.

Alphaviruses are emerging positive-stranded RNA viruses which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The nonstructural protein 1 (nsP1) is responsible for viral RNA capping and gates the replication organelles by assembling into monotopic membrane-associated dodecameric pores. The capping pathway is unique to Alphaviruses; beginning with the N7 methylation of a guanosine triphosphate (GTP) molecule, followed by the covalent linkage of an m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA. Here, we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and S-adenosyl methionine (SAM), how the enzyme reaches a metastable postmethylation state with SAH and m7GTP in the active site, and the subsequent covalent transfer of m7GMP to nsP1 triggered by the presence of RNA and postdecapping reaction conformational changes inducing the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of reaction intermediates. Our data identify the molecular determinants allowing each pathway transition, providing an explanation for the need for the SAM methyl donor all along the pathway and clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together, our results set ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.

Mechanical tomography of an archaeal lemon-shaped virus reveals membrane-like fluidity of the capsid and liquid nucleoprotein cargo.

Archaeal lemon-shaped viruses have unique helical capsids composed of highly hydrophobic protein strands which can slide past each other resulting in remarkable morphological reorganization. Here, using atomic force microscopy, we explore the biomechanical properties of the lemon-shaped virions of Sulfolobus monocaudavirus 1 (SMV1), a double-stranded DNA virus which infects hyperthermophilic (~80 °C) and acidophilic (pH ~ 2) archaea. Our results reveal that SMV1 virions are extremely soft and withstand repeated extensive deformations, reaching remarkable strains of 80% during multiple cycles of consecutive mechanical assaults, yet showing scarce traces of disruption. SMV1 virions can reversibly collapse wall-to-wall, reducing their volume by ~90%. Beyond revealing the exceptional malleability of the SMV1 protein shell, our data also suggest a fluid-like nucleoprotein cargo which can flow inside the capsid, resisting and accommodating mechanical deformations without further alteration. Our experiments suggest a packing fraction of the virus core to be as low as 11%, with the amount of the accessory proteins almost four times exceeding that of the viral genome. Our findings indicate that SMV1 protein capsid displays biomechanical properties of lipid membranes, which is not found among protein capsids of other viruses. The remarkable malleability and fluidity of the SMV1 virions are likely necessary for the structural transformations during the infection and adaptation to extreme environmental conditions.

The breathtaking world of human respiratory in vitro models: Investigating lung diseases and infections in 3D models, organoids, and lung-on-chip.

The COVID-19 pandemic illustrated an urgent need for sophisticated, human tissue models to rapidly test and develop effective treatment options against this newly emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Thus, in particular, the last 3 years faced an extensive boost in respiratory and pulmonary model development. Nowadays, 3D models, organoids and lung-on-chip, respiratory models in perfusion, or precision-cut lung slices are used to study complex research questions in human primary cells. These models provide physiologically relevant systems for studying SARS-CoV-2 and, of course, other respiratory pathogens, but they are, too, suited for studying lung pathologies, such as CF, chronic obstructive pulmonary disease, or asthma, in more detail in terms of viral infection. With these models, the cornerstone has been laid for further advancing the organs by, for example, inclusion of several immune cell types or humoral immune components, combination with other organs in microfluidic organ-on-chip devices, standardization and harmonization of the devices for reliable and reproducible drug and vaccine testing in high throughput.

All the single cells: if you like it then you should put some virus on it.

Across a rich 70-year history, single-cell virology has revealed the impact of host and pathogen heterogeneity during virus infections. Recent technological innovations have enabled higher-resolution analyses of cellular and viral heterogeneity. Furthermore, single-cell analysis has revealed extreme phenotypes and provided additional insights into host-pathogen dynamics. Using a single-cell approach to explore fundamental virology questions, contemporary researchers have contributed to a revival of interest in single-cell virology with increased insights and enthusiasm.

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

Harnessing preexisting influenza virus-specific immunity increases antibody responses against SARS-CoV-2.

In pandemic scenarios involving novel human pathogenic viruses, it is highly desirable that vaccines induce strong neutralizing antibodies as quickly as possible. However, current vaccine strategies require multiple immunization doses to produce high titers of neutralizing antibodies and are poorly protective after a single vaccination. We therefore wished to design a vaccine candidate that would induce increased protective immune responses following the first vaccine dose. We hypothesized that antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein could be increased by drawing upon immunity to a previous infection. We generated a fusion protein containing the influenza H1N1 PR8 virus nucleoprotein (NP) and the SARS-CoV-2 spike RBD. Mice with or without preexisting immunity to PR8 were then vaccinated with NP/RBD. We observed significantly increased SARS-CoV-2 neutralizing antibodies in mice with PR8 immunity compared to mice without preexisting PR8 immunity. Vaccination with NP/RBD protected mice from SARS-CoV-2-induced morbidity and mortality after a single dose. Additionally, we compared SARS-CoV-2 virus titers in the lungs and nasal turbinates 4 days post-challenge of mice vaccinated with NP/RBD. SARS-CoV-2 virus was detectable in the lungs and nasal turbinate of mice without preexisting PR8 immunity, while SARS-CoV-2 virus was completely undetectable in mice with preexisting PR8 immunity. We also found that CD4-positive T cells in mice with preexisting immunity to PR8 play an essential role in producing the increased antibody response against RBD. This vaccine strategy potentially can be modified to target other pathogens of concern and offers extra value in future pandemic scenarios.IMPORTANCEIncreased globalization and changes in human interactions with wild animals has increased the likelihood of the emergence of novel viruses with pandemic potential. Vaccines can be effective in preventing severe disease caused by pandemic viruses. However, it takes time to develop protective immunity via prime-boost vaccination. More effective vaccine designs should quickly induce protective immunity. We propose leveraging preexisting immunity to a different pathogen to boost protection against emerging viruses. We targeted SARS-CoV-2 as a representative pandemic virus and generated a fusion protein vaccine that combines the nucleoprotein from influenza A virus and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Our vaccine design significantly increased the production of RBD-specific antibodies in mice that had previously been exposed to influenza virus, compared to those without previous exposure. This enhanced immunity reduced SARS-CoV-2 replication in mice. Our results offer a vaccine design that could be valuable in a future pandemic setting.

Human-virus protein-protein interactions maps assist in revealing the pathogenesis of viral infection.

Many significant viral infections have been recorded in human history, which have caused enormous negative impacts worldwide. Human-virus protein-protein interactions (PPIs) mediate viral infection and immune processes in the host. The identification, quantification, localization, and construction of human-virus PPIs maps are critical prerequisites for understanding the biophysical basis of the viral invasion process and characterising the framework for all protein functions. With the technological revolution and the introduction of artificial intelligence, the human-virus PPIs maps have been expanded rapidly in the past decade and shed light on solving complicated biomedical problems. However, there is still a lack of prospective insight into the field. In this work, we comprehensively review and compare the effectiveness, potential, and limitations of diverse approaches for constructing large-scale PPIs maps in human-virus, including experimental methods based on biophysics and biochemistry, databases of human-virus PPIs, computational methods based on artificial intelligence, and tools for visualising PPIs maps. The work aims to provide a toolbox for researchers, hoping to better assist in deciphering the relationship between humans and viruses.

SARS-CoV-2, periodontal pathogens, and host factors: The trinity of oral post-acute sequelae of COVID-19.

COVID-19 as a pan-epidemic is waning but there it is imperative to understand virus interaction with oral tissues and oral inflammatory diseases. We review periodontal disease (PD), a common inflammatory oral disease, as a driver of COVID-19 and oral post-acute-sequelae conditions (PASC). Oral PASC identifies with PD, loss of teeth, dysgeusia, xerostomia, sialolitis-sialolith, and mucositis. We contend that PD-associated oral microbial dysbiosis involving higher burden of periodontopathic bacteria provide an optimal microenvironment for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. These pathogens interact with oral epithelial cells activate molecular or biochemical pathways that promote viral adherence, entry, and persistence in the oral cavity. A repertoire of diverse molecules identifies this relationship including lipids, carbohydrates and enzymes. The S protein of SARS-CoV-2 binds to the ACE2 receptor and is activated by protease activity of host furin or TRMPSS2 that cleave S protein subunits to promote viral entry. However, PD pathogens provide additional enzymatic assistance mimicking furin and augment SARS-CoV-2 adherence by inducing viral entry receptors ACE2/TRMPSS, which are poorly expressed on oral epithelial cells. We discuss the mechanisms involving periodontopathogens and host factors that facilitate SARS-CoV-2 infection and immune resistance resulting in incomplete clearance and risk for 'long-haul' oral health issues characterising PASC. Finally, we suggest potential diagnostic markers and treatment avenues to mitigate oral PASC.