Frontiers in Virology

With the continuous evolution of SARS-CoV-2, variants of concern (VOCs) and their mutations are a focus of rapid assessment. Vital mutations in the VOC are found in spike protein, particularly in the receptor binding domain (RBD), which directly interacts with ACE2 on the host cell membrane, a key determinant of the binding affinity and cell entry. With the reporting of the most recent VOC, omicron, we performed amino acid sequence alignment of the omicron spike protein with that of the wild type and other VOCs. Although it shares several conserved mutations with other variants, we found that omicron has a large number of unique mutations. We applied the Hopp-Woods scale to calculate the hydrophilicity scores of the amino acid stretches of the RBD and the entire spike protein, and found 3 new hydrophilic regions in the RBD of omicron, implying exposure to water, with the potential to bind proteins such as ACE2 increasing transmissibility and infectivity. However, careful analysis reveals that most of the exposed domains of spike protein can serve as antigenic epitopes for generating B cell and T cell-mediated immune responses. This suggests that in the collection of polyclonal antibodies to various epitopes generated after multiple doses of vaccination, some can likely still bind to the omicron spike protein and the RBD to prevent severe clinical disease. In summary, while the omicron variant might result in more infectivity, it can still bind to a reasonable repertoire of antibodies generated by multiple doses of current vaccines likely preventing severe disease. Effective vaccines may not universally prevent opportunistic infections but can prevent the sequelae of severe disease, as observed for the delta variant. This might still be the case with the omicron variant, albeit, with increased frequency of infection.

Three-dimensional structures of SARS-CoV-2 and other coronaviral proteins archived in the Protein Data Bank were used to analyze viral proteome evolution during the first six months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48,000 viral proteome sequences showed how each one of the 29 viral study proteins have undergone amino acid changes. Structural models computed for every unique sequence variant revealed that most substitutions map to protein surfaces and boundary layers with a minority affecting hydrophobic cores. Conservative changes were observed more frequently in cores versus boundary layers/surfaces. Active sites and protein-protein interfaces showed modest numbers of substitutions. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi-Gaussian distribution. Detailed results are presented for six drug discovery targets and four structural proteins comprising the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and functional interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure-based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.

Antibodies are powerful tools to detect expressed proteins. However off-target recognition can confound their use. Therefore, careful characterization is needed to validate specificity in distinct applications. Here we report the sequence and characterization of a mouse recombinant antibody that specifically detects ORF46 of murine gammaherpesvirus 68 (MHV68). This ORF encodes the viral uracil DNA glycosylase (vUNG). The antibody does not recognize murine uracil DNA glycosylase and is useful in detecting vUNG expressed in virally infected cells. It can detect expressed vUNG in cells via immunostaining and microscopy or flow cytometry analysis. The antibody can detect vUNG from lysates of expressing cells via immunoblot under native conditions but not denaturing conditions. This suggests it recognizes a confirmational based epitope. Altogether this manuscript describes the utility of the anti-vUNG antibody and suitability for use in studies of MHV68 infected cells.

Human (h) coronaviruses (CoVs) 229E, NL63, OC43, and HKU1 are less virulent and cause mild, self-limiting respiratory tract infections, while SARS-CoV, MERS-CoV, and SARS-CoV-2, are more virulent and have caused severe outbreaks. The CoV envelope (E) protein, an important contributor to the pathogenesis of severe hCoVs infections, may provide insight into this disparate severity of the disease. Topology prediction programs and 3D modelling software was used to predict and visualize structural aspects of the hCoV E protein related to its functions. All seven hCoV E proteins largely adopted different topologies, with some distinction between the more virulent and less virulent ones. The 3D models refined this distinction, showing the PDZ-binding motif (PBM) of SARS-CoV, MERS-CoV, and SARS-CoV-2 to be more flexible than the PBM of hCoVs 229E, NL63, OC43, and HKU1. We speculate that the increased flexibility of the PBM may provide the more virulent hCoVs with a greater degree of freedom, which can allow them to bind to different host proteins and can contribute to a more severe form of the disease. This is the first paper to predict the topologies and model 3D structures of all seven hCoVs E proteins, providing novel insights for possible drug and/or vaccine development.

The necrosis syndrome of freesia, first described in 1970 in Northern Europe, is still jeopardizing freesia cultivation all over the world. Although several viruses have been listed as possible causal agents, the etiology of the disease is still not clear and is possibly linked to a combination of different factors. In this study, a high-throughput sequencing virome analysis was performed on total RNA extracts derived from symptomatic freesia leaves; a novel virus putatively belonging to the recently ratified Konkoviridae family in the Bunyaviricetes class has been identified and characterized, for which we propose the name of freesia konkovirus 1 (FreKV-1). This family, officially listing only one genus and two species, has been expanded by exploring publicly available metatranscriptomic datasets through the Serratus Project Database and reconstructing new viral entities; the phylogenetic position of the Konkoviridae family has been investigated and new genera belonging to the family have been proposed. Moreover, a further previously unknown virus, putatively belonging to the Yueviridae family was partially characterized and its phylogenetic position was discussed. Overall, the analysis increased our knowledge of the number of viral agents infecting freesia and possibly involved in freesia necrosis syndrome.

The emergence of SARS-CoV-2 lineages derived from Omicron, including BA.2.86 (nicknamed "Pirola") and its relative, JN.1, has raised concerns about their potential impact on public and personal health due to numerous novel mutations. Despite this, predicting their implications based solely on mutation counts proves challenging. Empirical evidence of JN.1s increased immune evasion capacity in relation to previous variants is mixed. To improve predictions beyond what is possible based solely on mutation counts, we conducted extensive in silico analyses on the binding affinity between the RBD of different SARS-CoV-2 variants (Wuhan-Hu-1, BA.1/B.1.1.529, BA.2, XBB.1.5, BA.2.86, and JN.1) and neutralizing antibodies from vaccinated or infected individuals, as well as the human angiotensin-converting enzyme 2 (ACE2) receptor. We observed no statistically significant difference in binding affinity between BA.2.86 or JN.1 and other variants. Therefore, we conclude that the new SARS-CoV-2 variants have no pronounced immune escape or infection capacity compared to previous variants. However, minor reductions in binding affinity for both the antibodies and ACE2 were noted for JN.1. We discuss the implications of the in silico findings and highlight the need for modeling and docking studies to go above and beyond mutation and basic serological neutralization analysis. Future research in this area will benefit from increased structural analyses of memory B-cell derived antibodies and should emphasize the importance of choosing appropriate samples for in silico studies to assess protection provided by vaccination and infection. More-over, the fitness benefits of genomic variation outside of the RBD of BA.2.86 and JN.1 need to be investigated. This research contributes to understanding the BA.2.86 and JN.1 variants potential impact on public health. Taken together, this work introduces a paradigm for functional genomic epidemiology in ongoing efforts to combat the evolving SARS-CoV-2 pandemic and prepare for other hazards.

We report the first Sin Nombre virus (SNV) genome sequences from the Northwestern United States and the first SNV sequences recovered from voles. Analysis of samples collected from 189 individual rodents revealed high SNV prevalence in the region and evidence of viral reassortment, highlighting ongoing viral diversification in rodents.

The significantly greater infectivity of the SARS-CoV-2 Delta variants of concern (VOC) is hypothesized to be driven by key mutations that result in increased transmissibility, viral load and/or evasion of host immune response. We surveyed the mutational profiles of Delta VOC genomes between September 2020 and mid-August 2021 and identified a previously unreported mutation pattern at amino acid position 142 in the N-terminal domain (NTD) of the spike protein which demonstrated multiple rounds of mutation from G142 to D142 and back. This pattern of frequent back mutations was observed at multiple time points and across Delta VOC sub-lineages. The etiology for these recurrent mutations is unclear but raises the possibility of host-directed editing of the SARS-CoV-2 genome. Within Delta VOC this mutation is associated with higher viral load, further enhanced in the presence of another NTD mutation (T95I) which was also frequently observed in these cases. Protein modeling of both mutations predicts alterations of the surface topography of the NTD by G142D, specifically disturbance of the super site epitope that binds NTD-directed neutralizing antibodies (NAbs). The appearance of frequent and repeated G142D followed by D142G back mutations is previously unreported in SARS-CoV-2 and may represent viral adaptation to evolving host immunity characterized by increasing frequency of spike NAbs, from both prior infection and vaccine-based immunity. The emergence of alterations of the NTD in and around the main NAb epitope is a concerning development in the ongoing evolution of SARS-CoV-2 which may contribute to increased infectivity, immune evasion and breakthrough infections characteristic of Delta VOC. Future vaccine and therapy development may benefit by recognizing the emergence of these novel spike NTD mutations and considering their impact on antibody recognition, viral neutralization, infectivity, replication, and viral load.

The geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top disease in Iran and the newly established Becurtovirus genus type species. Although the biological features of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, and no information is available on the function of their viral genes. In this work, we demonstrate that BCTIV V2, as the curtoviral V2, is also a strong local silencing suppressor in Nicotiana benthamiana and can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector also causes the induction of necrotic lesions in N. benthamiana like the ones produced during the HR, both at local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant showed that V2 is required for systemic infection but not for viral replication in a local infection. Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 and plays a crucial role in viral pathogenicity and systemic movement.

With the rapid spread of SARS-CoV-2 variants, including those that are resistant to antibodies authorized for emergency use, it is apparent that new antibodies may be needed to effectively protect patients against more severe disease. Differences between the murine and human antibody repertoires may allow for the isolation of murine monoclonal antibodies that recognize a different or broader range of SARS-CoV-2 variants than the human antibodies that have been characterized so far. We describe mouse antibodies B13 and O24 that demonstrate neutralizing potency against SARS-CoV-2 Wuhan (D614G) and B.1.351 variants. Such murine antibodies may have advantages in protecting against severe symptoms when individuals are exposed to new SARS-CoV-2 variants.

Nucleotide binding Leucine-rich repeat (NLR) proteins play a crucial role in effector recognition and activation of Effector triggered immunity in plants following pathogen infection. Advances in genome sequencing have led to the identification of a myriad NLRs in numerous agriculturally important plant species. However, deciphering which NLR proteins recognize specific pathogen effectors remains a challenge. Predicting NLR-effector interactions in silico would provide a more targeted approach for experimental validation, critical for elucidating function, and advancing our understanding of NLR-triggered immunity. In this study, NLR-effector protein complex structures were predicted using AlphaFold2-Multimer for all experimentally validated NLR-effector interactions. Binding affinities- and energies were predicted using 97 machine learning models from Area-affinity. We show that predicted structures with an AlphaFold confidence score > 0.42 have acceptable accuracy, and can be used to investigate NLR-effector interactions in silico. Binding affinities for 58 NLR-effector complexes ranged between -8.5 and -10.6 log(K), and binding energies between -11.8 and -14.4 kcal/mol, depending on the Area-Affinity model used. For 2427 "forced" NLR-effector complexes, these estimates showed larger variability, enabling the identification of novel NLR-effector complexes with 99% accuracy using an Ensemble machine learning model. The narrow range of binding energies- and affinities for true interactions suggest a specific change in Gibbs free energy, and thus conformational change, is required for NLR activation. This is the first study to provide a method for predicting NLR-effector interactions, applicable to all plant-pathogen interactions. Finally, the NLR-Effector Interaction Classification (NEIC) resource can streamline research efforts by identifying NLRs important for providing resistance against plant pathogens, advancing our understanding of plant immunity.

Borealpox virus (BRPV; formerly Alaskapox) is an orthopoxvirus that has caused seven reported human infections in Alaska since 2015, including a fatal case in 2023. The natural reservoir of BRPV is unknown, although previous investigations have raised the possibility of wild small mammals transmitting the virus to humans, either through direct contact or via domestic cats and dogs. To understand which species may be involved in the maintenance and/or spillover of BRPV in Alaska, we trapped and sampled wild small mammals (including voles, shrews, and squirrels) in 2021 and 2024 near reported human case locations in Fairbanks and the Kenai Peninsula, respectively. We found evidence of previous exposure to orthopoxviruses in five species (including the House Mouse, Mus musculus) and detected BRPV DNA as well as viable virus in Northern Red-backed Voles (Clethrionomys rutilus). Further, screening of tissues from historical museum specimens revealed BRPV DNA in C. rutilus specimens collected in Denali National Park and Preserve in 1998 and 1999, 17 years before the first reported human case of BRPV. Phylogenomic analysis of all human and animal BRPV isolates strongly supports the hypothesis of local human infections through multiple spillover events. These findings suggest C. rutilus as a possible reservoir species for BRPV and indicate that BRPV has been present in Alaskan wild small-mammal populations for at least 25 years. Our study highlights the potential of museum collections to elucidate the temporal, spatial, and host ranges of emerging pathogens. Further museum- and field-based sampling will clarify the true geographic range of BRPV, which is closely related to Old World orthopoxviruses and may be circulating beyond North America.

Positive-sense single-stranded (+ss) RNA viruses such as Grapevine leafroll-associated virus 3 (GLRaV3) replicate their genomes within membrane-bound viral replication complexes (VRCs). The biogenesis of such VRCs is driven by viral "replicase" polyproteins containing several replication-related domains. Past electron microscopy evidence suggests that GLRaV3 forms VRCs from the outer mitochondrial membrane of host grapevine plants. Here, we report the subcellular localization of the replicase polyprotein encoded by GLRaV3 ORF1a (PP1a) towards understanding its putative role in VRC formation. Through confocal laser scanning microscopy analysis of distinct EGFP-tagged PP1a domains, interdomain regions and truncations expressed in model plants, we report the molecular signals responsible for the targeting and association of PP1a to the mitochondria. This signal, located in the "Iceberg" region downstream from the methyltransferase-guanylyltransferase (M/GTase) domain, comprises an amphipathic -helix and a downstream transmembrane domain (TMD). Mutagenesis studies suggest that the polar face of the amphipathic -helix functions as the targeting signal, whereas the non-polar face, together with the TMD, act in membrane-anchoring. Microscopy observations are confirmed through mitochondrial isolation via gradient centrifugation and Western blotting. Structure prediction of the GLRaV3 M/GTase domain and its downstream TMD suggests a putative dodecameric oligomeric state. This dodecamer may gate the neck of GLRaV3 VRCs and contribute to their biogenesis, a hypothesis to be tested in follow-up studies. SIGNIFICANCE STATEMENTPositive-sense RNA viruses such as GLRaV3 form specialized membrane-bound viral replication complexes (VRCs) within host cells to sequester viral genome replication. Past evidence suggests that GLRaV3 targets the outer mitochondrial membrane (OMM) of grapevine host cells for VRC formation, however the viral protein responsible remained unknown. Here, we report a newly identified amphipathic -helix and a downstream transmembrane domain in the replicase polyprotein encoded by GLRaV3 ORF1a that are crucial for OMM targeting and membrane association. Since OMM targeting mechanisms in plants are poorly understood, our findings not only shed light on how GLRaV3 assembles VRCs, but also provides insight into OMM targeting more broadly. This work lays the foundation towards elucidating the molecular mechanisms of GLRaV3 replication and host-pathogen interactions.

The animal reservoirs of sarbecoviruses represent a significant risk of emergent pandemics, as evidenced by the impact of SARS-CoV-2. Vaccines remain successful at limiting severe disease and death, however the continued emergence of SARS-CoV-2 variants, together with the potential for further coronavirus zoonosis, motivates the search for pan-coronavirus vaccines that induce broadly neutralizing antibodies. This necessitates a better understanding of the glycan shields of coronaviruses, which can occlude potential antibody epitopes on spike glycoproteins. Here, we compare the structure of several sarbecovirus glycan shields. Many N-linked glycan attachment sites are shared by all sarbecoviruses, and the processing state of certain sites is highly conserved. However, there are significant differences in the processing state at several glycan sites that surround the receptor binding domain. Our studies reveal similarities and differences in the glycosylation of sarbecoviruses and show how subtle changes in the protein sequence can have pronounced impacts on the glycan shield.

Feline coronavirus type 1 (FCoV-1) is widely known for causing feline infectious peritonitis (FIP), a systemic infection that is often fatal, with the virus known as the FIPV biotype. However, subclinical disease also occurs, in which cats may not show signs and intermittently shed the virus, including in feces, possibly for long periods of time. This virus is known as the FECV biotype. Progression of FECV to FIPV has been linked to several genomic changes, however a specific region of the viral spike protein at the interface of the spike S1 and S2 domains has been especially implicated. In this study, we followed a cat (#576) for six years from 2017, at which time FCoV-1 was detected in feces and conjunctival swabs, until 2022, when the animal was euthanized based on a diagnosis of alimentary small cell lymphoma. Over this time period, the cat was clinically diagnosed with inflammatory bowel disease and chronic rhinitis, and cardiac problems were also suspected. Using hybridization capture targeting the spike (S) gene of FCoV followed by next-generation sequencing, we screened 27 clinical samples. We detected FCoV-1 in 4 samples taken in 2017 (intestine and nasal tissue, feces, and conjunctiva), and 3 samples taken in 2022 (feces, and intestinal and heart tissue), but not in fecal samples taken in 2019 and 2020. Next, we focused on the S1/S2 region within S, which contains the furin cleavage site (FCS), a key regulator of viral transmission and pathogenesis. We show that the FCoV-1 variants obtained from feces in 2017 and 2022 were identical, while the ones from conjunctiva (2017), heart (2022), and intestine (2017 and 2022) were distinct. Sequence comparison of all the variants obtained showed that most of the non-synonymous changes in the S1/S2 region occur within the FCS. In the heart, we found two variants that differed by a single nucleotide, resulting in distinct FCS motifs that differ in one amino acid. It is predicted that one of these FCS motifs will down-regulate spike cleavability. The variant from the conjunctiva (2017) had a 6-nucleotide in-frame insertion that resulted in a longer and more exposed S1/S2 loop, which is predicted to be more accessible to the furin protease. Our studies indicate that FCoV-1 can independently persist in the gastrointestinal tract and heart of a cat over a long period of time without evidence of typical FIP signs, with intermittent viral shedding from the gastrointestinal and respiratory tracts.

In response to the COVID-19 pandemic, many life scientists are focused on SARS-CoV-2. To help them use available structural data, we systematically modeled all viral proteins using all related 3D structures, generating 872 models that provide detail not available elsewhere. To organise these models, we created a structural coverage map: a novel, one-stop visualization summarizing what is -- and is not -- known about the 3D structure of the viral proteome. The map highlights structural evidence for viral protein interactions, mimicry, and hijacking; it also helps researchers find 3D models of interest, which can then be mapped with UniProt, PredictProtein, or CATH features. The resulting Aquaria-COVID resource (https://aquaria.ws/covid) helps scientists understand molecular mechanisms underlying coronavirus infection. Based on insights gained using our resource, we propose mechanisms by which the virus may enter immune cells, sense the cell type, then switch focus from viral reproduction to disrupting host immune responses. SignificanceCurrently, much of the COVID-19 viral proteome has unknown molecular structure. To improve this, we generated [~]1,000 structural models, designed to capture multiple states for each viral protein. To organise these models, we created a structure coverage map: a novel, one-stop visualization summarizing what is -- and is not -- known about viral protein structure. We used these data to create an online resource, designed to help COVID-19 researchers gain insight into the key molecular processes that drive infection. Based on insights gained using our resource, we speculate that the virus may sense the type of cells it infects and, within certain cells, it may switch from reproduction to disruption of the immune system.

Reticuloendotheliosis retroviruses (REV) are known to cause immunosuppressive and oncogenic disease that affects numerous avian species. REV is present worldwide and recently has been reported in South America with cases of infected commercial flocks in Argentina. We surveyed for the presence of REV in birds from a state in the northern region of Brazil using real-time PCR. We report the first cases of REV in Brazil, detected in Muscovy ducks (Cairina moschata), wild turkeys (Meleagris gallopavo), and chickens (Gallus gallus) at a relatively high prevalence rate (16,8%). Phylogenetic analysis indicated a close relationship of this strain to variants in the United States. This study provides evidence of REV in the Amazon biome and provides a baseline for future surveillance of the virus in the region and throughout Brazil.

Natural history museum collections are valuable but underutilized resources for viral discovery, offering opportunities to test hypotheses about viral occurrence across space, time, and taxonomic groups. We developed machine learning models of bat host suitability to guide coronavirus and paramyxovirus screening of 1330 and 491 tissues, respectively, in a museum collection. For the first time, we recovered coronavirus (n = 16) and paramyxovirus (n = 3) sequences from archived museum tissues, confirming three novel coronavirus host species and three novel paramyxovirus host species (3% and 33% prediction success rate, respectively). These sequences included a SARS-like coronavirus and an orthoparamyxovirus from Angolan Rhinolophus fumigatus specimens collected in June 2019, suggesting that viruses with epidemic potential may be more widespread in sub-Saharan Africa than previously believed. Our study demonstrates the value of combining predictive modeling and collections-based viral discovery, particularly for filling outstanding sampling gaps and investigating changes in host-virus associations over time.

Members of the {beta}-coronavirus family such as SARS-CoV-2, SARS, and MERS have caused pandemics over the last 20 years. Future pandemics are likely and studying the coronavirus family members is necessary for their understanding and treatment. Coronaviruses possess 16 non-structural proteins, many of which are involved in viral replication and other vital functions. Non-structural protein 10 (nsp10) is an essential stimulator of nsp14 and nsp16, modulating RNA proofreading and viral RNA cap formation. Studying nsp10 of pathogenic coronaviruses is central to understanding its multifunctional role. We report the biochemical and biophysical characterisation of full-length nsp10 from MERS, SARS and SARS-CoV-2. Proteins were subjected to a combination of OmniSEC and SEC-MALS to characterise their oligomeric state. Full-length nsp10s were predominantly monomeric in solution, while truncated versions of nsp10 have a higher tendency to oligomerise. Small angle X-ray scattering (SAXS) experiments reveal a globular shape of nsp10 which is conserved in all three coronaviruses, including MERS nsp10, which diverges most from SARS and SARS-CoV-2 nsp10s. In conclusion, unbound nsp10 proteins from SARS, MERS, and SARS-CoV-2 are globular and predominantly monomeric in solution. Additionally, we describe for the first time a functional role of the C-terminus of nsp10 for tight binding to nsp14.

HIV-1 generates unspliced (US), partially spliced (PS), and completely spliced (CS) classes of RNAs; each playing distinct roles in viral replication. Elucidating their host protein "interactomes" is crucial to understanding virus-host interplay. Here, we present HyPR-MSSV for isolation of US, PS, and CS transcripts from a single population of infected CD4+ T-cells and mass spectrometric identification of their in vivo protein interactomes. Analysis revealed 212 proteins differentially associated with the unique RNA classes; including, preferential association of regulators of RNA stability with US- and PS-transcripts and, unexpectedly, mitochondria-linked proteins with US-transcripts. Remarkably, >80 of these factors screened by siRNA knock-down impacted HIV-1 gene expression. Fluorescence microscopy confirmed several to co-localize with HIV-1 US RNA and exhibit changes in abundance and/or localization over the course of infection. This study validates HyPR-MSSV for discovery of viral splice variant protein interactomes and provides an unprecedented resource of factors and pathways likely important to HIV-1 replication.