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Safe, effective, and low-cost oral antiviral therapies are needed to treat those at high risk for developing severe COVID-19. To that end, we performed a high-throughput screen to identify non-peptidic, non-covalent inhibitors of the SARS-CoV-2 main protease (Mpro), an essential enzyme in viral replication. NZ-804 was developed from a screening hit through iterative rounds of structure-guided medicinal chemistry. NZ-804 potently inhibits SARS-CoV-2 Mpro (0.009 μM IC 50 ) as well as SARS-CoV-2 replication in human lung cell lines (0.008 μM EC 50 ) and primary human airway epithelial cell cultures. Antiviral activity is maintained against distantly related sarbecoviruses and endemic human CoV OC43. In SARS-CoV-2 mouse and hamster disease models, NZ-804 therapy given once or twice daily significantly diminished SARS-CoV-2 replication and pathogenesis. NZ-804 synthesis is low cost and uncomplicated, simplifying global production and access. These data support the exploration of NZ-804 as a therapy for COVID-19 and future emerging sarbecovirus infections., Competing Interests: Declaration of interests J.C.S., S.T., X.B., I.V.K., J.L.W., N.E.Z., M.K.P., A.A., P.K.J., R.B., A.F., and Z.S. are listed as inventors on a patent for NZ-804. R.S.B. is a member of the advisory boards of VaxArt and Invivyd and has collaborations with Takeda, Pfizer, Moderna, Ridgeback Biosciences, Gilead, and Eli Lily. Y.K. has received unrelated funding support from Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Inc., Shionogi & Co., Ltd., Otsuka Pharmaceutical, KM Biologics, Kyoritsu Seiyaku, Shinya Corporation, and Fuji Rebio., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Coronavirus (CoV) cause considerable morbidity and mortality in humans and other mammals, as evidenced by the emergence of Severe Acute Respiratory CoV (SARS-CoV) in 2003, Middle East Respiratory CoV (MERS-CoV) in 2012, and SARS-CoV-2 in 2019. Although poorly characterized, natural genetic variation in human and other mammals modulate virus pathogenesis, as reflected by the spectrum of clinical outcomes ranging from asymptomatic infections to lethal disease. Using multiple human epidemic and zoonotic Sarbecoviruses, coupled with murine Collaborative Cross genetic reference populations, we identify several dozen quantitative trait loci that regulate SARS-like group-2B CoV pathogenesis and replication. Under a Chr4 QTL, we deleted a candidate interferon stimulated gene, Trim14 which resulted in enhanced SARS-CoV titers and clinical disease, suggesting an antiviral role during infection. Importantly, about 60 % of the murine QTL encode susceptibility genes identified as priority candidates from human genome-wide association studies (GWAS) studies after SARS-CoV-2 infection, suggesting that similar selective forces have targeted analogous genes and pathways to regulate Sarbecovirus disease across diverse mammalian hosts. These studies provide an experimental platform in rodents to investigate the molecular-genetic mechanisms by which potential cross mammalian susceptibility loci and genes regulate type-specific and cross-SARS-like group 2B CoV replication, immunity, and pathogenesis in rodent models. Our study also provides a paradigm for identifying susceptibility loci for other highly heterogeneous and virulent viruses that sporadically emerge from zoonotic reservoirs to plague human and animal populations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Ebola virus (EBOV), a major global health concern, causes severe, often fatal EBOV disease (EVD) in humans. Host genetic variation plays a critical role, yet the identity of host susceptibility loci in mammals remains unknown. Using genetic reference populations, we generate an F2 mapping cohort to identify host susceptibility loci that regulate EVD. While disease-resistant mice display minimal pathogenesis, susceptible mice display severe liver pathology consistent with EVD-like disease and transcriptional signatures associated with inflammatory and liver metabolic processes. A significant quantitative trait locus (QTL) for virus RNA load in blood is identified in chromosome (chr)8, and a severe clinical disease and mortality QTL is mapped to chr7, which includes the Trim5 locus. Using knockout mice, we validate the Trim5 locus as one potential driver of liver failure and mortality after infection. The identification of susceptibility loci provides insight into molecular genetic mechanisms regulating EVD progression and severity, potentially informing therapeutics and vaccination strategies., Competing Interests: Declaration of interests D.O.C. is employed by, has equity ownership in, and serves on the board of directors of TransViragen, the company that has been contracted by UNC-Chapel Hill to manage its Animal Models Core Facility. R.S.B. is a member of advisory boards for VaxArt, Takeda, and Invivyd, focused on unrelated projects., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

We developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases, including transmembrane protease serine 2 (TMPRSS2), matriptase, and hepsin. TMPRSS2 is a membrane-associated protease that is highly expressed in the upper and lower respiratory tracts and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell entry, replication, and dissemination of new virus particles. We have previously shown that compound MM3122 exhibited subnanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Furthermore, we evaluated MM3122 in a mouse model of COVID-19 and demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in proinflammatory cytokine and chemokine production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2-infected mice. Therefore, MM3122 is a promising lead candidate small-molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses., Importance: SARS-CoV-2 and other emerging RNA coronaviruses are a present and future threat in causing widespread endemic and pandemic infection and disease. In this paper, we have shown that the novel host cell protease inhibitor, MM3122, blocks SARS-CoV-2 viral replication and is efficacious as both a prophylactic and a therapeutic drug for the treatment of COVID-19 given intraperitoneally in mice. Targeting host proteins and pathways in antiviral therapy is an underexplored area of research, but this approach promises to avoid drug resistance by the virus, which is common in current antiviral treatments., Competing Interests: The Boon laboratory has received unrelated funding support in sponsored research agreements from AI Therapeutics, GreenLight Biosciences Inc., Moderna Inc., and Nano targeting & Therapy Biopharma Inc. The Boon laboratory has received funding support from AbbVie Inc., for the commercial development of SARS-CoV-2 mAb.

Among all RNA viruses, coronavirus RNA transcription is the most complex and involves a process termed "discontinuous transcription" that results in the production of a set of 3'-nested, co-terminal genomic and subgenomic RNAs during infection. While the expression of the classic canonical set of subgenomic RNAs depends on the recognition of a 6- to 7-nt transcription regulatory core sequence (TRS), here, we use deep sequence and metagenomics analysis strategies and show that the coronavirus transcriptome is even more vast and more complex than previously appreciated and involves the production of leader-containing transcripts that have canonical and noncanonical leader-body junctions. Moreover, by ribosome protection and proteomics analyses, we show that both positive- and negative-sense transcripts are translationally active. The data support the hypothesis that the coronavirus proteome is much vaster than previously noted in the literature., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Early antiviral treatments, including intravenous remdesivir (RDV), reduce hospitalization and severe disease caused by COVID-19. An orally bioavailable RDV analog may facilitate earlier treatment of non-hospitalized COVID-19 patients. Here we describe the synthesis and evaluation of alkyl glyceryl ether phosphodiesters of GS-441524 (RVn), lysophospholipid analogs which allow for oral bioavailability and stability in plasma. Oral treatment of SARS-CoV-2-infected BALB/c mice with 1- O -octadecyl-2- O -benzyl- sn -glyceryl-3- phospho -RVn (60 mg/kg orally, once daily for 5 days starting 12h after infection) reduced lung viral load by 1.5 log 10 units versus vehicle at day 2 and to below the limit of detection at day 5. Structure/activity evaluation of additional analogs that have hydrophobic ethers at the sn -2 of glycerol revealed improved in vitro antiviral activity by introduction of a 3-fluoro-4-methoxy-substituted benzyl or a 3- or 4-cyano-substituted benzyl. Collectively, our data support the development of RVn phospholipid prodrugs as oral antiviral agents for prevention and treatment of SARS-CoV-2 infections.

The four dengue virus serotypes co-circulate globally and cause significant human disease. Dengue vaccine development is challenging because some virus-specific antibodies are protective, while others are implicated in enhanced viral replication and more severe disease. Current dengue tetravalent vaccines contain four live attenuated serotypes formulated to theoretically induce balanced protective immunity. Among the number of vaccine candidates in clinical trials, only Dengvaxia is licensed for use in DENV seropositive individuals. To simplify live-virus vaccine design, we identify co-evolutionary constraints inherent in flavivirus virion assembly and design chimeric viruses to replace domain II (EDII) of the DENV2 envelope (E) glycoprotein with EDII from DENV4. The chimeric DENV2/4EDII virus replicates efficiently in vitro and in vivo. In male macaques, a single inoculation of DENV2/4EDII induces type-specific neutralizing antibodies to both DENV2 and DENV4, thereby providing a strategy to simplify DENV vaccine design by utilizing a single bivalent E glycoprotein immunogen for two DENV serotypes., (© 2023. The Author(s).)

Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to severe acute respiratory syndrome coronavirus (SARS-CoV) disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6 , that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2, and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species. IMPORTANCE Host genetic variation is an important determinant that predicts disease outcomes following infection. In the setting of highly pathogenic coronavirus infections genetic determinants underlying host susceptibility and mortality remain unclear. To elucidate the role of host genetic variation on sarbecovirus pathogenesis and disease outcomes, we utilized the Collaborative Cross (CC) mouse genetic reference population as a model to identify susceptibility alleles to SARS-CoV and SARS-CoV-2 infections. Our findings reveal that a multitrait loci found in chromosome 9 is an important regulator of sarbecovirus pathogenesis in mice. Within this locus, we identified and validated CCR9 and CXCR6 as important regulators of host disease outcomes. Specifically, both CCR9 and CXCR6 are protective against severe SARS-CoV, SARS-CoV-2, and SARS-related HKU3 virus disease in mice. This chromosome 9 multitrait locus may be important to help identify genes that regulate coronavirus disease outcomes in humans.

The number of mass spectrometry (MS)-based proteomics datasets in the public domain keeps increasing, particularly those generated by Data Independent Acquisition (DIA) approaches such as SWATH-MS. Unlike Data Dependent Acquisition datasets, the re-use of DIA datasets has been rather limited to date, despite its high potential, due to the technical challenges involved. We introduce a (re-)analysis pipeline for public SWATH-MS datasets which includes a combination of metadata annotation protocols, automated workflows for MS data analysis, statistical analysis, and the integration of the results into the Expression Atlas resource. Automation is orchestrated with Nextflow, using containerised open analysis software tools, rendering the pipeline readily available and reproducible. To demonstrate its utility, we reanalysed 10 public DIA datasets from the PRIDE database, comprising 1,278 SWATH-MS runs. The robustness of the analysis was evaluated, and the results compared to those obtained in the original publications. The final expression values were integrated into Expression Atlas, making SWATH-MS experiments more widely available and combining them with expression data originating from other proteomics and transcriptomics datasets., (© 2022. The Author(s).)

The p53 transcription factor plays a key role both in cancer and in the cell-intrinsic response to infections. The ORFEOME project hypothesized that novel p53-virus interactions reside in hitherto uncharacterized, unknown, or hypothetical open reading frames (orfs) of human viruses. Hence, 172 orfs of unknown function from the emerging viruses SARS-Coronavirus, MERS-Coronavirus, influenza, Ebola, Zika (ZIKV), Chikungunya and Kaposi Sarcoma-associated herpesvirus (KSHV) were de novo synthesized, validated and tested in a functional screen of p53 signaling. This screen revealed novel mechanisms of p53 virus interactions and two viral proteins KSHV orf10 and ZIKV NS2A binding to p53. Originally identified as the target of small DNA tumor viruses, these experiments reinforce the notion that all viruses, including RNA viruses, interfere with p53 functions. These results validate this resource for analogous systems biology approaches to identify functional properties of uncharacterized viral proteins, long non-coding RNAs and micro RNAs., Competing Interests: The authors have declared that no competing interests exist. Author Kathleen Corcoran was unable to confirm their authorship contributions. On their behalf, the corresponding author has reported their contributions to the best of their knowledge.

Zoonotic coronaviruses represent an ongoing threat, yet the myriads of circulating animal viruses complicate the identification of higher-risk isolates that threaten human health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered, highly pathogenic virus that likely evolved from closely related HKU2 bat coronaviruses, circulating in Rhinolophus spp. bats in China and elsewhere. As coronaviruses cause severe economic losses in the pork industry and swine are key intermediate hosts of human disease outbreaks, we synthetically resurrected a recombinant virus (rSADS-CoV) as well as a derivative encoding tomato red fluorescent protein (tRFP) in place of ORF3. rSADS-CoV replicated efficiently in a variety of continuous animal and primate cell lines, including human liver and rectal carcinoma cell lines. Of concern, rSADS-CoV also replicated efficiently in several different primary human lung cell types, as well as primary human intestinal cells. rSADS-CoV did not use human coronavirus ACE-2, DPP4, or CD13 receptors for docking and entry. Contemporary human donor sera neutralized the group I human coronavirus NL63, but not rSADS-CoV, suggesting limited human group I coronavirus cross protective herd immunity. Importantly, remdesivir, a broad-spectrum nucleoside analog that is effective against other group 1 and 2 coronaviruses, efficiently blocked rSADS-CoV replication in vitro. rSADS-CoV demonstrated little, if any, replicative capacity in either immune-competent or immunodeficient mice, indicating a critical need for improved animal models. Efficient growth in primary human lung and intestinal cells implicate SADS-CoV as a potential higher-risk emerging coronavirus pathogen that could negatively impact the global economy and human health., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Human norovirus (HuNoV) is the leading cause of global infectious acute gastroenteritis, causing ~20% of reported diarrheal episodes. Typically, GII.4 strains cause 50-70% of yearly outbreaks, and pandemic waves of disease approximately every 2-7 years due to rapid evolution. Importantly, GII.4 dominance is occasionally challenged by the sudden emergence of other GII strains, most recently by GII.2 strains which peaked in 2016-2017, dramatically increasing from 1% to 20% of total HuNoV outbreaks. To determine if viral capsid evolution may account for the sudden rise in GII.2 outbreaks, Virus Like Particles (VLPs) of two 2016-2017 GII.2 strains were compared by antigenic and histo blood group antigen (HBGA) binding profiles to the prototypic 1976 GII.2 Snow Mountain Virus (SMV) strain. Despite >50 years of GII.2 strain persistence in human populations, limited sequence diversity and antigenic differences were identified between strains. However, capsid microvariation did affect HBGA binding patterns, with contemporary strains demonstrating decreased avidity for type A saliva. Furthermore, bile salts increased GII.2 VLP avidity for HBGAs, but did not alter antigenicity. These data indicate that large changes in antigenicity or receptor binding are unlikely to explain GII.2 emergence, in contrast to the pandemic GII.4 strains, and indicate that host factors such as waning or remodeling of serum or mucosal immunity likely contributed to the surge in GII.2 prevalence.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the novel viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV) potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC 50  = 0.01 μM). Weaker activity is observed in Vero E6 cells (EC 50  = 1.65 μM) because of their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase of SARS-CoV-2. In mice infected with the chimeric virus, therapeutic RDV administration diminishes lung viral load and improves pulmonary function compared with vehicle-treated animals. These data demonstrate that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19., Competing Interests: Declaration of Interests The authors affiliated with Gilead Sciences, Inc. are employees of the company and may own company stock., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The emergence and rapid global spread of SARS-CoV-2 mark the third such identification of a novel coronavirus capable of causing severe, potentially fatal disease in humans in the 21 st century. As noted by Andersen et al. (Nature Medicine), the sequencing of proximal zoonotic ancestors to SARS-CoV-2 has aided in the identification of alleles that may contribute to the virus' virulence in humans., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog β-d-N 4 -hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (β-d-N 4 -hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Emerging coronaviruses (CoV) are constant global public health threats to society. Multiple ongoing clinical trials for vaccines and antivirals against CoVs showcase the availability of medical interventions to both prevent and treat the future emergence of highly pathogenic CoVs in human. However, given the diverse nature of CoVs and our close interactions with wild, domestic and companion animals, the next epidemic zoonotic CoV could resist the existing vaccines and antivirals developed, which are primarily focused on Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS CoV). In late 2019, the novel CoV (SARS-CoV-2) emerged in Wuhan, China, causing global public health concern. In this review, we will summarize the key advancements of current vaccines and antivirals against SARS-CoV and MERS-CoV as well as discuss the challenge and opportunity in the current SARS-CoV-2 crisis. At the end, we advocate the development of a "plug-and-play" platform technologies that could allow quick manufacturing and administration of broad-spectrum countermeasures in an outbreak setting. We will discuss the potential of AAV-based gene therapy technology for in vivo therapeutic antibody delivery to combat SARS-CoV-2 outbreak and the future emergence of severe CoVs., (Copyright © 2020 Tse, Meganck, Graham and Baric.)

Traditionally, the emergence of coronaviruses (CoVs) has been attributed to a gain in receptor binding in a new host. Our previous work with severe acute respiratory syndrome (SARS)-like viruses argued that bats already harbor CoVs with the ability to infect humans without adaptation. These results suggested that additional barriers limit the emergence of zoonotic CoV. In this work, we describe overcoming host restriction of two Middle East respiratory syndrome (MERS)-like bat CoVs using exogenous protease treatment. We found that the spike protein of PDF2180-CoV, a MERS-like virus found in a Ugandan bat, could mediate infection of Vero and human cells in the presence of exogenous trypsin. We subsequently show that the bat virus spike can mediate the infection of human gut cells but is unable to infect human lung cells. Using receptor-blocking antibodies, we show that infection with the PDF2180 spike does not require MERS-CoV receptor DPP4 and antibodies developed against the MERS spike receptor-binding domain and S2 portion are ineffective in neutralizing the PDF2180 chimera. Finally, we found that the addition of exogenous trypsin also rescues HKU5-CoV, a second bat group 2c CoV. Together, these results indicate that proteolytic cleavage of the spike, not receptor binding, is the primary infection barrier for these two group 2c CoVs. Coupled with receptor binding, proteolytic activation offers a new parameter to evaluate the emergence potential of bat CoVs and offers a means to recover previously unrecoverable zoonotic CoV strains. IMPORTANCE Overall, our studies demonstrate that proteolytic cleavage is the primary barrier to infection for a subset of zoonotic coronaviruses. Moving forward, the results argue that both receptor binding and proteolytic cleavage of the spike are critical factors that must be considered for evaluating the emergence potential and risk posed by zoonotic coronaviruses. In addition, the findings also offer a novel means to recover previously uncultivable zoonotic coronavirus strains and argue that other tissues, including the digestive tract, could be a site for future coronavirus emergence events in humans., (Copyright © 2020 American Society for Microbiology.)

The genetically diverse Orthocoronavirinae (CoV) family is prone to cross species transmission and disease emergence in both humans and livestock. Viruses similar to known epidemic strains circulating in wild and domestic animals further increase the probability of emergence in the future. Currently, there are no approved therapeutics for any human CoV presenting a clear unmet medical need. Remdesivir (RDV, GS-5734) is a monophosphoramidate prodrug of an adenosine analog with potent activity against an array of RNA virus families including Filoviridae, Paramyxoviridae, Pneumoviridae, and Orthocoronavirinae, through the targeting of the viral RNA dependent RNA polymerase (RdRp). We developed multiple assays to further define the breadth of RDV antiviral activity against the CoV family. Here, we show potent antiviral activity of RDV against endemic human CoVs OC43 (HCoV-OC43) and 229E (HCoV-229E) with submicromolar EC 50 values. Of known CoVs, the members of the deltacoronavirus genus have the most divergent RdRp as compared to SARS- and MERS-CoV and both avian and porcine members harbor a native residue in the RdRp that confers resistance in beta-CoVs. Nevertheless, RDV is highly efficacious against porcine deltacoronavirus (PDCoV). These data further extend the known breadth and antiviral activity of RDV to include both contemporary human and highly divergent zoonotic CoV and potentially enhance our ability to fight future emerging CoV., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human norovirus is the leading cause of viral acute onset gastroenteritis disease burden, with 685 million infections reported annually. Vulnerable populations, such as children under the age of 5 years, the immunocompromised, and the elderly show a need for inducible immunity, as symptomatic dehydration and malnutrition can be lethal. Extensive antigenic diversity between genotypes and within the GII.4 genotype present major challenges for the development of a broadly protective vaccine. Efforts have been devoted to characterizing antibody-binding interactions with dynamic human norovirus viral-like particles, which recognize distinct antigenic sites on the capsid. Neutralizing antibody functions recognizing these sites have been validated in both surrogate (ligand blockade of binding) and in vitro virus propagation systems. In this review, we focus on GII.4 capsid protein epitopes as defined by monoclonal antibody binding. As additional antibody epitopes are defined, antigenic sites emerge on the human norovirus capsid, revealing the antigenic landscape of GII.4 viruses. These data may provide a road map for the design of candidate vaccine immunogens that induce cross-protective immunity and the development of therapeutic antibodies and drugs.

Emergent strains of human norovirus seed pandemic waves of disease. These new strains have altered ligand binding and antigenicity characteristics. Study of viral variants isolated from immunosuppressed patients with long-term norovirus infection indicates that initial virus in vivo evolution occurs at the same antigenic sites as in pandemic strains. Here, cellular ligand binding and antigenicity of two cocirculating strains isolated from a patient with long-term norovirus infection were characterized. The isolated GII.4 viruses differed from previous strains and from each other at known blockade antibody epitopes. One strain had a unique sequence in epitope D, including loss of an insertion at residue 394, corresponding to a decreased relative affinity for carbohydrate ligands. Replacement of 394 with alanine or restoration of the contemporary strain epitope D consensus sequence STT improved ligand binding relative affinity. However, monoclonal antibody blockade of binding potency was only gained for the consensus sequence, not by the alanine insertion. In-depth study of unique changes in epitope D indicated that ligand binding, but not antibody blockade of ligand binding, is maintained despite sequence diversity, allowing escape from blockade antibodies without loss of capacity for binding cellular ligands. IMPORTANCE Human norovirus causes ∼20% of all acute gastroenteritis and ∼200,000 deaths per year, primarily in young children. Most epidemic and all pandemic waves of disease over the past 30 years have been caused by type GII.4 human norovirus strains. The capsid sequence of GII.4 strains is changing over time, resulting in viruses with altered ligand and antibody binding characteristics. The carbohydrate binding pocket of these strains does not vary over time. Here, utilizing unique viral sequences, we study how residues in GII.4 epitope D balance the dual roles of variable antibody binding site and cellular ligand binding stabilization domain, demonstrating that amino acid changes in epitope D can result in loss of antibody binding without ablating ligand binding. This flexibility in epitope D likely contributes to GII.4 strain persistence by both allowing escape from antibody-mediated herd immunity and maintenance of cellular ligand binding and infectivity., (Copyright © 2019 Lindesmith et al.)

Emerging and re-emerging zoonotic viral diseases are major threats to global health, economic stability, and national security. Vaccines are key for reducing coronaviral disease burden; however, the utility of live-attenuated vaccines is limited by risks of reversion or repair. Because of their history of emergence events due to their prevalence in zoonotic pools, designing live-attenuated coronavirus vaccines that can be rapidly and broadly implemented is essential for outbreak preparedness. Here, we show that coronaviruses with completely rewired transcription regulatory networks (TRNs) are effective vaccines against SARS-CoV. The TRN-rewired viruses are attenuated and protect against lethal SARS-CoV challenge. While a 3-nt rewired TRN reverts via second-site mutation upon serial passage, a 7-nt rewired TRN is more stable, suggesting that a more extensively rewired TRN might be essential for avoiding growth selection. In summary, rewiring the TRN is a feasible strategy for limiting reversion in an effective live-attenuated coronavirus vaccine candidate that is potentially portable across the Nidovirales order., Competing Interests: The authors declare no competing interests.

SynGAP is a Ras and Rap GTPase-activating protein (GAP) found in high concentration in the postsynaptic density (PSD) fraction from mammalian forebrain where it binds to PDZ domains of PSD-95. Phosphorylation of pure recombinant synGAP by Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) shifts the balance of synGAP's GAP activity toward inactivation of Rap1; whereas phosphorylation by cyclin-dependent kinase 5 (CDK5) has the opposite effect, shifting the balance toward inactivation of HRas. These shifts in balance contribute to regulation of the numbers of surface AMPA receptors, which rise during synaptic potentiation (CaMKII) and fall during synaptic scaling (CDK5). Polo-like kinase 2 (Plk2/SNK), like CDK5, contributes to synaptic scaling. These two kinases act in concert to reduce the number of surface AMPA receptors following elevated neuronal activity by tagging spine-associated RapGAP protein (SPAR) for degradation, thus raising the level of activated Rap. Here we show that Plk2 also phosphorylates and regulates synGAP. Phosphorylation of synGAP by Plk2 stimulates its GAP activity toward HRas by 65%, and toward Rap1 by 16%. Simultaneous phosphorylation of synGAP by Plk2 and CDK5 at distinct sites produces an additive increase in GAP activity toward HRas (∼230%) and a smaller, non-additive increase in activity toward Rap1 (∼15%). Dual phosphorylation also produces an increase in GAP activity toward Rap2 (∼40-50%), an effect not produced by either kinase alone. As we previously observed for CDK5, addition of Ca 2+ /CaM causes a substrate-directed doubling of the rate and stoichiometry of phosphorylation of synGAP by Plk2, targeting residues also phosphorylated by CaMKII. In summary, phosphorylation by Plk2, like CDK5, shifts the ratio of GAP activity of synGAP to produce a greater decrease in active Ras than in active Rap, which would produce a shift toward a decrease in the number of surface AMPA receptors in neuronal dendrites., (Copyright © 2018 Elsevier Inc. All rights reserved.)

With an ongoing threat posed by circulating zoonotic strains, new strategies are required to prepare for the next emergent coronavirus (CoV). Previously, groups had targeted conserved coronavirus proteins as a strategy to generate live attenuated vaccine strains against current and future CoVs. With this in mind, we explored whether manipulation of CoV NSP16, a conserved 2'O methyltransferase (MTase), could provide a broad attenuation platform against future emergent strains. Using the severe acute respiratory syndrome-CoV mouse model, an NSP16 mutant vaccine was evaluated for protection from heterologous challenge, efficacy in the aging host, and potential for reversion to pathogenesis. Despite some success, concerns for virulence in the aged and potential for reversion makes targeting NSP16 alone an untenable approach. However, combining a 2'O MTase mutation with a previously described CoV fidelity mutant produced a vaccine strain capable of protection from heterologous virus challenge, efficacy in aged mice, and no evidence for reversion. Together, the results indicate that targeting the CoV 2'O MTase in parallel with other conserved attenuating mutations may provide a platform strategy for rapidly generating live attenuated coronavirus vaccines. IMPORTANCE Emergent coronaviruses remain a significant threat to global public health and rapid response vaccine platforms are needed to stem future outbreaks. However, failure of many previous CoV vaccine formulations has clearly highlighted the need to test efficacy under different conditions and especially in vulnerable populations such as the aged and immunocompromised. This study illustrates that despite success in young models, the 2'O methyltransferase mutant carries too much risk for pathogenesis and reversion in vulnerable models to be used as a stand-alone vaccine strategy. Importantly, the 2'O methyltransferase mutation can be paired with other attenuating approaches to provide robust protection from heterologous challenge and in vulnerable populations. Coupled with increased safety and reduced pathogenesis, the study highlights the potential for 2'O methyltransferase attenuation as a major component of future live attenuated coronavirus vaccines., (Copyright © 2018 American Society for Microbiology.)

The recurrence of new human cases of Middle East respiratory syndrome coronavirus (MERS-CoV) underscores the need for effective therapeutic countermeasures. Nonhuman primate models are considered the gold standard for preclinical evaluation of therapeutic countermeasures. However, MERS-CoV-induced severe respiratory disease in humans is associated with high viral loads in the lower respiratory tract, which may be difficult to achieve in nonhuman primate models. Considering this limitation, we wanted to ascertain the effectiveness of using a MERS-CoV infectious clone (icMERS-0) previously shown to replicate to higher titers than the wild-type EMC 2012 strain. We observed respiratory disease resulting from exposure to the icMERS-0 strain as measured by CT in rhesus monkeys with concomitant detection of virus antigen by immunohistochemistry. Overall, respiratory disease was mild and transient, resolving by day 30 post-infection. Although pulmonary disease was mild, these results demonstrate for the first time the utility of CT imaging to measure disease elicited by a MERS-CoV infectious clone system in nonhuman primate models.

Emerging zoonotic viral diseases remain a challenge to global public health. Recent surveillance studies have implicated bats as potential reservoirs for a number of viral pathogens, including coronaviruses and Ebola viruses. Caliciviridae represent a major viral family contributing to emerging diseases in both human and animal populations and have been recently identified in bats. In this study, we blended metagenomics, phylogenetics, homology modeling, and in vitro assays to characterize two novel bat calicivirus (BtCalV) capsid sequences, corresponding to strain BtCalV/A10/USA/2009, identified in Perimyotis subflavus near Little Orleans, MD, and bat norovirus. We observed that bat norovirus formed virus-like particles and had epitopes and receptor-binding patterns similar to those of human noroviruses. To determine whether these observations stretch across multiple bat caliciviruses, we characterized a novel bat calicivirus, BtCalV/A10/USA/2009. Phylogenetic analysis revealed that BtCalV/A10/USA/2009 likely represents a novel Caliciviridae genus and is most closely related to "recoviruses." Homology modeling revealed that the capsid sequences of BtCalV/A10/USA/2009 and bat norovirus resembled human norovirus capsid sequences and retained host ligand binding within the receptor-binding domains similar to that seen with human noroviruses. Both caliciviruses bound histo-blood group antigens in patterns that overlapped those seen with human and animal noroviruses. Taken together, our results indicate the potential for bat caliciviruses to bind histo-blood group antigens and overcome a significant barrier to cross-species transmission. Additionally, we have shown that bat norovirus maintains antigenic epitopes similar to those seen with human noroviruses, providing further evidence of evolutionary descent. Our results reiterate the importance of surveillance of wild-animal populations, especially of bats, for novel viral pathogens. IMPORTANCE Caliciviruses are rapidly evolving viruses that cause pandemic outbreaks associated with significant morbidity and mortality globally. The animal reservoirs for human caliciviruses are unknown; bats represent critical reservoir species for several emerging and zoonotic diseases. Recent reports have identified several bat caliciviruses but have not characterized biological functions associated with disease risk, including their potential emergence in other mammalian populations. In this report, we identified a novel bat calicivirus that is most closely related to nonhuman primate caliciviruses. Using this new bat calicivirus and a second norovirus-like bat calicivirus capsid gene sequence, we generated virus-like particles that have host carbohydrate ligand binding patterns similar to those of human and animal noroviruses and that share antigens with human noroviruses. The similarities to human noroviruses with respect to binding patterns and antigenic epitopes illustrate the potential for bat caliciviruses to emerge in other species and the importance of pathogen surveillance in wild-animal populations., (Copyright © 2018 Kocher et al.)

Viral metagenomics, modeling of protein structure, and manipulation of viral genetics are key approaches that have laid the foundations of our understanding of coronavirus biology. In this review, we discuss the major advances each method has provided and discuss how future studies should leverage these strategies synergistically to answer novel questions., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC 50 ) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC 50 The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs. IMPORTANCE Coronaviruses (CoVs) cause severe human infections, but there are no approved antivirals to treat these infections. Development of nucleoside-based therapeutics for CoV infections has been hampered by the presence of a proofreading exoribonuclease. Here, we expand the known efficacy of the nucleotide prodrug remdesivir (GS-5734) to include a group β-2a CoV. Further, GS-5734 potently inhibits CoVs with intact proofreading. Following selection with the GS-5734 parent nucleoside, 2 amino acid substitutions in the nsp12 polymerase at residues that are identical across CoVs provide low-level resistance to GS-5734. The resistance mutations decrease viral fitness of MHV in vitro and attenuate pathogenesis in a SARS-CoV animal model of infection. Together, these studies define the target of GS-5734 activity and demonstrate that resistance is difficult to select, only partial, and impairs fitness and virulence of MHV and SARS-CoV, supporting further development of GS-5734 as a potential effective pan-CoV antiviral., (Copyright © 2018 Agostini et al.)

Extensive antigenic diversity within the GII.4 genotype of human norovirus is a major driver of pandemic emergence and a significant obstacle to development of cross-protective immunity after natural infection and vaccination. However, human and mouse monoclonal antibody studies indicate that, although rare, antibodies to conserved GII.4 blockade epitopes are generated. The mechanisms by which these epitopes evade immune surveillance are uncertain. Here, we developed a new approach for identifying conserved GII.4 norovirus epitopes. Utilizing a unique set of virus-like particles (VLPs) representing the in vivo -evolved sequence diversity within an immunocompromised person, we identify key residues within epitope F, a conserved GII.4 blockade antibody epitope. The residues critical for antibody binding are proximal to evolving blockade epitope E. Like epitope F, antibody blockade of epitope E was temperature sensitive, indicating that particle conformation regulates antibody access not only to the conserved GII.4 blockade epitope F but also to the evolving epitope E. These data highlight novel GII.4 mechanisms to protect blockade antibody epitopes, map essential residues of a GII.4 conserved epitope, and expand our understanding of how viral particle dynamics may drive antigenicity and antibody-mediated protection by effectively shielding blockade epitopes. Our data support the notion that GII.4 particle breathing may well represent a major mechanism of humoral immune evasion supporting cyclic pandemic virus persistence and spread in human populations. IMPORTANCE In this study, we use norovirus virus-like particles to identify key residues of a conserved GII.4 blockade antibody epitope. Further, we identify an additional GII.4 blockade antibody epitope to be occluded, with antibody access governed by temperature and particle dynamics. These findings provide additional support for particle conformation-based presentation of binding residues mediated by a particle "breathing core." Together, these data suggest that limiting antibody access to blockade antibody epitopes may be a frequent mechanism of immune evasion for GII.4 human noroviruses. Mapping blockade antibody epitopes, the interaction between adjacent epitopes on the particle, and the breathing core that mediates antibody access to epitopes provides greater mechanistic understanding of epitope camouflage strategies utilized by human viral pathogens to evade immunity.

During anti-retroviral therapy (ART) HIV-1 persists in cellular reservoirs, mostly represented by CD4+ memory T cells. Several approaches are currently being undertaken to develop a cure for HIV-1 infection through elimination (or reduction) of these reservoirs. Few studies have so far been conducted to assess the possibility of reducing the size of HIV-1 reservoirs through vaccination in virologically controlled HIV-1-infected children. We recently conducted a vaccination study with a combined hepatitis A virus (HAV) and hepatitis B virus (HBV) vaccine in 22 HIV-1-infected children. We assessed the size of the virus reservoir, measured as total HIV-1 DNA copies in blood cells, pre- and postvaccination. In addition, we investigated by immunostaining whether the frequencies of CD4+ and CD8+ T cells and parameters of immune activation and proliferation on these cells were modulated by vaccination. At 1 month from the last vaccination dose, we found that 20 out of 22 children mounted a serological response to HBV; a majority of children had antibodies against HAV at baseline. The number of HIV-1 DNA copies in blood at 1 month postvaccination was reduced in comparison to baseline although this reduction was not statistically significant. A significant reduction of HIV-1 DNA copies in blood following vaccination was found in 12 children. The frequencies of CD4+ (naïve, effector memory) and CD8+ (central memory) T-cell subpopulations changed following vaccinations and a reduction in the activation and proliferation pattern of these cells was also noticed. Multivariate linear regression analysis revealed that the frequency of CD8+ effector memory T cells prior to vaccination was strongly predictive of the reduction of HIV-1 DNA copies in blood following vaccination of the 22 HIV-1-infected children. The results of this study suggest a beneficial effect of vaccination to reduce the size of virus reservoir in HIV-1-infected children receiving ART. A reduced frequency of activated CD4+ cells and an increase in central memory CD8+ T cells were associated with this finding. Further studies should assess whether vaccination is a possible tool to reduce HIV-1 reservoirs.