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Antibodies perform both neutralizing and non-neutralizing effector functions that protect against certain pathogen-induced diseases. A human antibody directed at the SARS-CoV-2 Spike N-terminal domain (NTD), DH1052, was recently shown to be non-neutralizing, yet it protected mice and cynomolgus macaques from severe disease. The mechanisms of NTD non-neutralizing antibody-mediated protection are unknown. Here we show that Fc effector functions mediate NTD non-neutralizing antibody (non-nAb) protection against SARS-CoV-2 MA10 viral challenge in mice. Though non-nAb prophylactic infusion did not suppress infectious viral titers in the lung as potently as neutralizing antibody (nAb) infusion, disease markers including gross lung discoloration were similar in nAb and non-nAb groups. Fc functional knockout substitutions abolished non-nAb protection and increased viral titers in the nAb group. Fc enhancement increased non-nAb protection relative to WT, supporting a positive association between Fc functionality and degree of protection from SARS-CoV-2 infection. For therapeutic administration of antibodies, non-nAb effector functions contributed to virus suppression and lessening of lung discoloration, but the presence of neutralization was required for optimal protection from disease. This study demonstrates that non-nAbs can utilize Fc-mediated mechanisms to lower viral load and prevent lung damage due to coronavirus infection.

Mature DENVs represent the dominant form in vivo and are the target for vaccine development. Here, we used multiple strategies, including protein engineering and natural and directed evolution to generate DENV1, -2, -3, and -4 variants that are highly mature without compromising replication efficiency compared to the parental strains. Maturation of dengue viruses (DENVs) alters the structure, immunity, and infectivity of the virion and highly mature particles represent the dominant form in vivo . The production of highly mature virions principally relies on the structure and function of the viral premature membrane protein (prM) and its cleavage by the host protease furin. We redeveloped a reliable clonal cell line (VF1) which produces single-round mature DENVs without the need for DENV reverse genetics. More importantly, using protein engineering and directed evolution of the prM cleavage site, we engineered genetically stable mature DENVs in all serotypes independent of cell or host, usually with minimal impact on viral yield. Using these complementary strategies to regulate maturation, we demonstrate that the resulting mature DENVs are antigenically distinct from their isogenic partially mature forms. Given the clinical importance of mature DENVs in immunity, our study provides reliable strategies and reagents for the production of stable, high-titer mature DENVs for DENV antibody neutralization and vaccination immunity studies. Biologically, our data from directed evolution across host species reveals distinct maturation-dependent selective pressures between mammalian and insect cells, verifying the substrate preference between mammalian and insect furin, while hinting at an evolutionary equilibrium of DENV prM cleavage site between its host and vector in nature. IMPORTANCE Mature DENVs represent the dominant form in vivo and are the target for vaccine development. Here, we used multiple strategies, including protein engineering and natural and directed evolution to generate DENV1, -2, -3, and -4 variants that are highly mature without compromising replication efficiency compared to the parental strains. Given the clinical importance of mature DENVs in immunity, this work provides a roadmap for engineering highly mature DENV that could apply to future vaccine development. Our directed-evolution data also shed light on the divergent evolutionary relationship of DENVs between its host and vector.

SARS-CoV-2 (the causative agent of COVID-19) is a major public health threat and one of two related coronaviruses that have caused epidemics in modern history. A method of screening potential infectible hosts for preemergent and future emergent coronaviruses would aid in mounting rapid response and intervention strategies during future emergence events. The angiotensin-converting enzyme 2 (ACE2) receptor is a major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) host range determinant, and understanding SARS-CoV-2-ACE2 interactions will provide important insights into COVID-19 pathogenesis and animal model development. SARS-CoV-2 cannot infect mice due to incompatibility between its receptor binding domain and the murine ACE2 receptor. Through molecular modeling and empirical in vitro validation, we identified 5 key amino acid differences between murine and human ACE2 that mediate SARS-CoV-2 infection, generating a chimeric humanized murine ACE2. Additionally, we examined the ability of the humanized murine ACE2 receptor to permit infection by an additional preemergent group 2B coronavirus, WIV-1, providing evidence for the potential pan-virus capabilities of this chimeric receptor. Finally, we predicted the ability of these determinants to inform host range identification of preemergent coronaviruses by evaluating hot spot contacts between SARS-CoV-2 and additional potential host receptors. Our results identify residue determinants that mediate coronavirus receptor usage and host range for application in SARS-CoV-2 and emerging coronavirus animal model development. IMPORTANCE SARS-CoV-2 (the causative agent of COVID-19) is a major public health threat and one of two related coronaviruses that have caused epidemics in modern history. A method of screening potential infectible hosts for preemergent and future emergent coronaviruses would aid in mounting rapid response and intervention strategies during future emergence events. Here, we evaluated determinants of SARS-CoV-2 receptor interactions, identifying key changes that enable or prevent infection. The analysis detailed in this study will aid in the development of model systems to screen emergent coronaviruses as well as treatments to counteract infections.

A Correction to this paper has been published: https://doi.org/10.1038/s41586-020-03107-5

Coronaviruses are prone to transmission to new host species, as recently demonstrated by the spread to humans of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic 1 . Small animal models that recapitulate SARS-CoV-2 disease are needed urgently for rapid evaluation of medical countermeasures 2 , 3 . SARS-CoV-2 cannot infect wild-type laboratory mice owing to inefficient interactions between the viral spike protein and the mouse orthologue of the human receptor, angiotensin-converting enzyme 2 (ACE2) 4 . Here we used reverse genetics 5 to remodel the interaction between SARS-CoV-2 spike protein and mouse ACE2 and designed mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA), a recombinant virus that can use mouse ACE2 for entry into cells. SARS-CoV-2 MA was able to replicate in the upper and lower airways of both young adult and aged BALB/c mice. SARS-CoV-2 MA caused more severe disease in aged mice, and exhibited more clinically relevant phenotypes than those seen in Hfh4 - ACE2 transgenic mice, which express human ACE2 under the control of the Hfh4 (also known as Foxj1 ) promoter. We demonstrate the utility of this model using vaccine-challenge studies in immune-competent mice with native expression of mouse ACE2. Finally, we show that the clinical candidate interferon-λ1a (IFN-λ1a) potently inhibits SARS-CoV-2 replication in primary human airway epithelial cells in vitro—both prophylactic and therapeutic administration of IFN-λ1a diminished SARS-CoV-2 replication in mice. In summary, the mouse-adapted SARS-CoV-2 MA model demonstrates age-related disease pathogenesis and supports the clinical use of pegylated IFN-λ1a as a treatment for human COVID-19 6 . A model in mouse using a species-adapted virus recapitulates features of SARS-CoV-2 infection and age-related disease pathogenesis in humans, and provides a model system for rapid evaluation of medical countermeasures against coronavirus disease 2019 (COVID-19).

The aminotransferase gene family in the model plant Arabidopsis thaliana consists of 44 genes, eight of which are suggested to be alanine aminotransferases. One of the putative alanine aminotransferases genes, At3g08860, was attributed the function of alanine:glyoxylate aminotransferase/β‐alanine:pyruvate aminotransferase based on the analysis of gene expression networks and homology to other β‐alanine aminotransferases in plants. It was earlier demonstrated that At3g08860 is specifically upregulated in response to osmotic stress, but not other stresses (β‐alanine is an osmoprotectant in plants). Furthermore, it was shown that the expression of At3g08860 is highly coordinated with the genes of the uracil degradation pathway leading to the non‐proteinogenic amino acid β‐alanine. These evidence were suggestive of the involvement of At3g08860 in β‐alanine metabolism. However, direct experimental evidence for the function of At3g08860 was lacking, and therefore, the goal of this study was to elucidate the function of the uncharacterized aminotransferase annotated by the locus tag At3g08860. The cDNA of At3g08860 was demonstrated to functionally complement two E. coli mutants auxotrophic for the amino acids, L‐alanine (proteinogenic) and β‐alanine (non‐proteinogenic). Enzyme activity using purified recombinant At3g08860 further demonstrated that the enzyme is endowed with L‐alanine:glyoxylate aminotransferase activity.

The head of Salmonella virus SPN3US is composed of ~50 different proteins and is unusual because within its packaged genome there is a mass (>40 MDa) of ejection or E proteins that enter the Salmonella cell. The assembly mechanisms of this complex structure are poorly understood. Previous studies showed that eight proteins in the mature SPN3US head had been cleaved by the prohead protease. In this study, we present the characterization of SPN3US prohead protease mutants using transmission electron microscopy and mass spectrometry. In the absence of the prohead protease, SPN3US head formation was severely impeded and proheads accumulated on the Salmonella inner membrane. This impediment is indicative of proteolysis being necessary for the release and subsequent DNA packaging of proheads in the wild-type phage. Proteomic analyses of gp245- proheads that the normal proteolytic processing of head proteins had not occurred. Assays of a recombinant, truncated form of the protease found it was active, leading us to hypothesize that the C-terminal propeptide has a role in targeting the protease into the prohead core. Our findings provide new evidence regarding the essential role of proteolysis for correct head assembly in this remarkable parasite.

Zoonotic transmission of coronaviruses poses an ongoing threat to human populations. Endemic outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) have caused severe economic losses in the pig industry and have the potential to cause human outbreaks. Currently, there are no vaccines or specific antivirals against SADS-CoV, and our limited understanding of SADS-CoV host entry factors could hinder prompt responses to a potential human outbreak. Using a genomewide CRISPR knockout screen, we identified placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection. Knockout of PLAC8 abolished SADS-CoV infection, which was restored by complementing PLAC8 from multiple species, including human, rhesus macaques, mouse, pig, pangolin, and bat, suggesting a conserved infection pathway and susceptibility of SADS-CoV among mammals. Mechanistically, PLAC8 knockout does not affect viral entry; rather, knockout cells displayed a delay and reduction in viral subgenomic RNA expression. In a swine primary intestinal epithelial culture (IEC) infection model, differentiated cultures have high levels of PLAC8 expression and support SADS-CoV replication. In contrast, expanding IECs have low levels of PLAC8 expression and are resistant to SADS-CoV infection. PLAC8 expression patterns translate in vivo; the immunohistochemistry of swine ileal tissue revealed high levels of PLAC8 protein in neonatal compared to adult tissue, mirroring the known SADS-CoV pathogenesis in neonatal piglets. Overall, PLAC8 is an essential factor for SADS-CoV infection and may serve as a promising target for antiviral development for potential pandemic SADS-CoV.

Whole virus-based inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous coronavirus infection, the emergence of novel variants and the presence of large zoonotic reservoirs harboring novel heterologous coronaviruses provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes like vaccine-associated enhanced respiratory disease. Here, we use a female mouse model of coronavirus disease to evaluate inactivated vaccine performance against either homologous challenge with SARS-CoV-2 or heterologous challenge with a bat-derived coronavirus that represents a potential emerging disease threat. We show that inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide can cause enhanced respiratory disease during heterologous infection, while use of an alternative adjuvant does not drive disease and promotes heterologous viral clearance. In this work, we highlight the impact of adjuvant selection on inactivated vaccine safety and efficacy against heterologous coronavirus infection. Here, Dillard and Taft-Benz et al. show in a female mouse model how different adjuvants affect inactivated vaccine-mediated protection against homologous SARS-CoV-2 and heterologous SARS-CoV-1-like coronaviruses. They find that an aluminum hydroxide-adjuvanted vaccine can increase risk of adverse outcomes during heterologous infection.

ABSTRACT We report here the genome sequences of Salmonella enterica subsp. enterica serovar Typhimurium strains TT6675 and TT9097, which we utilize for genetic analyses of giant bacterial viruses. Our analyses identified several genetic variations between the two strains, most significantly confirming strain TT6675 as a serine suppressor and TT9097 as a nonsuppressor.