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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.

Background Coronavirus (CoV) nonstructural protein 14 (nsp14) has exoribonuclease (ExoN) activity, responsible for proofreading and contributing to replication fidelity. It has been reported that CoVs exhibit variable sensitivity to nsp14-ExoN deficiency. Betacoronavirus murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV were viable upon nsp14-ExoN deficiency. While betacoronavirus Middle East respiratory syndrome (MERS)-CoV and SARS-CoV-2 were non-viable with disabled nsp14-ExoN. In this study, we investigated the nsp14-ExoN deficiency of alphacoronavirus porcine epidemic diarrhea virus (PEDV) in viral pathogenesis using reverse genetics. Results Eight nsp14-ExoN deficient mutants, targeting the predicted active sites and the Zinc finger or mental-coordinating sites, of PEDV were designed. Only one mutant E191A with a mutation in the Mg 2+ -binding site was rescued using the infectious clone of PEDV PC22A strain (icPC22A). The passage no.1–3 (P1-3) of E191A grew to very low titers in Vero cells. To evaluate the pathogenesis of the E191A, 4 or 5-day-old gnotobiotic pigs were inoculated orally with 100 TCID 50 /pig of the E191A-P1, icPC22A, or mock. All mock pigs did not shed virus in feces or show clinical signs. All pigs inoculated with icPC22A shed high viral RNA levels, had severe diarrhea, and died by 6 days post-inoculation (dpi). In contrast, only 3 pigs (3/4, 75%) in the E191A-P1 group shed low levels of viral RNA and 2 pigs had moderate diarrhea at acute infection phase. At 22 dpi, each pig was challenged orally with 10 6 plaque forming unit of virulent icPC22A. All pigs in the mock group developed severe diarrhea and 2 of the 5 pigs died. Pigs in the E191A-P1 group had less severe diarrhea and no pigs died. Sanger sequencing analysis revealed that the viral genome in the fecal sample of one E191A-P1-inoculated pig and the P4 virus passaged in vitro lost the E191A mutation, suggesting the genetic instability of the E191A mutant. Conclusion The recombinant PEDV variants carrying mutations at the essential functional sites within nsp14-ExoN were either lethal or genetically unstable. Our finding further confirmed the critical role of nsp14-ExoN in CoV life cycle, suggesting that it may be a target for the design of universal anti-CoV drugs.

Monoclonal antibodies are a promising approach to treat COVID-19, however the emergence of SARS-CoV-2 variants has challenged the efficacy and future of these therapies. Antibody cocktails are being employed to mitigate these challenges, but neutralization escape remains a major challenge and alternative strategies are needed. Here we present two anti-SARS-CoV-2 spike binding antibodies, one Class 1 and one Class 4, selected from our non-immune human single-chain variable fragment (scFv) phage library, that are engineered into four, fully-human IgG-like bispecific antibodies (BsAb). Prophylaxis of hACE2 mice and post-infection treatment of golden hamsters demonstrates the efficacy of the monospecific antibodies against the original Wuhan strain, while promising in vitro results with the BsAbs demonstrate enhanced binding and distinct synergistic effects on neutralizing activity against circulating variants of concern. In particular, one BsAb engineered in a tandem scFv-Fc configuration shows synergistic neutralization activity against several variants of concern including B.1.617.2. This work provides evidence that synergistic neutralization can be achieved using a BsAb scaffold, and serves as a foundation for the future development of broadly reactive BsAbs against emerging variants of concern. COVID-19 can be treated with monoclonal antibodies against SARS-CoV-2, but emerging new variants might show resistance towards existing therapy. Here authors show that anti-SARS-CoV-2 spike human single-chain antibody fragments could gain neutralizing activity against variants of concern upon engineering into a human bispecific antibody.

Porcine deltacoronavirus (PDCoV) strain OH-FD22 infects poultry and shares high nucleotide identity with sparrow-origin deltacoronaviruses (SpDCoV) ISU73347 and HKU17 strains. We hypothesized that the spike (S) protein or receptor-binding domain (RBD) from these SpDCoVs would alter the host and tissue tropism of PDCoV. First, an infectious cDNA clone of PDCoV OH-FD22 strain (icPDCoV) was generated and used to construct chimeric icPDCoVs harboring the S protein of HKU17 (icPDCoV-SHKU17) or the RBD of ISU73347 (icPDCoV-RBDISU). To evaluate their pathogenesis, neonatal gnotobiotic pigs were inoculated orally/oronasally with the recombinant viruses or PDCoV OH-FD22. All pigs inoculated with icPDCoV or OH-FD22 developed severe diarrhea and shed viral RNA at moderate-high levels (7.62–10.56 log10 copies/mL) in feces, and low-moderate levels in nasal swabs (4.92–8.48 log10 copies/mL). No pigs in the icPDCoV-SHKU17 and icPDCoV-RBDISU groups showed clinical signs. Interestingly, low-moderate levels (5.07–7.06 log10 copies/mL) of nasal but not fecal viral RNA shedding were detected transiently at 1–4 days post-inoculation in 40% (2/5) of icPDCoV-SHKU17- and 50% (1/2) of icPDCoV-RBDISU-inoculated pigs. These results confirm that PDCoV infected both the upper respiratory and intestinal tracts of pigs. The chimeric viruses displayed an attenuated phenotype with the loss of tropism for the pig intestine. The SpDCoV S protein and RBD reduced viral replication in pigs, suggesting limited potential for cross-species spillover upon initial passage.

In this study, in a high-risk population of patients admitted for COVID-19, we found an earlier time to hospital discharge among participants receiving CCP with nAb titers of >1:640 compared with participants receiving CCP with a lower nAb titer and no CCP-related AEs. The significance of our research is in identifying a dose response of CCP and clinical outcomes based on nAb titer. COVID-19 convalescent plasma (CCP) was an early and widely adopted putative therapy for severe COVID-19. Results from randomized control trials and observational studies have failed to demonstrate a clear therapeutic role for CCP for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Underlying these inconclusive findings is a broad heterogeneity in the concentrations of neutralizing antibodies (nAbs) between different CCP donors. We conducted this study to evaluate the effectiveness and safety of nAb titer-defined CCP in adults admitted to an academic referral hospital. Patients positive by a SARS-CoV-2 nucleic acid amplification test and with symptoms for <10 days were eligible. Participants received either CCP with nAb titers of >1:640 (high-titer group) or ≥1:160 to 1:640 (standard-titer group) in addition to standard of care treatments. The primary clinical outcome was time to hospital discharge, with mortality and respiratory support evaluated as secondary outcomes. Adverse events were contrasted by CCP titer. Between 28 August and 4 December 2020, 316 participants were screened, and 55 received CCP, with 14 and 41 receiving high- versus standard-titer CCP, respectively. Time to hospital discharge was shorter among participants receiving high- versus standard-titer CCP, accounting for death as a competing event (hazard ratio, 1.94; 95% confidence interval [CI], 1.05 to 3.58; Gray’s P = 0.02). Severe adverse events (SAEs) (≥grade 3) occurred in 4 (29%) and 23 (56%) of participants receiving the high versus standard titer, respectively, by day 28 (risk ratio, 0.51; 95% CI, 0.21 to 1.22; Fisher’s P = 0.12). There were no observed treatment-related AEs. (This study has been registered at ClinicalTrials.gov under registration no. NCT04524507). IMPORTANCE In this study, in a high-risk population of patients admitted for COVID-19, we found an earlier time to hospital discharge among participants receiving CCP with nAb titers of >1:640 compared with participants receiving CCP with a lower nAb titer and no CCP-related AEs. The significance of our research is in identifying a dose response of CCP and clinical outcomes based on nAb titer. Although limited by a small study size, these findings support further study of high-nAb-titer CCP defined as >1:640 in the treatment of COVID-19.

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.

In this study, we found that neutralizing antibody responses in COVID-19-convalescent individuals vary in magnitude but are durable and correlate well with receptor binding domain (RBD) Ig binding antibody levels compared to other SARS-CoV-2 antigen responses. In our cohort, higher neutralizing antibody titers are independently and significantly associated with male sex compared to female sex. The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has now caused over 2 million deaths worldwide and continues to expand. Currently, much is unknown about functionally neutralizing human antibody responses and durability to SARS-CoV-2 months after infection or the reason for the discrepancy in COVID-19 disease and sex. Using convalescent-phase sera collected from 101 COVID-19-recovered individuals 21 to 212 days after symptom onset with 48 additional longitudinal samples, we measured functionality and durability of serum antibodies. We also evaluated associations of individual demographic and clinical parameters with functional neutralizing antibody responses to COVID-19. We found robust antibody durability out to 6 months, as well as significant positive associations with the magnitude of the neutralizing antibody response and male sex and in individuals with cardiometabolic comorbidities. IMPORTANCE In this study, we found that neutralizing antibody responses in COVID-19-convalescent individuals vary in magnitude but are durable and correlate well with receptor binding domain (RBD) Ig binding antibody levels compared to other SARS-CoV-2 antigen responses. In our cohort, higher neutralizing antibody titers are independently and significantly associated with male sex compared to female sex. We also show for the first time that higher convalescent antibody titers in male donors are associated with increased age and symptom grade. Furthermore, cardiometabolic comorbidities are associated with higher antibody titers independently of sex. Here, we present an in-depth evaluation of serologic, demographic, and clinical correlates of functional antibody responses and durability to SARS-CoV-2 which supports the growing literature on sex discrepancies regarding COVID-19 disease morbidity and mortality, as well as functional neutralizing antibody responses to SARS-CoV-2.

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).

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

Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5′ end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration. The frameshift stimulation element (FSE) of coronaviruses is an RNA structure that is required for balanced expression of viral proteins and is thus a promising drug target. A structure of the SARS-CoV-2 FSE serves as a guide for the development of antisense oligonucleotides that impair virus replication.