Entangling of Peptide Nanofibers Reduces the Invasiveness of SARS-CoV-2.

The viral spike (S) protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to angiotensin-converting enzyme 2 (ACE2) receptors on the host cells, facilitating its entry and infection. Here, functionalized nanofibers targeting the S protein with peptide sequences of IRQFFKK, WVHFYHK and NSGGSVH, which are screened from a high-throughput one-bead one-compound screening strategy, are designed and prepared. The flexible nanofibers support multiple binding sites and efficiently entangle SARS-CoV-2, forming a nanofibrous network that blocks the interaction between the S protein of SARS-CoV-2 and the ACE2 on host cells, and efficiently reduce the invasiveness of SARS-CoV-2. In summary, nanofibers entangling represents a smart nanomedicine for the prevention of SARS-CoV-2.

A SARS-CoV-2-Related Virus from Malayan Pangolin Causes Lung Infection without Severe Disease in Human ACE2-Transgenic Mice.

Coronavirus disease 2019 (COVID-19), which is caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the most severe emerging infectious disease in the current century. The discovery of SARS-CoV-2-related coronaviruses (SARSr-CoV-2) in bats and pangolins in South Asian countries indicates that SARS-CoV-2 likely originated from wildlife. To date, two SARSr-CoV-2 strains have been isolated from pangolins seized in Guangxi and Guangdong by the customs agency of China, respectively. However, it remains unclear whether these viruses cause disease in animal models and whether they pose a transmission risk to humans. In this study, we investigated the biological features of a SARSr-CoV-2 strain isolated from a smuggled Malayan pangolin (Manis javanica) captured by the Guangxi customs agency, termed MpCoV-GX, in terms of receptor usage, cell tropism, and pathogenicity in wild-type BALB/c mice, human angiotensin-converting enzyme 2 (ACE2)-transgenic mice, and human ACE2 knock-in mice. We found that MpCoV-GX can utilize ACE2 from humans, pangolins, civets, bats, pigs, and mice for cell entry and infect cell lines derived from humans, monkeys, bats, minks, and pigs. The virus could infect three mouse models but showed limited pathogenicity, with mild peribronchial and perivascular inflammatory cell infiltration observed in lungs. Our results suggest that this SARSr-CoV-2 virus from pangolins has the potential for interspecies infection, but its pathogenicity is mild in mice. Future surveillance among these wildlife hosts of SARSr-CoV-2 is needed to monitor variants that may have higher pathogenicity and higher spillover risk. IMPORTANCE SARS-CoV-2, which likely spilled over from wildlife, is the third highly pathogenic human coronavirus. Being highly transmissible, it is perpetuating a pandemic and continuously posing a severe threat to global public health. Several SARS-CoV-2-related coronaviruses (SARSr-CoV-2) in bats and pangolins have been identified since the SARS-CoV-2 outbreak. It is therefore important to assess their potential of crossing species barriers for better understanding of their risk of future emergence. In this work, we investigated the biological features and pathogenicity of a SARSr-CoV-2 strain isolated from a smuggled Malayan pangolin, named MpCoV-GX. We found that MpCoV-GX can utilize ACE2 from 7 species for cell entry and infect cell lines derived from a variety of mammalian species. MpCoV-GX can infect mice expressing human ACE2 without causing severe disease. These findings suggest the potential of cross-species transmission of MpCoV-GX, and highlight the need of further surveillance of SARSr-CoV-2 in pangolins and other potential animal hosts.

A triple-RBD-based mucosal vaccine provides broad protection against SARS-CoV-2 variants of concern.

The rapid mutation and spread of SARS-CoV-2 variants urge the development of effective mucosal vaccines to provide broad-spectrum protection against the initial infection and thereby curb the transmission potential. Here, we designed a chimeric triple-RBD immunogen, 3Ro-NC, harboring one Delta RBD and two Omicron RBDs within a novel protein scaffold. 3Ro-NC elicits potent and broad RBD-specific neutralizing immunity against SARS-CoV-2 variants of concern. Notably, intranasal immunization with 3Ro-NC plus the mucosal adjuvant KFD (3Ro-NC + KFDi.n) elicits coordinated mucosal IgA and higher neutralizing antibody specificity (closer antigenic distance) against the Omicron variant. In Omicron-challenged human ACE2 transgenic mice, 3Ro-NC + KFDi.n immunization significantly reduces the tissue pathology in the lung and lowers the viral RNA copy numbers in both the lung (85.7-fold) and the nasal turbinate (13.6-fold). Nasal virologic control is highly correlated with RBD-specific secretory IgA antibodies. Our data show that 3Ro-NC plus KFD is a promising mucosal vaccine candidate for protection against SARS-CoV-2 Omicron infection, pathology and transmission potential.

Lethal Swine Acute Diarrhea Syndrome Coronavirus Infection in Suckling Mice.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a recently emerging bat-borne coronavirus responsible for high mortality rates in piglets. In vitro studies have indicated that SADS-CoV has a wide tissue tropism in different hosts, including humans. However, whether this virus potentially threatens other animals remains unclear. Here, we report the experimental infection of wild-type BALB/c and C57BL/6J suckling mice with SADS-CoV. We found that mice less than 7 days old are susceptible to the virus, which caused notable multitissue infections and damage. The mortality rate was the highest in 2-day-old mice and decreased in older mice. Moreover, a preliminary neuroinflammatory response was observed in 7-day-old SADS-CoV-infected mice. Thus, our results indicate that SADS-CoV has potential pathogenicity in young hosts. IMPORTANCE SADS-CoV, which likely has originated from bat coronaviruses, is highly pathogenic to piglets and poses a threat to the swine industry. Little is known about its potential to disseminate to other animals. No efficient treatment is available, and the quarantine strategy is the only preventive measure. In this study, we demonstrated that SADS-CoV can efficiently replicate in suckling mice younger than 7 days. In contrast to infected piglets, in which intestinal tropism is shown, SADS-CoV caused infection and damage in all murine tissues evaluated in this study. In addition, neuroinflammatory responses were detected in some of the infected mice. Our work provides a preliminary cost-effective model for the screening of antiviral drugs against SADS-CoV infection.

The Glycan-Binding Trait of the Sarbecovirus Spike N-Terminal Domain Reveals an Evolutionary Footprint.

The spike protein on sarbecovirus virions contains two external, protruding domains: an N-terminal domain (NTD) with unclear function and a C-terminal domain (CTD) that binds the host receptor, allowing for viral entry and infection. While the CTD is well studied for therapeutic interventions, the role of the NTD is far less well understood for many coronaviruses. Here, we demonstrate that the spike NTD from SARS-CoV-2 and other sarbecoviruses binds to unidentified glycans in vitro similarly to other members of the Coronaviridae family. We also show that these spike NTD (S-NTD) proteins adhere to Calu3 cells, a human lung cell line, although the biological relevance of this is unclear. In contrast to what has been shown for Middle East respiratory syndrome coronavirus (MERS-CoV), which attaches sialic acids during cell entry, sialic acids present on Calu3 cells inhibited sarbecovirus infection. Therefore, while sarbecoviruses can interact with cell surface glycans similarly to other coronaviruses, their reliance on glycans for entry is different from that of other respiratory coronaviruses, suggesting sarbecoviruses and MERS-CoV have adapted to different cell types, tissues, or hosts during their divergent evolution. Our findings provide important clues for further exploring the biological functions of sarbecovirus glycan binding and adds to our growing understanding of the complex forces that shape coronavirus spike evolution. IMPORTANCE Spike N-terminal domains (S-NTD) of sarbecoviruses are highly diverse; however, their function remains largely understudied compared with the receptor-binding domains (RBD). Here, we show that sarbecovirus S-NTD can be phylogenetically clustered into five clades and exhibit various levels of glycan binding in vitro. We also show that, unlike some coronaviruses, including MERS-CoV, sialic acids present on the surface of Calu3, a human lung cell culture, inhibit SARS-CoV-2 and other sarbecoviruses. These results suggest that while glycan binding might be an ancestral trait conserved across different coronavirus families, the functional outcome during infection can vary, reflecting divergent viral evolution. Our results expand our knowledge on the biological functions of the S-NTD across diverse sarbecoviruses and provide insight on the evolutionary history of coronavirus spike.

Single-Cell Landscape of Lungs Reveals Key Role of Neutrophil-Mediated Immunopathology during Lethal SARS-CoV-2 Infection.

Due to the limitation of human studies with respect to individual difference or the accessibility of fresh tissue samples, how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in pathological complications in lung, the main site of infection, is still incompletely understood. Therefore, physiologically relevant animal models under realistic SARS-CoV-2 infection conditions would be helpful to our understanding of dysregulated inflammation response in lung in the context of targeted therapeutics. Here, we characterized the single-cell landscape in lung and spleen upon SARS-CoV-2 infection in an acute severe disease mouse model that replicates human symptoms, including severe lung pathology and lymphopenia. We showed a reduction of lymphocyte populations and an increase of neutrophils in lung and then demonstrated the key role of neutrophil-mediated lung immunopathology in both mice and humans. Under severe conditions, neutrophils recruited by a chemokine-driven positive feedback produced elevated "fatal signature" proinflammatory genes and pathways related to neutrophil activation or releasing of granular content. In addition, we identified a new Cd177high cluster that is undergoing respiratory burst and Stfahigh cluster cells that may dampen antigen presentation upon infection. We also revealed the devastating effect of overactivated neutrophil by showing the highly enriched neutrophil extracellular traps in lung and a dampened B-cell function in either lung or spleen that may be attributed to arginine consumption by neutrophil. The current study helped our understanding of SARS-CoV-2-induced pneumonia and warranted the concept of neutrophil-targeting therapeutics in COVID-19 treatment. IMPORTANCE We demonstrated the single-cell landscape in lung and spleen upon SARS-CoV-2 infection in an acute severe disease mouse model that replicated human symptoms, including severe lung pathology and lymphopenia. Our comprehensive study revealed the key role of neutrophil-mediated lung immunopathology in SARS-CoV-2-induced severe pneumonia, which not only helped our understanding of COVID-19 but also warranted the concept of neutrophil targeting therapeutics in COVID-19 treatment.

Inactivated SARS-CoV-2 Vaccine Shows Cross-Protection against Bat SARS-Related Coronaviruses in Human ACE2 Transgenic Mice.

Severe acute respiratory syndrome coronavirus (SARS-CoV-1) and SARS-CoV-2 are highly pathogenic to humans and have caused pandemics in 2003 and 2019, respectively. Genetically diverse SARS-related coronaviruses (SARSr-CoVs) have been detected or isolated from bats, and some of these viruses have been demonstrated to utilize human angiotensin-converting enzyme 2 (ACE2) as a receptor and to have the potential to spill over to humans. A pan-sarbecovirus vaccine that provides protection against SARSr-CoV infection is urgently needed. In this study, we evaluated the protective efficacy of an inactivated SARS-CoV-2 vaccine against recombinant SARSr-CoVs carrying two different spike proteins (named rWIV1 and rRsSHC014S, respectively). Although serum neutralizing assays showed limited cross-reactivity between the three viruses, the inactivated SARS-CoV-2 vaccine provided full protection against SARS-CoV-2 and rWIV1 and partial protection against rRsSHC014S infection in human ACE2 transgenic mice. Passive transfer of SARS-CoV-2-vaccinated mouse sera provided low protection for rWIV1 but not for rRsSHC014S infection in human ACE2 mice. A specific cellular immune response induced by WIV1 membrane protein peptides was detected in the vaccinated animals, which may explain the cross-protection of the inactivated vaccine. This study shows the possibility of developing a pan-sarbecovirus vaccine against SARSr-CoVs for future preparedness. IMPORTANCE The genetic diversity of SARSr-CoVs in wildlife and their potential risk of cross-species infection highlight the necessity of developing wide-spectrum vaccines against infection of various SARSr-CoVs. In this study, we tested the protective efficacy of the SARS-CoV-2 inactivated vaccine (IAV) against two SARSr-CoVs with different spike proteins in human ACE2 transgenic mice. We demonstrate that the SARS-CoV-2 IAV provides full protection against rWIV1 and partial protection against rRsSHC014S. The T-cell response stimulated by the M protein may account for the cross protection against heterogeneous SARSr-CoVs. Our findings suggest the feasibility of the development of pan-sarbecovirus vaccines, which can be a strategy of preparedness for future outbreaks caused by novel SARSr-CoVs from wildlife.

ACE2-independent infection of T lymphocytes by SARS-CoV-2.

SARS-CoV-2 induced marked lymphopenia in severe patients with COVID-19. However, whether lymphocytes are targets of viral infection is yet to be determined, although SARS-CoV-2 RNA or antigen has been identified in T cells from patients. Here, we confirmed that SARS-CoV-2 viral antigen could be detected in patient peripheral blood cells (PBCs) or postmortem lung T cells, and the infectious virus could also be detected from viral antigen-positive PBCs. We next prove that SARS-CoV-2 infects T lymphocytes, preferably activated CD4 + T cells in vitro. Upon infection, viral RNA, subgenomic RNA, viral protein or viral particle can be detected in the T cells. Furthermore, we show that the infection is spike-ACE2/TMPRSS2-independent through using ACE2 knockdown or receptor blocking experiments. Next, we demonstrate that viral antigen-positive T cells from patient undergone pronounced apoptosis. In vitro infection of T cells induced cell death that is likely in mitochondria ROS-HIF-1a-dependent pathways. Finally, we demonstrated that LFA-1, the protein exclusively expresses in multiple leukocytes, is more likely the entry molecule that mediated SARS-CoV-2 infection in T cells, compared to a list of other known receptors. Collectively, this work confirmed a SARS-CoV-2 infection of T cells, in a spike-ACE2-independent manner, which shed novel insights into the underlying mechanisms of SARS-CoV-2-induced lymphopenia in COVID-19 patients.

Antibody-Dependent Enhancement of SARS-CoV-2 Infection of Human Immune Cells: In Vitro Assessment Provides Insight in COVID-19 Pathogenesis.

Patients with COVID-19 generally raise antibodies against SARS-CoV-2 following infection, and the antibody level is positively correlated to the severity of disease. Whether the viral antibodies exacerbate COVID-19 through antibody-dependent enhancement (ADE) is still not fully understood. Here, we conducted in vitro assessment of whether convalescent serum enhanced SARS-CoV-2 infection or induced excessive immune responses in immune cells. Our data revealed that SARS-CoV-2 infection of primary B cells, macrophages and monocytes, which express variable levels of FcγR, could be enhanced by convalescent serum from COVID-19 patients. We also determined the factors associated with ADE, and found which showed a time-dependent but not viral-dose dependent manner. Furthermore, the ADE effect is not associated with the neutralizing titer or RBD antibody level when testing serum samples collected from different patients. However, it is higher in a medium level than low or high dilutions in a given sample that showed ADE effect, which is similar to dengue. Finally, we demonstrated more viral genes or dysregulated host immune gene expression under ADE conditions compared to the no-serum infection group. Collectively, our study provides insight into the understanding of an association of high viral antibody titer and severe lung pathology in severe patients with COVID-19.

Identification of ZDHHC17 as a Potential Drug Target for Swine Acute Diarrhea Syndrome Coronavirus Infection.

The recent emergence and spread of zoonotic viruses highlights that animal-sourced viruses are the biggest threat to global public health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an HKU2-related bat coronavirus that was spilled over from Rhinolophus bats to swine, causing large-scale outbreaks of severe diarrhea disease in piglets in China. Unlike other porcine coronaviruses, SADS-CoV possesses broad species tissue tropism, including primary human cells, implying a significant risk of cross-species spillover. To explore host dependency factors for SADS-CoV as therapeutic targets, we employed genome-wide CRISPR knockout library screening in HeLa cells. Consistent with two independent screens, we identified the zinc finger DHHC-type palmitoyltransferase 17 (ZDHHC17 or ZD17) as an important host factor for SADS-CoV infection. Through truncation mutagenesis, we demonstrated that the DHHC domain of ZD17 that is involved in palmitoylation is important for SADS-CoV infection. Mechanistic studies revealed that ZD17 is required for SADS-CoV genomic RNA replication. Treatment of infected cells with the palmitoylation inhibitor 2-bromopalmitate (2-BP) significantly suppressed SADS-CoV infection. Our findings provide insight on SADS-CoV-host interactions and a potential therapeutic application. IMPORTANCE The recent emergence of deadly zoonotic viral diseases, including Ebola virus and SARS-CoV-2, emphasizes the importance of pandemic preparedness for the animal-sourced viruses with potential risk of animal-to-human spillover. Over the last 2 decades, three significant coronaviruses of bat origin, SARS-CoV, MERS-CoV, and SARS-CoV-2, have caused millions of deaths with significant economy and public health impacts. Lack of effective therapeutics against these coronaviruses was one of the contributing factors to such losses. Although SADS-CoV, another coronavirus of bat origin, was only known to cause fatal diarrhea disease in piglets, the ability to infect cells derived from multiple species, including human, highlights the potential risk of animal-to-human spillover. As part of our effort in pandemic preparedness, we explore SADS-CoV host dependency factors as targets for host-directed therapeutic development and found zinc finger DHHC-type palmitoyltransferase 17 is a promising drug target against SADS-CoV replication. We also demonstrated that a palmitoylation inhibitor, 2-bromopalmitate (2-BP), can be used as an inhibitor for SADS-CoV treatment.

Protective Efficacy of Inactivated Vaccine against SARS-CoV-2 Infection in Mice and Non-Human Primates.

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused more than 96 million infections and over 2 million deaths worldwide so far. However, there is no approved vaccine available for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the disease causative agent. Vaccine is the most effective approach to eradicate a pathogen. The tests of safety and efficacy in animals are pivotal for developing a vaccine and before the vaccine is applied to human populations. Here we evaluated the safety, immunogenicity, and efficacy of an inactivated vaccine based on the whole viral particles in human ACE2 transgenic mouse and in non-human primates. Our data showed that the inactivated vaccine successfully induced SARS-CoV-2-specific neutralizing antibodies in mice and non-human primates, and subsequently provided partial (in low dose) or full (in high dose) protection of challenge in the tested animals. In addition, passive serum transferred from vaccine-immunized mice could also provide full protection from SARS-CoV-2 infection in mice. These results warranted positive outcomes in future clinical trials in humans.

Pathogenesis of SARS-CoV-2 in Transgenic Mice Expressing Human Angiotensin-Converting Enzyme 2.

COVID-19 has spread worldwide since 2019 and is now a severe threat to public health. We previously identified the causative agent as a novel SARS-related coronavirus (SARS-CoV-2) that uses human angiotensin-converting enzyme 2 (hACE2) as the entry receptor. Here, we successfully developed a SARS-CoV-2 hACE2 transgenic mouse (HFH4-hACE2 in C3B6 mice) infection model. The infected mice generated typical interstitial pneumonia and pathology that were similar to those of COVID-19 patients. Viral quantification revealed the lungs as the major site of infection, although viral RNA could also be found in the eye, heart, and brain in some mice. Virus identical to SARS-CoV-2 in full-genome sequences was isolated from the infected lung and brain tissues. Last, we showed that pre-exposure to SARS-CoV-2 could protect mice from severe pneumonia. Our results show that the hACE2 mouse would be a valuable tool for testing potential vaccines and therapeutics.

A pneumonia outbreak associated with a new coronavirus of probable bat origin.

Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats^1-4. Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans^5-7. Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor-angiotensin converting enzyme II (ACE2)-as SARS-CoV.