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.

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.

Ecological study of cave nectar bats reveals low risk of direct transmission of bat viruses to humans.

Bats are reservoirs of various viruses. The widely distributed cave nectar bat ( Eonycteris spelaea) is known to carry both filoviruses and coronaviruses. However, the potential transmission of theses bat viruses to humans is not fully understood. In this study, we tracked 16 E. spelaea bats in Mengla County, Yunnan Province, China, using miniaturized GPS devices to investigate their movements and potential contact with humans. Furthermore, to determine the prevalence of coronavirus and filovirus infections, we screened for the nucleic acids of the Menglà virus (MLAV) and two coronaviruses (GCCDC1-CoV and HKU9-CoV) in anal swab samples taken from bats and for antibodies against these viruses in human serum samples. None of the serum samples were found to contain antibodies against the bat viruses. The GPS tracking results showed that the bats did not fly during the daytime and rarely flew to residential areas. The foraging range of individual bats also varied, with a mean cumulative nightly flight distance of 25.50 km and flight speed of up to 57.4 km/h. Taken together, these results suggest that the risk of direct transmission of GCCDC1-CoV, HKU9-CoV, and MLAV from E. spelaea bats to humans is very low under natural conditions.

Novel sarbecovirus bispecific neutralizing antibodies with exceptional breadth and potency against currently circulating SARS-CoV-2 variants and sarbecoviruses.

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) has aroused concerns over their increased infectivity and transmissibility, as well as decreased sensitivity to SARS-CoV-2-neutralizing antibodies (NAbs) and the current coronavirus disease 2019 (COVID-19) vaccines. Such exigencies call for the development of pan-sarbecovirus vaccines or inhibitors to combat the circulating SARS-CoV-2 NAb-escape variants and other sarbecoviruses. In this study, we isolated a broadly NAb against sarbecoviruses named GW01 from a donor who recovered from COVID-19. Cryo-EM structure and competition assay revealed that GW01 targets a highly conserved epitope in a wide spectrum of different sarbecoviruses. However, we found that GW01, the well-known sarbecovirus NAb S309, and the potent SARS-CoV-2 NAbs CC12.1 and REGN10989 only neutralize about 90% of the 56 tested currently circulating variants of SARS-CoV-2 including Omicron. Therefore, to improve efficacy, we engineered an IgG-like bispecific antibody GW01-REGN10989 (G9) consisting of single-chain antibody fragments (scFv) of GW01 and REGN10989. We found that G9 could neutralize 100% of NAb-escape mutants (23 out of 23), including Omicron variant, with a geometric mean (GM) 50% inhibitory concentration of 8.8 ng/mL. G9 showed prophylactic and therapeutic effects against SARS-CoV-2 infection of both the lung and brain in hACE2-transgenic mice. Site-directed mutagenesis analyses revealed that GW01 and REGN10989 bind to the receptor-binding domain in different epitopes and from different directions. Since G9 targets the epitopes for both GW01 and REGN10989, it was effective against variants with resistance to GW01 or REGN10989 alone and other NAb-escape variants. Therefore, this novel bispecific antibody, G9, is a strong candidate for the treatment and prevention of infection by SARS-CoV-2, NAb-escape variants, and other sarbecoviruses that may cause future emerging or re-emerging coronavirus diseases.

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.

Genomic Characterization of Diverse Bat Coronavirus HKU10 in Hipposideros Bats.

Bats have been identified as natural reservoirs of a variety of coronaviruses. They harbor at least 19 of the 33 defined species of alpha- and betacoronaviruses. Previously, the bat coronavirus HKU10 was found in two bat species of different suborders, Rousettus leschenaultia and Hipposideros pomona, in south China. However, its geographic distribution and evolution history are not fully investigated. Here, we screened this viral species by a nested reverse transcriptase PCR in our archived samples collected over 10 years from 25 provinces of China and one province of Laos. From 8004 bat fecal samples, 26 were found to be positive for bat coronavirus HKU10 (BtCoV HKU10). New habitats of BtCoV HKU10 were found in the Yunnan, Guangxi, and Hainan Provinces of China, and Louang Namtha Province in Laos. In addition to H. pomona, BtCoV HKU10 variants were found circulating in Aselliscus stoliczkanus and Hipposideros larvatus. We sequenced full-length genomes of 17 newly discovered BtCoV HKU10 strains and compared them with previously published sequences. Our results revealed a much higher genetic diversity of BtCoV HKU10, particularly in spike genes and accessory genes. Besides the two previously reported lineages, we found six novel lineages in their new habitats, three of which were located in Yunnan province. The genotypes of these viruses are closely related to sampling locations based on polyproteins, and correlated to bat species based on spike genes. Combining phylogenetic analysis, selective pressure, and molecular-clock calculation, we demonstrated that Yunnan bats harbor a gene pool of BtCoV HKU10, with H. pomona as a natural reservoir. The cell tropism test using spike-pseudotyped lentivirus system showed that BtCoV HKU10 could enter cells from human and bat, suggesting a potential interspecies spillover. Continuous studies on these bat coronaviruses will expand our understanding of the evolution and genetic diversity of coronaviruses, and provide a prewarning of potential zoonotic diseases from bats.

Discovery of Bat Coronaviruses through Surveillance and Probe Capture-Based Next-Generation Sequencing.

Coronaviruses (CoVs) of bat origin have caused two pandemics in this century. Severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV both originated from bats, and it is highly likely that bat coronaviruses will cause future outbreaks. Active surveillance is both urgent and essential to predict and mitigate the emergence of these viruses in humans. Next-generation sequencing (NGS) is currently the preferred methodology for virus discovery to ensure unbiased sequencing of bat CoVs, considering their high genetic diversity. However, unbiased NGS is an expensive methodology and is prone to missing low-abundance CoV sequences due to the high background level of nonviral sequences present in surveillance field samples. Here, we employ a capture-based NGS approach using baits targeting most of the CoV species. Using this technology, we effectively reduced sequencing costs by increasing the sensitivity of detection. We discovered nine full genomes of bat CoVs in this study and revealed great genetic diversity for eight of them.IMPORTANCE Active surveillance is both urgent and essential to predict and mitigate the emergence of bat-origin CoV in humans and livestock. However, great genetic diversity increases the chance of homologous recombination among CoVs. Performing targeted PCR, a common practice for many surveillance studies, would not reflect this diversity. NGS, on the other hand, is an expensive methodology and is prone to missing low-abundance CoV sequences. Here, we employ a capture-based NGS approach using baits targeting all CoVs. Our work demonstrates that targeted, cost-effective, large-scale, genome-level surveillance of bat CoVs is now highly feasible.

Identification of a novel lineage bat SARS-related coronaviruses that use bat ACE2 receptor.

Severe respiratory disease coronavirus-2 (SARS-CoV-2) has been the most devastating disease COVID-19 in the century. One of the unsolved scientific questions of SARS-CoV-2 is the animal origin of this virus. Bats and pangolins are recognized as the most probable reservoir hosts that harbour highly similar SARS-CoV-2 related viruses (SARSr-CoV-2). This study identified a novel lineage of SARSr-CoVs, including RaTG15 and seven other viruses, from bats at the same location where we found RaTG13 in 2015. Although RaTG15 and the related viruses share 97.2% amino acid sequence identities with SARS-CoV-2 in the conserved ORF1b region, it only shows less than 77.6% nucleotide identity to all known SARSr-CoVs at the genome level, thus forming a distinct lineage in the Sarbecovirus phylogenetic tree. We found that the RaTG15 receptor-binding domain (RBD) can bind to ACE2 from Rhinolophus affinis, Malayan pangolin, and use it as an entry receptor, except for ACE2 from humans. However, it contains a short deletion and has different key residues responsible for ACE2 binding. In addition, we showed that none of the known viruses in bat SARSr-CoV-2 lineage discovered uses human ACE2 as efficiently as the pangolin-derived SARSr-CoV-2 or some viruses in the SARSr-CoV-1 lineage. Therefore, further systematic and longitudinal studies in bats are needed to prevent future spillover events caused by SARSr-CoVs or to understand the origin of SARS-CoV-2 better.

Comparative Antiviral Efficacy of Viral Protease Inhibitors against the Novel SARS-CoV-2 In Vitro.

The recent outbreak of novel coronavirus pneumonia (COVID-19) caused by a new coronavirus has posed a great threat to public health. Identifying safe and effective antivirals is of urgent demand to cure the huge number of patients. Virus-encoded proteases are considered potential drug targets. The human immunodeficiency virus protease inhibitors (lopinavir/ritonavir) has been recommended in the global Solidarity Trial in March launched by World Health Organization. However, there is currently no experimental evidence to support or against its clinical use. We evaluated the antiviral efficacy of lopinavir/ritonavir along with other two viral protease inhibitors in vitro, and discussed the possible inhibitory mechanism in silico. The in vitro to in vivo extrapolation was carried out to assess whether lopinavir/ritonavir could be effective in clinical. Among the four tested compounds, lopinavir showed the best inhibitory effect against the novel coronavirus infection. However, further in vitro to in vivo extrapolation of pharmacokinetics suggested that lopinavir/ritonavir could not reach effective concentration under standard dosing regimen [marketed as Kaletra®, contained lopinavir/ritonavir (200 mg/50 mg) tablets, recommended dosage is 400 mg/10 mg (2 tablets) twice daily]. This research concluded that lopinavir/ritonavir should be stopped for clinical use due to the huge gap between in vitro IC50 and free plasma concentration. Nevertheless, the structure-activity relationship analysis of the four inhibitors provided further information for de novel design of future viral protease inhibitors of SARS-CoV-2.

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.

The SARS-CoV-2 subgenome landscape and its novel regulatory features.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global pandemic. CoVs are known to generate negative subgenomes (subgenomic RNAs [sgRNAs]) through transcription-regulating sequence (TRS)-dependent template switching, but the global dynamic landscapes of coronaviral subgenomes and regulatory rules remain unclear. Here, using next-generation sequencing (NGS) short-read and Nanopore long-read poly(A) RNA sequencing in two cell types at multiple time points after infection with SARS-CoV-2, we identified hundreds of template switches and constructed the dynamic landscapes of SARS-CoV-2 subgenomes. Interestingly, template switching could occur in a bidirectional manner, with diverse SARS-CoV-2 subgenomes generated from successive template-switching events. The majority of template switches result from RNA-RNA interactions, including seed and compensatory modes, with terminal pairing status as a key determinant. Two TRS-independent template switch modes are also responsible for subgenome biogenesis. Our findings reveal the subgenome landscape of SARS-CoV-2 and its regulatory features, providing a molecular basis for understanding subgenome biogenesis and developing novel anti-viral strategies.