Design of customized coronavirus receptors.

Although coronaviruses use diverse receptors, the characterization of coronaviruses with unknown receptors has been impeded by a lack of infection models1,2. Here we introduce a strategy to engineer functional customized viral receptors (CVRs). The modular design relies on building artificial receptor scaffolds comprising various modules and generating specific virus-binding domains. We identify key factors for CVRs to functionally mimic native receptors by facilitating spike proteolytic cleavage, membrane fusion, pseudovirus entry and propagation for various coronaviruses. We delineate functional SARS-CoV-2 spike receptor-binding sites for CVR design and reveal the mechanism of cell entry promoted by the N-terminal domain-targeting S2L20-CVR. We generated CVR-expressing cells for 12 representative coronaviruses from 6 subgenera, most of which lack known receptors, and show that a pan-sarbecovirus CVR supports propagation of a propagation-competent HKU3 pseudovirus and of authentic RsHuB2019A3. Using an HKU5-specific CVR, we successfully rescued wild-type and ZsGreen-HiBiT-incorporated HKU5-1 (LMH03f) and isolated a HKU5 strain from bat samples. Our study demonstrates the potential of the CVR strategy for establishing native receptor-independent infection models, providing a tool for studying viruses that lack known susceptible target cells.

Close relatives of MERS-CoV in bats use ACE2 as their functional receptors.

Middle East respiratory syndrome coronavirus (MERS-CoV) and several bat coronaviruses use dipeptidyl peptidase-4 (DPP4) as an entry receptor1-4. However, the receptor for NeoCoV-the closest known MERS-CoV relative found in bats-remains unclear5. Here, using a pseudotype virus entry assay, we found that NeoCoV and its close relative, PDF-2180, can efficiently bind to and use specific bat angiotensin-converting enzyme 2 (ACE2) orthologues and, less favourably, human ACE2 as entry receptors through their receptor-binding domains (RBDs) on the spike (S) proteins. Cryo-electron microscopy analysis revealed an RBD-ACE2 binding interface involving protein-glycan interactions, distinct from those of other known ACE2-using coronaviruses. We identified residues 337-342 of human ACE2 as a molecular determinant restricting NeoCoV entry, whereas a NeoCoV S pseudotyped virus containing a T510F RBD mutation efficiently entered cells expressing human ACE2. Although polyclonal SARS-CoV-2 antibodies or MERS-CoV RBD-specific nanobodies did not cross-neutralize NeoCoV or PDF-2180, an ACE2-specific antibody and two broadly neutralizing betacoronavirus antibodies efficiently inhibited these two pseudotyped viruses. We describe MERS-CoV-related viruses that use ACE2 as an entry receptor, underscoring a promiscuity of receptor use and a potential zoonotic threat.

Sarbecovirus RBD indels and specific residues dictating multi-species ACE2 adaptiveness.

Our comprehensive understanding of the multi-species ACE2 adaptiveness of sarbecoviruses remains elusive, particularly for those with various receptor binding motif (RBM) insertions/deletions (indels). Here, we analyzed RBM sequences from 268 sarbecoviruses categorized into four RBM indel types. We examined the ability of 20 representative sarbecovirus Spike glycoproteins (S) and derivatives in utilizing ACE2 from various bats and several other mammalian species. We reveal that sarbecoviruses with long RBMs (type-I) can achieve broad ACE2 tropism, whereas viruses with single deletions in Region 1 (type-II) or Region 2 (type-III) exhibit narrower ACE2 tropism. Sarbecoviruses with double region deletions (type-IV) completely lost ACE2 usage, which is restricted by clade-specific residues within and outside RBM. Lastly, we propose the evolution of sarbecovirus RBM indels and illustrate how loop lengths, disulfide, and residue determinants shape multi-species ACE2 adaptiveness. This study provides profound insights into the mechanisms governing ACE2 usage and spillover risks of sarbecoviruses.

Novel Intercellular Spread Mode of Respiratory Syncytial Virus Contributes to Neutralization Escape.

Developing widely used respiratory syncytial virus (RSV) vaccines remains a significant challenge, despite the recent authorization of two pre-F vaccines for elderly adults. Previous reports have suggested that even when vaccine-induced immunity generates high titers of potent neutralizing antibodies targeting the pre-F protein, it may not fully inhibit breakthrough of RSV infections. This incomplete inhibition of RSV breakthrough infections can lead to an increased risk of enhanced respiratory disease (ERD) in vaccinated individuals. The reasons why potent neutralizing antibodies cannot fully prevent RSV breakthrough infections are not yet clear. In an attempt to explain this phenomenon, we investigated the effect of potent neutralizing antibodies on the intercellular spread of RSV. Our findings indicated that a specific titer of potent neutralizing antibodies, such as 5C4, could block certain modes of intercellular spread, such as the diffusion of cell-free virions and the delivery of virions through filopodia. However, these antibodies did not fully inhibit the entire process of intercellular spread. Through the use of super-resolution imaging techniques, we observed a novel and efficient spread mode called the transition of viral materials through intercellular nanotubes (TVMIN), independent of virions and insensitive to the presence of antibodies. TVMIN allowed RSV-infected cells to directly transfer viral materials to neighboring cells via intercellular nanotubes that are rich in microfilaments. TVMIN began as early as 5 hours post-infection (h.p.i.) and rapidly initiated infection in recipient cells. Our data provided new insights into the intercellular spread of RSV and might help explain the occurrence of breakthrough infections.

Broad host tropism of ACE2-using MERS-related coronaviruses and determinants restricting viral recognition.

Recently, two Middle East respiratory syndrome coronavirus (MERS-CoV) closely related to bat merbecoviruses, NeoCoV and PDF-2180, were discovered to use angiotensin-converting enzyme 2 (ACE2) for entry. The two viruses cannot use human ACE2 efficiently, and their host range and cross-species transmissibility across a wide range of mammalian species remain unclear. Herein, we characterized the species-specific receptor preference of these viruses by testing ACE2 orthologues from 49 bats and 53 non-bat mammals through receptor-binding domain (RBD)-binding and pseudovirus entry assays. Results based on bat ACE2 orthologues revealed that the two viruses were unable to use most, but not all, ACE2 from Yinpterochiropteran bats (Yin-bats), which is distinct from NL63 and SARS-CoV-2. Besides, both viruses exhibited broad receptor recognition spectra across non-bat mammals. Genetic and structural analyses of bat ACE2 orthologues highlighted four crucial host range determinants, all confirmed by subsequent functional assays in human and bat cells. Notably, residue 305, participating in a critical viral receptor interaction, plays a crucial role in host tropism determination, particularly in non-bat mammals. Furthermore, NeoCoV and PDF-2180 mutants with enhanced human ACE2 recognition expanded the potential host range, especially by enhancing their interaction with an evolutionarily conserved hydrophobic pocket. Our results elucidate the molecular basis for the species-specific ACE2 usage of MERS-related viruses and shed light on their zoonotic risks.

An Optimized FI-RSV Vaccine Effectively Protects Cotton Rats and BALB/c Mice without Causing Enhanced Respiratory Disease.

BACKGROUND: Despite considerable efforts toward vaccine development in past decades, no effective vaccines against respiratory syncytial virus (RSV) are available. Recently, we showed that an optimized formalin concentration can preserve prefusion protein (pre-F) on RSV-infected cells and protect mice against RSV infection without causing enhanced respiratory disease (ERD). Here, we sought to further stabilize pre-F on RSV virions by optimizing the production of FI-RSV. METHODS: Freshly produced RSV virions were treated with formalin under different concentrations to obtained an opti-FI-RSV vaccine with high pre-F level. Immunogenicity and safety of opti-FI-RSV were evaluated in Balb/c mice and cotton rats. RESULTS: Using 0.0156-0.1778% formalin, we successfully preserved pre-F on virions. This opti-FI-RSV exhibited improved immunogenicity and efficacy without causing ERD. Surprisingly, opti-FI-RSV, with a pre-F-dominant immunogen, still caused ERD after immunization with a suboptimal dose or when the neutralizing antibody titers declined. ERD was avoided by coadministering opti-FI-RSV with CpG + MPLA adjuvant, which subsequently induced a Th1-biasing immune response and, more importantly, significantly improved antibody avidity. CONCLUSIONS: Our study provides a new method to obtain a novel FI-RSV vaccine with a high pre-F level and may provide a reference for developing other inactivated vaccines. Our findings also emphasize that appropriate adjuvants are critical for nonreplicating vaccines.

A novel point-of-care test of respiratory syncytial viral RNA based on cellulose-based purification and convective PCR.

BACKGROUND: Respiratory syncytial virus (RSV) infection is a global public-health problem. Timely diagnostics are needed for high-risk patients. Several methods have been used for RSV detection but not suitable for on-site detection due to the requirement of specialized laboratories and expensive equipment. METHODS: We developed a convenient, rapid and low-cost method of nucleic acids (NA) extraction based on cellulose paper, which could extract NA from nasopharyngeal swabs (NPSs) within 1 min. This extraction method was integrated with fluorescence convection polymerase chain reaction (CPCR), which easily affordable and easy-to-use NA detection of the RSV in 33 min. RESULTS: The developed cellulose-based NA purification combine with CPCR (CP-CPCR) reliably detected as little as 0.01 TCID50/mL of RSV cultures. CP-CPCR performance was tested further using NPSs: it showed sensitivity of 100% and a specificity of 100% compared with traditional extraction and amplification methods. CONCLUSIONS: Our evaluation confirmed high specificity, sensitivity and efficient of the CP-CPCR, which can be used widely for RSV testing in resource-limited settings.

[Preparation and identification of mouse anti-human monoclonal antibody against P protein of respiratory syncytial virus].

Objective To prepare the monoclonal antibodies specifically against P protein of human respiratory syncytial virus (HRSV) and identify it. Methods HRSV P protein prepared by prokaryotic expression in the form of overlapping peptides was used as the immunogen, and monoclonal antibodies (mAbs) were screened by hybridoma technology. Western blot analysis was used to verify the binding activity of the screened mAbs and P protein, and immunofluorescence cytochemical staining was used to determine whether the obtained mAbs could be used to detect the expression of P protein in HEp-2 cells infected with HRSV. Results P181-15A3 and P211-16D8 with great reactivity and specific recognition of HRSV P protein were screened. Both mAbs could bind to P protein by Western blot analysis and could be used for immunocytochemical detection of P protein in HEp-2 cells after HRSV infection. Conclusion We have successfully prepared the mAbs against HRSV P protein.

The Optimal Concentration of Formaldehyde is Key to Stabilizing the Pre-Fusion Conformation of Respiratory Syncytial Virus Fusion Protein.

BACKGROUND: To date, there is no licensed vaccine available to prevent respiratory syncytial virus (RSV) infection. The valuable pre-fusion conformation of the fusion protein (pre-F) is prone to lose high neutralizing antigenic sites. The goals of this study were to stabilize pre-F protein by fixatives and try to find the possibility of developing an inactivated RSV vaccine. METHODS: The screen of the optimal fixative condition was performed with flow cytometry. BALB/c mice were immunized intramuscularly with different immunogens. The serum neutralizing antibody titers of immunized mice were determined by neutralization assay. The protection and safety of these immunogens were assessed. RESULTS: Fixation in an optimal concentration of formaldehyde (0.0244%-0.0977%) or paraformaldehyde (0.0625%-1%) was able to stabilize pre-F. Additionally, BALB/c mice inoculated with optimally stabilized pre-F protein (opti-fixed) induced a higher anti-RSV neutralization (9.7 log2, mean value of dilution rate) than those inoculated with unstable (unfixed, 8.91 log2, p < 0.01) or excessively fixed (exce-fixed, 7.28 log2, p < 0.01) pre-F protein. Furthermore, the opti-fixed immunogen did not induce enhanced RSV disease. CONCLUSIONS: Only the proper concentration of fixatives could stabilize pre-F and the optimal formaldehyde condition provides a potential reference for development of an inactivated RSV vaccine.

An optimized high-throughput fluorescence plate reader-based RSV neutralization assay.

A licensed vaccine for respiratory syncytial virus (RSV) has yet to be developed, and a reliable and repeatable neutralizing assay is indispensable for vaccine development. Here, we demonstrated an optimized high-throughput RSV neutralization assay that utilizes a fluorescence plate reader (reader) as a substitute for flow cytometry to detect fluorescent signals in RSV-A2 mKate-infected cells. Furthermore, this study tested the influence of virus input and infectivity on the neutralizing assay and highlighted critical factors (together with a suggested protocol) for obtaining stable data using this assay.

[Comparison of Respiratory Syncytial Virus Infection on Different Week-ages BALB/c Mice].

Respiratory syncytial virus(RSV)is a leading cause of lower respiratory tract disease. The major high risk population for RSV infection are<6month infants and elders with age older than 65 years. At present, BALB/c mice were wildly used as animal model for RSV infection, however there has no report about the comparison of different week-ages BALB/c mice after RSV infection. A different week-ages BALB/c mice model was described in this study to compare their susceptibility after RSV infection. Young(10weeks),middle aged(30weeks)and aged(60weeks)mice were intranasally infected with 106 or 107plaque-forming units (PFU) RSV, then clinical symptom, weight, RSV titer in nose/lung, histology and immunohistochemistry was examined. And age-related susceptibility was analyzed. A high-titer virus(107PFU)infection showed significant weight loss at 6-11 day post infection while 106 PFU didn't lead to obvious weight change. In 10(7) PFU infected group, replication of virus in nose and lung was detected, the virus in lung located around pulmonary alveoli, and the hematoxylin eosin stain showed significant infiltration of inflammatory cells and pathological tissue damage. Mice trended to be more susceptible to RSV infection as the growth of age. Older mice experience more weight loss. Lung histology of older mice showed more serious bronchiolitis and increased number of inflammatory cells in alveolar spaced, and 60week-old mice tended to be the most significant. In this study, we have successfully established a different week-ages BALB/c mice model, which will serve as the basis for investigating antibody or vaccine and further infection mechanism research of RSV.