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.

[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.