Lethal Infection of Human ACE2-Transgenic Mice Caused by SARS-CoV-2-related Pangolin Coronavirus GX_P2V(short_3UTR) (preprint)

SARS-CoV-2-related pangolin coronavirus GX_P2V(short_3UTR) is highly attenuated, but can cause mortality in a specifically designed human ACE2-transgenic mouse model, making it an invaluable surrogate model for evaluating the efficacy of drugs and vaccines against SARS-CoV-2.

Ultrastructural modifications induced by an attenuated SARS-CoV-2-related pangolin coronavirus GX_P2V(short_3UTR) in Vero cells (preprint)

Background Positive-strand RNA viruses, such as SARS-CoV-2, manipulate host cell endomembranes to form viral replication organelles (vROs) for replication and protection. Pangolin coronavirus GX_P2V(short_3UTR), a cell-culture-adapted SARS-CoV-2-related coronavirus with a 104-nucleotide deletion in its 3´-terminus untranslated region, is highly attenuated in both in vitro and in vivo infection models. The mechanism underlying this attenuation remains unclear.Methods Vero cells were infected with GX_P2V(short_3UTR) and analyzed using transmission electron microscopy at various time points post-infection.Results Our study demonstrated that GX_P2V(short_3UTR) enters cells via endocytosis, leading to the formation of delayed vROs, composed of double-membrane vesicle, convoluted membranes, and double-membrane spherules. These structures were only observed after 12 hours post-infection. At 24 hours post-infection, vROs were readily identifiable, including the formation of annular lamellae due to nuclear pore stacking. By 48 hours post-infection, infected cells exhibited a characteristic feature of a complex reticulovesicular network. Similar to SARS-CoV-2, GX_P2V(short_3UTR) were found to bud within endoplasmic reticulum-Golgi compartments, accumulate in autophagy-like vesicles and multivesicular bodies, and egress via the lysosomal pathway. Notably, we did not observe any large vacuoles containing highly dense viral particles, which had been reported in SARS-CoV-2-infected cells.Conclusions Pangolin coronavirus GX_P2V(short_3UTR) undergoes a typical SARS-CoV-2-like life cycle in Vero cells. The delayed formation of vROs and the sparsely populated viral vacuoles in infected cells could contribute to the attenuation of pangolin coronavirus GX_P2V(short_3UTR).

The recombinant receptor-binding domain of a pangolin coronavirus spike by prokaryotic expression can partially induce neutralizing antibodies (preprint)

We previously reported that a SARS-CoV-2 related pangolin coronavirus GX_P2V can induce neutralizing antibodies against SARS-CoV-2 in a golden hamster model. The two viruses GX_P2V and SARS-CoV-2 have genomes with high homology and both use ACE2 as their receptor, but have distinct receptor-binding domains (RBDs) of their spike proteins. The shared neutralizing epitopes of these two viruses are unknown. Here we describe a novel method of soluble expression of GX_P2V RBD in E. coli by tagging RBD with a carboxy-terminal fragment of the SADS coronavirus nucleocapsid protein. The recombinant GX_P2V RBD have human ACE2-binding activities. Mice immunized with this recombinant RBD produced significant titers of neutralizing antibodies against GX_P2V pseudoviruses, not SARS-CoV-2 pseudoviruses. Our data suggest that the GX_P2V RBD has none or limited neutralizing epitopes against SARS-CoV-2, thus neutralizing antibodies against SARS-CoV-2 from GX_P2V infected golden hamsters is likely RBD-independent.

Pathogen-Host Adhesion between SARS-CoV-2 S Proteins from Different Variants and Human ACE2 Probed at Single-Molecule and Single-Cell Levels (preprint)

Pathogen-Host adhesion is considered the first step of infection for many pathogens such as bacteria and virus. The binding of the receptor binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) onto human angiotensin-converting enzyme 2 (ACE2) is considered as the first step for the SARS-CoV-2 to adhere onto the host cells during the infection. Within three years, a number of variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been found all around the world. Here, we investigated the adhesion of S Proteins from different variants and ACE2 using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) and single-cell force spectroscopy (SCFS). We found that the unbinding force and binding probability of the S protein from Delta variant to the ACE2 was the highest among the variants tested in our study at both single-molecule and single-cell levels. Molecular dynamics simulation showed that ACE2-RBD (Omicron) complex is destabilized by the E484A and Y505H mutations and stabilized by S477N and N501Y mutations, when compared with Delta variant. In addition, a neutralizing antibody, produced by immunization with wild type RBD of S protein, could effectively inhibit the binding of S proteins from wild type, Delta and Omicron variants onto ACE2. Our results provide new insight for the molecular mechanism of the adhesive interactions between S protein and ACE2 and suggest effective monoclonal antibody can be prepared using wild type S protein against the Delta and Omicron variants by inhibit the pathogen-host adhesion.

N-glycoproteomic Profiling Revealing New Coronavirus Therapeutic Targets That Maybe Involved in Cepharanthine’s Intervention (preprint)

N-glycosylation is an important post-translational modification involved in protein folding, signal transduction, extracellular matrix organization and immune response. Evidence showed that glycosylated SARS-CoV-2 Spike protein may be a potential target in viral pathogenesis and drug/vaccine design. To investigate the mechanism of coronavirus infestation and drug targets from glycosylation perspective, we constructed a SARS-CoV-2 cellular model using GX_P2V-infected VeroE6 cells to study the effects of GX_P2V on glycoproteins in presence or absence of Cepharanthine (CEP) through N-glycoproteomics profiling. The results showed that coronavirus GX_P2V could cause aberrant protein glycosylation, whereas CEP can partially maintain GX_P2V-induced aberrant glycoproteins at homeostasis. Further study revealed that proteins LAMB1 and FN1 were pivotal in counteracting coronavirus-induced aberrant protein glycosylation by CEP. Furthermore, CEP can dramatically regulate the glycosylation of viral proteins S, M and N. Our results suggest that despite the strong anti-coronavirus effects of CEP, drug combinations need be considered to achieve optimal therapeutic strategies.

Development of a dual-gene loop-mediated isothermal amplification (LAMP) detection assay for SARS-CoV-2: A preliminary study (preprint)

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as a rapidly spreading global pathogen stressing the need for development of rapid testing protocols ever than before. The aim of present study was to develop a SARS-CoV-2 detection protocol which can be performed within minimal resources and timeframe. For this purpose, we implemented the reverse transcription loop-mediated isothermal amplification (RT-LAMP) methodology for the qualitative detection of SARS-CoV-2 RNA. In order to improve the detection capability, the RT-LAMP assay was developed to simultaneously amplify two viral genes: ORF1a and N. A total of 45 SARS-CoV-2 associated coronavirus disease 2019 (COVID-19) cases were enrolled. Viral RNA was extracted from the nasopharyngeal swab samples and analyzed simultaneously using PCR and RT-LAMP protocols. Overall, our SARS-CoV-2 dual gene RT-LAMP assay was found to be 95% accurate in detecting positive cases and showed no cross-reactivity or false-positive result in non-COVID-19 samples. Further evaluation on larger and multi-centric cohorts is currently underway to establish the diagnostic accuracy and subsequent implementation into clinical practice and at point-of-care settings.