High-accuracy mapping of human and viral direct physical protein-protein interactions using the novel computational system AlphaFold-pairs (preprint)

Protein-protein interactions are central, highly flexible components of regulatory mechanisms in all living cells. Over the years, diverse methods have been developed to map protein-protein interactions. These methods have revealed the organization of protein complexes and networks in numerous cells and conditions. However, these methods are also time consuming, costly and sensitive to various experimental artifacts. To avoid these caveats, we have taken advantage of the AlphaFold-Multimer software, which succeeded in predicting the structure of many protein complexes. We designed a relatively simple algorithm based on assessing the physical proximity of a test protein with other AlphaFold structures. Using this method, named AlphaFold-pairs, we have successfully defined the probability of a protein-protein interaction forming. AlphaFold-pairs was validated using well-defined protein-protein interactions found in the literature and specialized databases. All pairwise interactions forming within the 12-subunit transcription machinery RNA Polymerase II, according to available structures, have been identified. Out of 66 possible interactions (excluding homodimers), 19 specific interactions have been found, and an additional previously unknown interaction has been unveiled. The SARS-CoV-2 surface glycoprotein Spike (or S) was confirmed to interact with high preference with the human ACE2 receptor when compared to other human receptors. Notably, two additional receptors, INSR and FLT4, were found to interact with S. For the first time, we have successfully identified protein-protein interactions that are likely to form within the reassortant Eurasian avian-like (EA) H1N1 swine G4 genotype Influenza A virus, which poses a potential zoonotic threat. Testing G4 proteins against human transcription factors and molecular chaperones (a total of 100 proteins) revealed strong specific interactions between the G4 HA and HSP90B1, the G4 NS and the PAQosome subunit RPAP3, as well as the G4 PA and the POLR2A subunit. We predict that AlphaFold-pairs will revolutionize the study of protein-protein interactions in a large number of healthy and diseased systems in the years to come.

Simultaneous Detection of Seven Human Coronaviruses by Multiplex PCR and MALDI-TOF MS

Human coronaviruses (HCoVs) are associated with a range of respiratory symptoms. The discovery of severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome, and SARS-CoV-2 pose a significant threat to human health. In this study, we developed a method (HCoV-MS) that combines multiplex PCR with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), to detect and differentiate seven HCoVs simultaneously. The HCoV-MS method had high specificity and sensitivity, with a 1–5 copies/reaction detection limit. To validate the HCoV-MS method, we tested 163 clinical samples, and the results showed good concordance with real-time PCR. Additionally, the detection sensitivity of HCoV-MS and real-time PCR was comparable. The HCoV-MS method is a sensitive assay, requiring only 1 μL of a sample. Moreover, it is a high-throughput method, allowing 384 samples to be processed simultaneously in 30 min. We propose that this method be used to complement real-time PCR for large-scale screening studies.

A Reactive Copper-Organophosphate-MXene Heterostructure Enabled Antibacterial, Self-Extinguishing and Mechanically Robust Polymer Nanocomposites

The ongoing Covid-19 pandemic has raised the need for urgent antibacterial requirements for many commercially important polymers, e.g., epoxy resins (EPs). Meanwhile, intrinsic flammability and poor impact toughness are two big obstacles that greatly impede the practical applications of EPs. Hence, it has been imperative but highly challenging to create advanced EPs combining satisfactory antibacterial, fire-retardant and mechanically robust performances so far. Here, we report a reactive multifunctional heterostructure, copper-organophosphate-MXene (CuP-MXene) by rational design. Our results show that with 5.0 wt% of CuP-MXene, in addition to achieving a high antibacterial efficiency above 99.9%, the resultant EP nanocomposite exhibits satisfactory flame retardancy (UL-94 V-0 rating, peak heat release rate decreased by 64.4%) and improved mechanical properties (tensile strength, elastic modulus and impact strength increased by 31.7%, 38.9%, and 25.0%, respectively) relative to virgin EP, outperforming its previous counterparts. Such a desirable performance portfolio arises from multiple synergistic effects between CuP and MXene. This work provides a general strategy for the design of multifunctional nanoadditives and advanced functional polymers, and creates more opportunities for industrial applications of EP in the areas of coatings, medical devices and furniture.

Identification of novel bat coronaviruses sheds light on the evolutionary origins of SARS-CoV-2 and related viruses (preprint)

Although a variety of SARS-CoV-2 related coronaviruses have been identified, the evolutionary origins of this virus remain elusive. We describe a meta-transcriptomic study of 411 samples collected from 23 bat species in a small (~1100 hectare) region in Yunnan province, China, from May 2019 to November 2020. We identified coronavirus contigs in 40 of 100 sequencing libraries, including seven representing SARS-CoV-2-like contigs. From these data we obtained 24 full-length coronavirus genomes, including four novel SARS-CoV-2 related and three SARS-CoV related genomes. Of these viruses, RpYN06 exhibited 94.5% sequence identity to SARS-CoV-2 across the whole genome and was the closest relative of SARS-CoV-2 in the ORF1ab, ORF7a, ORF8, N, and ORF10 genes. The other three SARS-CoV-2 related coronaviruses were nearly identical in sequence and clustered closely with a virus previously identified in pangolins from Guangxi, China, although with a genetically distinct spike gene sequence. We also identified 17 alphacoronavirus genomes, including those closely related to swine acute diarrhea syndrome virus and porcine epidemic diarrhea virus. Ecological modeling predicted the co-existence of up to 23 Rhinolophus bat species in Southeast Asia and southern China, with the largest contiguous hotspots extending from South Lao and Vietnam to southern China. Our study highlights both the remarkable diversity of bat viruses at the local scale and that relatives of SARS-CoV-2 and SARS-CoV circulate in wildlife species in a broad geographic region of Southeast Asia and southern China. These data will help guide surveillance efforts to determine the origins of SARS-CoV-2 and other pathogenic coronaviruses.

Serological diagnostic kit of SARS-CoV-2 antibodies using CHO-expressed full-length SARS-CoV-2 S1 proteins (preprint)

WHO has declared COVID-19 a pandemic with more than 300,000 confirmed cases and more than 14,000 deaths. There is urgent need for accurate and rapid diagnostic kits. Here we report the development and validation of a COVID-19/SARS-CoV-2 S1 serology ELISA kit for the detection of total anti-virus antibody (IgG+IgM) titers in sera from either the general population or patients suspected to be infected. For indirect ELISA, CHO-expressed recombinant full length SARS-CoV-2-S1 protein with 6*His tag was used as the coating antigen to capture the SARS-CoV-2-S1 antibodies specifically. The specificity of the ELISA kit was determined to be 97.5%, as examined against total 412 normal human sera including 257 samples collected prior to the outbreak and 155 collected during the outbreak. The sensitivity of the ELISA kit was determined to be 97.5% by testing against 69 samples from hospitalized and/or recovered COVID-19 patients. The overall accuracy rate reached 97.3%. Most importantly, in one case study, the ELISA test kit was able to identify an infected person who had previously been quarantined for 14 days after coming into contact with a confirmed COVID-19 patient, and discharged after testing negative twice by nucleic acid test. With the assays developed here, we can screen millions of medical staffs in the hospitals and people in residential complex, schools, public transportations, and business parks in the epidemic centers of the outbreaks to fish out the "innocent viral spreaders", and help to stop the further spreading of the virus.

A novel bat coronavirus reveals natural insertions at the S1/S2 cleavage site of the Spike protein and a possible recombinant origin of HCoV-19 (preprint)

The unprecedented epidemic of pneumonia caused by a novel coronavirus, HCoV-19, in China and beyond has caused public health concern at a global scale. Although bats are regarded as the most likely natural hosts for HCoV-191,2, the origins of the virus remain unclear. Here, we report a novel bat-derived coronavirus, denoted RmYN02, identified from a metagenomics analysis of samples from 227 bats collected from Yunnan Province in China between May and October, 2019. RmYN02 shared 93.3% nucleotide identity with HCoV-19 at the scale of the complete virus genome and 97.2% identity in the 1ab gene in which it was the closest relative of HCoV-19. In contrast, RmYN02 showed low sequence identity (61.3%) to HCoV-19 in the receptor binding domain (RBD) and might not bind to angiotensin-converting enzyme 2 (ACE2). Critically, however, and in a similar manner to HCoV-19, RmYN02 was characterized by the insertion of multiple amino acids at the junction site of the S1 and S2 subunits of the Spike (S) protein. This provides strong evidence that such insertion events can occur in nature. Together, these data suggest that HCoV-19 originated from multiple naturally occurring recombination events among those viruses present in bats and other wildlife species.

Generation of antibodies against COVID-19 virus for development of diagnostic tools (preprint)

The COVID-19 China coronavirus started in Dec 2019 was challenged by the lack of accurate serological diagnostic tool for this deadly disease to quickly identify and isolate the infected patients. The generation of COVID-19-specific antibodies is essential for such tasks. Here we report that polyclonal and monoclonal antibodies were generated by immunizing animals with synthetic peptides corresponding to different areas of Nucleoprotein (N) of COVID-19. The specificities of the COVID-19 antibodies were assessed by Western Blot analysis against NPs from COVID-19, MERS and SARS. Antibodies were used for immunohistochemistry staining of the tissue sections from COVID-19 infected patient, as a potential diagnostic tool. A Sandwich ELISA kit was quickly assembled for quantitation of the virus/NP of COVID-19 concentrations in the vaccine preparations. Development of POCT is also aggressively undergoing.