Simultaneous Detection of SARS-CoV-2 and Six Other Human Coronaviruses by Multiplex PCR and MALDI-TOF MS (preprint)

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is challenging the health systems worldwide, and large population testing is a vital step to control this pandemic. Here, we developed a new method (named HCoV-MS), which combines multiplex PCR with matrix-assisted laser desorption/ionization-time of flight mass spectrometry to simultaneously detect and differentiate seven human coronaviruses (HCoVs). The HCoV-MS method had good specificity and sensitivity, with a detection limit of 1-5 copies/reaction. To validate the HCoV-MS method, we tested 151 clinical samples, and the results showed good concordance with real-time PCR. In addition, 41 D614G variants were identified, which were consistent with the sequencing results. This method was also used in EQAE-SARS-COV in 2020, and all the samples were accurately identified. Taken together, HCoV-MS could be used as an effective method for large-scale detection. It was also capable of detecting key single nucleotide polymorphism about variants.

A comprehensive overview on the transmission, pathogenesis, diagnosis, treatment, and prevention of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a single positive-strand RNA virus that is responsible for the current pandemic that the world has been facing since 2019. The primary route of transmission of SARS-CoV-2 is through respiratory tract transmission. However, other transmission routes such as fecal-oral, vertical transmission, and aerosol-eye also exist. In addition, it has been found that the pathogenesis of this virus involves the binding of the virus's S protein to its host cell surface receptor angiotensin-converting enzyme 2, which results in the subsequent membrane fusion that is required for SARS-CoV-2 to replicate and complete its entire life. The clinical symptoms of patients infected with SARS-CoV-2 can range from asymptomatic to severe. The most common symptoms seen include fever, dry cough, and fatigue. Once these symptoms are observed, a nucleic acid test is done using reverse transcription-polymerase chain reaction. This currently serves as the main confirmatory tool for COVID-19. Despite the fact that no cure has been found for SARS-CoV-2, prevention methods such as vaccines, specific facial mask, and social distancing have proven to be quite effective. It is imperative to have a complete understanding of the transmission and pathogenesis of this virus. To effectively develop new drugs as well as diagnostic tools, more knowledge about this virus would be needed.

Heterologous boost with mRNA vaccines against SARS-CoV-2 Delta/Omicron variants following an inactivated whole-virus vaccine.

The coronavirus SARS-CoV-2 has mutated quickly and caused significant global damage. This study characterizes two mRNA vaccines ZSVG-02 (Delta) and ZSVG-02-O (Omicron BA.1), and associating heterologous prime-boost strategy following the prime of a most widely administrated inactivated whole-virus vaccine (BBIBP-CorV). The ZSVG-02-O induces neutralizing antibodies that effectively cross-react with Omicron subvariants. In naïve animals, ZSVG-02 or ZSVG-02-O induce humoral responses skewed to the vaccine's targeting strains, but cellular immune responses cross-react to all variants of concern (VOCs) tested. Following heterologous prime-boost regimes, animals present comparable neutralizing antibody levels and superior protection against Delta and Omicron BA.1variants. Single-boost only generated ancestral and omicron dual-responsive antibodies, probably by "recall" and "reshape" the prime immunity. New Omicron-specific antibody populations, however, appeared only following the second boost with ZSVG-02-O. Overall, our results support a heterologous boost with ZSVG-02-O, providing the best protection against current VOCs in inactivated virus vaccine-primed populations.

An automated nucleic acid detection platform using digital microfluidics with an optimized Cas12a system.

Outbreaks of both influenza virus and the novel coronavirus SARS-CoV-2 are serious threats to human health and life. It is very important to establish a rapid, accurate test with large-scale detection potential to prevent the further spread of the epidemic. An optimized RPA-Cas12a-based platform combined with digital microfluidics (DMF), the RCD platform, was established to achieve the automated, rapid detection of influenza viruses and SARS-CoV-2. The probe in the RPA-Cas12a system was optimized to produce maximal fluorescence to increase the amplification signal. The reaction droplets in the platform were all at the microliter level and the detection could be accomplished within 30 min due to the effective mixing of droplets by digital microfluidic technology. The whole process from amplification to recognition is completed in the chip, which reduces the risk of aerosol contamination. One chip can contain multiple detection reaction areas, offering the potential for customized detection. The RCD platform demonstrated a high level of sensitivity, specificity (no false positives or negatives), speed (≤30 min), automation and multiplexing. We also used the RCD platform to detect nucleic acids from influenza patients and COVID-19 patients. The results were consistent with the findings of qPCR. The RCD platform is a one-step, rapid, highly sensitive and specific method with the advantages of digital microfluidic technology, which circumvents the shortcomings of manual operation. The development of the RCD platform provides potential for the isothermal automatic detection of nucleic acids during epidemics. Electronic Supplementary Material: Supplementary material is available in the online version of this article at 10.1007/s11426-021-1169-1.

Current Status and Future Perspective of Artificial Intelligence in the Management of Peptic Ulcer Bleeding: A Review of Recent Literature.

With the decreasing incidence of peptic ulcer bleeding (PUB) over the past two decades, the clinician experience of managing patients with PUB has also declined, especially for young endoscopists. A patient with PUB management requires collaborative care involving the emergency department, gastroenterologist, radiologist, and surgeon, from initial assessment to hospital discharge. The application of artificial intelligence (AI) methods has remarkably improved people's lives. In particular, AI systems have shown great potential in many areas of gastroenterology to increase human performance. Colonoscopy polyp detection or diagnosis by an AI system was recently introduced for commercial use to improve endoscopist performance. Although PUB is a longstanding health problem, these newly introduced AI technologies may soon impact endoscopists' clinical practice by improving the quality of care for these patients. To update the current status of AI application in PUB, we reviewed recent relevant literature and provided future perspectives that are required to integrate such AI tools into real-world practice.

Identification and functional analysis of the SARS-COV-2 nucleocapsid protein.

BACKGROUND: A severe form of pneumonia, named coronavirus disease 2019 (COVID-19) by the World Health Organization is widespread on the whole world. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was proved to be the main agent of COVID-19. In the present study, we conducted an in depth analysis of the SARS-COV-2 nucleocapsid to identify potential targets that may allow identification of therapeutic targets. METHODS: The SARS-COV-2 N protein subcellular localization and physicochemical property was analyzed by PSORT II Prediction and ProtParam tool. Then SOPMA tool and swiss-model was applied to analyze the structure of N protein. Next, the biological function was explored by mass spectrometry analysis and flow cytometry. At last, its potential phosphorylation sites were analyzed by NetPhos3.1 Server and PROVEAN PROTEIN. RESULTS: SARS-COV-2 N protein composed of 419 aa, is a 45.6 kDa positively charged unstable hydrophobic protein. It has 91 and 49% similarity to SARS-CoV and MERS-CoV and is predicted to be predominantly a nuclear protein. It mainly contains random coil (55.13%) of which the tertiary structure was further determined with high reliability (95.76%). Cells transfected with SARS-COV-2 N protein usually show a G1/S phase block company with an increased expression of TUBA1C, TUBB6. At last, our analysis of SARS-COV-2 N protein predicted a total number of 12 phosphorylated sites and 9 potential protein kinases which would significantly affect SARS-COV-2 N protein function. CONCLUSION: In this study, we report the physicochemical properties, subcellular localization, and biological function of SARS-COV-2 N protein. The 12 phosphorylated sites and 9 potential protein kinase sites in SARS-COV-2 N protein may serve as promising targets for drug discovery and development for of a recombinant virus vaccine.

A selective sweep in the Spike gene has driven SARS-CoV-2 human adaptation (preprint)

While SARS-CoV-2 likely has animal origins, the viral genetic changes necessary to adapt this animal-derived ancestral virus to humans are largely unknown, mainly due to low levels of sequence polymorphism and the notorious difficulties in experimental manipulations of coronavirus genomes. We scanned more than 182,000 SARS-CoV-2 genomes for selective sweep signatures and found that a distinct footprint of positive selection is located around a non-synonymous change (A1114G; T372A) within the Receptor-Binding Domain of the Spike protein, which likely played a critical role in overcoming species barriers and accomplishing interspecies transmission from animals to humans. Structural analysis indicated that the substitution of threonine with alanine in SARS-CoV-2 concomitantly removes a predicted glycosylation site at N370, resulting in more favorable binding predictions to human ACE2, the cellular receptor. Using a novel bacteria-free cloning system for manipulating RNA virus genomes, we experimentally validated that this SARS-CoV-2-unique substitution significantly increases replication in human cells relative to its putative ancestral variant. Notably, this mutation's impact on virus replication in human cells was much more significant than that of the Spike D614G mutant, which has been widely reported to have been selected for during human-to-human transmission.

Inhibition of PIKfyve kinase prevents infection by EBOV and SARS-CoV-2 (preprint)

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric VSV containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or SARS-CoV-2 (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small molecule inhibitors of the main endosomal Phosphatidylinositol-3-Phosphate/Phosphatidylinositol 5-Kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define new tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.