Human Alphacoronavirus Universal Primers for Genome Amplification and Sequencing.

Rapid and accurate sequencing covering the entire genome is essential to identify genetic variations of viral pathogens. However, due to the low viral titers in clinical samples, certain amplification steps are required for viral genome sequencing. At present, there are no universal primers available for alphacoronaviruses and that, since these viruses have diverse strains, new primers specific to the target strain must be continuously developed for sequencing. Thus, in this study, we aimed to develop a universal primer set valid for all human alphacoronaviruses and applicable to samples containing trace amounts of the virus. To this aim, we designed overlapping primer pairs capable of amplifying the entire genome of all known human alphacoronaviruses. The selected primers, named the AC primer set, were composed of 10 primer pairs stretching over the entire genome of alphacoronaviruses, and produced PCR products of the expected size (3-5 kb) from both the HCoV-229E and HCoV-NL63 strains. After genome amplification, an evaluation using various sequencing platforms was carried out. The amplicon library sequencing data were assembled into complete genome sequences in all sequencing strategies examined in this study. The sequencing accuracy varied depending on the sequencing technology, but all sequencing methods showed a sequencing error of less than 0.01%. In the mock clinical specimen, the detection limit was 10-3 PFU/ml (102 copies/ml). The AC primer set and experimental procedure optimized in this study may enable the fast diagnosis of mutant alphacoronaviruses in future epidemics.

Receptor-binding domain of SARS-CoV-2 spike protein efficiently inhibits SARS-CoV-2 infection and attachment to mouse lung.

COVID-19, caused by a novel coronavirus, SARS-CoV-2, poses a serious global threat. It was first reported in 2019 in China and has now dramatically spread across the world. It is crucial to develop therapeutics to mitigate severe disease and viral spread. The receptor-binding domains (RBDs) in the spike protein of SARS-CoV and MERS-CoV have shown anti-viral activity in previous reports suggesting that this domain has high potential for development as therapeutics. To evaluate the potential antiviral activity of recombinant SARS-CoV-2 RBD proteins, we determined the RBD residues of SARS-CoV-2 using a homology search with RBD of SARS-CoV. For efficient expression and purification, the signal peptide of spike protein was identified and used to generate constructs expressing recombinant RBD proteins. Highly purified RBD protein fused with the Fc domain of human IgG showed potent anti-viral efficacy, which was better than that of a protein fused with a histidine tag. Intranasally pre-administrated RBD protein also inhibited the attachment of SARS-COV-2 to mouse lungs. These findings indicate that RBD protein could be used for the prevention and treatment of SARS-CoV-2 infection.

A Novel Frameshifting Inhibitor Having Antiviral Activity against Zoonotic Coronaviruses.

Recent outbreaks of zoonotic coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have caused tremendous casualties and great economic shock. Although some repurposed drugs have shown potential therapeutic efficacy in clinical trials, specific therapeutic agents targeting coronaviruses have not yet been developed. During coronavirus replication, a replicase gene cluster, including RNA-dependent RNA polymerase (RdRp), is alternatively translated via a process called -1 programmed ribosomal frameshift (-1 PRF) by an RNA pseudoknot structure encoded in viral RNAs. The coronavirus frameshifting has been identified previously as a target for antiviral therapy. In this study, the frameshifting efficiencies of MERS-CoV, SARS-CoV and SARS-CoV-2 were determined using an in vitro -1 PRF assay system. Our group has searched approximately 9689 small molecules to identify potential -1 PRF inhibitors. Herein, we found that a novel compound, 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline (KCB261770), inhibits the frameshifting of MERS-CoV and effectively suppresses viral propagation in MERS-CoV-infected cells. The inhibitory effects of 87 derivatives of furo[2,3-b]quinolines were also examined showing less prominent inhibitory effect when compared to compound KCB261770. We demonstrated that KCB261770 inhibits the frameshifting without suppressing cap-dependent translation. Furthermore, this compound was able to inhibit the frameshifting, to some extent, of SARS-CoV and SARS-CoV-2. Therefore, the novel compound 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline may serve as a promising drug candidate to interfere with pan-coronavirus frameshifting.

Real-Time Mild and Moderate COVID-19 Human Body Temperature Detection Using Artificial Intelligence Coronavirus Antigens: Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens (Adv. Biology 4/2021)

Summary Day by day, COVID-19 cases are increasing all over the world. Without a proper vaccine to control the disease, the only solution so far is social distancing and identifying the disease at an early stage. In more than 80% of confirmed cases there are only mild symptoms, like fever;therefore, we have to check the body temperature of people in public places like shopping malls, hotels, airports, schools and universities, etc. In this chapter we propose contactless temperature (CT) measurement utilizing thermal (TS), RGB, and 3D sensors. We also propose a fever location camera (FLC) which gives high-quality estimates from up to 2 or 3 meters away. Using cutting-edge technology, the fever location framework (FLF) estimates the internal heat level of individuals in groups of three or four by checking and filtering their face temperatures. If a high temperature is identified, the framework sounds an alarm or cautioning message, which has propelled face recognition technology. The framework, which is based on the investigation of face temperature, guarantees high-quality estimations. Using facial recognition (FR) likewise limits false readings;for example, an individual carrying a hot beverage. Using a devoted programming stage, a signal can be set to inform us of unusual temperatures. It can precisely recognize the facial temperature (FT) of numerous individuals quickly, with an exactness of ≤ 0.3 °C. Temperature recognition range can be set with the ideal location of up to 3 meters in the framework highlighted by a bi-directional double-channel (infrared light + visible light) camera utilizing a heated sensor and low level interference signals. The production of biomolecules that require human-specific lipid environments is extremely useful for basic research and medical applications. In article number 2000154, Seong-Jun Kim, Jae-Sung Woo, Sangsu Bae, and co-workers integrate multiple proteins or virus antigens into defined transcriptional hotspots in the human genome via a homology-independent targeted insertion method using CRISPR nucleases. This system is similar to a production pipeline of biomolecules in a factory controlled by CRISPR.

Coronavirus Antigens: Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens (Adv. Biology 4/2021)

The production of biomolecules that require human-specific lipid environments is extremely useful for basic research and medical applications. In article number 2000154, Seong-Jun Kim, Jae-Sung Woo, Sangsu Bae, and co-workers integrate multiple proteins or virus antigens into defined transcriptional hotspots in the human genome via a homology-independent targeted insertion method using CRISPR nucleases. This system is similar to a production pipeline of biomolecules in a factory controlled by CRISPR.

Comparison of Digital PCR and Quantitative PCR with Various SARS-CoV-2 Primer-Probe Sets.

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.

Development of a SARS-CoV-2-specific biosensor for antigen detection using scFv-Fc fusion proteins.

Coronavirus disease 2019 (COVID-19) is a newly emerged human infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In a global pandemic, development of a cheap, rapid, accurate, and easy-to-use diagnostic test is necessary if we are to mount an immediate response to this emerging threat. Here, we report the development of a specific lateral flow immunoassay (LFIA)-based biosensor for COVID-19. We used phage display technology to generate four SARS-CoV-2 nucleocapsid protein (NP)-specific single-chain variable fragment-crystallizable fragment (scFv-Fc) fusion antibodies. The scFv-Fc antibodies bind specifically and with high affinity to the SARS-CoV-2 NP antigen, but not to NPs of other coronaviruses. Using these scFv-Fc antibodies, we screened three diagnostic antibody pairs for use on a cellulose nanobead (CNB)-based LFIA platform. The detection limits of the best scFv-Fc antibody pair, 12H1 as the capture probe and 12H8 as the CNB-conjugated detection probe, were 2 ng antigen protein and 2.5 × 104 pfu cultured virus. This LFIA platform detected only SARS-CoV-2 NP, not NPs from MERS-CoV, SARS-CoV, or influenza H1N1. Thus, we have successfully developed a SARS-CoV-2 NP-specific rapid diagnostic test, which is expected to be a simple and rapid diagnostic test for COVID-19.

Repurposing Screens of FDA-Approved Drugs Identify 29 Inhibitors of SARS-CoV-2.

COVID-19, caused by the novel coronavirus SARS-CoV-2, has spread globally and caused serious social and economic problems. The WHO has declared this outbreak a pandemic. Currently, there are no approved vaccines or antiviral drugs that prevent SARS-CoV-2 infection. Drugs already approved for clinical use would be ideal candidates for rapid development as COVID-19 treatments. In this work, we screened 1,473 FDA-approved drugs to identify inhibitors of SARS-CoV-2 infection using cell-based assays. The antiviral activity of each compound was measured based on the immunofluorescent staining of infected cells using anti-dsRNA antibody. Twenty-nine drugs among those tested showed antiviral activity against SARS-CoV-2. We report this new list of inhibitors to quickly provide basic information for consideration in developing potential therapies.

Progress and Challenges in the Development of COVID-19 Vaccines and Current Understanding of SARS-CoV-2- Specific Immune Responses.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading globally, and the WHO has declared this outbreak a pandemic. Vaccines are an effective way to prevent the rapid spread of COVID-19. Furthermore, the immune response against SARS-CoV-2 infection needs to be understood for the development of an efficient and safe vaccine. Here, we review the current understanding of vaccine targets and the status of vaccine development for COVID-19. We also describe host immune responses to highly pathogenic human coronaviruses in terms of innate and adaptive immunities.

Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor.

Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously called 2019-nCoV). Based on the rapid increase in the rate of human infection, the World Health Organization (WHO) has classified the COVID-19 outbreak as a pandemic. Because no specific drugs or vaccines for COVID-19 are yet available, early diagnosis and management are crucial for containing the outbreak. Here, we report a field-effect transistor (FET)-based biosensing device for detecting SARS-CoV-2 in clinical samples. The sensor was produced by coating graphene sheets of the FET with a specific antibody against SARS-CoV-2 spike protein. The performance of the sensor was determined using antigen protein, cultured virus, and nasopharyngeal swab specimens from COVID-19 patients. Our FET device could detect the SARS-CoV-2 spike protein at concentrations of 1 fg/mL in phosphate-buffered saline and 100 fg/mL clinical transport medium. In addition, the FET sensor successfully detected SARS-CoV-2 in culture medium (limit of detection [LOD]: 1.6 × 101 pfu/mL) and clinical samples (LOD: 2.42 × 102 copies/mL). Thus, we have successfully fabricated a promising FET biosensor for SARS-CoV-2; our device is a highly sensitive immunological diagnostic method for COVID-19 that requires no sample pretreatment or labeling.