Ocular tropism of SARS-CoV-2 in animal models with retinal inflammation via neuronal invasion following intranasal inoculation.

Although ocular manifestations are reported in patients with COVID-19, consensus on ocular tropism of SARS-CoV-2 is lacking. Here, we infect K18-hACE2 transgenic mice with SARS-CoV-2 using various routes. We observe ocular manifestation and retinal inflammation with production of pro-inflammatory cytokines in the eyes of intranasally (IN)-infected mice. Intratracheal (IT) infection results in dissemination of the virus from the lungs to the brain and eyes via trigeminal and optic nerves. Ocular and neuronal invasions are confirmed using intracerebral (IC) infection. Notably, the eye-dropped (ED) virus does not cause lung infection and becomes undetectable with time. Ocular and neurotropic distribution of the virus in vivo is evident in fluorescence imaging with an infectious clone of SARS-CoV-2-mCherry. The ocular tropic and neuroinvasive characteristics of SARS-CoV-2 are confirmed in wild-type Syrian hamsters. Our data can improve the understanding regarding viral transmission and clinical characteristics of SARS-CoV-2 and help in improving COVID-19 control procedures.

Immunological and Pathological Peculiarity of Severe Acute Respiratory Syndrome Coronavirus 2 Beta Variant.

Diverse severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have emerged since the beginning of the COVID-19 pandemic. We investigated the immunological and pathological peculiarity of the SARS-CoV-2 beta variant of concern (VoC) compared to the ancestral strain. Comparative analysis of phenotype and pathology revealed that the beta VoC induces slower disease progression and a prolonged presymptomatic period in the early stages of SARS-CoV-2 infection but ultimately causes sudden death in the late stages of infection in the K18-hACE2 mouse model. The beta VoC induced enhanced activation of CXCL1/2-CXCR2-NLRP3-IL-1ß signal cascade accelerating neutrophil recruitment and lung pathology in beta variant-infected mice, as evidenced by multiple analyses of SARS-CoV-2-induced inflammatory cytokines and transcriptomes. CCL2 was one of the most highly secreted cytokines in the early stages of infection. Its blockade reduced virus-induced weight loss and delayed mortality. Our study provides a better understanding of the variant characteristics and need for treatment. IMPORTANCE Since the outbreak of COVID-19, diverse SARS-CoV-2 variants have been identified. These variants have different infectivity and transmissibility from the ancestral strains. However, underlying molecular mechanisms have not yet been fully elucidated. In our study, the beta variant showed distinct pathological conditions and cytokine release kinetics from an ancestral strain in a mouse model. It was associated with higher neutrophil recruitment by increased levels of CXCL1/2, CXCR2, and interleukin 1ß (IL-1ß) at a later stage of viral infection. Our study will provide a better understanding of SARS-CoV-2 pathogenesis.

Current Updates on COVID-19 Vaccines and Therapeutics: As of June 2022.

Since COVID-19, caused by SARS-CoV-2 infection, has become a global issue, many vaccines and therapeutic candidates have been developed or are being developed against the COVID-19 endemic and the next wave. However, it is difficult to overcome the spread and mutation rate of SARS-CoV-2 in the COVID-19 pandemic because development of vaccines and therapeutics involves considerable social cost and time, as well as research capabilities. Thus, assessing the development status of these agents is important for advancing efficient research strategies. In this review, we summarize the status of 141 vaccines and 345 therapeutic candidates under development worldwide, according to their development stage and characteristics. As of June 2022, 32 vaccines and 12 therapeutics have been approved for emergency use. Although the development of four of these therapeutics was terminated owing to their low efficacy against various variants of SARS-CoV-2, many new candidates that have completed phase 3 clinical trials have been awaiting phase 4 clinical trials or full approval by the Food and Drug Administration (FDA). These efforts are expected to contribute to establishing an efficient research strategy to overcome the COVID-19 pandemic and facilitate its transition toward an endemic phase. Electronic Supplementary Material ESM: The online version of this article (doi: 10.1007/s12257-022-0188-4) contains supplementary material, which is available to authorized users.

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.

Escape and Over-Activation of Innate Immune Responses by SARS-CoV-2: Two Faces of a Coin.

In the past 20 years, coronaviruses (CoVs), including SARS-CoV-1, MERS-CoV, and SARS-CoV-2, have rapidly evolved and emerged in the human population. The innate immune system is the first line of defense against invading pathogens. Multiple host cellular receptors can trigger the innate immune system to eliminate invading pathogens. However, these CoVs have acquired strategies to evade innate immune responses by avoiding recognition by host sensors, leading to impaired interferon (IFN) production and antagonizing of the IFN signaling pathways. In contrast, the dysregulated induction of inflammasomes, leading to uncontrolled production of IL-1 family cytokines (IL-1ß and IL-18) and pyroptosis, has been associated with COVID-19 pathogenesis. This review summarizes innate immune evasion strategies employed by SARS-CoV-1 and MERS-CoV in brief and SARS-CoV-2 in more detail. In addition, we outline potential mechanisms of inflammasome activation and evasion and their impact on disease prognosis.

Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern.

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alpha's small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic.

In Vitro Replication Inhibitory Activity of Xanthorrhizol against Severe Acute Respiratory Syndrome Coronavirus 2.

In spite of the large number of repositioned drugs and direct-acting antivirals in clinical trials for the management of the ongoing COVID-19 pandemic, there are few cost-effective therapeutic options for severe acute respiratory syndrome (SARS) coronavirus 2 (SCoV2) infection. In this paper, we show that xanthorrhizol (XNT), a bisabolane-type sesquiterpenoid compound isolated from the Curcuma xanthorrhizza Roxb., a ginger-line plant of the family Zingiberaceae, displays a potent antiviral efficacy in vitro against SCoV2 and other related coronaviruses, including SARS-CoV-1 (SCoV1) and a common cold-causing human coronavirus. XNT reduced infectious SCoV2 titer by ~3-log10 at 20 µM and interfered with the replication of the SCoV1 subgenomic replicon, while it had no significant antiviral effects against hepatitis C virus and noroviruses. Further, XNT exerted similar antiviral functions against SCoV2 variants, such as a GH clade strain and a delta strain currently predominant worldwide. Neither SCoV2 entry into cells nor the enzymatic activity of viral RNA polymerase (Nsp12), RNA helicase (Nsp13), or the 3CL main protease (Nsp5) was inhibited by XNT. While its CoV replication inhibitory mechanism remains elusive, our results demonstrate that the traditional folk medicine XNT could be a promising antiviral candidate that inhibits a broad range of SCoV2 variants of concern and other related CoVs.

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.

Colorimetric RT-LAMP Methods to Detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Standard diagnostic methods of Coronavirus Disease 2019 (COVID-19) rely on RT-qPCR technique which have limited point-of-care test (POCT) potential due to necessity of dedicated equipment and specialized personnel. LAMP, an isothermal nucleic acid amplification test (NAAT), is a promising technique that may substitute RT-qPCR for POCT of genomic materials. Here, we provide a protocol to perform reverse transcription LAMP targeting SARS-CoV-2. We adopted both real-time fluorescence detection and end-point colorimetric detection approaches. Our protocol would be useful for screening diagnosis of COVID-19 and be a baseline for development of improved POCT NAAT.

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.

Robust and persistent SARS-CoV-2 infection in the human intestinal brush border expressing cells.

Studies on patients with the coronavirus disease-2019 (COVID-19) have implicated that the gastrointestinal (GI) tract is a major site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We established a human GI tract cell line model highly permissive to SARS-CoV-2. These cells, C2BBe1 intestinal cells with a brush border having high levels of transmembrane serine protease 2 (TMPRSS2), showed robust viral propagation, and could be persistently infected with SARS-CoV-2, supporting the clinical observations of persistent GI infection in COVID-19 patients. Ectopic expression of viral receptors revealed that the levels of angiotensin-converting enzyme 2 (ACE2) expression confer permissiveness to SARS-CoV-2 infection, and TMPRSS2 greatly facilitates ACE2-mediated SARS-CoV-2 dissemination. Interestingly, ACE2 but not TMPRSS2 expression was significantly promoted by enterocytic differentiation, suggesting that the state of enterocytic differentiation may serve as a determining factor for viral propagation. Thus, our study sheds light on the pathogenesis of SARS-CoV-2 in the GI tract.

Comparative Analysis of Primer-Probe Sets for RT-qPCR of COVID-19 Causative Virus (SARS-CoV-2).

Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also previously known as 2019-nCoV). Within 8 months of the outbreak, more than 10,000,000 cases of COVID-19 have been confirmed worldwide. Since human-to-human transmission occurs easily and the rate of human infection is rapidly increasing, sensitive and early diagnosis is essential to prevent a global outbreak. Recently, the World Health Organization (WHO) announced various primer-probe sets for SARS-CoV-2 developed at different institutions: China Center for Disease Control and Prevention (China CDC, China), Charité (Germany), The University of Hong Kong (HKU, Hong Kong), National Institute of Infectious Diseases in Japan (Japan NIID, Japan), National Institute of Health in Thailand (Thailand NIH, Thailand), and US CDC (USA). In this study, we compared the ability to detect SARS-CoV-2 RNA among seven primer-probe sets for the N gene and three primer-probe sets for the Orf1 gene. The results revealed that "NIID_2019-nCOV_N" from the Japan NIID and "ORF1ab" from China CDC represent a recommendable performance of RT-qPCR analysis for SARS-CoV-2 molecular diagnostics without nonspecific amplification and cross-reactivity for hCoV-229E, hCoV-OC43, and MERS-CoV RNA. Therefore, the appropriate combination of NIID_2019-nCOV_N (Japan NIID) and ORF1ab (China CDC) sets should be selected for sensitive and reliable SARS-CoV-2 molecular diagnostics.

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.

Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

The coronavirus disease 2019 (COVID-19) pandemic now has >2,000,000 confirmed cases worldwide. COVID-19 is currently diagnosed using quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. We developed and evaluated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. RT-LAMP assays reported in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay to enable higher throughput.