Distinct Molecular Mechanisms Characterizing Pathogenesis of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has continued for over 2 years, following the outbreak of coronavirus-19 (COVID-19) in 2019. It has resulted in enormous casualties and severe economic crises. The rapid development of vaccines and therapeutics against SARS-CoV-2 has helped slow the spread. In the meantime, various mutations in the SARS-CoV-2 have emerged to evade current vaccines and therapeutics. A better understanding of SARS-CoV-2 pathogenesis is a prerequisite for developing efficient, advanced vaccines and therapeutics. Since the outbreak of COVID-19, a tremendous amount of research has been conducted to unveil SARSCoV-2 pathogenesis, from clinical observations to biochemical analysis at the molecular level upon viral infection. In this review, we discuss the molecular mechanisms of SARS-CoV-2 propagation and pathogenesis, with an update on recent advances.

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.

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.

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.

Current Status of Epidemiology, Diagnosis, Therapeutics, and Vaccines for Novel Coronavirus Disease 2019 (COVID-19).

Coronavirus disease 2019 (COVID-19), which causes serious respiratory illness such as pneumonia and lung failure, was first reported in Wuhan, the capital of Hubei, China. The etiological agent of COVID-19 has been confirmed as a novel coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is most likely originated from zoonotic coronaviruses, like SARS-CoV, which emerged in 2002. Within a few months of the first report, SARS-CoV-2 had spread across China and worldwide, reaching a pandemic level. As COVID-19 has triggered enormous human casualties and serious economic loss posing global threat, an understanding of the ongoing situation and the development of strategies to contain the virus's spread are urgently needed. Currently, various diagnostic kits to test for COVID-19 are available and several repurposing therapeutics for COVID-19 have shown to be clinically effective. In addition, global institutions and companies have begun to develop vaccines for the prevention of COVID-19. Here, we review the current status of epidemiology, diagnosis, treatment, and vaccine development for COVID-19.