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.

SARS-CoV-2 Infection of Microglia Elicits Proinflammatory Activation and Apoptotic Cell Death.

Accumulating evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes various neurological symptoms in patients with coronavirus disease 2019 (COVID-19). The most dominant immune cells in the brain are microglia. Yet, the relationship between neurological manifestations, neuroinflammation, and host immune response of microglia to SARS-CoV-2 has not been well characterized. Here, we reported that SARS-CoV-2 can directly infect human microglia, eliciting M1-like proinflammatory responses, followed by cytopathic effects. Specifically, SARS-CoV-2 infected human microglial clone 3 (HMC3), leading to inflammatory activation and cell death. RNA sequencing (RNA-seq) analysis also revealed that endoplasmic reticulum (ER) stress and immune responses were induced in the early, and apoptotic processes in the late phases of viral infection. SARS-CoV-2-infected HMC3 showed the M1 phenotype and produced proinflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor α (TNF-α), but not the anti-inflammatory cytokine IL-10. After this proinflammatory activation, SARS-CoV-2 infection promoted both intrinsic and extrinsic death receptor-mediated apoptosis in HMC3. Using K18-hACE2 transgenic mice, murine microglia were also infected by intranasal inoculation of SARS-CoV-2. This infection induced the acute production of proinflammatory microglial IL-6 and TNF-α and provoked a chronic loss of microglia. Our findings suggest that microglia are potential mediators of SARS-CoV-2-induced neurological problems and, consequently, can be targets of therapeutic strategies against neurological diseases in patients with COVID-19. IMPORTANCE Recent studies reported neurological and cognitive sequelae in patients with COVID-19 months after the viral infection with several symptoms, including ageusia, anosmia, asthenia, headache, and brain fog. Our conclusions raise awareness of COVID-19-related microglia-mediated neurological disorders to develop treatment strategies for the affected patients. We also indicated that HMC3 was a novel human cell line susceptible to SARS-CoV-2 infection that exhibited cytopathic effects, which could be further used to investigate cellular and molecular mechanisms of neurological manifestations of patients with COVID-19.

Computational design of a neutralizing antibody with picomolar binding affinity for all concerning SARS-CoV-2 variants.

Coronavirus disease 2019, caused by SARS-CoV-2, remains an on-going pandemic, partly due to the emergence of variant viruses that can "break-through" the protection of the current vaccines and neutralizing antibodies (nAbs), highlighting the needs for broadly nAbs and next-generation vaccines. We report an antibody that exhibits breadth and potency in binding the receptor-binding domain (RBD) of the virus spike glycoprotein across SARS coronaviruses. Initially, a lead antibody was computationally discovered and crystallographically validated that binds to a highly conserved surface of the RBD of wild-type SARS-CoV-2. Subsequently, through experimental affinity enhancement and computational affinity maturation, it was further developed to bind the RBD of all concerning SARS-CoV-2 variants, SARS-CoV-1 and pangolin coronavirus with pico-molar binding affinities, consistently exhibited strong neutralization activity against wild-type SARS-CoV-2 and the Alpha and Delta variants. These results identify a vulnerable target site on coronaviruses for development of pan-sarbecovirus nAbs and vaccines.

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.

Computational Design of Potent D-Peptide Inhibitors of SARS-CoV-2.

Blocking the association between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) is an attractive therapeutic approach to prevent the virus from entering human cells. While antibodies and other modalities have been developed to this end, d-amino acid peptides offer unique advantages, including serum stability, low immunogenicity, and low cost of production. Here, we designed potent novel D-peptide inhibitors that mimic the ACE2 α1-binding helix by searching a mirror-image version of the PDB. The two best designs bound the RBD with affinities of 29 and 31 nM and blocked the infection of Vero cells by SARS-CoV-2 with IC50 values of 5.76 and 6.56 µM, respectively. Notably, both D-peptides neutralized with a similar potency the infection of two variants of concern: B.1.1.7 and B.1.351 in vitro. These potent D-peptide inhibitors are promising lead candidates for developing SARS-CoV-2 prophylactic or therapeutic treatments.

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.

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.