SARS-CoV-2의 종간 전파 양상, 괴사성 세포사멸 (Necroptosis)에 의한 신경병증 발생 기전 및 Dieckol의 항바이러스 효능에 관한 통합적 연구 = An Integrated Study on SARS-CoV-2: Interspecies Transmission, Necroptosis-Mediated Neuropathogenesis, and Antiviral Efficacy of Dieckol

다국어 초록 (Multilingual Abstract)

The global spread of coronavirus disease 2019 (COVID-19) has resulted in an unprecedented pandemic and is caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 has zoonotic characteristics, requring a comprehensive understanding of its pathogenicity and tissue tropism mechanisms in various species. This study aimed to the integration of the pathophysiology of COVID-19 by investigating the susceptibility and characteristics of various animal species to SARS-CoV-2, identifying cell death mechanisms related to central nervous system (CNS), and suggesting the therapeutic potential of natural products extracted from seaweed.
As a result of SARS-CoV-2 antibodies in domestic cats, dogs, cattle and black goats using ELISA, SARS-CoV-2 antibodies were detected positive in about 2-4% of cats, dogs, cattle, and black goats. qRT-PCR was confirmed positive for one individual each in a cat, cattle, and black goat. This suggests that various animal species in environments close to humans can become hosts for SARS-CoV-2, and emphasizes the need to expand the surveillance system for zoonotic infectious diseases. Additionally, the virus was isolated and characterized from cats that showed highly positive results in qRT-PCR and ELISA. The results obtained cross-species infection through isolated virus and whole-genome analysis. This supports that cross-species spillover from humans to animals can realistically occur, and increases the importance of infection surveillance and epidemiology in household animals.
Moreover, using transgenic mice expressing the human ACE2 receptor (K18-hACE2), it was confirmed that SARS-CoV-2 infection induces severe pathological changes, inflammatory response and necroic cell death in brain tissue. These characteristics were particularly evident in brain neurons, and increased expression of genes in the necroptosis pathway, such as ZBP1, RIPK3, and MLKL, was observed. These features were particularly revealed in brain neurons, where increased expression of genes within the necroptosis pathway, such as ZBP1, RIPK3, and MLKL, was observed. This shows that SARS-CoV-2 can induce not only respiratory pathology but also pathological damage to nervous tissue. Finally, dieckol, a polyphenol compound derived from marine brown algae, was confirmed to have antiviral and neuroprotective effects. Dieckol showed high binding affinity to the major enzyme protein (RdRp, 3CLpro) of SARS-CoV-2, and simultaneously showed inhibitory effects on virus proliferation and SARS-CoV-2 induced cell death in vivo and in vitro. In particular, Dieckol has shown therapeutic potential in alleviating necroptosis-based neuropathy by inhibiting the expression of SARS-CoV-2 induced p-MLKL and inflammatory cytokines. Collectively, this study provides insights into the interspecies transmission dynamics and CNS infection mechanisms of SARS-CoV-2 and identifies a natural-product-based antiviral candidate. These findings are expected to contribute to a multifaceted understanding of COVID-19 and establishment of SARS-CoV-2–associated neurological complications.

목차 (Table of Contents)

• Chapter Ⅰ. Backgrounds & overview 1
• 1. Structure and genome of SARS-CoV-2 2
• 2. Transmission of SARS-CoV-2 4
• 3. SARS-CoV-2 symptom and neuroinvasion 6
• 4. SARS-CoV-2 induced cell death 8
• 5. SARS-CoV-2 therapeutic strategy 10
• Reference 12
• Chapter Ⅱ. Demonstration of SARS-CoV-2 exposure in cats, dogs, Korean native cattle and Korean native black goats in South Korea 23
• 1. Introduction 24
• 2. Materials and methods 27
• 2.1. Sample collection methodology 27
• 2.2 Virus and cells 27
• 2.3 RNA extraction and quantitative PCR (qPCR) for reverse transcription 27
• 2.4 Enzyme-linked immunosorbent assay (ELISA) for antibody detection 28
• 2.5 Plaque reduction neutralization test (PRNT) for assessing neutralizing antibodies 29
• 3. Results 31
• 3.1. Detection of SARS-CoV-2 antigen in nasal samples of cats, dogs, cattle and goats 31
• 3.2. Detection of antibodies to SARS-CoV-2 in serum samples of cats, dogs, Korean native cattle and goats 31
• 4. Discussion 33
• Reference 37
• Chapter Ⅲ. Suspected Human-To-Cat spillover of SARS-CoV-2 omicron variant in South Korea 47
• 1. Introduction 48
• 2. Materials and methods 50
• 2.1. Study design and Sample Collection 50
• 2.2. RNA extraction and reverse Transcription Real-Time qPCR 50
• 2.3. Serological analysis 51
• 2.4. Virus isolation and propagation 51
• 2.5. Wohle-Genome Sequencing and phylogenetic analysis 52
• 3. Results 54
• 3.1.Regional screening and isolation of SARS-CoV-2 from a domestic Cat 54
• 3.2. Phylogenetic analysis and spike protein characterization of JBNU-Cat SARS-CoV-2 55
• 4. Discussion 56
• Reference 61
• Chapter Ⅳ. SARS-CoV-2 induces RIPK3 dependent cell death and severe neuroinflammation following neuroinvasion 72
• 1. Introduction 73
• 2. Materials and methods 75
• 2.1. Virus and cells 75
• 2.2. Overexpression model of ACE2 in the SH-SY5Y cell line 75
• 2.3. Mouse experiment 76
• 2.4. Measurement of viral burden and viral protein levels 76
• 2.5. Histopathological analysis and immunohistochmistry 76
• 2.6. TCID50 assay 77
• 2.7. RNA-seq analysis 78
• 2.8. RT2-profiler PCR array 78
• 3. Results 80
• 3.1. Differential organ suceptibility and progressive neurotropism of SARS-CoV-2 in K18-hACE2 mice 80
• 3.2. Differences of neuropathological complications in the brains of K18-hACE2 mice after SARS-CoV-2 infection 81
• 3.3. The early cellular responses to SARS-CoV-2 infection 82
• 3.4. The necrotic pathway is activated during the early brain response to SARS-CoV-2 infection 84
• 3.5. SARS-CoV-2 infection leads to neuronal death via stimulation of necroptosis-related genes 84
• 4. Discussion 86
• Reference 92
• Chapter Ⅴ. A proof study in SARS-CoV-2-infected models for preventing neuronal death events of dieckol-based treatment 120
• 1. Introduction 121
• 2. Materials and methods 123
• 2.1. Virus and cells 123
• 2.2. Chemical extraction 123
• 2.3. Docking simulation 123
• 2.4. Mouse experiment 124
• 2.5. Measurement of viral burden and viral protein levels 124
• 2.6. Histopathological analysis and immunofluorescence analysis 125
• 2.7. TUNEL assay 126
• 2.8. Plaque assay 126
• 2.9. RT2-profiler PCR array 127
• 3. Results 128
• 3.1. Docking prediction and antiviral activity of dieckol 128
• 3.2. Dieckol suppresses SARS-CoV-2 induced cell death in vitro 128
• 3.3. Dieckol prevents SARS-CoV-2-Induced neurotropism in K18-hACE2 mice 129
• 4. Discussion 131
• Reference 133
• 국문초록 153
• Acknowledgement 155