DOI: 10.25442/hku.20976358.v1
title: Supporting data for “INVESTIGATION ON THE PATHOGENESIS AND NOVEL INTERVENTION TARGETS OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 INFECTION”

description: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first emerged in late 2019 and rapidly disseminated globally. Despite being similar in their genomic compositions and the ability to cause pneumonia, SARS-CoV-2 and SARS-CoV-1 differ in their capability to cause extrapulmonary (such as neurological and gastrointestinal) manifestations. Importantly, the mortality rate of COVID-19 has been generally less than that of SARS. The virological characteristics of SARS-CoV-2 that contribute to these clinical differences were unknown at the early stage of the pandemic. In Chapter 2, the differential cellular susceptibilities, replication kinetics, and cell damage profiles of SARS-CoV-2 and SARS-CoV-1 were systematically investigated in 25 different cell lines. SARS-CoV-2 infected and replicated to comparable levels in human Calu3 (lung) and Caco2 (colon) cells, whereas SARS-CoV-1 replicated more efficiently in Caco2 cells than in Calu3 cells (P&lt;0.01). Moreover, SARS-CoV-2 replicated in U251 (neuron) cells (P&lt;0.05). SARS-CoV-2 consistently caused significantly delayed and lower levels of cell damage than did SARS-CoV-1 in VeroE6 and FRhK4 (non-human primate kidney) cells (P&lt;0.050). These <em>in vitro</em> findings were then validated in our newly established <em>ex vivo</em> human lung and intestinal tissue organ culture models. These results helped to explain the lower incidence of diarrhea, decreased disease severity, and reduced mortality in COVID-19 patients than SARS patients. In Chapter 3, a combination of <em>in vitro</em> and <em>in vivo</em> models was used to investigate the virological characteristics of emerging SARS-CoV-2 variants with potentially altered species tropism, transmissibility, virulence, and/or immunoevasivness. The results showed that SARS-CoV-2 variants containing the spike N501Y mutation, but not wild-type SARS-CoV-2, could efficiently utilize murine angiotensin converting enzyme 2 (ACE2) for virus entry and infect wild-type C57B6 mice (<em>Mus musculus</em>) and street rats (<em>Rattus norvegicus</em>). These findings have important implications for the potential animal species that facilitate SARS-CoV-2 transmission. In Chapter 4, the <em>in vitro</em> and <em>in vivo</em> characteristics of the emerging Omicron (B.1.1.529) variant were investigated. Compared with wild-type and preceding variants of SARS-CoV-2, the replication of Omicron was significantly attenuated in Calu-3 and Caco-2 cells, and in both the upper and lower respiratory tracts of K18-human ACE2-transgenic mice which was associated with attenuated pathogenicity <em>in vivo</em>. Mechanistically, Omicron was inefficient in utilizing the host protease transmembrane serine protease 2 for virus entry. These findings helped to explain the lower disease severity caused by Omicron clinically compared with previous SARS-CoV-2 strains. Building on the <em>in vitro, ex vivo,</em> and <em>in vivo</em> models in Chapter 2 to 4, potential intervention strategies for COVID-19 were explored in Chapter 5. Metabolomics analysis identified adenosine triphosphate citrate lyase (ACLY) as a key host factor involved in the replication of SARS-CoV-2. Inhibition of ACLY by either siRNA knockdown or the ACLY inhibitor SB 204990 resulted in significant reduction in SARS-CoV-2 viral loads <em>in vitro</em> and/or <em>in vivo.</em> These results highlighted the potential of targeting ACLY as a potential anti-SARS-CoV-2 strategy. In summary, the novel findings in this thesis advance the understanding on the pathogenesis and potential interventions of SARS-CoV-2 infection.