Investigation on the pathogenesis and novel intervention targets of severe acute respiratory syndrome coronavirus 2 infection

Abstract

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<0.01). Moreover, SARS-CoV-2 replicated in U251 (neuron) cells (P<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<0.050). These in vitro findings were then validated in our newly established ex vivo 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 in vitro and in vivo 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 (Mus musculus) and street rats (Rattus norvegicus). These findings have important implications for the potential animal species that facilitate SARS-CoV-2 transmission.

In Chapter 4, the in vitro and in vivo 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 in vivo. 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 in vitro, ex vivo, and in vivo 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 in vitro and/or in vivo. 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.