Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 mice

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. To date, it is estimated that over 113 million individuals have been infected with SARS-CoV-2 and over 2.5 million human deaths have been recorded world-wide. Currently, three vaccines have been approved by the Food and Drug Administration (FDA) for emergency use. However much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse genetics system approach to successfully engineer recombinant (r)SARS-CoV-2 where we individually removed the viral 3a, 6, 7a, 7b, and 8 ORF proteins, and characterized these recombinant viruses in vitro and in vivo. We observed differences in plaque morphology, with the ORF deficient (delORF) viruses producing smaller plaques that than those of the wild-type (WT) rSARS-CoV-2. However, growth kinetics of the delORF viruses were similar to those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin converting enzyme 2 (hACE2) mice with the delORF rSARS-CoV-2 identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while deltaORF7a, delORF7b and delORF8 rSARS-CoV-2 induced comparable pathology than to rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse genetics system to generate rSARS-CoV-2 and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live-attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.