Activation of SARS-CoV-2 by trypsin-like proteases in the clinical specimens of patients with COVID-19

SARS-CoV-2 enters host cells through the angiotensin converting enzyme 2 (ACE2) receptor and/or transmembrane protease, serine 2 (TMPRSS2). Serine proteases, such as TMPRSS2 and trypsin, promote viral entry. In this study, we investigated whether proteases increased SARS-CoV-2 infectivity using pseudotyped viruses and clinical specimens from patients with COVID-19. First, we investigated how trypsin increased infectivity using the pseudotyped virus. Our findings revealed that trypsin increased infectivity after the virus was adsorbed on the cells, but no increase in infectivity was observed when the virus was treated with trypsin. We examined the effect of trypsin on SARS-CoV-2 infection in clinical specimens and found that the infectivity of the SARS-CoV-2 delta variant increased 36,000-fold after trypsin treatment. By contrast, the infectivity of SARS-CoV-2 omicron variant increased to less than 20-fold in the clinical specimens. Finally, infectivity of clinical specimens containing culture supernatants of Fusobacterium necrophorum was increased from several- to 10-fold. Because SARS-CoV-2 infectivity increases in the oral cavity, which may contain anaerobic bacteria, keeping the oral cavities clean may help prevent SARS-CoV-2 infection. Importance In this study, we examined whether trypsin-like proteases increased the infectivity of SARS-CoV-2. We found that trypsin-like proteases increased the infectivity of both the pseudotyped viruses and the live virus in the clinical specimens. The increase in infectivity was significantly higher for the delta than the omicron variant. A large amount of protease in the oral cavity during SARS-CoV-2 infection is expected to increase infectivity. Therefore, keeping the oral cavity clean is important for preventing infection. ### Competing Interest Statement The authors have declared no competing interest.