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

Abstract

SARS-CoV-2 enters host cells through the angiotensin converting enzyme 2 (ACE2) receptor and/or transmembrane protease, serine 2 (TMPRSS2). 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, using five clinical specimens containing delta variants, enhancement of viral infectivity was evaluated in the presence of the culture supernatant of several anaerobic bacteria. As a result, viral infectivities of all the clinical specimens containing culture supernatants of Fusobacterium necrophorum were significantly increased from several- to tenfold. 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.

Figure 1

Effect of trypsin treatment on SARS-CoV-2pv infectivity. (A) The increased infectivity of viruses treated with trypsin at the indicated concentration after inoculation of cells with the Wuhan, alpha, delta, and omicron variants of SARS-CoV-2pv or VSVpv was calculated based on the infectivity of the trypsin-untreated virus. (B) The viral supernatants of Wuhan, alpha, delta, and omicron variants of SARS-CoV-2pv or VSVpv were inoculated into cells after treatment with trypsin at the indicated concentrations, and the increased infectivity of viruses was calculated based on the infectivity of the trypsin-untreated virus. The results shown are from three independent assays, with error bars representing standard deviations. Significance was determined by the Dunnett’s test in comparison to the data of no trypsin treatment: *P < 0.05; **P < 0.01.

Figure 2

Effect of trypsin treatment on syncytium formation in cells expressing S protein of SARS-CoV-2. Syncytium formation in (A) VeroE6 and (B) 293 T cells transiently expressing S protein of SARS-CoV-2. The eGFP-expressing plasmid was co-transfected for visualization. Syncytium formation was imaged using a fluorescence microscope.

Figure 3

Effect of trypsin treatment on SARS-CoV-2 infection using clinical specimens. Comparison of clinical specimens of the (A) delta (n = 56) and (B) omicron (n = 44) variants by Ct value at PCR testing. The load of viral genome after viral amplification with trypsin treatment is expressed as the infection enhancement ratio, where the amount of viral genome after viral amplification without trypsin treatment is set as 1. Filled circle; Relative viral genome quantity of trypsin (400 μg/mL)-treated specimens, open triangle; Relative viral genome quantity of specimens not treated with trypsin. (C) Boxplot analysis combining the results of (A) and (B). Regarding the ratio, the same as (A) and (B) without trypsin treatment is set as 1. Significance was determined by the Mann–Whitney U-test: ***P < 0.001.

Figure 4

Effect of anaerobic oral bacterial culture supernatant on the infectivity of SARS-CoV-2 using clinical specimens. After adding the five clinical specimens (A–E) containing the SARS-CoV-2 delta variant to the cells, the increase in infectivity of viruses was calculated after treatment with culture supernatants of (A) Prevotella melaninogenica, (B) Prevotella intermedia, and (C) Fusobacterium necrophorum based on the infectivity of the virus treated in the control medium. The results shown are from three independent assays, with error bars representing standard deviations. Significance was determined by the Student’s t-test in comparison to the data of no treatment (Ctrl): *P < 0.05; **P < 0.01.