Trypsin promotes porcine deltacoronavirus mediating cell-to-cell fusion in a cell type-dependent manner

Abstract

Porcine deltacoronavirus (PDCoV) is a newly emerging threat to the global porcine industry. PDCoV has been successfully isolated using various medium additives including trypsin, and although we know it is important for viral replication, the mechanism has not been fully elucidated. Here, we systematically investigated the role of trypsin in PDCoV replication including cell entry, cell-to-cell membrane fusion and virus release. Using pseudovirus entry assays, we demonstrated that PDCoV entry is not trypsin dependent. Furthermore, unlike porcine epidemic diarrhea virus (PEDV), in which trypsin is important for the release of virus from infected cells, PDCoV release was not affected by trypsin. We also demonstrated that trypsin promotes PDCoV replication by enhancing cell-to-cell membrane fusion. Most importantly, our study illustrates two distinct spreading patterns from infected cells to uninfected cells during PDCoV transmission, and the role of trypsin in PDCoV replication in cells with different virus spreading types. Overall, these results clarify that trypsin promotes PDCoV replication by mediating cell-to-cell fusion transmission but is not crucial for viral entry. This knowledge can potentially contribute to improvement of virus production efficiency in culture, not only for vaccine preparation but also to develop antiviral treatments.

Tissue-Specific Immunopathology in Fatal COVID-19

“…Although SARS-CoV-2 S-protein expression within lung alveolar epithelial cells was patchy in nature, consistent with possible aspiration or inhalation of virus from the upper respiratory tract ( 20 ), expression at nonpulmonary sites frequently revealed several well-demarcated areas of confluent SARS-CoV-2 S-protein expression within adjacent cells, surrounded by cells with no detectable protein ( Figure 1E ). These “foci of infection,” with numerous affected cells adjacent to unaffected cells, are suggestive of cell-to-cell spread, as reported in other coronavirus and respiratory viruses ( 21 , 22 ).…”

Tissue-specific tolerance in fatal Covid-19

“…These ‘foci of infection’, with numerous affected cells adjacent to unaffected cells, are suggestive of cell-to-cell spread as reported in other coronavirus and respiratory viruses. 14,15…”

The porcine deltacoronavirus accessory protein NS6 is expressed in vivo and incorporated into virions

“…The PDCoV Chinese “Hunan” strain (GenBank accession no. KY513724 ) was used ( Pan et al, 2017 ; Wang et al, 2018 ; Yang et al, 2020a ), which was propagated in LLC-PK1 cells with 5 μg/ml of trypsin at 37 °C with 5% CO2.…”

“…For all CoVs, M and S make up the majority of protein incorporated into the viral envelope. Trimers of S, a type I membrane glycoprotein, form the unique spike structure found on the surface of virions that mediates attachment to the host receptor as well as subsequent membrane fusion ( Wang et al, 2018 ; Yang et al, 2020a ). The S protein is incorporated into virions through noncovalent interactions with the M protein ( Godeke et al, 2000 ).…”