Characterizing replication kinetics and plaque production of type I feline infectious peritonitis virus in three feline cell lines

Abstract

Investigating type I feline coronaviruses (FCoVs) in tissue culture is critical for understanding the basic virology, pathogenesis, and virus-host interactome of these important veterinary pathogens. This has been a perennial challenge as type I FCoV strains do not easily adapt to cell culture. Here we characterize replication kinetics and plaque formation of a model type I strain FIPV Black in Fcwf-4 cells established at Cornell University (Fcwf-4 CU). We determined that maximum virus titers (>10 pfu/mL) were recoverable from infected Fcwf-4 CU cell-free supernatant at 20 h post-infection. Type I FIPV Black and both biotypes of type II FCoV formed uniform and enumerable plaques on Fcwf-4 CU cells. Therefore, these cells were employable in a standardized plaque assay. Finally, we determined that the Fcwf-4 CU cells were morphologically distinct from feline bone marrow-derived macrophages and were less sensitive to exogenous type I interferon than were Fcwf-4 cells purchased from ATCC.

Generating and evaluating type I interferon receptor-deficient and feline TMPRSS2-expressing cells for propagating serotype I feline infectious peritonitis virus

“…1C). In AK-D 2.2 cells, plaques were more easily identified at 72 hpi, and plaque sizes were comparable to those on Fcwf-4 CU cells at 48 hpi as we recently reported (O'Brien et al, 2018). It was difficult to accurately determine virus titer using wildtype AK-D cells, as the plaques were too small to count accurately at both time points and led to an underestimation of PFU/mL.…”

“…To our knowledge, such IFN-nonresponsive feline cell lines were not available and studies using gene-modifying tools such as Crispr/Cas had not been performed in feline cells permissive to FCoVs. In a previous study, we characterized the growth kinetics of FIPV Black in AK-D and Fcwf-4 CU cells and demonstrated that rapid growth of wild-type serotype I FIPV could be achieved given the right cell type and conditions (O'Brien et al, 2018). However, these cells still produced IFN upon stimulation suggesting that neither cell line would be appropriate for recovery of viruses attenuated by deletion on IFN antagonists.…”

“…In addition to disrupting IFN signaling in AK-D cells, we also targeted the IFNαR2 in the feline macrophage-like cell line, Fcwf-4 CU. These cells are a more physiologically relevant cell type in which to study FIPV and grow the Black strain to high, cell-free titers (O'Brien et al, 2018). To knock down the IFNαR2, parental Fcwf-4 CU cells were transduced with lentiviruses carrying pLentiCRISPRv2 encoding an sgRNA targeting the second exon of the feline ifnαr2 gene (2.2), the same sequence targeted in AK-D cells.…”

“…In this study, we sought to eliminate IFN-responsiveness in two serotype I FCoV-permissive cell lines: AK-D, immortalized cells derived from feline lung tissue, and Fcwf-4 Cornell University (CU) (O'Brien et al, 2018), a feline macrophage-like cell line. To this end, we employed the Crispr/Cas gene editing system to disrupt domain 2 of the type I IFN receptor (IFNαR2) in both AK-D and Fcwf-4 CU cell lines.…”

Entry of Scotophilus Bat Coronavirus-512 and Severe Acute Respiratory Syndrome Coronavirus in Human and Multiple Animal Cells

“…In this study, entry efficiencies of Scotophilus bat CoV-512 and SARS-CoV in 11 different cells were evaluated using a lentivirus-based pseudovirus system. HEK-293T cells were selected as they have been used for the production of pseudoviruses; additionally, several cell lines were selected because they were used for the isolation and maintenance of viruses in other studies, such as Vero and PK15 cells for PEDV [17], MDCK cells for influenza viruses [18], and Fcwf-4 cells for FCoV [19]. Given that CoVs tend to replicate in the epithelial cells of enteric and respiratory tracts, Caco-2 and IEC-6 were selected.…”