Fig 1.
Carollia perspicillata primary cultures are susceptible to entry of diverse viruses.
(A) Primary cells were isolated from brain, lung, kidney, liver, and spleen of a single C. perspicillata bat and (B) infected with pseudotypes bearing glycoproteins from the indicated viruses. (C) Cell entry phenotypes of common laboratory cell lines to viral entry with pseudotype panel. Shown are results for six infection replicates per virus. Bat silhouette image produced by Roberto Díaz Sibaja (PyloPic). The data underlying this figure can be found in the S1 Data file available from the journal.
Fig 2.
Carollia perspicillata cell lines immortalized through diverse methods are susceptible to viral entry.
(A) Select primary cells were further transduced with lentiviral vectors representative of different strategies to immortalize cell lines. Transduced (B) brain, (C) lung, (D) kidney, and (E) spleen cells were screened for entry with pseudotyped particles bearing glycoproteins from the indicated viruses. Shown are the data from infection experiments performed in six replicates. The data underlying this figure can be found in the S1 Data file available from the journal.
Fig 3.
Carollia perspicillata cells support infection with replication-competent recombinant VSV (rVSV).
(A) Cp kidney cells immortalized by SV40 T-antigen (CaPsm-K SV40T) were infected with replication-competent VSV (rVSV) pseudotyped with indicated orthohantavirus glycoproteins. Neutralization of rVSV ANDV and HTNV with soluble PCDH1 receptor (sEC1–2) on Cp kidney cells (B), and human U2OS cells for comparison (C). (D) Neutralization of rVSV-ANDV and HTNV on Cp kidney cells by preincubation of cells with an anti-PCDH1 (mAb-3305) antibody. Neutralization of rVSV ANDV by mAb-3305 on human pulmonary microvascular endothelial cells (HPMEC) is shown as a positive control. (E) To rule out the possibility that mAb-3305 fails to block rVSV-ANDV infection in Cp kidney cells due to rapid uptake/clearance of the antibody, the experiment was performed on ice. The data underlying this figure can be found in the S1 Data file available from the journal.
Fig 4.
Immortalized Carollia kidney cells respond to surrogate virus infection.
(A) CaPsm-K primary and immortalized cells were transfected with 10 μg of poly (I:C) chemically labeled with rhodamine (pIC-Rho) for 16 h. Following fixation, cells were stained with antibodies against GAPDH and DAPI and visualized by confocal microscopy. Scale bars represent 25 μm. (B) CaPsm-K cells were transfected with 100 ng of poly (I:C) for 8 h. The upregulation of IFN-β and IFIT1 transcripts was assessed by qPCR. Data are represented as a mean ± SD, n = 4 replicates. (C) CaPsm-K cells transfected with 100 ng of poly (I:C) for 8 h were infected with VSV-GFP (MOI 1) for 16 h (n = 4). Viral replication was visualized by fluorescent microscopy. Scale bars represent 100 μm. (D) Green fluorescent protein (GFP) signal was quantified using ImageJ (Two-way ANOVA with Šídák’s multiple comparisons test). (E) Representative western blot of VSV-GFP infected cells, where IFIT1, VSV-M, and ACTB were probed for. L = molecular weight ladder. (F) CaPsm-K cells transfected with 100 ng of poly(I:C) for 8 h were infected with MERS-CoV (MOI 0.1) for 48 h (n = 3). Supernatant was collected and TCID50 assay was performed to assess viral titer. (G) Representative western blot of MERS-CoV infected cells, where IFIT1, MERS-CoV nucleocapsid (N), and GAPDH were probed for. L = molecular weight ladder. The data underlying this figure can be found in the S1 Data file available from the journal.
Fig 5.
Immortalized clonal cell lines retain susceptibility to viral entry.
(A) Select transduced cells were further plated for clonal isolation and 20 clones were propagated from single cells plated in 96-well format. (B) Clones that continuously grew were further screened for suppressibility to viral entry with pseudotypes bearing glycoproteins of the indicated viruses. (C) Clones that grew past 10 passages were re-screened for susceptibility to viral entry. Shown are the results from infection experiments performed in six replicates. The data underlying this figure can be found in the S1 Data file available from the journal.
Fig 6.
Immortalized Carollia cells support whole virus replication.
(A) Primary and immortalized kidney (CaPsm-K) cells were infected with VSV-GFP (MOI 1), MERS-CoV (MOI 0.1), or SARS-CoV-2 (MOI 0.01) for up to 72 h. Replication was monitored using TCID50 limiting dilution assays. (B) Clonal transduced kidney cells (CKg9) cells and CaPsmK-SV40T cells infected with Andes virus were monitored by qPCR. (C) CKg9 cells infected with Seoul virus (SEOV) were monitored by end-point microscopy for Seoul nucleocapsid protein. The data underlying this figure can be found in the S1 Data file available from the journal.