A Look inside the Replication Dynamics of SARS-CoV-2 in Blyth's Horseshoe Bat (Rhinolophus lepidus) Kidney Cells

Abstract

Bats are considered the natural reservoir of numerous emerging viruses such as severe acute respiratory syndrome coronaviruses (SARS-CoVs). There is a need for immortalized bat cell lines to culture and investigate the pathogenicity, replication kinetics, and evolution of emerging coronaviruses. We illustrate the susceptibility and permissiveness of a spontaneously immortalized kidney cell line (Rhileki) from Blyth's horseshoe bat (R. lepidus) to SARS-CoV-2 virus, including clinical isolates, suggesting a possible virus-host relationship. We were able to observe limited SARS-CoV-2 replication in Rhileki cells compared with simian VeroE6 cells. Slower viral replication in Rhileki cells was indicated by higher ct values (RT-PCR) at later time points of the viral culture and smaller foci (foci forming assay) compared with those of VeroE6 cells. With this study we demonstrate that SARS-CoV-2 replication is not restricted to R. sinicus and could include more Rhinolophus species. The establishment of a continuous Rhinolophus lepidus kidney cell line allows further characterization of SARS-CoV-2 replication in Rhinolophus bat cells, as well as isolation attempts of other bat-borne viruses. IMPORTANCE The current COVID-19 pandemic demonstrates the significance of bats as reservoirs for severe viral diseases. However, as bats are difficult to establish as animal models, bat cell lines can be an important proxy for the investigation of bat-virus interactions and the isolation of bat-borne viruses. This study demonstrates the susceptibility and permissiveness of a continuous kidney bat cell line to SARS-CoV-2. This does not implicate the bat species Rhinolophus lepidus, where these cells originate from, as a potential reservoir, but emphasizes the usefulness of this cell line for further characterization of SARS-CoV-2. This can lead to a better understanding of emerging viruses that could cause significant disease in humans and domestic animals.

Keywords: SARS-CoV; SARS-CoV-2; bat; bat cells; coronavirus; primary cell; reservoir; viral replication; virus isolation; zoonoses.

FIG 1

SARS-CoV-2 infection of Rhileki cells. (A) SARS-CoV-2 quantified Rhileki cell supernatants. Mean ct values with SEM of Rhileki cell culture supernatants infected with SARS-CoV-2 MOI 1 (merged results of isolates 1775, 2018, and 2310) previously passaged six times in Vero cells. Replication was determined by E gene (red) and RdRp expression (blue). Cytopathic effect (CPE) of cells was estimated through observation by bright field microscopy at ×10 magnification (gray dotted lines). (B) Cytopathic effect upon SARS-CoV-2 infection of Rhileki and VeroE6 cells 4 dpi. Cell monolayers were inoculated with the Cambodian SARS-CoV-2 isolated 1775 at MOI of 1 or 5 for 1 h; afterwards, cells were washed and grown in infection medium containing 5% FCS (for Vero cells) or 0.5% BSA (for Rhileki cells), respectively. CPE was documented by bright field microscopy at a ×10 magnification. Images were taken with Cytation5 multi-mode reader (BioTek). (C) Rhileki cells were inoculated with SARS-CoV-2 MOI 0.1 (merged results of isolates 1775, 2018, and 2310) using different media compositions either with 0.5% BSA (orange circles) or with 5% FCS (green squares). Replication was determined by E gene expression in the culture supernatants (unfilled shapes) and cell lysates (filled shapes). Cytopathic effect (CPE) of cells was estimated through observation by bright field microscopy at ×10 magnification (gray dotted lines). (D) Focus forming assay on Rhileki and VeroE6 cells infected with SARS-CoV-2 MOI 0.1 (isolate 2310, passaged six times in Vero cells). Staining of infected cells was carried out with pooled sera from confirmed COVID-19 patients. (E) Rhileki and VeroE6 cells were infected with the same amount of 47 Wuhan-like virus stock samples. Virus titers were determined by FFA staining, expressing the amount of initial infection events in focus forming units (ffu/mL). Wilcoxon test P < 0.0001. (F) Isolation attempts of Wuhan strains. Combined oro-nasopharyngeal swab samples of confirmed COVID-19 patients were used for inoculation and culture supernatants of both cell lines were analyzed 7 dpi for the presence of SARS-CoV-2 by E gene RT-PCR. (G) Isolation of α-VoC strains. SARS-CoV-2 in swabs used for inoculation was determined by IP4 RT-PCR. Supernatant of isolation on Vero cells were taken as soon as 50% CPE was observed (5 to 7 dpi). For isolation on Rhileki cells, supernatant was taken and analyzed from 3 dpi on every 2 days. Additionally at 15 dpi supernatant and lyzed cells were tested for presence of SARS-CoV-2 by RT-PCR targeting E gene.