Isolation and genetic characterization of human coronavirus NL63 in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells

Abstract

Background: Cryopreserved primary human renal proximal tubule epithelial cells (RPTEC) were obtained from a commercial supplier for studies of Simian virus 40 (SV40). Within twelve hrs after cell cultures were initiated, cytoplasmic vacuoles appeared in many of the RPTEC. The RPTEC henceforth deteriorated rapidly. Since SV40 induces the formation of cytoplasmic vacuoles, this batch of RPTEC was rejected for the SV40 study. Nevertheless, we sought the likely cause(s) of the deterioration of the RPTEC as part of our technology development efforts. Methods: Adventitious viruses in the RPTEC were isolated and/or detected and identified by isolation in various indicator cell lines, observation of cytopathology, an immunoflurorescence assay, electron microscopy, PCR, and sequencing. Results: Cytomegalovirus (CMV) was detected in some RPTEC by cytology, an immunofluorescence assay, and PCR. Human Herpesvirus 6B was detected by PCR of DNA extracted from the RPTEC, but was not isolated. Human coronavirus NL63 was isolated and identified by RT-PCR and sequencing, and its replication in a fresh batch of RPTEC and another type of primary human kidney cells was confirmed.

Antiviral Action of Tryptanthrin Isolated from Strobilanthes cusia Leaf against Human Coronavirus NL63

“…Anti-HCoV-NL63 activity of S. cusia leaf extract at the concentrations of 0.1, 1, and 10 μg/mL was further evaluated using the CPE reduction and virus yield inhibition assays (Figure 1). HCoV-NL63 induced an observable vacuolation in LLC-MK2 cells at 37 °C 48 h post-infection, as described in the prior report [24]. The S. cusia leaf methanol extract reduced HCoV-NL63-induced CPE in LLC-MK2 cells in a concentration-dependent manner ( Figure 1A).…”

“…After 24, 36 and 48 h of incubation at 37 °C and 5% CO2, HCoV-NL63-induced CPEs such as cell swelling, rounding, vacuoles, and eventual detachment were imaged using a microscope, in which vacuoles in infected cells were predominant [24]. Images of CPE in infected cells were captured using a microscope.…”

Antiviral activity of Sambucus FormosanaNakai ethanol extract and related phenolic acid constituents against human coronavirus NL63

“…The cells were simultaneously added with HCoV NL63, and treated with Sambucus Formosana Nakai stem ethanol extract at the concentrations of 0, 1, 10, and 50 μg/ml or the phenolic acid constituents at the concentrations of 0, 10, 50, and 100 μM, respectively. After the 36-h incubation at 37 ℃ and 5％ CO 2 , HCoV NL63-induced cytopathic effect (CPE) with cell swelling, rounding, vacuoles, and eventual detachment was photographed using microscope, in which vacuoles in CPE from HCoV NL63-infected LLC-MK2 cells were more predominant at 37°C (Lednicky et al, 2013). In the cell cycle assay, the cells were harvested 36 h post infection, stained with propidium iodide, and then examined using flow cytometry, as described in our prior study .…”

“…After the addition of HCoV NL63, the cells were simultaneously treated with caffeic acid at the concentrations of 0, 10, and 50 μM. After the 36-h incubation at 32 ℃ in a 5％ CO 2 incubator, swelling, rounding, and eventual de-attachment in HCoV-NL63-induced CPE were more predominant at 32°C (Lednicky et al, 2013), and then the images were recorded by microscope. Subsequently, the cells were fixed with 4% paraformaldehyde solution in PBS for 30 min, incubated with the quench buffer (50 mM NH 4 CI) for 15 min, permeabilized and blocked using the cell perforation and blocking solution containing 1% albumin bovine (Affymetrix) plus triton X-100 (ThermoFisher) for 4 h at 4°C, and then reacted with HCoV-NL63-immunized sera in 1% BSA (1:2000) overnight at 4°C and secondary antibody Alexa Fluor anti-mouse IgG in 1% BSA (1:3000) for 1 h at 4°C (ThermoFisher).…”

Development of transgenic mouse model expressing porcine aminopeptidase N and its susceptibility to porcine epidemic diarrhea virus

“…They are classified into four genera, Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus, based on genetic similarities (Adams et al, 2014). The primary replication of the coronaviruses is often confined to respiratory-or gastrointestinal-tract epithelial cells, so they usually induce respiratory or enteric diseases, but also hepatic, renal and neuronal infections (Lednicky et al, 2013;Masters, 2006;Weiss and Navas-Martin, 2005).…”