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The response of primary human epithelial cells to stringent virus amplification conditions refutes the existence claims of all viruses and SARS-CoV-2

Exosomes are small extracellular vesicles containing cargo RNA, DNA, and cellular proteins. They are produced by all cell types, serve cell-cell communication, and offer promising therapeutic opportunities. To study RNA species and extracellular vesicles under harsh protocols routinely used in virology, healthy primary human epithelial cells were cultured over three passages with stress protocols for virus (virion) amplification. Despite the absence of virus inoculation, the cells developed severe cytopathic effects (CPE), resulting in visible subtotal destruction and plaque formation in the cell lawn. Blind inspection of cells under control and viral amplification conditions allowed identification of the different morphologies with a hit rate of 100%. Total RNA from cells and cell culture supernatants of three biological and two technical replicates per stress group was dissected by next-generation sequencing together with total RNA from the same optimally cultured cells. Sequence and extracellular vesicle analyses are in progress.

Viruses from isolates, e.g., bats, are propagated in cell cultures under harsh culture conditions by depriving them of much of their food by reducing fetal calf serum (FCS) from 10% to 2% or 1% in Dulbecco's Modified Eagle's Medium (DMEM), consistent with ATCC recommendations. Food deprivation is also routinely combined with high concentrations of Gibco's triple antibiotics (penicillin/streptomycin antibiotics with amphotericin B antifungal) and sequential "blind passaging" of cell culture supernatants onto the next cell culture⁽¹⁾. Morphologically, virion amplification leads to cytopathic effects (CPE), which ends in rounding of cells, ballooning of the same and cellular degeneration, which is finally visible by plaque formation in a confluent cell culture. Accordingly, viral particles enriched from these cell culture supernatants can be imaged by electron microscopy. To rule out the hypothesis that harsh stress conditions without viral inoculation may lead to the formation of exosomes⁽²⁾ that are virion-like, we subjected healthy primary human epithelial cells to routine viral amplification protocols. We then isolated total RNA from starved or control cells and supernatants using viral RNA isolation kits or routine TRIzol extraction and subjected the RNA to next-generation sequencing.

Healthy, primary human epithelial cells were grown over four passages (P3-P6) under optimal culture conditions in defined epithelial control medium containing 1x triple antibiotics (CM). After the first passage, the cell pool was divided into four groups. After 3 days in CM, cultures were transferred to either fresh CM (CM, Control 1), DMEM/GlutaMAX with 10% FCS, 1x triple antibiotics (Control 2) or stress medium (Starvation 1 & 2). During the first stress treatment, the stress medium contained DMEM, 1% FCS and 3x triple antibiotics. The second and third passages were "blind" passages in which 50% of the culture supernatant from the last passage was transferred to the next passage in DMEM, 1% FCS and 3x triple antibiotics. The second stress group was additionally treated with total yeast RNA (yRNA) at each passage for one hour before the addition of the stress medium (Starvation 2). After transfer to DMEM containing 10% FCS, epithelial cells adopted a flatter morphology than in CM and formed a continuous cell lawn, which may be attributed to the high calcium concentrations in DMEM. Otherwise, the cells continued to divide normally (Figure 1A). In contrast, the cell lawns in the stress media shrank to small islands with reduced growth and incipient cell degeneration. During the next two passages, cells incubated with the supernatant of the stressed cells from the previous passage showed increasing CPE with cell-free areas reminiscent of virion-induced plaques in the cell lawn, and more dead cells floating in the supernatant (Figure 1B). Confluent cultures under stress (Figure 1C) stained with crystal violet (Figure 1D) confirmed the distinct CPE. Pyknotic cells with condensed nuclei or ballooning cells were predominantly present in the Starvation 1 group, and areas of total cell destruction or plaques were also observed in the Starvation 1 but predominantly in the Starvation 2 group. Experiments were performed in three biological replicates and two technical duplicates. All cultures were inspected blindly with stressed cultures easily recognized by drastic changes in morphology. After three passages, RNA was isolated from control 1 and the two stressed cell groups and supernatants using viral RNA kits or TRIzol and subjected to next-generation sequencing. The amount of total RNA isolated was most productive in control group 1 (Table 1) and was of perfect quality in all groups (data not shown). Additional supernatants were further used for extracellular particle analysis. Experiments are in progress.

Commercial human primary passage 3 epithelial cells were thawed and seeded at 4,000 cells/cm2 in 75cm2 flasks for expansion at 37°C with 5% CO2 in defined epithelial low calcium medium (without FCS) and 1x triple antibiotic (Gibco) (control medium, CM). At >80% confluence, expansion cells were detached with 5mL accutase enzyme at 37°C for 10 minutes. Accutase was neutralized with 10mL CM, cells were centrifuged at 400G for 5 minutes, resuspended in 1mL CM, live cells were counted by trypan blue staining in Countess II instrument (ThermoFisher). Cells were sawn out for the experiment or parallel expansion rounds for subsequent experiments.

For each experiment, four groups of healthy primary epithelial cells were seeded from the same expanded pool in CM at 4000 cells/cm2 in 25cm2 culture flasks and cultured to >50% confluence. Medium was then replaced by four experimental conditions; for control cells by fresh CM (Control 1) or commercial DMEM supplemented with GlutaMAX, 10% heat-inactivated FCS and 1x triple antibiotic (Control 2). Food was deprived by replacing CM with DMEM, with 1% FCS and 3x triple antibiotics, essentially corresponding to virion amplification1 protocols (Starvation 1 & 2). The stressed Starvation group 2 was additionally treated with 10 μg total yeast RNA (yRNA) per culture flask for 1h and washed thoroughly with phosphate-buffered saline (PBS) before changing medium group 1 & 2. Two "blind passages" were then performed, in which 50% of the supernatant from Starvation Groups 1 and 2 was transferred to the next cell culture. The supernatants were cleaned of dead cells by centrifugation at 400G for 5 minutes. Control groups received 100% fresh medium. The experiments were repeated three times in duplicates. The length of culture under stress defined in the first biological replicate, were kept constant for all experiments. No medium change was performed during the stress period.

P4: Media change in control and stressed cells at circa 50 confluency; control cells cultured to >80% confluency, stressed cells cultured for 5 days after media change.

P5: Media change in control and stressed cells >50 confluency, control cells cultured to >80% confluency, stressed cells cultured for 8 days after media change.

P6/RNA isolation: media change in control and stressed cells at circa 50 confluency; control cells cultured to >80% confluency, stressed cells cultured for 5 days after media change.

P6/Crystal violet: media change in control and stressed cells at 100% confluency; stress induction for 3 days.

One representative image of all cell cultures was taken daily at room temperature using a Nikon Eclipse TS100 brightfield microscope with a Nikon 1J5 camera, a Nikon FT1 adapter, and a 4x objective.

At the end of passage 6, half of the total cellular RNA was isolated using the Promega miRNA- kit (Promega, Z6211), which is recommended for small and long RNA samples, according to the manufacturer's protocol. The other half of total cellular RNA was isolated using the standard TRIzol protocol. Total RNA from cell culture supernatant was isolated using the routinely used Qiagen viral RNA kit (Qiagen, 52904) according to the manufacturer's protocol. All RNA samples were treated with DNase. Total RNA concentration and 260/280 and 260/230 ratios were determined using a NanoDrop 2000 (ThermoFisher). RNA amounts were highest in samples cultured in CM and lowest in stressed groups 1 and 2, while supernatants had very low but similar RNA amounts (Table 1). 8.3 mg of high quality total RNA, assessed by Bioanalyzer, from control group 1 and stressed groups 1 & 2 were sequenced by "next generation RNA sequencing".

At the final passage, a second set of 25cm2 culture flasks were seeded at 8000 cells/cm2 (set 2) to visualize cytopathic effects. At 100% confluence, these cells were exposed to one of the four media conditions. Three days after exposure, cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature and then stained with 1% Crystal Violet for an additional 30 minutes at room temperature before being washed thoroughly with tap water at room temperature. Nikon Eclipse TS100 brightfield microscope with a Nikon 1J5 camera, a Nikon FT1 adapter, and a 4x or 20x objective was used to image the stained cultures.

1. 1 Ge, X. Y. et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535-538, doi:10.1038/nature12711 (2013).
2. Gurung, S., Perocheau, D., Touramanidou, L. & Baruteau, J. The exosome journey: from biogenesis to uptake and intracellular signalling. Cell Commun Signal 19, 47, doi:10.1186/s12964-021-00730-1 (2021).

A. Passage 4 - Expansion

B. Passage 6 - Expansion, Set 1, RNA harvest

C. Passage 6 - Set 2, CPE in confluent cultures

D. Passage 6 - Set 2, CPE in confluent cultures (Crystal Violet)

Figure 1. stress of epithelial cells. Representative microscopic images of the 4 experimental groups of epithelial cells at passage 4 and 6.

From left to right: healthy control cells with 1x triple antibiotics in control medium (CM) or DMEM/GlutaMAX with 10% FCS; stressed cells with 3x triple antibiotics and 1% FCS in DMEM.

Cells in the right panel were treated with total yeast RNA (yRNA) for 1h before media change.

(A), (B) Cells in expansion for the purpose of RNA isolation. Note that CPE becomes more prominent across the three passages. (B) Top row: Cells before medium change.

(C), (D) Confluent cells visualizing CPE; (C) Top row: confluent cells before medium change.

(D) Cell cultures from 3 biological replicates stained with crystal violet at the time of harvest.

Note that cells in the two left panels form a continuous cell lawn while cells in the two right panels show a high number of plaques (arrows) compatible with significant cytopathic effects, increasing from day 1 to day 5. Cultures treated with yeast RNA show a significantly higher number of larger plaques.

Insets: 20× magnification; some rare pyknotic and ballooning cells were observed in control cultures; ballooning cells with empty cytoplasm are most abundant under stress conditions 1. Cultures were inspected blindly by 2 experimenters daily with a 100% hit rate. Bars; 500 μm.All cultures: n=3 in duplicates.

Tabel 1: RNA Isolation