Fig 2: Cytopathic effect of SARS-CoV-2 variant of concern Gamma (P.1) from a placenta sample in Vero E6 cell culture. (A) Vero E6 cell cultures (negative control). (B) Cytopathic effects consisting of rounding and detachment of cells in VERO E6 cultures 3 days after the third passage. EVOS™ XL Core Imaging System—Thermo Fisher Scientific (10×).

Fig 3: The effect of L15 EPS on primary osteoblast precursors and bone formation. A Photomicrographs of ALP staining of primary osteoblast precursor obtained from the calvaria of ICR neonates with or without L15 EPS for 7 days. Inverted imaging system (EVOS™ XL Core Imaging System; Thermo Scientific™, Waltham, MA, USA). Scale bar = 200 μm. B The expression of osteogenic differentiation marker genes (RUNX2, COL1A1, and ALP) in primary osteoblast precursor were analyzed by real time-PCR. C The ICR neonates calvarial ex vivo organ culture was performed with or without L15 EPS. Calvaria was cultured for 7 days and then processed for histological examination. Thickness of the calvaria was assessed by H&E staining (Olympus BX50 microscope; Scale bar = 25 μm). *p < 0.05. N = 3

Fig 4: L15 EPS enhances osteogenic differentiation of hDPSCs. A Photomicrograph of ALP staining of hDPSCs with or without L15 EPS. Inverted imaging system (EVOS™ XL Core Imaging System; Thermo Scientific™, Waltham, MA, USA). Scale bar = 200 µm B Photograph showing calcium deposition of hDPSCs with or without L15 EPS by ARS staining. C The expression of osteogenic differentiation marker genes (RUNX2, ALP, and COL1A1) were analyzed by real time-PCR. D The expression of osteogenic differentiation marker proteins (BMP4, RUNX2, and ALP) expression were analyzed by western blot analysis. (E) Quantitative analysis of intensity in (D) relative to GAPDH. Scale bar = 200 µm. *p < 0.05, N = 3

Fig 5: Histological examination of the control and infected tracheas. Cross sections of trachea samples from control chickens and chickens infected with H9N2 AIV A/chicken/Pakistan/UDL01/08 stained with haematoxylin and eosin (A,B). (A) Healthy section of the inner epithelial layer (arrows); (B) section of the inner epithelial layer that has degraded (arrows). Samples collected at day 4 post-challenge. Images were captained with an imaging microscope (EVOS XL Core Imaging System, Thermo Fisher). (C) Estimated percentage damage to the inner epithelial layer in tracheas of control and H9N2-AIV-infected chickens. Control group is represented by six samples that were taken from birds at 2–10 days of experiment, whereas samples from infected birds (N = 5 each) were collected at days 2, 4 and 10 post-challenge (post-ch). The percentage of the inner epithelial layer that had either been degraded or had the cilia on its surface stripped away was estimated independently by two people, and the mean percentage damage of the two estimates was used in the statistical analysis. Two-tailed t-tests were used to assess significant differences between the control and infected groups * p < 0.05.