An animal model of SARS produced by infection of Macaca mulatta with SARS coronavirus.

A new SARS animal model was established by inoculating SARS coronavirus (SARS-CoV) into rhesus macaques (Macaca mulatta) through the nasal cavity. Pathological pulmonary changes were successively detected on days 5-60 after virus inoculation. All eight animals showed a transient fever 2-3 days after inoculation. Immunological, molecular biological, and pathological studies support the establishment of this SARS animal model. Firstly, SARS-CoV-specific IgGs were detected in the sera of macaques from 11 to 60 days after inoculation. Secondly, SARS-CoV RNA could be detected in pharyngeal swab samples using nested RT-PCR in all infected animals from 5 days after virus inoculation. Finally, histopathological changes of interstitial pneumonia were found in the lungs during the 60 days after viral inoculation: these changes were less marked at later time points, indicating that an active healing process together with resolution of an acute inflammatory response was taking place in these animals. This animal model should provide insight into the mechanisms of SARS-CoV-related pulmonary disease and greatly facilitate the development of vaccines and therapeutics against SARS

Cell blebbing upon addition of cryoprotectants: a self-protection mechanism.

In this work, the mechanism of cell bleb formation upon the addition of cryoprotectants (CPAs) was investigated, and the role of cell blebs in protecting cells was determined. The results show that after adding CPAs, the hyperosmotic stress results in the breakage of the cortical cytoskeleton and the detachment of the cell membrane from the cortical cytoskeleton, causing the formation of cell blebs. Multiple blebs decrease the intracellular hydrostatic pressure induced by the extracellular hyperosmotic shock and alleviate the osmotic damage to cells, which reduces the cell mortality rate. In the presence of a low concentration of CPAs, cell blebs can effectively protect cells. In contrast, in the presence of a high concentration of CPAs, the protective effect is limited because of severe disruption in the cortical cytoskeleton. To determine the relationship between blebs and the mortality rate of cells, we defined a bleb index and found that the bleb index of 0.065 can be regarded as a reference value for the safe addition of DMSO to HeLa cells. The bleb index can also explain why the stepwise addition of CPAs is better than the single-step addition of CPAs. Moreover, the mechanism of the autophagy of cells induced by the hyperosmotic stress was studied, and the protective effect associated with the autophagy was compared with the effect of the blebbing. The findings reported here elucidate a self-protection mechanism of cells experiencing the hyperosmotic stress in the presence of CPAs, and they provide significant evidence for cell tolerance in the field of cryopreservation

Stepwise addition of DMSO.

<p><b>(A)</b> Comparison of the mortality rate of cells between stepwise and single-step addition. <b>(B)</b> Bleb index in the stepwise addition method. HeLa cells were treated with 20% DMSO for 30 minutes, and the solution was removed quickly and changed to 40% DMSO for 30 minutes. It was then changed to 60% DMSO for 30 minutes and, finally, to 80% DMSO. The inverted fluorescence microscope was used to observe dead cells labeled by PI and Hoechst. For <b>(A)</b>, the number of cells used was approximately 500 and the experiment was repeated 5 times. For <b>(B)</b>, the number of cells used was approximately 40. **p<0.01 was considered statistically significant.</p

Cell blebs and cytoskeleton under different DMSO concentrations.

<p><b>(A)</b> The bleb and cytoskeleton were observed by an inverted fluorescence microscope (membrane: red; cytoskeleton: green). <b>(B)</b> The bleb and cytoskeleton were observed by a confocal microscope (cytoskeleton: green; nucleus: blue). <b>(C)</b> The fluid flows in the formation of blebs under a hypoosmotic condition (0.1×PBS) and a hyperosmotic condition (25% DMSO in PBS). The experiments were repeated 3 times.</p

Cell blebs induced by the addition of CPAs.

<p>Various concentrations of <b>(A)</b> DMSO and <b>(B)</b> glycerol were applied to HeLa cells for 30 minutes. The development of cell blebs during the first 3 minutes was observed as the initial state and after 30 minutes as the stable state. Initiate: 3 minutes, and Stabilized: 30 minutes. The experiments were repeated 3 times.</p

Life cycle of a dynamic bleb.

<p><b>(A)</b> the inflation and retraction of one bleb (black arrows); <b>(B)</b> the actin microfilament reorganization during the bleb inflation and retraction; <b>(C)</b> the comparison of the inflation and retraction time between DMSO and glycerol. For <b>(A)</b> and <b>(B)</b>, the experiments were repeated 3 times. For <b>(C)</b>, the number of cells used was approximately 20.</p

The autophagy induced by the addition of CPAs.

<p><b>(A)</b> GFP-LC3/HeLa cells were treated with various concentrations of DMSO, and GFP green fluorescence dots appeared in cells. <b>(B)</b> LC3 conversion was determined by western blot in HeLa cells treated with different concentrations of DMSO. <b>(C)</b> Effect of DMSO on the autophagy rate. <b>(D)</b> GFP-LC3 /HeLa cells were inhibited by 3-MA, and then stimulated by 30% DMSO. Shrinkage of cell nuclei is a hallmark of apoptosis. <b>(E)</b> Autophagy reduced the apoptosis in the presence of 30% DMSO. **p<0.01 was considered statistically significant. The experiments were repeated 5 times. The number of cells used was approximately 500.</p

Effect of the concentration of CPAs: (A) number of cell blebs; (B) total area of cell blebs; (C) bleb index; (D) mortality rate of cells; (E) schematic of A_lip and A_cyto.

<p>HeLa cells were treated with a series of solutions containing different amounts of DMSO or glycerol, as well as the fluorochromes Hoechst and PI. After 30 minutes, when cells were stable, an inverted fluorescence microscope was used to observe cell death. For <b>(A)</b>, <b>(B)</b> and <b>(C)</b>, the cell number was approximately 40. For <b>(D)</b>, the cell number was approximately 500 and the experiment was repeated 5 times. For <b>(E)</b>, the red boundary denotes the lipid bilayer and the green boundary denotes the cortical cytoskeleton.</p

An animal model of SARS produced by infection of Macaca mulatta with SARS coronavirus.

A new SARS animal model was established by inoculating SARS coronavirus (SARS-CoV) into rhesus macaques (Macaca mulatta) through the nasal cavity. Pathological pulmonary changes were successively detected on days 5-60 after virus inoculation. All eight animals showed a transient fever 2-3 days after inoculation. Immunological, molecular biological, and pathological studies support the establishment of this SARS animal model. Firstly, SARS-CoV-specific IgGs were detected in the sera of macaques from 11 to 60 days after inoculation. Secondly, SARS-CoV RNA could be detected in pharyngeal swab samples using nested RT-PCR in all infected animals from 5 days after virus inoculation. Finally, histopathological changes of interstitial pneumonia were found in the lungs during the 60 days after viral inoculation: these changes were less marked at later time points, indicating that an active healing process together with resolution of an acute inflammatory response was taking place in these animals. This animal model should provide insight into the mechanisms of SARS-CoV-related pulmonary disease and greatly facilitate the development of vaccines and therapeutics against SARS