# Multipotent Antiviral Effects of Deacetylated Chitosan
## 3 Results
### Antiviral Activity of Deacetylated Chitosan in Human A549 Cells  
The composition of deacetylated chitosan was further explored by determining its antiviral potential in A549 human lung cancer cells. Consistent with the plaque formation in MDCK cells, western blot analyses indicated that HA protein levels were reduced in both virus-infected A549 cells with 85% deacetylated chitosan, but not 60% (Figures 4A,B and S2). In comparison, GSE alone reduced the HA protein level of H1N1. Next, H1N1 and HPIV3 viral RNAs in A549 cells treated with deacetylated chitosan were examined via qRT-PCR. The gene expression of H1N1 viral matrix _M1_ and nucleoprotein _NP_ was reduced in H1N1-infected A549 cells with 85% deacetylated chitosan, but not 60%, suggesting that chitosan inhibits viral infection deacetylation-dependently (Figures 4C and S3). In HPIV3-infected A549 cells, deacetylated chitosan also attenuated the expression of nucleoprotein (N) and phosphoprotein (P) (Figures 4D and S3). These data suggest the antiviral potential of deacetylated chitosan in human cells.  
Figure 4: Assessment of antiviral activity of deacetyl-chitosan in human A549 cells. (**A**,**B**) HA protein levels of H1N1-infected (**A**) or HPIV3-infected (**B**) A549 cells treated with or without deacetyl-chitosan. Whole-cell lysates were analyzed by western blot using antibodies on the right. (**C**) qRT-PCR of _H1N1_M1_ and _H1N1_NP_ expression in H1N1-infected A549 cells. (**D**) qRT-PCR of _HPIV3-N_ and _HPIV3-P_ expression in HPIV3-infected A549 cells. Quantitative data for gene expression are presented as the mean \(\pm\) SEM from three biological replicates (n = 3). A (60% deacetylated chitosan), B (85% deacetylated chitosan), C (GSE), D (60% deacetylated chitosan + GSE), and E (85% deacetylated chitosan + GSE) are shown in Table 1. Control group was compared to treatment group from three biological replicates (n = 3). * \(p\) < 0.05, **_p_ < 0.01.  
Deacetylated Chitosan Prevents Virus-Induced P38 MAPK Activation and Mitochondrial Fragmentation in A549 Cells  
The antiviral effects of deacetylated chitosan may stem from cellular stress signaling and mitochondrial integrity. Therefore, several stress-signaling pathways potentially affected by the composition of deacetylated chitosan were screened to test this hypothesis. Western blot analyses revealed that the deacetylated chitosan composition inhibited phosphorylation of p38 MAPK but not that of NFxB p105 and ERK (Figures 5A and S4). Next, we determined whether the composition of deacetylated chitosan affected virus-mediated mitochondrial integrity. Interestingly, mitochondrial fragmentation was significantly inhibited (Figures 5B and S5), indicating that deacetylated chitosan obstructed HPIV3-induced p38 MAPK signaling and mitochondrial fragmentation in A549 cells.  
Figure 5: Deacetyl-chitosan prevents virus-induced p38 MAPK activation and mitochondrial fragmentation in A549 cells. (**A**) Western blot analysis of protein levels of phospho or total NFxB p105, ERK and p38 MAPK using whole-cell lysates from HPIV3-infected A549 cells treated with or without deacetyl-chitosan. HSP90 was used as a loading control. (**B**) HPIV3-infected A549 cells treated with or without 85% deacetyl-chitosan with GSE, followed by treatment with Mito-Tracker Green dye for 1 h. Mitochondria were observed via fluorescence microscopy.