# Multipotent Antiviral Effects of Deacetylated Chitosan
## 4 Discussion  
Chitosan is a linear polymer and polysaccharide derivative of deacetylated chitin. It is associated with numerous biological activities and is influenced by the degree of N-deacetylation, positive charge, polymerization degree, and other chemical modifications [24]. Notably, chitosan derivatives exhibit antiviral effects against various viral infections, including HIV, human papillomavirus, influenza A, and human coronavirus [12; 25; 26; 27]. Recent reports have indicated that chitosan and its derivatives can bind to the pocket formed by the spike protein of SARS-CoV-2 [28; 29]. Moreover, another study reported that sulfated chitoligosaccharides may block viral adsorption and membrane fusion by targeting the influenza A HA protein [15]. Similarly, our findings demonstrate that deacetylated chitosan combined with GSE reduced H1N1 HA protein levels in A549 cells. Chitosan, with a degree of deacetylation of 85%, was found to be more effective in reducing both H1N1- and HPIV3-induced plaque formation, while GSE alone reduced H1N1-induced plaque formation but not HPIV3-induced plaque formation. However, further studies are required to elucidate the underlying biological mechanisms. Treatment with high concentrations of deacetylated chitosan had significant antiviral effects on H1N1 and HPIV, whereas GSE did not affect HPIV. GSE alone effectively blocked the H1N1 virus, and high-dose deacetylated chitosan inhibited HPIV infection. However, combining GSE with high-dose deacetylated chitosan proved to be the most effective against H1N1. Studies on the treatment of avian influenza with grapefruit extract also found that GSE was effective against enveloped viruses, such as avian influenza virus and Newcastle disease virus, but not against nonenveloped infectious burstal disease virus (IBDV). Grapefruit extract alone has no bactericidal effect. Instead, it likely destroys the cytoplasmic membrane, which induces a bactericidal effect [16]. A previous study reported that GSE in corn starch-chitosan bio-nanocomposite films could offer an innovative, eco-friendly, active packaging solution for the confectionery industry, prolonging shelf life and maintaining food safety and quality [30]. However, studies on the antiviral activity and food safety of the combination of chitosan and GSE are lacking. Further in vivo studies exploring this combination are required. Chitosan is characterized by its degree of deacetylation or MW; thus, its solubility, hydrophilicity, crystallinity, and cell response predict its potential for various biological applications [31]. A recent in vitro study concluded that low-MW chitoligosaccharides other than chitosan also inhibit SARS-CoV-2 infection [22]. Deacetylation of chitosan is an important chemical modification that affects its performance and use in biological applications [31]. In addition, chitosan can restore immunity which is initially lowered by obesity [32; 33; 34]. Obesity intensities insulin resistance generated by an immune system imbalance between M1 and M2 macrophages [35]. This response augments the susceptibility to H1N1 influenza virus infection in patients with obesity owing to their low immunity [36]. The weight-reducing effect of chitosan can help prevent obesity and maintain immunity, effectively averting influenza viral infections. The antiviral activity of chitosan, which has been tested and proven in practice, has been studied against several human viruses such as herpes simplex virus type 1, human cytomegalovirus, and Rift Valley fever virus [37]. In this study, we examined the antiviral activities of deacetylated chitosan and GSE against representative human respiratory viruses, specifically H1N1 and HPIV3. HPIV infection causes mitochondrial fragmentation in the lung cells, resulting in cell death and severe lung damage [16; 38]. These inflammation-related factors are expressed in response to mitochondrial destruction, which exacerbates the severity of lung damage [39].