# Multipotent Antiviral Effects of Deacetylated Chitosan
## 1 Introduction  
Influenza epidemics and the severe acute respiratory syndrome coronavirus (SARS-CoV-2) pandemic are important issues threatening human health. Influenza is an enveloped, segmented, single-stranded RNA virus categorized into subtypes based on hemagglutinin (HA) and neuraminidase (N): A, B, C, and D. Among the subtypes, influenza A is predominantly prevalent during winter (e.g., H1N1 pdm09, the 2009 pandemic H1N1 strain) [1]. The Centers for Disease Control and Prevention in the United States reported 60.8 million cases, 274,304 hospitalizations, and 12,469 deaths caused by H1N1 pdm09 between 2009 and 2010 [2]. In contrast, the human parainfluenza virus (HPIV) is an enveloped, nonsegmented, single-stranded RNA virus classified into four serotypes and two subtypes (HPIV1, 2, 3, 4a, and 4b) [3]. As an influenza-like respiratory pathogen, HPIV primarily targets lung epithelial cells in the respiratory tract. Respiratory viral infections are the foremost causes of asthma and pneumonia, causing arduous social and financial burden [4]. The raw materials and manufacturing methods used affect the characteristics of chitosan. For instance, comparative studies on chitosan yields from various raw materials have revealed high concentrations of chitosan in crab and shrimp shells. Chitosan, a modified natural cationic polymer comprising \(\beta\)-(1\(\rightarrow\)4)-linked D-glucosamine residues, is obtained via a partial N-deacetylation of chitin. This poly-aminosaccharide polymer has excellentchemical and biological properties owing to the presence of several amino groups [5; 6]. Chitosan can be produced using chemical or enzymatic methods that differ according to the degree of acetylation, acetyl group distribution along the chains, molecular weight (MW), and chitosan viscosity in solution [7; 8]. Chitosan is a natural biopolymer obtained through the enzymatic deacetylation of chitin to different degrees [9], and its derivatives inhibit several prominent pathogenic viruses, such as the human immunodeficiency virus (HIV), influenza A, and SARS-CoV-2 [10; 11; 12]. Interestingly, the degree of deacetylation, MW, and derivatives make chitosan a promising antiviral drug candidate [10; 13]. A recent study showed that covalent conjugation of the influenza A antigen to chitosan could be an effective nasal vaccine candidate against the influenza A H1N1 virus [14]. Sulfated dithioligosaccharide showed potent, low-toxicity anti-influenza activity owing to its sulfation modification, targeting of HA protein, and oral treatment efficacy similar to that of oseltamivir [15]. These findings suggest that chitosan has potential for antiviral and medical applications in combating influenza viruses. Grapefruit seed extract (GSE) is a natural antioxidant compound containing tocopherols, citric and ascorbic acids, and flavonoids, with potential effects against viruses and bacteria [16; 17; 18; 19]. A recent study reported that quercetin and naringenin, which are representative GSE flavonoids, exhibit potent anti-inflammatory and antiviral effects through nuclear factor kappa B (NF\(\kappa\)B), Toll-like receptor (TLR), and interleukin-6 (IL-6) signaling [20; 21]. This study aimed to assess the antiviral efficacy of chitosan and grapefruit seed extract against H1N1 and HPIV3. Since the transformation of chitin to chitosan only occurs at over 50% deacetylation, we procured samples exhibiting more than 85% deacetylation through time-controlled settings for high-level deacetylation. To discern the differences brought about by varying degrees of deacetylation, we utilized chitosan that had been subjected to 60% and 85% deacetylation. While the antiviral efficacy and various physiological activities of chitosan and grapefruit seed extract are known, the antiviral efficacy of chitosan based on its degree of acetylation remains unexplored. This study verified the antiviral effects according to the degree of deacetylation of chitosan against human influenza and paranafluenza viruses in vitro.