According to many studies, sulfated polysaccharides are a promising source of antiviral molecules. In fact, in this work, Chi-S could inhibit the in vitro viral infection of PCV2, indicating that the presence of sulfate groups specifically improves the antiviral activity of Chi. Other factors influencing the antiviral activity would be: the presence of anionic groups, the degree of sulfation, charged density and molecular weight, affecting the virus penetration by alterating intracellular signals and delaying the replicative cycle of viruses . Other studies indicated that hypersulphated polysaccharides interfere with electrostatic interactions between the positively charged region of the viral envelope glycoproteis and the negative charges of the heparan sulfate surface receptor chains .
It was observed in this study that the LMW Chi-S produces the lowest viral copy number among all polymers tested (Figure 3). A possible explanation of this effect, is that the depolymerization of the polysaccharide during the sulfation process, allow it a better interaction with the virus particle, generating greater antiviral effects, thus inhibiting the spread of the virus through the cell. However, at present there is no uniform statement in the literature on the effect of molecular weight on the antimicrobial properties of Chi, and some conclusions are contradictory . According to  the antiviral activity of Chi increases as its molecular weight decreases and as its degree of acetylation increases. Thus, data from other studies indicate that HMW Chi possesses higher antiviral properties [31, 32].
Additionally, it is possible to determine that the inhibition of the virus using Chi-S, occurs in the initial stages of infection, determining it as an effective antiviral agent. Since Chi-S appears to prevent the attachment of the virus to the host cell receptor by modifying relevant subsequent processes and delaying intracellular events that occur during post-entry and replication process. Therefore, if the virus-receptor interaction is hindered, processes such as attachment, signaling, internalization, endocytosis, and replication would be affected . These ideas are inferred, since the reduction of viral copy number was observed when the infection was simultaneous upon Chi-S application (Figure 4). However, the "preventive" application in the cells before the infection does not exert a protective effect on them or a long-lasting antiviral state. As a final point, although it was not significant, to apply Chi-S, 1 and 2 h after infection (+1 and +2), reduced viral DNA copies. Therefore, the Chi-S would also affect the later stages, such as the penetration of the virus.
The results obtained are similar to those reported for other polysaccharides, where it is deduced that the most acceptable mechanism of action would be by impeding the viral attachment of PCV2 to sulfated polysaccharide receptors. Thus, when heparan sulfate and Chi-S are chemically compared, it is possible to observe that they present a structural analogy, composed of alternating linear sulfated polymers of uronic acid- (1 → 4) -D-glucosamine and consequently they can mimic their biological functions [7, 8].
It was also observed that pre-incubating the virus with Chi-S produced a greater reduction in viral copy number, but it was also reduced, although less significant, when applying the Chi-S at the time of infection (Figure 5). Therefore, it could be established that there is a blockage of viral adsorption through direct interference with the virus particle. It has been reported that 3,6-sulfated Chi directly inhibits human papillomavirus (HPV) by binding to viral capsid proteins and, therefore, blocking viral adsorption . Other sulfated Chi-oligosaccharide derivatives block the interaction between HIV-1gp120 and CD4 + cell surface receptors, thereby inhibiting virus-cell fusion and subsequent virus entry. This induces a masking effect that eventually inhibits the attachment and subsequent penetration of the virus into host cells . Recently, it was demostred that sulfated polysaccharides possess a strong antiviral effect due to their direct interaction with SARS-CoV spike protein . Therefore, our results indicate that Chi-S could interact directly with viral capsid of PCV2 to inactivate it, and thus allowing strong antiviral responses. To further support our evidence, , have also investigated low-molecular-weight sulfated chitosan naturally obtained from the squid Sepia pharaonis, demonstrating that the antiviral activity of this polymer against Newcastle disease virus is by binding to the surface glycoprotein. Therefore, its antiviral activity would be mediated by a competitive inhibition of the attachment of the viral particle to avian erythrocytes, thus functioning as a potential antiviral agent. Our results suggest that the Chi-S chemically obtained from industrial waste has great potential as an anti-PCV2 product that can be further validated in vivo.
According to our results, significant reductions of viral copy number were observed in the early stages of infection, therefore, Chi-S interferes with the penetration of the virus, since a reduction in viral DNA was also observed when viral adsorption in cell culture was allowed (Figure 6). These reductions were important in the first 40 min, however at 60 min a reduction in viral DNA was still observed, but not as efficient as it was for early post-infection stages (Figure 6). Recent research indicates that sulfated polysaccharides interfere with the internalization of the virus by interacting with its membrane proteins, that is, they bind with carbohydrate groups linked to the polypeptide chains of the virus to inhibit their penetration . Additionally, sulfated polysaccharides bind to the allosteric site of the viral capsid, preventing the virus from initiating its cycle within the host cell. Indeed, Lee et al., 2004 investigated sulfated polysaccharides from green algae and synthetic sulfated xylan exhibit potent antiviral activities against HSV-1 (herpes simplex virus) . The authors showed that some sulfated polysaccharides not only inhibited the early stages of viral replication, such as viral attachment and penetration into host cells, but also interfered with the late stages of replication.
We have previously described the ability of generic Chi-C (LMW) to bind with PCV2 virus-like particles and that this conjugate would be useful and efficient for developing mucosal vaccines against PCV2 virus, since Chi, additionally, is an excellent adjuvant of the mucosa-associated immune system .
Here, we have shown that Chi-S have significant anti PCV2 activity. The mechanism of action would be through inhibition of viral adsorption, acting as a mimetic of the heparan sulfate receptor, binding to the viral capsid proteins and therefore blocking the attachment of the virus to the host cell, which reflects that Chi-S can be considered a “clone” coreceptor for virus binding, which would compete in the attachment with heparan sulfate or chondroitin sulfate, the virus's natural receptors on the cell surface. It has been well demonstrated that PCV2 uses these GAGs for attachment to porcine monocytic 3D4/31 and porcine kidney epithelial PK-15 cells .