实验海洋生物学重点实验室知识库

Avian leucosis virus subgroup J (ALV-J) is a kind of virus that can induce myeloid leukemia in chickens. It can cause immunosuppression, neoplastic death and secondary infection in chickens. ALV-J has caused severe losses to the world poultry industry since discovered. Currently, there is still no drug or commercial vaccine against ALV-J infection. In recent years, algae polysaccharides have been found to have inhibitory effects on various viruses. And algae polysaccharides has drawn much attention because of its high safety, antiviral and immunomodulatory activity. However, the high molecular weight, high viscosity and poor solubility limit the application of natural algae polysaccharides. The low molecular algae polysaccharides, characterized by well water solubility, high stability and easy absorption, overcome the disadvantages of the natural algae polysaccharides. Therefore, it is of great significance to apply low molecular weight algae polysaccharides to prevent ALV-J. Algae polysaccharides were the firstly used to resist ALV-J in our laboratory. And in our previous study, it was found that the anti-ALV-J activity of different molecular weight of Sargassum fusiforme polysaccahrides was better than those of Grateloupia filicina and Ulva perva polysaccharides, among which S. fusiforme polysaccharides with the molecular weight of 9 kDa had the best antiviral effect. While, the antiviral activity was not stable, meanwhile, its mechanism and the in vivo antiviral effect were still not clear. Nanomicelles are kinds of amphiphilic molecular aggregates composed of hydrophilic and hydrophobic regions. Due to the advantages of convenient preparation, good biocompatibility and high drug loading efficiency, nanomicelles are often used as carriers for drug transport. Some studies have shown that the drugs can be directly modified by hydrophilic or hydrophobic molecule and self-assembled to nanomicelles. This kind of modification, to a certain extent, reduce the introduction of hydrophobic and hydrophilic segments and the side effects that the segments may bring about. Meanwhile, the original activity of the drug might be improved. Therefore, in this article, on the basis of previous studies, we screened a 9 kDa low molecular weight S. fusiforme polysaccharide (SFP) to study its anti-ALV-J activity mechanism in vitro, and investigated its antiviral activity in vivo. Hydrophobic modification of the polysaccharide was also carried out, and the derivatives was self-assembled to nano-micelles to evaluate their antiviral effect in vitro. The main results are as follows:

1) The ALV-J gp85 protein, which could bind to cell surface, was successfully prepared in vitro. It was found that SFP could bind to gp85 protein by using the surface plasma resonance technology, indicating that SFP inhibited the adsorption of ALV-J to host cells through binding with gp85 protein. The association rate (k_a), dissociation rate (k_d) and dissociation constants (K_D) were 7.020 (1/Ms), 0.001489 (1/s) and 3.940×10^-5 (M), respectively, and the binding was related to sulfate content. The in vivo experiments showed that the SFP can significantly inhibit the ALV-J. It could improve body weight and immune organ index of ALV-J infected chicken, promote the peripheral blood lymphocyte proliferation, increase the CD4⁺ and CD8⁺ T lymphocytes proportions, promote the secretion of IL-2 and IFN-gamma, at the same time, it could effectively increase the chicken NDV antibody production, significantly reduce the viral shedding, and relieve the immunosuppression of ALV-J induced in chickens.

2) The SFP-lauric acid, myricic acid and palmitic acid derivatives were prepared successfully, and self-assembled into micelles (SFP-C12M, SFP-C14M and SFP-C16M). The average particle sizes of the three micelles were around 200nm, and the polydispersity indexes were 0.15±0.041, 0.096±0.03 and 0.153±0.015, respectively. Zeta potential were all around -40mv. The critical micelle concentration was low (0.0088, 0.005 and 0.0121mg/mL, respectively), indicating that the micelles were stable. Scanning electron microscope results showed that the three kinds of micelles were almost spherical with smooth surface, dispersed and uniform, and the particle size was consistent with that of the particle size meter, which further proved that the micelles were synthesized successfully. In vitro anti-ALV-J experiments showed that the expression of ALV-J gene and p27 protein could be significantly inhibited by the micelles, and the ALV-J gene relative expression of the SFP-C14M and SFP-C16M groups were significantly lower than that of the SFP group, indicated that their antiviral activity was better than that of the SFP. Among them, SFP-C16M had the best anti-ALV-J activity.

3) Compared with SFP, SFP-C16M could note noly inhibit virus adsorption onto the host cells, but also have a supression on virus reproduction, indicating that the hydrophobic modified SFP nanomicelles had a new anti-ALV-J mechanism. The phospholipid vesicle model was used further to study the mechanism of the interaction between nanomicelles and virus. Results suggestted that 100μg/mL SFP-C16M could completely destroy the phospholipid membranes within 24h, and 300μg/mL SFP-C16M disrupted the membranes within 6h. On the contrary, SFP did not have such effect. The results indicated that the SFP micelles might disrupt the virus envelope to exert the new antiviral effect.

In summary, SFP could bind to ALV-J gp85 protein to inhibit the virus absorption onto the host cells, and it can significantly inhibit ALV-J in vivo, reduce the growth inhibition and organ damage, alleviate the immune suppression induced by ALV-J, and improve the immunity of chicken. In addition, after modified with long chain fatty acids self-assembled into nanomicelles, the anti-ALV-J activity of SFP micelles was greatly improved. And the micelles also showed antiviral activity in the stage of virus propagation, which may be achieved by disrupting the viral envelope. This study provides a theoretical basis for the research and application of seaweed polysaccharide as a novel antiviral veterinary drug.