Preparation of New Sargassum fusiforme Polysaccharide Long-Chain Alkyl Group Nanomicelles and Their Antiviral Properties against ALV-J.

Avian leukosis virus subgroup J (ALV-J) is an immunosuppressive virus which has caused heavy losses to the poultry breeding industry. Currently, there is no effective medicine to treat this virus. In our previous experiments, the low-molecular-weight Sargassum fusiforme polysaccharide (SFP) was proven to possess antiviral activity against ALV-J, but its function was limited to the virus adsorption stage. In order to improve the antiviral activity of the SFP, in this study, three new SFP long-chain alkyl group nanomicelles (SFP-C12M, SFP-C14M and SFP-C16M) were prepared. The nanomicelles were characterized according to their physical and chemical properties. The nanomicelles were characterized by particle size, zeta potential, polydispersity index, critical micelle concentration and morphology. The results showed the particle sizes of the three nanomicelles were all approximately 200 nm and SFP-C14M and SFP-C16M were more stable than SFP-C12M. The newly prepared nanomicelles exhibited a better anti-ALV-J activity than the SFP, with SFP-C16M exhibiting the best antiviral effects in both the virus adsorption stage and the replication stage. The results of the giant unilamellar vesicle exposure experiment demonstrated that the new virucidal effect of the nanomicelles might be caused by damage to the phospholipid membrane of ALV-J. This study provides a potential idea for ALV-J prevention and development of other antiviral drugs.

Screening active fractions from Pinus massoniana pollen for inhibiting ALV-J replication and their structure activity relationship investigation.

The objective was to identify the active fractions of polysaccharide against replication of ALV-J and elucidate their structure activity relationship. The optimal extraction conditions were extracting temperature 90℃, pH 9 and the ratio of liquid to solid 30:1. Under these conditions, extraction yield of total polysaccharide was 6.5 % ± 0.19 %. Total polysaccharide was then purified by DEAE-52 cellulose and Sephadex G-200 gel. Three fractions, PPP-1, PPP-2, and PPP-3, were identified with molecular weight of 463.70, 99.41, and 26.97 kDa, respectively. Three polysaccharide fractions were all composed of 10 monosaccharides in different proportions. Compared with PPP-1, which was mainly composed of glucose, PPP-2 and PPP-3 contained a higher proportion of galactose, glucuronic acid and galacturonic acid. The Congo red assay indicated that the PPP-2 may have a triple helical structure, while PPP-1 and PPP-3 were absent. In vitro assay showed that there was no significant cytotoxicity among the polysaccharide fractions under the concentration of 800 µg mL-1 (P > 0.05). The antiviral test showed that PPP-2 had the strongest activity, indicating PPP-2 was the major antiviral component. The structure-activity relationship showed that the antiviral activities of polysaccharide fractions were affected by their monosaccharide composition, molecular weight, and triple helical structure, which was a result of a combination of multiple molecular structural factors. These results showed that the PPP-2 could be exploited as a valued product for replacing synthetic antiviral drugs, and provided support for future applications of polysaccharide from Pinus massoniana pollen as a useful source for antiviral agent.

A novel Bursin-like peptide as a potential virus inhibitor and immunity regulator in SPF chickens infected with recombinant ALV.

BACKGROUND: Avian leukosis viruses (ALVs) are important contagious suppressive factors of chicken immunity and growth performance, resulted in enormous economic loss. Although virus eradication programs are applied in breeder flocks, ALVs are still widespread globally. Therefore, other valuable adjunct to reduce the negative effect of ALVs should be considered. Bursin-like peptide (BLP) showed remarkable immunomodulatory effects, whereas their influence on ALV-infected avian groups has not been reported. Here, a designed hybrid BLP was expressed in E. coli. The purified BLP was injected subcutaneously weekly in SPF chickens congenitally infected with a natural ALV strain. Then the influences of this BLP on the growth performance, immune response and virus titer of ALV-infected chickens were determined. RESULTS: This BLP injection significantly improved the body weights of ALV-infected birds (P < 0.05). BLP injection significantly enhanced organ index in the BF in ALV-infected birds (P < 0.05). The weekly injection of BLP significantly lengthened the maintenance time of antibodies against Newcastle disease virus (NDV) attenuated vaccine of ALV-infected birds (P < 0.05) and boosted the antibody titer against avian influenza virus (AIV) H5 inactive vaccine of mock chicken (P < 0.05). BLP injection in mock chickens enhanced the levels of serum cytokines (IL-2, IL-4 and interferon-γ) (P < 0.05). Surprisingly, the novel BLP significantly inhibited expression of the ALV gp85 gene in the thymus (P < 0.05), kidney (P < 0.05) and bursa of Fabricius (BF) (P < 0.01) of ALV-infected chickens. Both viral RNA copy number and protein level decreased significantly with BLP (50 µg/mL) inoculation before ALV infection in DF1 cells (P < 0.05). CONCLUSIONS: This is the first report investigating the influence of BLP on the growth and immunity performance of chickens infected by ALV. It also is the first report about the antiviral effect of BLP in vivo and in vitro. This BLP expressed in E. coli showed potential as a vaccine adjuvant, growth regulator and antiretroviral drug in chickens to decrease the negative effects of ALV infection.

Taishan Pinus Massoniana pollen polysaccharide inhibits the replication of acute tumorigenic ALV-J and its associated tumor growth.

Avian leukosis virus subgroup J (ALV-J) has resulted in considerable economic losses in the poultry industry. In recent years, fibrosarcoma induced by ALV-J, which contains the v-fps oncogene, has gained momentum, and this has brought about new challenges to the poultry industry. To study the inhibitory effects of Taishan Pinus Massoniana pollen polysaccharide (TPPPS) on acute ALV-J infection and tumor development, antiviral and antitumor models of the Fu-J (SDAU1005) strain of ALV-J were established in vitro and in vivo. The results of in vitro experiments showed that TPPPS significantly inhibited viral replication in a dose-dependent manner during adsorption and pretreatment stages. The results of in vivo experiments have shown that TPPPS significantly reduced the viral load in the plasma and tumor tissues, as well as inhibited tumor growth. We further examined the difference in transcriptome expression by using RNA-Seq technology. A total of 560 differentially expressed genes were identified that included 329 up-regulated genes and 231 down-regulated genes. The up-regulated genes were mainly immune-related genes, whereas the down-regulated genes were mainly tumor-regulated genes. Gene Ontology (GO) term enrichment included immune system processes, positive regulation of immune system processes, regulation of immune system processes, leukocyte activation, cell activation, and protein binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the main immune and tumor-related pathways included T-cell receptor signaling pathway, cytokine-cytokine receptor interactions, natural killer cell-mediated cytotoxicity, PI3K-Akt signaling pathway, JAK-STAT signaling pathway, NF-κB signaling pathway, and Ras signaling pathway. In summary, our results preliminarily point to the antiviral and antitumor mechanism of TPPPS in vivo and in vitro.

The antiviral property of Sargassum fusiforme polysaccharide for avian leukosis virus subgroup J in vitro and in vivo.

Avian Leukosis Virus Subgroup J (ALV-J) is an oncogenic retrovirus, mainly spread by vertical and horizontal transmission, which have caused severe losses in world poultry industry. Sargassum fusiforme polysaccharide (SFP), a marine algae sulfated polysaccharide, has attracted more attention due to its variously biological activities. In this study, the anti-ALV-J property of SFP was assessed in vivo and in vitro. The results demonstrated that different Mw of SFPs showed virustatic activity to ALV-J in vitro by combining with the virus when ALV-J adsorbed onto the host cells. When treated with SFPs, the ALV-J gene and protein expression reduced clearly and SFP-3 (Molecular weight 9 kDa) had the best antiviral effect. Results in vivo showed that the immunosuppression of the ALV-J infected chickens were relieved by SFP-3. Moreover, SFP-3 obviously inhibit the viral shedding and alleviated the organs damage caused by ALV-J. This study offered a new method for ALV-J treatment and enriched the potential application of SFP.

Joint treatment with azidothymidine and antiserum for eradication of avian leukosis virus subgroup a contamination in vaccine virus seeds.

Contamination of avian attenuated vaccines by avian leukosis virus (ALV) is considered to be a specific horizontal transmission of ALV. Eradication of ALV contamination in vaccine virus seeds is thus a precondition of qualified vaccine production. In this study, we used the nucleoside reverse transcriptase (RT) inhibitor azidothymidine (AZT) together with monofactor antiserum against avian leukosis virus subgroup A (ALV-A) to remove ALV-A from vaccine virus seeds. Different doses of ALV-A were artificially added to the Newcastle disease virus (NDV) vaccine seeds, then the ALV-contaminated attenuated virus vaccine virus seeds were cultured in DF-1 cells. Single-drug treatment with 5 µg/mL AZT or 5% (v/v) as well as combined treatment with AZT and antiserum significantly suppressed ALV-A replication (P < 0.001) in the vaccine virus seeds. Complete absence of virus replication was observed in cells exposed to joint treatment with AZT and antiserum. The treated virus seeds met the requirements of the Chinese Ministry of Agriculture. Therefore, combined treatment with AZT and antiserum can be used to eradicate contaminating ALV-A from vaccine virus seeds, thus providing a new approach for improving vaccine safety.

Antiviral Activity against Avian Leucosis Virus Subgroup J of Degraded Polysaccharides from Ulva pertusa.

Avian Leukosis Virus Subgroup J (ALV-J), a retrovirus of avian, has caused enormous economics losses to poultry industry around the world. Polysaccharides from marine algae are featured diversity bioactivities. To find the potential effect to prevent ALV-J spread, in this study, polysaccharides from Ulva pertusa (UPPs) and four low molecular weight (Mw) U. pertusa polysaccharides (LUPPs) were prepared and their functions on ALV-J were investigated. Firstly, LUPPs were obtained by hydrogen peroxide (H2O2) oxidative degradation. The effects of degradation conditions on Mw of the UPP were also investigated. Results showed that the H2O2 oxidative degradation method could degrade UPP effectively, and the degradation was positively related to H2O2 concentration and temperature and negatively to pH. The chemical characteristics of UPP and LUPPs were also determined. Afterwards, the anti-ALV-J activity of the polysaccharides were carried out in vitro. Results showed that UPP and LUPPs could inhibit ALV-J and LUPP-3 and Mw of 4.3 kDa exerted the strongest suppression. The action phase assay showed that LUPP-3 could bind with the viral particles and prevented ALV-J adsorption onto the host cells. And the ALV-J relative gene and gp85 protein expression were significantly suppressed after being administration with LUPP-3. Therefore, the low Mw polysaccharides from U. pertusa have great potential as an anti-ALV-J drug alternative.

Baicalin is an inhibitor of subgroup J avian leukosis virus infection.

Avian leukosis virus subgroup J (ALV-J) can cause great economic losses to the poultry industry worldwide. Baicalin, one of the flavonoids present in S.baicalensis Georgi, has been shown to have antiviral activities. To investigate whether baicalin has antiviral effects on the infection of ALV-J in DF-1 cells, the cells were treated with baicalin at different time points. We found that baicalin could inhibit viral mRNA, protein levels and overall virus infection in a dose- and time-dependent manner using a variety of assays. Baicalin specifically targeted virus internalization and reduced the infectivity of ALV-J particles, but had no effect on the levels of major ALV-J receptor and virus binding to DF-1 cells. Collectively, these results suggest that baicalin might have potential to be developed as a novel antiviral agent for ALV-J infection.

Antiviral effect of lithium chloride on replication of avian leukosis virus subgroup J in cell culture.

Lithium chloride (LiCl) has been reported to possess antiviral activity against several viruses. In the current study, we assessed the antiviral activity effect of LiCl on ALV-J infection in CEF cells by using real-time PCR, Western blot analysis, IFA and p27 ELISA analysis. Our results showed that both viral RNA copy number and protein level decreased significantly in a dose and time dependent manner. Time-course analysis revealed that the antiviral effect was more pronounced when CEFs were treated at the post infection stage rather than at early absorption or pre-absorption stages. Further experiments demonstrated that LiCl did not affect virus attachment or entry, but rather affected early virus replication. We also found that inhibition of viral replication after LiCl treatment was associated with reduced mRNA levels of pro-inflammatory cytokines. These results demonstrate that LiCl effectively blocked ALV-J replication in CEF cells and may be used as an antiviral agent against ALV-J.

Degradation of Polysaccharides from Grateloupia filicina and Their Antiviral Activity to Avian Leucosis Virus Subgroup J.

In this study, polysaccharides from Grateloupia filicinia (GFP) were extracted and several low molecular weight (Mw) G. filicina polysaccharides (LGFPs) were prepared by the hydrogen peroxide (H2O2) oxidation method. Additionally, the effect of different experimental conditions on the degradation of GFP was determined. Results showed that the GFP degradation rate was positively related to H2O2 concentration and temperature, and negatively related to pH. Chemical analysis and Fourier transform infrared spectra (FT-IR) of GFP and LGFPs showed that the degradation caused a slight decrease of total sugar and sulfate content. However, there was no obvious change for monosaccharide contents. Then, the anti-ALV-J activity of GFP and LGFPs were determined in vitro. Results revealed that all of the samples could significantly inhibit ALV-J and lower Mw LGFPs exhibited a stronger suppression, and that the fraction LGFP-3 with Mw 8.7 kDa had the best effect. In addition, the reaction phase assays showed that the inhibition effect was mainly because of the blocking virus adsorption to host cells. Moreover, real-time PCR, western-blot, and IFA were further applied to evaluate the blocking effects of LGFP-3. Results showed that the gene relative expression and gp85 protein for LGFPS-3 groups were all reduced. Data from IFA showed that there was less virus infected cells for 1000 and 200 µg/mL LGFPS-3 groups when compared to virus control. Therefore, lower Mw polysaccharides from G. filicina might supply a good choice for ALV-J prevention and treatment.