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.

Taishan Pinus massoniana Pollen Polysaccharides Enhance Immune Responses in Chickens Infected by Avian Leukosis Virus Subgroup B.

Immunosuppressive virus, which can cause suppressed immunity and vaccination failure, frequently occurs in chicken flocks and seriously destroys the poultry industry. Our previous studies have reported that Taishan Pinus massoniana pollen polysaccharide (TPPPS) possess immunomodulatory effects and improve the immune effects of vaccines. In this study, avian leukosis virus subgroup B (ALV-B) was chosen as immunosuppressive virus to artificially establish immunosuppressive models in chickens, and the immune modulatory ability of TPPPS on the immune response of chickens was evaluated. Four randomly assigned groups (Group I-IV) of these immunosuppressed chickens were administered with TPPPS at doses of 0, 100, 200, and 400 mg/kg (every kilogram chick), respectively. Group V was administered with saline as control. At seven day old, 10 chickens randomly selected from Group I-V were inoculated with the attenuated Newcastle disease (ND) vaccine. The results showed that during the monitoring period, TPPPS significantly enhanced weight of immune organs, peripheral lymphocyte proliferation, the percentage of CD4+ and the ratio of CD4+/CD8+, IL-2 and IFN-γ production, and ALV-B antibody positive rate of chickens in a dose-dependent manner, with 400 mg/kg TPPPS being the most effective. In addition, the antibody titer against Newcastle disease virus (NDV) in Group IV with 400 mg/kg was significantly higher than those in other groups. We observed the stronger immunity in the TPPPS group, which indicates that TPPPS could be used as an immunoenhancer to relieve immunosuppression caused by ALV-B in the poultry industry.

Taishan Pinus massoniana pollen polysaccharide inhibits subgroup J avian leucosis virus infection by directly blocking virus infection and improving immunity.

Subgroup J avian leucosis virus (ALV-J) generally causes neoplastic diseases, immunosuppression and subsequently increases susceptibility to secondary infection in birds. The spread of ALV-J mainly depends on congenital infection and horizontal contact. Although ALV-J infection causes enormous losses yearly in the poultry industry worldwide, effective measures to control ALV-J remain lacking. In this study, we demonstrated that Taishan Pinus massoniana pollen polysaccharide (TPPPS), a natural polysaccharide extracted from Taishan Pinus massoniana pollen, can significantly inhibit ALV-J replication in vitro by blocking viral adsorption to host cells. Electron microscopy and blocking ELISA tests revealed that TPPPS possibly blocks viral adsorption to host cells by interacting with the glycoprotein 85 protein of ALV-J. Furthermore, we artificially established a congenitally ALV-J-infected chicken model to examine the anti-viral effects of TPPPS in vivo. TPPPS significantly inhibited viral shedding and viral loads in immune organs and largely eliminated the immunosuppression caused by congenital ALV-J infection. Additionally, pre-administration of TPPPS obviously reduced the size and delayed the occurrence of tumors induced by acute oncogenic ALV-J infection. This study revealed the prominent effects and feasible mechanisms of TPPPS in inhibiting ALV-J infection, thereby providing a novel prospect to control ALV-J spread.

Quantitative evaluation of DNA methylation patterns for ALVE and TVB genes in a neoplastic disease susceptible and resistant chicken model.

Chicken endogenous viruses, ALVE (Avian Leukosis Virus subgroup E), are inherited as LTR (long terminal repeat) retrotransposons, which are negatively correlated with disease resistance, and any changes in DNA methylation may contribute to the susceptibility to neoplastic disease. The relationship between ALVE methylation status and neoplastic disease in the chicken is undefined. White Leghorn inbred lines 7(2) and 6(3) at the ADOL have been respectively selected for resistance and susceptibility to tumors that are induced by avian viruses. In this study, the DNA methylation patterns of 3 approximately 6 CpG sites of four conserved regions in ALVE, including one unique region in ALVE1, the promoter region in the TVB (tumor virus receptor of ALV subgroup B, D and E) locus, were analyzed in the two lines using pyrosequencing methods in four tissues, i.e., liver, spleen, blood and hypothalamus. A significant CpG hypermethylation level was seen in line 7(2) in all four tissues, e.g., 91.86 +/- 1.63% for ALVE region2 in blood, whereas the same region was hemimethylated (46.16 +/- 2.56%) in line 6(3). CpG methylation contents of the ALVE regions were significantly lower in line 6(3) than in line 7(2) in all tissues (P < 0.01) except the ALVE region 3/4 in liver. RNA expressions of ALVE regions 2 and 3 (PPT-U3) were significantly higher in line 6(3) than in line 7(2) (P < 0.01). The methylation levels of six recombinant congenic strains (RCSs) closely resembled to the background line 6(3) in ALVE-region 2, which imply the methylation pattern of ALVE-region 2 may be a biomarker in resistant disease breeding. The methylation level of the promoter region in the TVB was significantly different in blood (P < 0.05) and hypothalamus (P < 0.0001), respectively. Our data disclosed a hypermethylation pattern of ALVE that may be relevant for resistance against ALV induced tumors in chickens.