The inhibitory effect of phosphorylated Codonopsis pilosula polysaccharide on autophagosomes formation contributes to the inhibition of duck hepatitis A virus replication.

Duck hepatitis A virus type 1 (DHAV) infection causes duck viral hepatitis and results in enormous loss to poultry farming industry. We reported that phosphorylated Codonopsis pilosula polysaccharide (pCPPS) inhibited DHAV genome replication. Here we further explored its underlying antiviral mechanisms. Autophagosomes formation is essential for the genome replication of picornaviruses. In this study, Western blot, confocal microscopy observation, and ELISA methods were performed to analyze polysaccharides' effects on autophagy by the in vitro and in vivo experiments. Results obtained from in vitro and in vivo experiments showed that Codonopsis pilosula polysaccharide did not play a role in regulating autophagy and had no therapeutic effects on infected ducklings. However, pCPPS treatment downregulated LC3-II expression level activated by DHAV and rapamycin, indicating the inhibition of autophagosomes formation. The interdiction of autophagosomes formation resulted in the inhibition of DHAV genome replication. Further study showed that pCPPS treatment reduced the concentration of phosphatidylinositol-3-phosphate (PI3P), an important component of membrane, in cells and serum, and consequently, autophagosomes formation was downregulated. In vivo experiments also verified the therapeutic effect of pCPPS. Phosphorylated Codonopsis pilosula polysaccharide treatment increased the infected ducklings' survival rate and alleviated hepatic injury. Our studies verified the effects of pCPPS against DHAV infection in duck embryo hepatocytes and ducklings and confirmed that phosphorylated modification enhanced the bioactivities of polysaccharides. The results also stated pCPPS's antiviral mechanisms, provided fundamental basis for the development of new anti-DHAV agents.

Assessment of the hepatocyte protective effects of gypenoside and its phosphorylated derivative against DHAV-1 infection on duck embryonic hepatocytes.

BACKGROUND: Duck viral hepatitis (DVH) is an acute disease of young ducklings with no effective veterinary drugs for treatment. Gynostemma pentaphyllum is a well-known traditional Chinese medicine that plays an important role in the treatment of various diseases. Gypenoside (GP), one of the main ingredients of Gynostemma pentaphyllum, was reported with good hepatoprotective effects. However, its low solubility limits its application in the clinics. To improve its solubility and bioactivity, a phosphorylated derivative of gypenoside (pGP) was prepared by the sodium trimetaphosphate-sodium tripolyphosphate (STMP-STPP) method. An infrared spectroscopy method was applied to analyse the structures of GP and pGP. Then, a methyl thiazolyl tetrazolium (MTT) colorimetric assay was applied to study the hepatocyte protective efficacy of these two drugs against duck hepatitis A virus type 1 (DHAV-1) infection, and qPCR, TUNEL labelling and flow cytometry methods were used to study the relevant hepatocyte protective in vitro. RESULTS: The infrared spectroscopy detection results showed that the phosphorylation modification of GP was successful. The MTT colorimetric assay results showed that both GP and pGP possessed good hepatocyte protective efficacy in vitro, and pGP performed better than GP when the drug was added before or after virus inoculation. Furthermore, the qPCR results revealed that both drugs could effectively inhibit the adsorption (when adding GP and pGP pre-virus inoculation), replication and release of DHAV-1, and the viral inhibition rate of pGP was greater than that of GP. The subsequent TUNEL labelling and flow cytometry assays showed that both GP and pGP could significantly inhibit duck embryo hepatocyte apoptosis induced by DHAV-1, and the inhibition effect of pGP was much stronger than that of GP. CONCLUSIONS: GP exerts good hepatocyte protective efficacy not only by inhibiting the proliferation of DHAV-1 but also by inhibiting duck embryonic hepatocyte apoptosis induced by DHAV-1, and phosphorylation modification significantly improves the antiviral and the anti-apoptotic effects of GP. Therefore, pGP has the potential to be developed into a novel drug against DHAV-1 infection.

Effect of fetal calf serum on propagation of duck hepatitis A virus genotype 3 in duck embryo fibroblast cells.

BACKGROUND: Duck viral hepatitis (DVH) is a highly contagious viral disease affecting ducks. It can be caused by five agents, including duck hepatitis A virus genotypes 1 (DHAV-1), 2 (DHAV-2), and 3 (DHAV-3), as well as duck hepatitis virus 2 and duck hepatitis virus 3. Since 2007, DHAV-3 has been known to be the most prevalent in East and South Asia. So far, the information regarding the propagation of DHAV-3 in cultured cells is limited. In this study, we describe the comparative studies on the growth properties of DHAV-3 in primary duck embryo fibroblast (DEF) cells using two different strains: a virulent strain C-GY and an attenuated strain YDF120. The effect of fetal calf serum (FCS) and chick serum (CS) on DHAV-3 replication and the mechanism of the inhibitory effect conferred by FCS were also investigated. RESULTS: Following serial passages, both C-GY and YDF120 failed to produce cytopathic effect and plaques. The combined quantitative real-time PCR and indirect immunofluorescence staining methods showed that the two viruses could be propagated productively in DEF cells. Investigation of the viral growth kinetics revealed that the two viruses replicated in DEF cells with similar efficiencies, while the viral load of the virulent C-GY strain peaked more rapidly when compared with the attenuated YDF120 strain. Neutralization assay and time-of-drug-addition study indicated that FCS displayed inhibitory effect on DHAV-3 replication. Analysis on the mechanism of action of FCS against DHAV-3 demonstrated that the inhibitory effect was reflected at three steps of the DHAV-3 life cycle including adsorption, replication, and release. CONCLUSIONS: Both virulent and attenuated DAHV-3 strains can establish noncytocidal, productive infections in DEF cells. The virulent strain replicates more rapidly than the attenuated strain in early infection period. FCS has an inhibitory effect on DHAV-3 replication, which may be attributed to action of a non-specific inhibitory factor present in FCS directly on the virus. These findings may provide new insights into the development of potential antiviral agents.

Assessment of the Effect of Baicalin on Duck Virus Hepatitis.

BACKGROUND: Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is a malignant disease in ducklings, causing economic losses in the duck industry. However, there is still no antiviral drug against DHAV-1 in the clinic. OBJECTIVE: Our aim is to investigate the anti-DHAV-1 effect of baicalin, which is a flavonoid derived from the Chinese medicinal herb huangqin (Scutellaria baicalensis Georgi). METHODS: Here, we first detected its anti-DHAV-1 ability in vitro and in vivo. At the same time, the inhibition of baicalin on DHAV-1 reproduction was determined. Finally, we tested and verified the anti-oxidative and immuno-enhancing roles of baicalin on its curative effect on DVH. RESULTS: Baicalin possessed anti-DHAV-1 effect. It improved the cytoactive of DEH which was infected by DHAV-1 as well as reduced the DHAV-1 reproduction in DEH. Under baicalin treatment, mortality of ducklings infected by DHAV-1 decreased, additionally the DHAV-1 level and liver injury in such ducklings were significantly reduced or alleviated. The in vitro mechanism study indicated baicalin inhibited DHAV-1 reproduction via interfering the viral replication and release. Furthermore, the in vivo mechanism study manifested both the anti-oxidative and immuno-enhancing abilities of baicalin, which played crucial roles in its curative effect on DVH. CONCLUSION: This study may provide a scientific basis for developing baicalin as one or a part of the anti-DHAV-1 drugs.

PI3KC3-dependent autophagosomes formation pathway is of crucial importance to anti-DHAV activity of Chrysanthemum indicum polysaccharide.

We previously reported that Chrysanthemum indicum polysaccharide (CIPS) effectively inhibited the replication of duck hepatitis A virus (DHAV). However, the inhibition mechanisms are still unclear. Autophagy plays important role in virus genomic replication. Therefore, in present study, the effect of autophagy on DHAV genome replication as well as the influence of CIPS on autophagy were studied. qPCR, western blot, and ELISA methods were applied to observe the autophagy and analyze the inhibition mechanisms of CIPS on DHAV. Results showed that DHAV infection increased the expression level of LC3-II and interdicted the degradation of p62. Treating with rapamycin benefited DHAV gene expression level. What's more, DHAV infection and rapamycin treatment also promoted the expression of PI3KC3 and increased the concentration of PI3P. However, CIPS treatment significantly downregulated the expressions of LC3-II and PI3KC3 induced by DHAV and rapamycin, and consequently inhibited autophagosomes formation. As a result, DHAV replication was inhibited.

Inhibition mechanisms of baicalin and its phospholipid complex against DHAV-1 replication.

Duck hepatitis A virus type 1 (DHAV-1) is a serious infectious virus of ducklings. Recent study showed baicalin (BA) and baicalin phospholipid complex (BAPC) possessed anti-DHAV-1 effect. However, the antiviral mechanism is not clear. Therefore, the aim of the present work is to study influences and mechanisms of BA and BAPC on DHAV-1. The effects of BA and BAPC on DHAV-1 replication were analyzed by CCK-8 and RT-qPCR methods. And the results showed BA inhibited the replication of DHAV-1, and BAPC was more effective. Then, the influences of BA and BAPC on DHAV-1 protein translation and RNA synthesis were detected by western blot and RT-qPCR. Both BA and BAPC inhibited the protein translation, and BAPC did better. Furthermore, BAPC also inhibited the RNA synthesis. Afterwards, DHAV-1 IRES activity, DHAV-1 3D protein stability, and cellular Hsp70 expression were studied to in-depth understand the inhibition effects of BA and BAPC on DHAV-1 replication. The results indicated BA and BAPC dropped the protein translation via suppressing DHAV-1 IRES activity. Additionally, BAPC dropped the RNA synthesis via reducing the 3D protein stability and inhibiting cellular Hsp70 expression.

RAW REHMANNIA RADIX POLYSACCHARIDE CAN EFFECTIVELY RELEASE PEROXIDATIVE INJURY INDUCED BY DUCK HEPATITIS A VIRUS.

BACKGROUND: Duck viral hepatitis (DVH), caused by duck hepatitis A virus (DHAV), is a fatal contagious infectious disease which spreads rapidly with high morbidity and high mortality, and there is no effective clinical drug against DVH. MATERIALS AND METHODS: Raw Rehmannia Radix Polysaccharide (RRRP), Lycii Fructus polysaccharides and Astragalus Radix polysaccharides were experimented in vitro and in vivo. Mortality rate, livers change, liver lesion scoring, peroxidative injury evaluation indexes in vitro and in vivo, and hepatic injury evaluation indexes of optimal one were detected and observed in this experiment. RESULTS: RRRP could reduce mortality with the protection rate about 20.0% compared with that of the viral control (VC) group, finding that RRRP was the most effective against DHAV. The average liver scoring of the VC, blank control (BC), RRRP groups were 3.5, 0, 2.1. Significant difference (P<0.05) appeared between any two groups, demonstrating that it can alleviate liver pathological change. RRRP could make the hepatic injury evaluation indexes similar to BC group while the levels of the VC group were higher than other two groups in general. The levels of SOD, GSH-Px, CAT of RRRP group showed significant higher than that of VC group while the levels of NOS and MDA showed the opposite tendency, thus, RRRP could release peroxidative injury. CONCLUSION: RRRP was the most effective against duck hepatitis A virus (DHAV). RRRP could reduce mortality, alleviate liver pathological change, down-regulate liver lesion score, release peroxidative injury and hepatic injury. The antiviral and peroxidative injury releasing activity of RRRP for DHAV provided a platform to test novel drug strategies for hepatitis A virus in human beings.

Effects of Chrysanthemum indicum polysaccharide and its phosphate on anti-duck hepatitis a virus and alleviating hepatic injury.

To explore new effective anti-duck hepatitis A virus drugs, Chrysanthemum indicum polysaccharide (CIPS) was phosphorylation modified using STMP-STPP method, and phosphorylated Chrysanthemum indicum polysaccharide (pCIPS) was obtained. Characteristic absorption peaks were observed in pCIPS using IR spectrum, suggested that CIPS was successfully modified. In addition, field emission scanning electron micro-scope (FE-SEM) was used to observe the polysaccharides' surface features. In vitro, we found that the survival rate of DHAV-infected hepatocytes increased after the two drugs treatment, indicated that the two drugs possess good anti-DHAV activity. The results of real-time PCR showed that pCIPS inhibited the virus gene replication more effectively than CIPS. Reed-Muench assay was used to observe the changes of the virulence, and the expression level of IFN-ß was observed to verify the changes of virulence. In vivo experiment, the blood virus content reduced after CIPS and pCIPS treatment. To evaluate the ducklings' hepatic injury, the serum ALT, AST, TP and ALB levels were detected. Results showed that both CIPS and pCIPS could alleviate the hepatic injury of ducklings infected DHAV, especially for pCIPS. All the results above mentioned demonstrated that the anti-DHAV activity of CIPS was enhanced after phosphorylation modification.

Anti-DHAV-1 reproduction and immuno-regulatory effects of a flavonoid prescription on duck virus hepatitis.

CONTEXT: The flavonoid prescription baicalin-linarin-icariin-notoginsenoside R1 (BLIN) has a curative effect on duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1). However, the mechanism of this curative effect is not understood. OBJECTIVE: This study investigates the mechanism of the curative effect of BLIN on DVH caused by DHAV-1. We analyzed the anti-DHAV-1 reproduction mechanism and immuno-regulatory effect of BLIN. MATERIALS AND METHODS: The anti-DHAV-1 reproduction effects of BLIN at 20, 10, 5 and 2.5 µg/mL in vitro, as well as the influence of BLIN at 20 µg/mL on DHAV-1 adsorption, replication and release were tested using the qRT-PCR method. The promotion abilities of BLIN at 20, 10, 5 and 2.5 µg/mL on T- and B-lymphocyte proliferation were investigated by the MTT method. IL-2 and IFN-γ levels and total anti-DHAV-1 antibody secretion after treatment with DHAV-1 for 4, 8 and 54 h were determined by ELISA. RESULTS: BLIN showed a dose-dependent DHAV-1 reproduction inhibitory effect. The inhibitory effect was highest at 20 µg/mL, where DHAV-1 adsorption and release were significantly lower. Meanwhile, BLIN at 5 µg/mL significantly increased T and B lymphocyte proliferation. BLIN stimulated total anti-DHAV-1 antibody secretion in ducklings at the dosage of 4 mg per duckling, but did not stimulate IL-2 and IFN-γ secretion significantly. CONCLUSIONS: BLIN inhibits DHAV-1 reproduction by suppressing its adsorption and release. Additionally, BLIN promoted the duckling antiviral response.

Treatment effect of a flavonoid prescription on duck virus hepatitis by its hepatoprotective and antioxidative ability.

CONTEXT: Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is an acute and lethal disease of young ducklings. However, there is still no effective drug to treat DVH. OBJECTIVE: This study assessed the curative effect on DVH of a flavonoid prescription baicalin-linarin-icariin-notoginsenoside R1 (BLIN) as well as the hepatoprotective and antioxidative effects of BLIN. MATERIALS AND METHODS: MTT method was used to test the anti-DHAV-1 ability of BLIN in vitro. We then treated ducklings by BLIN (3 mg per duckling, once a day for 5 days) to evaluate the in vivo efficacy. To study the hepatoprotective and antioxidative roles of BLIN in its curative effect on DVH, we investigated the hepatic injury evaluation biomarkers and the oxidative stress evaluation indices of the ducklings. RESULTS: On duck embryonic hepatocytes, DHAV-1 inhibitory rate of BLIN at 20 µg/mL was 69.3%. The survival rate of ducklings treated by BLIN was about 35.5%, which was significantly higher than that of virus control (0.0%). After the treatment of BLIN, both the hepatic injury and the oxidative stress of infected ducklings alleviated. At the same time, a significant positive correlation (p < 0.05) existed between the hepatic injury indices and the oxidative stress indices. CONCLUSIONS: BLIN showed a significant curative effect on DVH. The antioxidative and hepatoprotective effects of BLIN made great contributions to the treatment of DVH. Furthermore, BLIN is expected to be exploited as a new drug for the clinical treatment of DVH.

Comparison of the anti-duck hepatitis A virus activities of phosphorylated and sulfated Astragalus polysaccharides.

Duck hepatitis A virus (DHAV) (Picornaviridae) causes an infectious disease in ducks which results in severe losses in duck industry. However, the proper antiviral supportive drugs for this disease have not been discovered. Polysaccharide is the main ingredient of Astragalus that has been demonstrated to directly and indirectly inhibit RNA of viruses replication. In this study, the antiviral activities of Astragalus polysaccharide (APS) and its derivatives against DHAV were evaluated and compared. APS was modified via the sodium trimetaphosphate and sodium tripolyphosphate (STMP-STPP) method and chlorosulfonic acid-pyridine method to obtain its phosphate (pAPS) and sulfate (sAPS), respectively. The infrared structures of APS, pAPS, and sAPS were analyzed with the potassium bromide disc method. Additionally, the antiviral activities were evaluated with the MTT ((4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) method in vitro and the artificial inoculation method in vivo. The clinical therapy effects were evaluated by mortality rate, liver function-related biochemical indicators, and visual changes in pathological anatomy. The anti-DHAV proliferation effects of APS, pAPS, and sAPS on the viral multiplication process in cell and blood were observed with the reverse transcription-polymerase chain reaction method. The results revealed that pAPS inhibited DHAV proliferation more efficiently in the entire process of viral multiplication than APS and sAPS. Moreover, only pAPS significantly improved the survival rate to 33.5% and reduced the DHAV particle titer in the blood as well as liver lesions in clinical trials. The results indicated that pAPS exhibited greater anti-DHAV activity than APS and sAPS both in vitro and in vivo.

Phosphorylated Codonopsis pilosula polysaccharide could inhibit the virulence of duck hepatitis A virus compared with Codonopsis pilosula polysaccharide.

To screen effective anti-duck hepatitis A virus (DHAV) drugs, we applied STMP-STPP method to prepare phosphorylated Codonopsis pilosula polysaccharide (pCPPS), the phosphorylation-modified product of Codonopsis pilosula polysaccharide (CPPS). The IR spectrum and field emission scanning electron microscope (FE-SEM) were subsequently used to analyze the structure of pCPPS. Several tests were conducted to compare the anti-DHAV activities of CPPS and pCPPS. The MTT method was used to compare the effect of the drugs on DHAV-infected duck embryonic hepatocytes (DEHs), and the Reed-Muench assay was employed to observe changes in the virulence of DHAV. We also applied real-time PCR to examine the relationship between virus replication and the expression of IFN-ß. The results indicated that CPPS could not inhibit the replication of DHAV. In contrast, pCPPS increased the virus TCID50, inhibited viral replication and, accordingly, increased the survival rate of DEHs infected with DHAV. Because DHAV induced the expression of IFN-ß, and the IFN-ß expression level was positively associated with the number of DHAV, the reduction of IFN-ß expression levels after pCPPS treatment demonstrated a decrease in the number of virus particles. These results indicated that pCPPS, which reduces the number of DHAV, was more effective than CPPS in anti-DHAV activity.

Assessment of a Flavone-Polysaccharide Based Prescription for Treating Duck Virus Hepatitis.

Because polysaccharide and flavone ingredients display good antiviral activity, we developed a flavone/polysaccharide-containing prescription that would be effective against duck viral hepatitis (DVH) and investigated its hepatoprotective effects. Flavones were derived from Hypericum japonicum (HJF) (entire herb of Hypericum japonicum Thunb) and Salvia plebeia (SPF) (entire herb of Salvia plebeia R. Br.), and polysaccharides were derived from Radix Rehmanniae Recens (RRRP) (dried root of Rehmannia glutinosa Libosch). This prescription combination was based on the theory of syndrome differentiation and treatment in traditional Chinese veterinary medicine. In vitro and in vivo experiments were conducted using the three single ingredients compared to the combined HRS prescription to determine their anti-duck hepatitis A viral (anti-DHAV) activity. The results showed that all experimental conditions displayed anti-DHAV activity, but the HRS prescription presented the best effect. To further investigate the hepatoprotective effect of the HRS prescription on DHAV-induced hepatic injury, we tested the mortality rate, the hepatic pathological severity score, plasma biochemical indexes of hepatic function, blood DHAV gene expression levels and peroxidation damage evaluation indexes and then analyzed correlations among these indexes. The results demonstrated that the HRS prescription significantly decreased the mortality rate, reduced the severity of hepatic injury, decreased the hepatic pathological severity score, depressed blood DHAV gene expression levels, and returned the indexes of hepatic function and peroxidation almost to a normal level. These results indicate that the HRS prescription confers an outstanding hepatoprotective effect, and we expect that it will be developed into a new candidate anti-DHAV drug.

Determine the structure of phosphorylated modification of icariin and its antiviral activity against duck hepatitis virus A.

BACKGROUND: Our previous research showed that icariin (1) and its phosphorylated structural modification (2) improved the survival and attenuated oxidative stress and liver dysfunction induced by duck virus hepatitis. In this paper, we were one step closer to determine the structure of phosphorylation icariin (2) by the FT-IR, HRESIMS and (13)C NMR. Anti-DHAV activities of 1 and 2 were compared in duck embryonic hepatocytes (DEHs) cultured in vitro and by artificial infection method in vivo. Additionally, the antiviral mechanisms of replication/release in vitro and the DHAV gene expression in vivo of 1 and 2 were analyzed. RESULTS: Compound 2's molecular formula was C33H42O18P. The results indicated that 1 and 2 effectively resisted DHAV invading DEHs, that they decreased the mortality of ducklings challenged with DHAV, and that 2 performed more effectively. 1 and 2 performed evenly on DHAV release; however, 2 restrained virus replication far more effectively. Since the anti-DHAV mechanisms of 1 and 2 in vitro probably involve suppression of replication and release, 2's better performance in anti-DHAV may result from its far more effectively inhibiting virus replication. CONCLUSIONS: The compound 2's chemical structure was defined as 8-prenylkaempferol-4'-methylether-3-rhamnosyl-7-(6'''-phosphate)-glycoside. 1 and 2 exhibited anti-virus activity on DHAV. Our results suggest that 1 and 2 might become an anti-virus plant material candidate.

Effects of Bush Sophora Root polysaccharide and its sulfate on immuno-enhancing of the therapeutic DVH.

Bush Sophora Root polysaccharide (BSRPS) and its sulfate, sulfated Bush Sophora Root polysaccharide (sBSRPS), possess the antiviral activities against duck hepatitis A virus. However their antiviral mechanisms are still not clear. This paper reported their immuno-enhancing roles in the therapeutic effects for duck virus hepatitis (DVH). The effects of BSRPS and sBSRPS on stimulating lymphocyte proliferation were investigated by MTT methods. After that, ducklings were challenged with DHAV and treated with BSRPS and sBSRPS. Meanwhile, the total antibody (Ab), cytokines including interferon gamma (IFN-γ), hepatocyte growth factor (HGF), interleukin (IL)-2, IL-6 and IL-8 were determined by enzyme-linked immuno sorbent assay methods. The results showed that BSRPS owned a fine hepatoprotective effect with stable HGF producing ability. Sulfated modification was able to increase the proliferation rates of B and T lymphocytes and the secretions of total Ab, IFN-γ and IL-2, as comparison with those of BSRPS group. In summary, both of them exhibited immuno-enhancing effects on the therapeutic effects for DVH, and the capacity of sBSRPS was stronger than that of BSRPS.

Anti-duck virus hepatitis mechanisms of Bush Sophora Root polysaccharide and its sulfate verified by intervention experiments.

In our previous study, Bush Sophora Root polysaccharide (BSRPS) and its sulfate (sBSRPS) exhibited anti-duck virus hepatitis (DVH) abilities as well as anti-oxidative and immuno-enhancement effects. The aim of this paper was to ulteriorly investigate the exact anti-DVH mechanisms of BSRPS and sBSRPS by intervention experiments. Hinokitiol and FK506 were used as the pro-oxidant and immunosuppressant, respectively. The dynamic deaths, oxidative and immune evaluation indexes and hepatic pathological change scores were detected. When was intervened by hinokitiol, sBSRPS still possessed therapeutic effect while BSPRS was useless. Under the condition of immunosuppression, BSRPS lost a part role in treating DVH; however such a role of sBSRPS completely exhausted. These results suggested both anti-oxidative and immuno-enhancement effects of BSRPS played roles in healing DVH, and the former was more crucial; unlike BSRPS, only immuno-enhancement ability of sBSRPS was imperative for its curative effect on DVH.

The anti-DHAV activities of Astragalus polysaccharide and its sulfate compared with those of BSRPS and its sulfate.

This paper studied the anti-duck hepatitis A virus (DHAV) activities of Astragalus polysaccharide (APS) and its sulfate (sAPS) compared with those of Bush Sophora Root polysaccharide (BSRPS) and its sulfate (sBSRPS). The antiviral activities of APS and sAPS were measured by MTT and real-time PCR methods, in vitro. In vivo experiment, the mortality rate and the evaluation indexes of hepatic injury, peroxidative injury and immune level were measured. Just like the condition of BSRPS and sBSRPS, the anti-DHAV activities of sAPS were stronger than those of APS, both in vitro and in vivo. It indicated sulfated modification could enhance the antiviral ability of polysaccharide. But unlike the antiviral effects of BSPRS and sBSRPS in vivo, APS and sAPS did not reduce the mortality rates as their abilities of scavenging free radicals and alleviating the hepatic injuries were weaker than those of BSRPS and sBSRPS. And they even did not enhance the immune levels.

Bush Sophora Root polysaccharide and its sulfate can scavenge free radicals resulted from duck virus hepatitis.

In order to study the antioxidant effect of Bush Sophora Root polysaccharide (BSRPS) and its sulfate on anti-duck virus hepatitis (DVH), sulfated Bush Sophora Root polysaccharide (sBSRPS) was prepared by chlorosulfonic acid-pyridine method. Ducklings were fed with BSRPS and sBSRPS after challenged DHAV. Death was monitored, evaluation indexes of peroxidative and hepatic injury at the initial (4th and 8th hour) and later (54th hour) stages were detected. The results showed a fine treatment effect of BSRPS and sBSRPS. Visual hepatic pathological injury severities were less serious after the treatment. At the initial stage, free radical levels in all groups were the same, and BSRPS and sBSRPS reduced the hepatic injury through inhibiting virus replication. At the later stage, mass free radicals were detected in VC group while free radical levels in BSRPS and sBSRPS groups were significantly lower than VC group. The antioxidant effect of BSRPS and sBSRPS might alleviate the hepatic injury.

Comparison of Bush Sophora Root polysaccharide and its sulfate's anti-duck hepatitis A virus activity and mechanism.

In order to research the sulfating modification in enhancing the anti-duck hepatitis A virus (DHAV) activity of Bush Sophora Root polysaccharide (BSRPS), sulfated Bush Sophora Root polysaccharide (sBSRPS) was prepared by chlorosulfonic acid-pyridine method. KBr pellets method was applied to analyze their different structures. Anti-DHAV activity was studied by duck embryonic hepatocytes culture in vitro and artificial inoculation method in vivo. Direct immunofluorescence method and Real-time PCR were applied to study the antiviral mechanism of adsorption, replication and release in vitro and the dynamic change of virus content of blood in vivo. The results showed at the most effective content, sBSRPS (7.813 µg/mL) could inhibit both replication and release of DHAV in vitro, BSRPS (500 µg/mL) only inhibit replication. The relative expression of DHAV gene at the 8thh and the mortality rate of sBSRPS group were significantly reduced. These results indicated sBSRPS performed more effectively in anti-DHAV activity than BSRPS.

Identification, expression and activity analyses of five novel duck beta-defensins.

In the current study, five novel avian ß-defensins (AvBDs) were identified and characterized in tissues from Peking ducks (Anas platyrhynchos). The nucleotide sequences of these cDNAs comprised 198 bp, 182 bp, 201 bp, 204 bp, and 168 bp, and encoded 65, 60, 66, 67, and 55 amino acids, respectively. Homology, characterization and comparison of these genes with AvBD from other avian species confirmed that they were Apl_AvBD1, 3, 5, 6, and 16. Recombinant AvBDs were produced and purified by expressing these genes in Escherichia coli. In addition, peptides were synthesized according to the respective AvBD sequences. Investigation of the antibacterial activity of the Apl_AvBDs showed that all of them exhibited antibacterial activity against all 12 bacteria investigated (P<0.05 or P<0.01). In addition, the antibacterial activity of all of the AvBDs against M. tetragenus and P. multocida decreased significantly in the presence of 150 mM NaCl (P<0.01). None of the AvBDs showed hemolytic activity. Consistent with their broad-spectrum antibacterial activity, the five novel Apl_AvBDs inhibited replication of duck hepatitis virus (DHV) in vitro significantly (P<0.05). The mRNA expression of all five Apl_AvBD in most tissues, including immune organs and the liver, was upregulated in response to DHV infection at different time points. These findings provide evidence that these defensins activate the immune response to combat microbial infection.