In vitro antiviral activity of sulfated Auricularia auricula polysaccharides

Total Auricularia auricula polysaccharide (AAP(t)) was prepared by extracting and removing the proteins. Column chromatography was used to further graded it into AAP(1) and AAP(2). Three AAPs were modified by chlorosulfonic acid-pyridine method to obtain three sulfated AAPs (sAAPs), sAAP(t), sAAP(1) and sAAP(2), respectively. Three sAAPs and Newcastle disease virus (NDV) were added into cultivation system of chicken embryo fibroblast (CEF) in three manners, pre-, post- and simultaneous-adding polysaccharide with NDV respectively, taking three non-modified AAPs as control. Their anti-viral activities were compared by MTT method. The results showed that sAAPs and AAPs at a certain concentration could significantly inhibit the cellular infectivity of NDV in three manners. The effects of sAAPs were better than that of AAPs. It indicated that sulfated modification could enhance the antiviral activity of AAP. sAAP(1) and sAAP(t) possessed stronger activity and would be as the component of a new-type antiviral drug.     

Optimization of sulfated modification conditions of tremella polysaccharide and effects of modifiers on cellular infectivity of NDV

Based on our previous research, sulfated modification conditions of Tremella polysaccharide (TPS), the chlorosulfonic acid to pyridine (CSA-Pry) ratio, reaction temperature and time, were optimized by L₉ (3⁴) orthogonal design taking the yield and degree of sulfation (DS) of modifiers as indexes. Two TPSs, TPS_tp and TPS_70c, were modified under optimized conditions. The effects of two modifiers, sTPS_tp and sTPS_70c, on cellular infectivity of NDV were determined by MTT method taking the non-modified TPS_tp, TPS_tc and TPS_70c as controls. The results showed that the optimized modification conditions were reaction temperature of 80 °C, CSA-Pry ratio of 1:6 and reaction time of 1.5 h. Five polysaccharides at proper concentrations could significantly inhibit the infectivity of NDV to CEF. The virus inhibitory rates of sTPS_tp at 1.563 μg mL⁻¹ group were the highest and significantly higher than those of other three non-modified polysaccharide groups in three sample-adding modes. This indicated that sulfated modification could significantly improve the antiviral activity of TPS. sTPS_tp possessed the best efficacy and would be as a component of antiviral polysaccharide drug.     

Liposome and epimedium polysaccharide-propolis flavone can synergistically enhance immune effect of vaccine

Three preparations of epimedium polysaccharide-propolis flavone immunopotentiator (EPI), EPI liposome, EPI suspension and EPI watery solution were prepared. In immune response test, their adjuvanticities were compared in 14-day-old chickens vaccinated with Newcastle disease (ND) vaccine. In immune protection test, the effects of the three preparations on Newcastle disease virus (NDV) infection were compared in chickens vaccinated with ND vaccine then challenged with NDV. The results displayed that EPI liposome could enhance the antibody titer, T lymphocyte proliferation and the concentrations of interferon-γ and interleukin-6, when compared with the other two preparations. In EPI liposome group, the antibody titers, lymphocyte proliferation and protective rate were the highest, while the mortality and morbidity were the lowest, in comparison with the other groups. These results indicated that liposome could enhance the immune effect of EPI on ND vaccine and would be expected as the suitable dosage form of this immunopotentiator.     

Solomonseal Polysaccharide and Sulfated Codonopsis pilosula Polysaccharide Synergistically Resist Newcastle Disease Virus

Five combinations of three ratios (PS₉-sPS₁, PS₇-sPS₃ and PS₆-sPS₄) were prepared with polysaccharide (PS) and sulfated polysaccharide (sPS). The antiviral activities of these compounds were subsequently compared in vitro using the MTT assay, observation of the virus structure and immunofluorescence. The results demonstrated that SP₉-sCP₁, CP₇-sCA₃, EP₇-sAP₃, CA₉-sEP₁ and EP₇-sCA₃ presented higher activities, and SP₉-sCP₁ displayed the highest virus inhibition rate and clearly killed the virus and inhibited viral antigen expression. In an in vivo test, 28-day-old chickens were challenged with Newcastle disease virus (NDV) and were administered the five drug combinations. On day 14 after the challenge, the morbidity, mortality and cure rate in each group were calculated. The results indicated that SP₉-sCP₁ presented the lowest morbidity and mortality and the highest cure rate. These results indicate that Solomonseal polysaccharide and sulfated Codonopsis pilosula polysaccharide synergistically resist NDV. Moreover, SP₉-sCP₁ had the highest efficacy and may be used as a new antiviral drug.     

表达新城疫病毒F48E8株融合蛋白基因的重组鸡痘病毒及其免？…

将将城疫病毒（ＮＤＶ）Ｆ４８Ｅ８株融合蛋白基因导入鸡痘病毒（ＦＰＶ）插入载体ｐＥＧＦ１１７５－１的Ｐ７．５启动子下游,得到转移载体ｐＦＧ１１７５－１重组质粒。采用脂质体转染技术,将该质粒转染ＦＰＶ２８２Ｅ株感染的鸡胚成纤维细胞（ＣＥＦ）。,经过多次蓝斑筛选纯化,获稳定的重组病毒ｒＦＰＶ－ＮＤＦ。间接免疫荧光试验表明,ｒＦＰＶ－ＮＤＦ感染的ＣＥＦ中表达了ＮＤＶ的融合蛋白。用ｒＦＰＶ－ＮＤＦ免疫的ＳＦ     

Partial antiviral activities of the Asn631 chicken Mx against newcastle disease virus and vesicular stomatitis virus

Conflicting data existed for the antiviral potential of the chicken Mx protein and the importance of the Asn631 polymorphism in determination of the antiviral activity. In this study we modified the chicken Mx cDNA from the Ser631 to Asn631 genotype and transfected them into COS-I cells, chicken embryonic fibroblast (CEF) or NIH 3T3 cells. The Mx protein was mainly located at the cytoplasm. The transfected cell cultures were challenged with newcastle disease virus (NDV) or vesicular stomatitis virus (VSV), cytopathic affect (CPE) inhibition assay showed that the times for development of visible and full CPE were significantly postponed by the Asn631 cDNA transfection at 48 h transfection, but not by the Ser631 cDNA transfection. Viral titration assay showed that the virus titers were significantly reduced before 72 h postinfection. CEF cells was incubated by the cell lysates extracted from the COS-I cells transfected with pcDNA-Mx/Asn631, could resist and delayed NDV infection. These data suggested the importance of the Asn631 polymorphism of the chicken Mx in determination of the antiviral activities against NDV and VSV at early stage of viral infection, which were relatively weak and not sufficient to inhibit the viral replication at late stage of viral infection.     

枯草芽孢杆菌fmbJ产生的新型抗微生物物质体外抗NDV和IBDV的活性分析

测定了枯草芽孢杆菌fmbJ株产生的新型抗微生物物质的体外抗新城疫病毒(Newcastle disease virus,NDV)lasota株、传染性法式囊病病毒(Infectious Bursal Disease Virus,IBDV)哈尔滨(H)株作用。结果表明该新型抗微生物物质对鸡胚成纤维(Chicken Embryo Fibroblasts,CEF)细胞的TD50和TD0分别为128.95mg/L、25.79mg/L;对NDVlasota株、IBDV H株所致细胞病变效应有明显的抑制作用,可使细胞存活率显著升高;该抗微生物物质具有抗NDVlasota株、IBDV H株作用;并具有预防其感染及抑制其复制的作用。其抗病毒作用效果和病毒唑相当,由于其对CEF细胞的毒性较弱,可作为一种抗病毒药物进行开发研究。     

Inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of Marek's disease virus

The replication of Marek's disease herpesvirus (MDV) and herpesvirus of turkeys (HVT) in chicken embryo fibroblast (CEF) cultures was inhibited by the addition of S-nitroso-N-acetylpenicillamine, a nitric oxide (NO)-generating compound, in a dose-dependent manner. Treatment of CEF culture, prepared from 11-day-old embryos, with recombinant chicken gamma interferon (rChIFN-gamma) and lipopolysaccharide (LPS) resulted in production of NO which was suppressed by the addition of N(G)-monomethyl L-arginine (NMMA), an inhibitor of inducible NO synthase (iNOS). Incubation of CEF cultures for 72 h prior to treatment with rChIFN-gamma plus LPS was required for optimal NO production. Significant differences in NO production were observed in CEF derived from MDV-resistant N2a (major histocompatibility complex [MHC], B(21)B(21)) and MDV-susceptible S(13) (MHC, B(13)B(13)) and P2a (MHC, B(19)B(19)) chickens. N2a-derived CEF produced NO earlier and at higher levels than CEF from the other two lines. The lowest production of NO was detected in P2a-derived CEF. NO production in chicken splenocyte cultures followed a similar pattern, with the highest levels of NO produced in cultures from N2a chickens and the lowest levels produced in cultures from P2a chickens. Replication of MDV and HVT was significantly inhibited in CEF cultures treated with rChIFN-gamma plus LPS and producing NO. The addition of NMMA to CEF treated with rChIFN-gamma plus LPS reduced the inhibition. MDV infection of chickens treated with S-methylisothiourea, an inhibitor of iNOS, resulted in increased virus load compared to nontreated chickens. These results suggest that NO may play an important role in control of MDV replication in vivo.     

Astragalus polysaccharide and sulfated epimedium polysaccharide synergistically resist the immunosuppression

The immunoenhancement of compound polysaccharides, APS-sEPS composed with astragalus polysaccharide (APS) and sulfated epimedium polysaccharide (sEPS), was observed in immunosuppressed model chicken induced by cyclophosphamide (Cy). 11-day-old chickens were injected with Cy once a day for three successive days except vaccine control group. At day-14-old, all chickens were vaccinated with ND vaccine, and in experimental groups simultaneously administrated with APS-sEPS at three dosages, APS and sEPS once a day for three successive days. On days 7, 14, 21 and 28 after the administration, the peripheral T-lymphocyte proliferation, serum antibody titers, IFN-γ, IL-2, IgG and IgM were determined. The results displayed that APS-sEPS could overcome Cy-induced immunosuppression, significantly promote T-lymphocyte proliferation and raised serum antibody titers, IFN-γ, IL-2, IgG and IgM levels, its high and medium doses were superior to single APS or sEPS. This demonstrated that APS and sEPS could synergistically resist the immunosuppression and APS-sEPS was an effective immunopotentiator.     

Autophagy Benefits the Replication of Newcastle Disease Virus in Chicken Cells and Tissues

Newcastle disease virus (NDV) is an important avian pathogen. We previously reported that NDV triggers autophagy in U251 glioma cells, resulting in enhanced virus replication. In this study, we investigated whether NDV triggers autophagy in chicken cells and tissues to enhance virus replication. We demonstrated that NDV infection induced steady-state autophagy in chicken-derived DF-1 cells and in primary chicken embryo fibroblast (CEF) cells, evident through increased double- or single-membrane vesicles, the accumulation of green fluorescent protein (GFP)-LC3 dots, and the conversion of LC3-I to LC3-II. In addition, we measured autophagic flux by monitoring p62/SQSTM1 degradation, LC3-II turnover, and GFP-LC3 lysosomal delivery and proteolysis, to confirm that NDV infection induced the complete autophagic process. Inhibition of autophagy by pharmacological inhibitors and RNA interference reduced virus replication, indicating an important role for autophagy in NDV infection. Furthermore, we conducted in vivo experiments and observed the conversion of LC3-I to LC3-II in heart, liver, spleen, lung, and kidney of NDV-infected chickens. Regulation of the induction of autophagy with wortmannin, chloroquine, or starvation treatment affects NDV production and pathogenesis in tissues of both lung and intestine; however, treatment with rapamycin, an autophagy inducer of mammalian cells, showed no detectable changes in chicken cells and tissues. Moreover, administration of the autophagy inhibitor wortmannin increased the survival rate of NDV-infected chickens. Our studies provide strong evidence that NDV infection induces autophagy which benefits NDV replication in chicken cells and tissues.     

Egyptian propolis: 2. Chemical composition,antiviral and antimicrobial activities of East Nile Delta propolis

Three propolis samples from East Nile Delta, Egypt were collected. Propolis samples were investigated by GC/MS,103 compounds were identified, 20 being new for propolis. Dakahlia propolis was a typical poplar propolis but it contained two new caffeate esters and two new triterpenoids. Ismailia propolis was characterized by the presence of new triterpenic acid methyl esters and it did not contain any aromatic acids, esters and flavonoids. Sharkia propolis was characterized by the presence of caffeate esters only, some di- and triterpenoids. The antiviral (Infectious Bursal Disease Virus and Reo-Virus) and antimicrobial (Staphylococcus aureus; Escherichia coli and Candida albicans) activities of propolis samples were investigated. Dakahlia propolis showed the highest antiviral activity against Infectious Bursal Disease Virus (IBDV) and the highest antibacterial activity against Escherichia coli and the highest antifungal activity against Candida albicans. While Ismailia propolis had the highest antiviral activity against Reo-virus. Sharkia propolis showed the highest antibacterial activity against Staphylococcus aureus and moderate antiviral activity against infectious bursal disease virus and reovirus.     

Comparative Studies on Large- and Small-Plaque-Forming Clones of Newcastle Disease Virus (Miyadera Strain)

The Miyadera strain of Newcastle disease virus (NDV) consisted predominantly of virus particles forming small plaques on monolayers of chick embryo fibroblasts (CEF), and contained small amounts of virus particles forming large plaques. These large- and small-plaque-forming clones of this virus (NDV-L and NDV-S) were isolated. The small size of the NDV-S plaques did not appear to be due to an agar inhibitor. NDV-L produced a much higher yield of infective virus particles in CEF and they were released more completely from the infected cells than were those produced by NDV-S. The yield of infective virus of NDV-L per cell from cultures of CEF was comparable to the yield from the allantoic cells. The infectivity/hemagglutinin ratio for NDV-L from CEF was as high as the ratio for virus from the allantoic cells, but the ratio for NDV-S from CEF was lower. NDV-S demonstrated an autointerference phenomenon in CEF when infected at high multiplicities, but NDV-L did not. Contrary to virus multiplication, NDV-S exhibited a more rapid and marked cytopathic effect on monolayers of CEF than NDV-L. In the allantoic cavity of eggs NDV-S produced slightly higher virus yields than NDV-L. No correlation existed between plaque size of the two viruses and the capacity to induce interferon synthesis or the susceptibility to the action of interferon. The properties of both distinctive plaque isolates were stable on egg passage.     

Rescue and preliminary application of a recombinant newcastle disease virus expressing green fluorescent protein gene

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain, seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of the NDV ZJI strain. The pNDV/ZJI, with three helper plasmids, pCIneoNP, pCIneoP and pCIneoL, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, an infectious NDV ZJI strain was successfully rescued. Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP. After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIGFP, was rescued. Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection, indicating that the GFP gene was expressed at a relatively high level. NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route. Four days post-infection, strong green fluorescence could be detected in the kidneys and tracheae, indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.     

Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain, seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of the NDV ZJI strain. The pNDV/ZJI, with three helper plasmids, pCIneoNP, pCIneoP and pCIneoL, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, an infectious NDV ZJI strain was successfully rescued. Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP. After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIGFP, was rescued. Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection, indicating that the GFP gene was expressed at a relatively high level. NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route. Four days post-infection, strong green fluorescence could be detected in the kidneys and tracheae, indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.     

短发夹结构RNA干扰新城疫病毒的增殖

 以新城疫病毒（NDV）NP基因为标靶,构建3个细胞内表达发夹样结构小干扰RNA（shRNA）的质粒载体,在鸡胚成纤维细胞（CEF）和鸡胚上进行了RNAi试验,筛选出一个有效抑制病毒复制的小分子ndv1.用阳离子脂质体转染试剂Silent-fect 将ndv1转染CEF,以不相关shRNA质粒载体HK为阴性对照,4 h后接种NDV,与对照相比,干涉组在病毒感染后3 h NP基因的表达量降低2.3倍,6 h 降低21.1倍,9 h降低9.8倍;ndv1能在48 h内完全阻断NDV在CEF中的增殖,延缓病变出现时间,减轻病变程度.将Silent-fect-ndv1混合物与NDV同时注入10日龄SPF鸡胚绒毛尿囊腔,能使10⁵ ELD₅₀NDV感染后17 h鸡胚尿囊液中病毒增殖量减少94.4%,使10⁶ ELD₅₀NDV感染后17　h鸡胚尿囊液中病毒增殖量减少62.5%.实验结果证实,在CEF中存在RNAi机制,抑制NDV NP基因的表达能有效阻断该病毒增殖,说明NP基因在NDV复制过程中起重要作用.实验结果为进一步利用RNAi技术在CEF和鸡胚中研究病毒基因组功能及筛选抗病毒小分子奠定了基础.     

Effect of Egyptian propolis on the susceptibility of LDL to oxidative modification and its antiviral activity with special emphasis on chemical composition

The antioxidant activity of eight Egyptian propolis samples from different localities was evaluated by the antioxidative potential and capacity of the DPPH-ESR signal, superoxide anion generated in the xanthine-xanthine oxidase (XOD) system and low density lipoprotein (LDL) peroxidation assay. As, F, Is and D samples showed the highest antioxidative capacity and potential, respectively. The El, IsR, Is, D and So samples exhibited highly significant antioxidant activity in the XOD system and in LDL peroxidation assays. The antiviral activity of propolis samples was investigated. They showed variations in their activity; sample D induced the highest antiviral activity against Newcastle disease virus and infectious bursal disease virus. 42 Polyphenolic compounds were identified by HPLC; 13 aromatic acids, esters and alcohols were present, 29 flavonoids were identified, 6 of them being new to propolis.     

表面展示新城疫病毒部分F蛋白的重组干酪乳杆菌的构建及其免疫原性

本研究旨在构建重组干酪乳杆菌pLA-Newcastlediseasevirus (NDV)-F/Lactobacillus casei,获得表达产物,并探讨其免疫效果。利用PCR扩增携带部分主要抗原表位的NDV F基因,与穿梭质粒pLA连接转化至大肠杆菌BL21 (DE3)中,筛选阳性重组质粒,将其电转化至干酪乳杆菌中,构建重组干酪乳杆菌pLA-NDV-F/L. casei,应用PCR鉴定阳性菌株,Western blotting鉴定重组菌反应原性,间接免疫荧光、流式细胞术和激光共聚焦检测蛋白表达情况。试验选用14日龄雏鸡,各组免疫方式为口服+滴鼻。设立pLA-NDV-F/L. casei两次免疫组和三次免疫组、弱毒疫苗组、 pLA/L.casei、未攻毒PBS组和攻毒PBS组。间接ELISA方法检测雏鸡血清IgG、肠道、鼻腔、肺脏中sIgA抗体效价,评价试验组雏鸡攻毒保护率。结果表明,有94.10%的重组菌表达了F蛋白,且高效表达在干酪乳杆菌细胞表面,蛋白大小为62kDa,并能与抗NDV阳性血清特异性结合。各免疫组anti-F IgG和s Ig A抗体水平显著高于对照组,p LA-NDV-F/L. casei三次免疫组抗体持续时间比两次免疫组延长28 d,抗体峰值没有显著差异。免疫pLA-NDV-F/L. casei三次、两次、弱毒疫苗、pLA/L. casei和PBS的攻击保护率分别为80%、80%、90%、0%和0%。因此,利用干酪乳杆菌表达体系成功表达了携带部分抗原表位的NDVF基因,具备良好的反应原性和免疫原性,可诱导机体产生保护性免疫应答。     

Effect of sulfated astragalus polysaccharide on cellular infectivity of infectious bursal disease virus

Four kinds of astragalus polysaccharides (APSs), APS(t), APS(40), APS(50) and APS(60), were extracted by water decoction and one-step or stepwise ethanol precipitation methods, and modified by chlorosulfonic acid-pyridine method to obtain four sulfated APSs (sAPSs) (sAPS(t), sAPS(40), sAPS(50), sAPS(60)), respectively. The effects of four sAPSs on cellular infectivity of bursal disease virus (IBDV) were compared by MTT method taking non-modified APS(t) as control. The results showed that modified sAPSs inhibited IBDV to infect CEF significantly in comparison with non-modified APS(t), which indicated that sulfated modification could enhance the antiviral activity of the APS, by which it would be expected to develop a new-type antiviral drug.