高效液相尺寸排阻色谱-多角度激光散射定量检测灭活禽流感病毒抗原中完整病毒颗粒

本研究旨在建立一种灭活禽流感病毒原料液中完整病毒颗粒准确定量的方法。针对直接采用高效液相尺寸排阻色谱法（high performance size exclusion chromatography，HPSEC）检测灭活禽流感病毒原液存在杂质干扰的问题，首先以H5N8型抗原为对象，分别考察了聚乙二醇（polyethylene glycol，PEG）沉淀和离子交换色谱法（ion exchange chromatography，IEC）进行预处理。在优化条件下，经DEAE FF阴离子交换层析纯化预处理，杂蛋白去除率为86.87%，病毒血凝回收率为100%。HPSEC分析预处理后的样品，8.5–10.0 min处色谱峰样品电泳检测显示主要为H5N8病毒蛋白，动态光散射分析平均粒径为127.7 nm，推测为完整病毒特征峰；在IEC预处理后的样品中加入抗体进行HPSEC检测，8.5-10.0 min处特征峰消失，显示IEC预处理有效去除了杂质干扰。通过将HPSEC与多角度激光散射技术（multi-angle laser scattering technique，MALLS）联用，可以准确获得样品中完整病毒颗粒的数量，且病毒颗粒数与色谱峰面积具有良好线性关系（R²=0.997）。建立的IEC预处理-HPSEC-MALLS检测方法应用于其他亚型（H7N9）、批次和浓度的病毒原液中完整病毒颗粒数的准确检测，均具有良好适用性，且重复性好，相对标准偏差（relative standard deviation，RSD）<5%，n=3。     

腺相关病毒载体工程毒株精细化纯化

[背景] 大量制备高纯度的重组腺相关病毒（Recombinant Adeno-Associated Virus,rAAV）,是以腺相关病毒（AAV）为投递载体的基因靶向治疗或基因工程药物研发过程中需要解决的重要问题。[目的] 利用层析柱法大量纯化质粒和rAAV细胞培养液,以获得高纯度的rAAV颗粒。[方法] 对组装rAAV的3种质粒用HiPrep^TM10 Sepharose 6FF和HiTrap PlasmidSelect Xtra凝胶层析柱纯化,目的是得到组装AAV的超螺旋质粒DNA,然后对组装rAAV过程中的AAV-293细胞培养方法进行优化,组装成功病毒后,再对收集到的细胞提取液用HiLoad^TM10Q和HiLoad^TM10SP阴阳离子交换层析柱纯化,最后用纯化浓缩后的rAAV感染HT1080细胞,通过流式细胞仪检测荧光表达细胞数,计算浓缩后活病毒感染滴度。[结果] HiPrep和HiTrap层析柱纯化后得到大量高纯度的超螺旋质粒DNA,组装病毒后,用HiLoad柱纯化获得大量高纯度的rAAV颗粒,通过测定,rAAV基因组滴度达到（2.46±0.37）×10¹² TCID₅₀/mL （MOI=1.0×10⁴）。[结论] 通过一系列精细化组装纯化制备出高纯度、高滴度rAAV病毒颗粒。     

Occurrence and characterization of a nucleopolyhedrovirus from <Emphasis Type="Italic">Maruca vitrata</Emphasis> (Lepidoptera,Pyralidae) isolated in Taiwan

A nucleopolyhedrovirus (MaviMNPV) was isolated from diseased larvae of legume pod borer (LPB), Maruca vitrata, at Tainan in Taiwan. Electron microscopical studies on the ultrastructure of MaviMNPV occlusion bodies (OBs) showed several virions (up to 19) with multiple nucleocapsids (up to 6) packaged within a single viral envelope. The diameter of OBs was 0.9 to 1.3 μm with a mean of 1.152±0.116 μm. The complete sequence of the MaviMNPV polyhedrin (Polh) gene contained 735 nucleotides (GenBank accession number DQ399596). Phylogenetic analyses using the complete sequence of the Polh gene of MaviMNPV indicated that this virus clusters with Group I NPVs. The genome size of MaviMNPV estimated with restriction enzymes viz., HindIII, EcoRI, BglII and PstI was 113.41 ± 1.50 kbp. First instar LPB larvae were the most susceptible stage (LC₅₀ 2.053 × 10² OBs/ml) followed by second, third and fourth instars with the median lethal concentrations (LC₅₀s) 1.410 × 10³, 2.390 × 10³ and 2.636 × 10³ OBs/ml, respectively. This is the first record of this virus from this region. The first and second authors have equal contributions in this paper     

猪肠病毒G (EV-G)荧光定量PCR检测方法的建立与四川地区分子流行病学调查

【背景】猪肠病毒G (Enterovirus G,EV-G)在猪群中普遍存在,可引起猪的皮肤损伤、肌肉麻痹、肺炎、发热、腹泻等,也存在无症状感染。【目的】建立检测EV-G的荧光定量PCR (real-time quantitative PCR,RT-qPCR)方法并开展四川地区分子流行病学调查。【方法】根据EV-G的5ʹUTR基因保守序列设计特异性引物,建立可检测EV-G各基因型的RT-qPCR,并评估其灵敏性、特异性和重复性,开展四川地区EV-G的流行调查。【结果】标准品在1.89×10²-1.89×10⁸ copies/μL浓度范围内线性关系良好;在特异性试验中,其他9种猪病毒(猪德尔塔冠状病毒、猪流行性腹泻病毒、传染性胃肠炎病毒、日本乙型脑炎病毒、猪萨佩罗病毒、猪细小病毒、猪圆环病毒、猪伪狂犬病病毒和猪繁殖与呼吸综合征病毒)检测均为阴性;最低检测浓度为1.89×10¹ copies/μL;组内和组间变异系数分别小于1%和2%。检测2013-2021年四川省431份猪腹泻样品,EV-G总体阳性率为31.1%,表明该病毒在四川地区的感染较普遍。随机选取9份四川的EV-G阳性样品扩增VP1基因并测序分析,发现四川EV-G流行的基因型包括G1、G3、G4和G9,但以EV-G1为优势基因型。【结论】本研究建立了一种检测EV-G各基因型的RT-qPCR方法,并初步掌握了四川EV-G的流行现状,为后续深入开展该病毒的研究奠定了基础。     

The effect of benthic sediments on dissolved nutrient concentrations and fluxes

The Ria Formosa is a meso-tidal coastal lagoon experiencing enhanced nutrient concentrations. Assessment of sediment–seawater interaction is essential if nutrient dynamics and the risk of eutrophication are to be fully understood. Pore water concentrations of dissolved inorganic and organic phosphorus, ammonium, nitrate and nitrite were determined in cores from six sites. Changes in nutrients concentrations were measured in intertidal pools on sand and mud between tides. Dissolved inorganic phosphorus (DIP) concentrations (~200 μmol l⁻¹) and effluxes (123 ± 14 μmol m⁻² h⁻¹) were greater from sand than mud (37 ± 10 μmol m⁻² h⁻¹), possibly due to the binding of P with the <63 μm fraction. NH₄⁺ effluxes were high outside the Anc?o Basin (821 ± 106 μmol m⁻² h⁻¹) and were associated with Enteromorpha sp. mats. The greatest NO₃⁻ efflux was from sediments near a salt marsh (170 ± 67 μmol m⁻² h⁻¹). These sediment fluxes of P were not sufficient to account for elevated P concentrations seen by other workers on the ebb tide from the Anc?o Basin. Intertidal pools were sinks for Dissolved Inorganic Nitrogen (DIN) and DIP over the 6 h exposure period. Thus, tidepools may be an important route of nutrients into sediments that enhances the effects of sediments on seawater nutrient concentrations.     

金属离子抑制细菌整合子捕获耐药基因盒的机制研究

【目的】探索金属离子对整合子捕获耐药基因盒的影响及其相关机制。【方法】在大肠埃希菌中构建一个1类整合子捕获耐药基因盒的体内模型。通过实时荧光定量PCR (real-time fluorescence quantitative PCR,RT-qPCR)测定不同浓度银(0.3、0.9、1.5 μg/mL的Ag⁺)和铜离子(5、50、100、150、210 μg/mL的Cu²⁺)干预后实验组和无金属离子干预对照组整合子整合频率,并用表型筛选法验证。利用质谱法测量细菌吸收的金属离子浓度,并进一步通过转录组测序方法分析银离子抑制细菌整合子捕获耐药基因盒的分子机制。【结果】qPCR和表型筛选法的结果表明,0.9 μg/mL银离子组整合频率为9.42×10^‒5 (6.49×10^‒5,1.44×10^‒4),1.5 μg/mL银离子组整合频率为7.29×10^‒5 (4.45×10^‒5,9.03×10^‒5),与对照组2.59×10^‒4 (2.24×10^‒4,3.33×10^‒4)相比整合频率明显降低,差异有统计学意义(P<0.001)。而不同浓度铜离子组与对照组没有明显差异。转录组测序方法对银离子作用前后大肠杆菌基因表达水平进行对比分析,通过GO功能和KEGG通路富集发现,差异表达基因与甲基半乳糖苷转运(methylgalactoside transport)、麦芽糖转运(maltose transport)、磷酸烯醇式丙酮酸-甘油磷酸转移酶活性(phosphoenolpyruvate-glycerone phosphotransferase activity)和甘油激酶活性(glycerone kinase activity)等功能有关以及涉及氨基酸糖和核苷酸糖代谢(amino sugar and nucleotide sugar metabolism)、鞭毛组装(flagellar assembly)、阳离子抗菌肽(CAMP)耐药性[cationicantimicrobial peptide (CAMP) resistance]、果糖和甘露糖代谢和磷酸糖转移酶系统[phosphotransferase system (PTS)]等代谢通路。通过蛋白互作网络筛选出前12个枢纽基因(ptsG、malE、lamB、lacZ、malK、basR、ais、ugd、nagE、metN、malQ和malF)。【结论】一定浓度银离子可以抑制整合子整合耐药基因盒。铜离子对整合频率影响不明显,因此不是所有具有杀菌性的金属离子都能对整合频率产生影响。银离子抑制细菌整合子捕获耐药基因盒可能是通过影响麦芽糖转运和碳分解代谢来调控。然而,碳分解代谢和麦芽糖转运过程很复杂,需要进一步研究。目前尚无细菌整合子转录组学相关研究,这为解决细菌耐药性问题提供了一个新的途径。     

竹茶混交模式对表层土壤有机碳储量及组分的影响

为探究毛竹林下种植茶树对土壤有机碳储量与碳组分的影响,该研究以毛竹纯林、竹茶混交林和常绿阔叶林为研究对象,采集这3种林分类型的表层(0～10 cm)土壤,测定土壤有机碳(SOC)、碳组分、生物与非生物因素指标。结果表明:(1)竹茶混交林林下植物多样性相较于毛竹纯林显著降低,但其土壤有机碳密度(22.54±2.09)t·hm^-2、碳组分与毛竹纯林无显著差异(P>0.05)。竹茶混交林的矿物结合态有机碳(MOC)为(20.13±1.83)g·kg^-1,占总有机碳的92.66%。常绿阔叶林土壤有机碳密度比竹茶混交林和毛竹纯林高土壤有机碳密度分别高41.15%和41.00%(P<0.05)。(2)3种林分类型土壤微生物量碳(MBC)含量范围为0.58～3.08 g·kg^-1,土壤16S rRNA丰度范围为2.18×10¹⁰ ～5.65×10¹⁰copies·g^-1,固碳基因cbbL丰度范围为0.37×10⁸～ 1.10 ×10⁸ copies·g^-1,土壤微生物碳利用效率范围为0.03～0.28; 3种林分类型之间微生物相关指标不存在显著差异(P>0.05)。(3)3种林分类型SOC与土壤pH、砂粒含量和地上凋落物生物量呈显著负相关,与土壤黏粒含量、粉粒含量、总氮、C:N、总磷和铵态氮含量呈显著正相关(P<0.05)。(4)就不同碳组分而言,颗粒有机碳(POC)和MOC均与土壤pH、砂粒含量和根系生物量呈显著负相关,与土壤含水量、黏粒含量、粉粒含量、总氮、C:N、总磷和铵态氮含量呈显著正相关(P<0.05)。综上表明,竹茶混交改造会造成原生毛竹纯林林下植被多样性下降,但并未造成土壤碳储量下降; 而相较于常绿阔叶林,毛竹经营措施需要改进,以提升其碳汇效益。     

猪链球菌4型疫苗用菌株的筛选

【背景】猪链球菌4型（Streptococcus suis serotype 4,SS4）分离率日益升高,对养殖业和公共卫生安全造成严重危害,目前尚无有效的SS4疫苗。【目的】筛选致病力强、抗原性好、遗传性状稳定的SS4疫苗菌种。【方法】以7株SS4分离株（代号为A1—A7）为受试菌株,通过累积法测定菌株半数致死量（LD₅₀）,ELISA测定免疫小鼠血清中IgG效价,攻毒保护试验测定免疫保护率,并采集小鼠脏器观察病理组织学变化。再连续传代培养受试菌株,分别对第10、20、30代菌株进行致病性和抗原性试验。【结果】A1—A7菌株对小鼠的LD₅₀分别为2.19×10⁸、1.76×10⁸、1.83×10⁸、1.01×10⁸、4.05×10⁸、1.19×10⁸和9.03×10⁷ CFU。二免7 d后,A1、A2、A3、A4、A6、A7免疫组IgG效价分别为1：1 600、1：1 600、1：3 200、1：6 400、1：3 200和1：6 400,免疫保护率分别为30%、30%、50%、70%、60%和80%,而且A4、A7免疫组小鼠组织病变较其余4组轻微。体外传至30代后,A4菌株的LD₅₀上升至3.81×10⁸ CFU,IgG效价下降至1：1 600,免疫保护率下降至40%,而A7菌株的LD₅₀上升至2.49×10⁸ CFU,IgG效价和免疫保护率稳定保持为1：6 400和80%,而且A7免疫组小鼠的组织病变较A4免疫组轻微。【结论】A7菌株（原始编号为HBgu18-4）具有强致病力和良好的抗原性,而且遗传性状均一、稳定,可作为SS4制苗候选菌株。     

Carbon-nanotube-modified electrodes for the direct bioelectrochemistry of pseudoazurin

The bioelectrochemistry of the blue copper protein, pseudoazurin, at glassy carbon and platinum electrodes that were modified with single-wall carbon nanotubes (SWNTs) was investigated by multiple scan rate cyclic voltammetry. The protein showed reversible electrochemical behavior at both bare glassy carbon electrodes (GCEs) and SWNT-modified GCEs (SWNT|GCEs); however, direct electrochemistry was not observed at any of the platinum electrodes. The effect of the carbon nanotubes at the GCE was to amplify the current response 1000-fold (nA at bare GCE to μA at SWNT|GCE), increase the apparent diffusion coefficient D _app of the solution-borne protein by three orders of magnitude, from 1.35 × 10⁻¹¹ at bare GCE to 7.06 × 10⁻⁸ cm² s-1 at SWNT|GCE, and increase the heterogeneous electron transfer rate constant k _s threefold, from 1.7 × 10⁻² cm s⁻¹ at bare GCE to 5.3 × 10⁻² cm s⁻¹ at SWNT|GCE. Pseudoazurin was also found to spontaneously adsorb onto the nanotube-modified GCE surface. Well-resolved voltammograms indicating quasi-reversible faradaic responses were obtained for the adsorbed protein in phosphate buffer, with I _pc and I _pa values now greater than corresponding values for solution-borne pseudoazurin at SWNT|GCEs and with significantly reduced ΔE _p values. The largest electron transfer rate constant of 1.7 × 10⁻¹ cm s⁻¹ was achieved with adsorbed pseudoazurin at the SWNT|GCE surface in deaerated buffer solution consistent with its presumed role in anaerobic respiration of some bacteria.     

Phytoplankton primary production in a mesotrophic pond in sub-tropical Bangladesh

Annual gross primary productivity in mesotrophic Shahidullah Hall pond (Dhaka, Bangladesh) was 1383.35 g C m⁻² y⁻¹ (arithmetic mean). Daily primary productivity (between 1.6 and 6.8 g C m⁻² d⁻¹ was correlated with chlorophylla, day length and dissolved silica. Chlorophylla related significantly withk, incident light, SRP, alkalinity and conductivity. A negative correlation existed between biomass and rainfall. Productivity, biomass, conductivity, alkalinity, and SRP increased after mid-winter.k, I _k andZ _eu varied according seasonally.P _max related directly with temperature. Seasonal variation of ∝ ^B was 0.0049–0.0258 mg C (mg chla mmol PAR)⁻¹ m⁻². Q₁₀ was 2.12, community respiration 1334.99 g C m⁻² y⁻¹, and the underwater light climate 186.43μE m⁻² s⁻¹.     

Investigation of porcine circovirus contamination in human vaccines

DNA from porcine circovirus type 1 (PCV1) and 2 (PCV2) has recently been detected in two vaccines against rotaviral gastroenteritis from manufacturers A and B. We investigated if PCV1 sequences are present in other viral vaccines. We screened seeds, bulks and final vaccine preparations from ten manufacturers using qRT-PCR. We detected 3.8 × 10³ to 1.9 × 10⁷ PCV1 DNA copies/milliliter in live poliovirus seeds for inactivated polio vaccine (IPV) from manufacturer A, however, following inactivation and purification, the finished IPV was PCV1-negative. PCV1 DNA was not detectable in live polio preparations from other vaccine producers. There was no detectable PCV1 DNA in the measles, mumps, rubella and influenza vaccines analysed including material supplied by manufacturer A. We confirmed that the PCV1 genome in the rotavirus vaccine from manufacturer A is near full-length. It contains two mutations in the PCV cap gene, which may result from viral adaptation to Vero cells. Bulks of this vaccine contained 9.8 × 10¹⁰ to 1.8 × 10¹¹ PCV1 DNA copies/millilitre and between 4.1 × 10⁷ and 5.5 × 10⁸ DNA copies were in the final doses. We found traces of PCV1 and PCV2 DNA in the rotavirus vaccine from manufacturer B. This highlights the issue of vaccine contamination and may impact on vaccine quality control.     

Studies on water transport through the sweet cherry fruit surface: characterizing conductance of the cuticular membrane using pericarp segments

Water conductance of the cuticular membrane (CM) of mature sweet cherry fruit (Prunus avium L. cv. Sam) was investigated by monitoring water loss from segments of the outer pericarp excised from the cheek of the fruit. Segments consisted of epidermis, hypodermis and several cell layers of the mesocarp. Segments were mounted in stainless-steel diffusion cells with the mesocarp surface in contact with water, while the outer cuticular surface was exposed to dry silica (22 ± 1 °C). Conductance was calculated by dividing the amount of water transpired per unit area and time by the difference in water vapour concentration across the segment. Conductance values had a log normal distribution with a median of 1.15 × 10⁻⁴ m s⁻¹ (n=357). Transpiration increased linearly with time. Conductance remained constant and was not affected by metabolic inhibitors (1 mM NaN₃ or 0.1 mM carbonylcyanide m-chlorophenylhydrazone) or thickness of segments (range 0.8–2.8 mm). Storing fruit (up to 42 d, 1 °C) used as a source of segments had no consistent effect on conductance. Conductance of the CM increased from cheek (1.16 ± 0.10 × 10⁻⁴ m s⁻¹) to ventral suture (1.32 ± 0.07 × 10⁻⁴ m s⁻¹) and to stylar end (2.53 ± 0.17 × 10⁻⁴ m s⁻¹). There was a positive relationship (r²=0.066**; n=108) between conductance and stomatal density. From this relationship the cuticular conductance of a hypothetical astomatous CM was estimated to be 0.97 ± 0.09 × 10⁻⁴ m s⁻¹. Removal of epicuticular wax by stripping with cellulose acetate or extracting epicuticular plus cuticular wax by dipping in CHCl₃/methanol increased conductance 3.6- and 48.6-fold, respectively. Water fluxes increased with increasing temperature (range 10–39 °C) and energies of activation, calculated for the temperature range from 10 to 30 °C, were 64.8 ± 5.8 and 22.2 ± 5.0 kJ mol⁻¹ for flux and vapour-concentration-based conductance, respectively. Received: 23 March 2000 / Accepted: 28 July 2000     

Characterisation of acidic sites of Pseudomonas biomass capable of binding protons and cadmium and removal of cadmium via biosorption

Summary The ability of Pseudomonas aeruginosa to accumulate Cd(II) ions from wastewater industries was experimentally investigated and mathematically modelled. From the potentiometric titration and non-ideal competitive analysis (NICA) model, it was found that the biomass contains three acidic sites. The values of proton binding (pK _i =1.66±3.26×10⁻³, 1.92±1.63×10⁻⁴ and 2.16±3.79×10⁻⁴) and binding constant of cadmium metal ions (pK _M1=1.99±2.45×10⁻³ and pK _M2=1.67±4.08×10⁻³) on the whole surface of biomass showed that protonated functional groups and biosorption of Cd(II) ions could be attributed to a monodentate binding to one acidic site, mainly the carboxylic group. From the isothermal sorption experimental data and Langmuir model, it was also found that the value of Langmuir equilibrium (pK _f) constant is 2.04±2.1×10⁻⁵ suggesting that the carboxyl group is the main active binding site. In addition, results showed that the maximum cadmium capacity (q _max) and affinity of biomass towards cadmium metal ions (b) at pH 5.1 and 20 min were 96.5±0.06 mg/g and 3.40×10⁻³± 2.10×10⁻³, respectively. Finally, interfering metal ions such as Pb(II), Cu(II), Cr(III), Zn(II), Fe(II), Mn(II), Ca(II) and Mg(II) inhibited Cd(II) uptake. Comparing the biosorption of Cd(II) by various Pseudomonas isolates from contaminated environment samples (soil and sewage treatment plant) showed that maximum capacities and equilibrium times were different, indicating that there was a discrepancy in the chemical composition between biomasses of different strains.     

Efficacy of a plant‐produced virus‐like particle vaccine in chickens challenged with Influenza A H6N2 virus

The efficacy, safety, speed, scalability and cost‐effectiveness of producing hemagglutinin‐based virus‐like particle (VLP) vaccines in plants are well‐established for human influenza, but untested for the massive poultry influenza vaccine market that remains dominated by traditional egg‐grown oil‐emulsion whole inactivated virus vaccines. For optimal efficacy, a vaccine should be closely antigenically matched to the field strain, requiring that influenza A vaccines be updated regularly. In this study, an H6 subtype VLP transiently expressed in Nicotiana benthamiana was formulated into a vaccine and evaluated for efficacy in chickens against challenge with a heterologous H6N2 virus. A single dose of the plant‐produced H6 VLP vaccine elicited an immune response comparable to two doses of a commercial inactivated H6N2 vaccine, with mean hemagglutination inhibition titres of 9.3 log₂ and 8.8 log₂, respectively. Compared to the non‐vaccinated control, the H6 VLP vaccine significantly reduced the proportion of shedders and the magnitude of viral shedding by >100‐fold in the oropharynx and >6‐fold in the cloaca, and shortened oropharyngeal viral shedding by at least a week. Despite its potency, the cost of the antigenic mismatch between the inactivated H6N2 vaccine and challenge strain was evident not only in this vaccine's failure to reduce viral shedding compared to the non‐vaccinated group, but its apparent exacerbation of oropharyngeal viral shedding until 21 days post‐challenge. We estimate that a kilogram of plant leaf material can produce H6 VLP vaccines sufficient for between 5000 and 30 000 chickens, depending on the effective dose and whether one or two immunizations are administered.     

A neutral carrier-based liquid membrane microelectrode for divalent putrescine cations

A new ion-selective liquid membrane microelectrode, based on the neutral carrier 1,1′-bis(2,3-naphtho-18-crown-6), is described that shows the dependence of EMF on the activity of divalent putrescine cations a _Put, with the linear slope s _Put = 26 ± 3 mV/decade (mean ± SD, N = 18), in the range 10⁻⁴–10⁻¹ M at 25 ± 1 °C. Values of potentiometric putrescine cation selectivity coefficients of logK ^Pot _{Put j} (mean ± SD, N) are obtained by the separate solution method for the ions K⁺ (1.0 ± 0.4, 10), Na⁺ (−1.2 ± 0.4, 8), Ca²⁺ (−2.3 ± 0.5, 10) and Mg²⁺ (−2.5 ± 0.5, 7). The microelectrode can be applied for the direct analysis of the activities of free divalent putrescine cations in the range 5 × 10⁻⁴ to 10⁻¹ M in an extracellular ionic environment. Established analytical methods, e.g. high performance liquid chromatography, determine the total concentration of the derivatives of free and bound putrescine. Received: 20 December 1998 / Revised version: 7 May 1999 / Accepted: 27 May 1999     

A Novel Virus-Like Particle Based Vaccine Platform Displaying the Placental Malaria Antigen VAR2CSA

Placental malaria caused by Plasmodium falciparum is a major cause of mortality and severe morbidity. Clinical testing of a soluble protein-based vaccine containing the parasite ligand, VAR2CSA, has been initiated. VAR2CSA binds to the human receptor chondroitin sulphate A (CSA) and is responsible for sequestration of Plasmodium falciparum infected erythrocytes in the placenta. It is imperative that a vaccine against malaria in pregnancy, if administered to women before they become pregnant, can induce a strong and long lasting immune response. While most soluble protein-based vaccines have failed during clinical testing, virus-like particle (VLP) based vaccines (e.g., the licensed human papillomavirus vaccines) have demonstrated high efficacy, suggesting that the spatial assembly of the vaccine antigen is a critical parameter for inducing an optimal long-lasting protective immune response. We have developed a VLP vaccine display platform by identifying regions of the HPV16 L1 coat protein where a biotin acceptor site (AviTag^TM) can be inserted without compromising VLP-assembly. Subsequent biotinylation of Avi-L1 VLPs allow us to anchor monovalent streptavidin (mSA)-fused proteins to the biotin, thereby obtaining a dense and repetitive VLP-display of the vaccine antigen. The mSA-VAR2CSA antigen was delivered on the Avi-L1 VLP platform and tested in C57BL/6 mice in comparison to two soluble protein-based vaccines consisting of naked VAR2CSA and mSA-VAR2CSA. The mSA-VAR2CSA Avi-L1 VLP and soluble mSA-VAR2CSA vaccines induced higher antibody titers than the soluble naked VAR2CSA vaccine after three immunizations. The VAR2CSA Avi-L1 VLP vaccine induced statistically significantly higher endpoint titres compared to the soluble mSA-VAR2CSA vaccine, after 1^st and 2^nd immunization; however, this difference was not statistically significant after 3^rd immunization. Importantly, the VLP-VAR2CSA induced antibodies were functional in inhibiting the binding of parasites to CSA. This study demonstrates that the described Avi-L1 VLP-platform may serve as a versatile system for facilitating optimal VLP-display of large and complex vaccine antigens.     

Virus and microbial loop dynamics over an annual cycle in three contrasting Antarctic lakes

1. Viral and microbial loop dynamics were investigated over an annual cycle in three contrasting saline Antarctic lakes – Highway Lake (salinity 4‰), Pendant Lake (salinity 19‰) and Ace Lake, a meromictic system (with a mixolimnion salinity of 18‰) in order to assess the importance of viruses in extreme, microbially dominated systems. 2. Virus like particles (VLP) showed no clear seasonal pattern, with high concentrations occurring in both winter and summer (range 0.89 × 10⁷ ± 0.038 to 12.017 × 10⁷ ± 1.28 mL^?1). VLP abundances reflected lake productivity based on chlorophyll a concentrations. Bacterial abundances and biomass did not correlate with VLP numbers except in Pendant Lake, the most productive of the three lakes studied. 3. Pendant Lake supported the highest bacterial biomass (range Highway: 18.44 ± 1.35 to 59.43 ± 2.80 ng C mL^?1; Ace: 14.42 ± 2.69 to 68.39 ± 2.95 ng C mL^?1; Pendant: 31.36 ± 3.94 to 115.95 ± 4.49 ng C mL^?1) so that virus to bacteria ratios (VBR) (range 30.48 ± 7.96 to 96.67 ± 8.21) were higher in Ace Lake (range 30.58 ± 3.98 to 80.037 ± 1.60) and Highway Lake (range 18.63 ± 3.12 to 126.74 ± 6.50). 4. Negative correlations occurred between VLP and cryptophytes (dominant phototrophic nanoflagellates), suggesting that they were not hosts to lytic viruses. Among the other protists only the heterotrophic nanoflagellates of Highway Lake (dominated by the marine choanoflagellate Diaphanoeca grandis) showed a positive correlation with VLP. 5. The VLP was negatively correlated with photosynthetically active radiation (PAR) and temperature, both of which increased with ice thinning and breakout, increasing viral decay. In winter VLP probably persisted in cold, dark water. 6. High VLP concentrations and high VBR (values at the upper end of those reported for marine and lacustrine systems) indicated that viruses, most of which were probably bacteriophage, are a major element within the microbial communities in extreme, saline lakes.     

Exploiting cholera vaccines as a versatile antigen delivery platform

The development of safe, immunogenic and protective cholera vaccine candidates makes possible their use as a versatile antigen delivery platform. Foreign antigens can be delivered to the immune system with cholera vaccines by expressing heterologous antigens in live attenuated vectors, as fusion proteins with cholera toxin subunits combined with inactivated Vibrio cholerae whole cells or by exposing them on the surface of V. cholerae ghosts. Progress in our understanding of the genes expressed by V. cholerae during infection creates unprecedented opportunities to develop an improved generation of vaccine vectors to induce immune protection against a broad range of pathogenic organisms.     

The Double-Water-Film Electrode: A Device for Measuring the Resistance and the Capacitance of the Internode/Node Interface of Chara as Functions of Time and Temperature

Abstract A ``double-water-film electrode technique' has been developed for the long-term characterization of the electrical properties across the interface between the nodal (N) and internodal (A or B) cells and the vacuole along the length of an internode of Chara as a function of time and temperature. The electrode unit consisted of a pair of the water-film electrodes described elsewhere (Chilcott 1988; Chilcott and others 1983; Coster and others 1984; Lucas 1985; and Ogata 1983). The distance between two water-film probes was fixed at 1.0 cm. By scanning the electrode unit, the spatial variations in electrical resistance and capacitance along the longitudinal axis of Chara were observed. Analysis was performed by applying an electrical equivalent circuit for the biomembrane (Philippson 1921). Across the internode (−A or −B)/central nodal cells interface, the specific parallel resistance (Rm) and the parallel capacitance (Cm) at 20°C were 30 ± 5 × 10⁻³Ωm² and 1.5 ± 0.5 × 10⁻¹Fm⁻² (at 30 Hz), respectively. And the series resistance, corresponding to the vacuole of the internode was 8 × 10⁻³Ωm². Study of temperature dependencies of Rm and Cm suggested that a dynamic homeostatic regulation was operating at the interface where numerous plasmodesmata were observed with an electron microscope (Pickett-Heaps 1967; Spanswick and Costerton 1967). Assuming that the individual cylinder of plasmodesma was filled only with cytoplasm, the number of plasmodesma per interface was estimated at 2.6 × 10⁵. Received 19 January 2000; accepted 16 March 2000