Use of a dialyzable short-chain phospholipid for efficient solubilization and reconstitution of influenza virus envelopes

Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of various macromolecules. To further advance the use of virosomes, we developed a novel dialysis procedure for the reconstitution of influenza virus membranes that is easily applicable to industrial production and compatible with encapsulation of a variety of compounds. This procedure relies on the use of 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) as a solubilizing agent. DCPC is a short-chain lecithin with detergent-like properties and with a critical micelle concentration of 14 mM. DCPC effectively dissolved the influenza virus membranes after which the nucleocapsids could be removed by ultracentrifugation. The solubilized membrane components were reconstituted either by removal of DCPC by dialysis or by a procedure involving initial dilution of the solubilized membrane components followed by dialysis. Both protocols resulted in removal of 99.9% of DCPC and simultaneous formation of virosomes. Analysis of the virosome preparations by equilibrium sucrose density gradient centrifugation revealed co-migration of phospholipid and protein for virosomes produced by either method. Moreover, both virosome preparations showed morphological and fusogenic characteristics similar to native influenza virus. Size, homogeneity and spike density of the virosomes varied with the two different reconstitution procedures employed. The recovery of viral membrane proteins and phospholipids in the virosomes was found to be higher for the dilution/dialysis procedure than for the simple dialysis protocol. This novel procedure for the production of virosomes is straightforward and robust and allows further exploitation of virosomes as vaccines or as drug delivery vehicles not only in academia, but also in industrial settings.     

Fusion of reconstituted influenza virus envelopes with liposomes mediated by streptavidin/biotin interactions

Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) represent an efficient fusogenic cellular delivery system. By interaction of HA with its natural receptors, sialylated lipids (gangliosides) or proteins, virosomes bind to cells and, following endocytic uptake, deliver their contents to the cytosol through fusion from within acidic endosomes. Here, we show that binding to sialic acid is not necessary for fusion. In the presence of streptavidin, virosomes containing a biotinylated lipid fused with liposomes lacking sialic acid if these liposomes also had a biotinylated lipid in their membranes. Moreover, fusion characteristics corresponded well with fusion of virosomes with ganglioside-containing liposomes.     

Protection of inactivated influenza virus vaccine against lethal influenza virus infection in diabetic mice

Influenza virus infection frequently causes complications and some excess mortality in the patients with diabetes. Vaccination is an effective measure to prevent influenza virus infection. In this paper, antibody response and protection against influenza virus infection induced by vaccination were studied in mouse model of diabetes. Healthy and diabetic BALB/c mice were immunized once or twice with inactivated influenza virus vaccine at various dosages. Four weeks after the first immunization or 1 week after the second immunization, the mice were challenged with influenza virus at a lethal dose. The result showed that the antibody responses in diabetic mice were inhibited. Immunization once with high dose or twice with low dose of vaccine provided full protection against lethal influenza virus challenge in diabetic mice, however, in healthy mice, immunization only once with low dose provided a full protection.     

甲型流感病毒流行毒株检测和分型基因芯片的研制

【目的】研制一种可同时对甲型流感病毒H1N1、H1N2、H3N2、H5N1和H9N2等5种流行亚型进行检测和分型的基因芯片。【方法】根据National Center for Biotechnology Information中Influenza Virus Resource数据库,针对H1N1、H1N2、H3N2、H5N1和H9N2等5种亚型甲型流感病毒的HA和NA基因设计46条特异性寡核苷酸探针和1条质控探针,点制成基因芯片。利用通用引物扩增流感病毒HA和NA基因,使用Klenow酶对扩增产物进行荧光标记和片段化,将标记后产物和芯片杂交,清洗、扫描后根据荧光信号判定检测结果。用18株不同种属来源的甲型流感病毒分离毒株和186份咽拭子对芯片特异性、敏感性和临床应用进行初步评价。【结果】所有18株分离毒株均能被芯片准确检测并分型,芯片检测灵敏度能达约1×104个病毒基因拷贝。同时8份咽拭子检测结果为H1N1阳性,4份咽拭子为H3N2阳性。【结论】研究表明该芯片具有较高的特异性和灵敏度,可为甲型流感病毒的监测提供一种有效的方法。     

A new method for reconstitution of highly fusogenic sendai virus envelopes

A new way for reconstituting highly fusogenic Sendai virus envelopes is described. As opposed to previously described methods, in the present one the detergent (Triton X-100) is removed by direct addition of SM-2 Bio-beads to the detergent solubilized mixture of the viral phospholipids and glycoproteins, thus avoiding the long dialysis step. The vesicles obtained in the present work resemble, in their composition, size and features, envelopes of intact Sendai virus particles. The present method allows the enclosure of low and high molecular weight material within the reconstituted viral envelopes.     

Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance.     

流行性感冒裂解疫苗的研制

作为换代产品,流行性感冒裂解疫苗的研制已取得突破性进展,根据WHO有关规程的规定和大量的试验研究结果。完整建立了该疫苗的生产工艺和生产质量控制系统,完成了“流行性感冒裂解疫苗制造及检定规程”等规定性文件的起草和审核工作,以中试规模连续生产了三批疫苗并全部自检合格,通过疫苗稳定性试验,效力试验,异常毒性试验及过敏性试验等观察。进一步肯定了疫苗的质量。     

Search for additional influenza virus to cell interactions

Sialyl oligosaccharides have long been considered to be the sole receptors for influenza virus. However, according to [1] some viruses are able to grow in sialic-free MDCK cells. Here we attempted to reveal a possible second, non-sialic receptor, hypothesizing the involvement of additional carbohydrate lectin recognition in influenza virus reception process, first of all in situations when a lectin of the host cell could recognize the viral carbohydrate ligand. We tested the presence of galactose- and sialic acid-binding lectins, as well as mannoside- and sulfo-N-acetyllactosamine-recognizing properties of MDCK and Vero cells using polyacrylamide neoglycoconjugates and antibodies. MDCK cells bind galactoside probes stronger than Vero cells, whereas Vero cells bind preferentially sialoside, mannoside and various sulfo-oligosaccharide probes. The probing of viruses with the neoglycoconjugates revealed specific 6′-HSO ₃ LacNAc (but not other sulfated oligosaccharides) binding property of A and B human strains. Affinity of 6′-HSO ₃ LacNAc probe was comparable with affinity of 6′-SiaLac probe but the binding was not inhibited by the sialooligosaccharide.     

流感病毒血凝素蛋白裂解机制的研究进展

血凝素(Hemagglutinin,HA)是流感病毒的主要表面抗原之一,诱导机体产生中和抗体,介导病毒囊膜与靶细胞膜融合,从而启动病毒对宿主细胞的感染过程。HA蛋白以前体形式合成,需经宿主蛋白酶水解为HA1、HA2两个亚单位,并以二硫键连接,病毒才获得感染性。研究表明宿主蛋白酶的分布与流感病毒感染后的致病力和组织嗜性有直接关系。潜在的裂解酶及其抑制因子的发现为流感的防治提供了新的思路,成为干预治疗的新潜在靶点。就当前国内外关于流感病毒血凝素的结构与功能、裂解机制及其应用的研究进展进行综述。     

Effect of adenovirus and influenza virus infection on obesity

The purpose of this review is to provide an overview of the effects of adenovirus and influenza virus infections on obesity in various experimental models. We reviewed studies that were conducted within the past 10 years and were related to virus infection and obesity prevalence. Here, we discuss a different causal relationship between adenovirus and influenza infections with obesity. Adenovirus infection can cause obesity, whereas obesity can be a risk factor for increasing influenza virus infection and increases the risk of morbidity and mortality. The prevalence of obesity due to adenovirus infections may be due to an increase in glucose uptake and reduction in lipolysis caused by an increase in corticosterone secretion. Adenovirus infections may lead to increases in appetite by decreasing norepinephrine and leptin levels and also cause immune dysfunction. The relationship between obesity and influenza virus infection could be summarized by the following features: decreases in memory T-cell functionality and interferon (IFN)-α, IFN-β, and IFN-γ mRNA expression, increases in viral titer and infiltration, and impaired dendritic cell function in obese individuals. Moreover, leptin resistance may play an important role in increasing influenza virus infections in obese individuals. In conclusion, prevention of adenovirus infections could be a good approach for reducing obesity prevalence, and prevention of obesity could reduce influenza virus infections from the point of view of viral infections and obesity.     

细胞适应性流感病毒高产株的制备研究

与鸡胚培养制备的流感疫苗相比,细胞制备的疫苗具有免疫原性好、生产不受鸡胚限制等优点。但目前流感病毒株在细胞上产量较低,成为疫苗生产的主要限制因素。现就用于制备细胞适应性高产株主要的3种方法,即连续传代、随机突变构建病毒突变体和病毒重配的研究进展,以及突变位点对病毒增殖的影响作一概述。     

流感病毒对福尔马林敏感性的研究

采用三种流感病毒株：A1/京防/1/86、A3/济防/15/90、A3/武汉/359/95,进行敏感性试验。用不浓度的福尔马林灭活病毒,并在不同间隔时间检测血凝交价及灭活效果。结果显示,三种毒株经福尔马林灭活后,血凝滴度均有下降,病毒灭活时间也随着福尔马林浓度的降低而延长,说明相同亚型不同株别及不同亚型的流感病毒,对福尔马林的敏感程度均有差异。该试验对今后流感病毒的研究及其疫苗生产均有重要意义。     

跨膜蛋白CD82在流感病毒感染初期的表达

了解细胞跨膜蛋白CD82在流感病毒感染初期的表达变化,分析CD82在流感病毒感染中的作用。

采用逆转录−聚合酶链式反应（RT-PCR）方法对流感病毒感染4 h内的HEP-2细胞中CD82蛋白mRNA表达量进行检测。

流感病毒感染HEP-2细胞后1 h−3 h细胞 CD82的mRNA表达量降低,并且随着流感病毒接种量增加CD82表达量降低持续时间延长。

流感病毒可以在转录水平调节宿主细胞CD82的表达。CD82在流感病毒感染初期表达下降,提示CD82可能在流感病毒与宿主细胞粘附和入侵过程中发挥作用。

     

Design of a set of probes with high potential for influenza virus epidemiological surveillance

An Influenza Probe Set (IPS) consisting in 1,249 9-mer probes for genomic fingerprinting of closely and distantly related Influenza Virus strains was designed and tested in silico. The IPS was derived from alignments of Influenza genomes. The RNA segments of 5,133 influenza strains having diverse degree of relatedness were concatenated and aligned. After alignment, 9-mer sites having high Shannon entropy were searched. Additional criteria such as: G+C content between 35 to 65%, absence of dimer or trimer consecutive repeats, a minimum of 2 differences between 9mers and selecting only sequences with Tm values between 34.5 and 36.5oC were applied for selecting probes with high sequential entropy. Virtual Hybridization was used to predict Genomic Fingerprints to assess the capability of the IPS to discriminate between influenza and related strains. Distance scores between pairs of Influenza Genomic Fingerprints were calculated, and used for estimating Taxonomic Trees. Visual examination of both Genomic Fingerprints and Taxonomic Trees suggest that the IPS is able to discriminate between distant and closely related Influenza strains. It is proposed that the IPS can be used to investigate, by virtual or experimental hybridization, any new, and potentially virulent, strain.     

Development of tetraphenylethylene-based fluorescent oligosaccharide probes for detection of influenza virus

Tetraphenylethylene (TPE) derivatives have strong fluorescence in aggregated state. We designed and synthesized a tetraphenylethylene derivative bearing alkyne groups which were used for combination by click chemistry. The new TPE compound bearing alkyne groups was used to synthesize fluorescence oligosaccharide probes which have lactosyl and 6′-sialyllactosyl moieties as ligands. We found that the TPE compounds bearing lactosyl and 6′-sialyllactosyl moieties were useful for detection of RCA120 and SSA lectins, respectively. Moreover, we have shown that TPE-based fluorescent oligosaccharide probe bearing 6′-sialyllactose moiety can be utilized as a “turn-on” fluorescent sensor for detection of influenza virus.     

流行性感冒病毒灭活疫苗规模化生产工艺的建立

本文报告了流行性感冒病毒疫苗规模化生产工艺的建立及结果,用鸡胚分别接种A1,A3,和B型病毒株,培养后进行了纯化,配苗及检定,对培养时间,收获量、离心分离及纯化、灭活、脱糖等重要步骤进行了优化比较。结果表明,培养66小时病毒滴度及收获量达到最高;采用蔗糖密度梯度离心可收到理想的分离效果;超滤脱糖优于透析法;β-丙内酯在生产中是一种理想的灭活剂,并依此筛选出最优化工艺配置,建立了切实可行的疫苗规模生产工艺,成功制备了质量稳定,安全可靠的疫苗制剂。     

Comparative aspects of infectious salmon anemia virus,an orthomyxovirus of fish,to influenza viruses

Infectious salmon anaemia (ISA) is a viral disease that was first recorded in 1984 in farmed Atlantic salmon. The infectious salmon anaemia virus (ISAV) is classified as the type species of the genus Isavirus in the Orthomyxoviridae family and is evolutionary remote to the influenza viruses. The genome consists of eight negative single-stranded RNA segments, and it utilises the same mechanisms as influenza viruses to enter and exit cells. Although a common ancestor of ISAV and other genera of Orthomyxoviruses could be dated back several millions of years, there are still many similarities between ISAV and the influenza viruses regarding morphology, replication cycles and interactions with their respective hosts.     

流感病毒基因的密码子偏好性及聚类分析

流行性感冒病毒是一种造成人类及动物患流行性感冒的RNA病毒,它造成急性上呼吸道感染,并由空气迅速传播,在世界各地常有周期性的大流行。根据该病毒的基因组CDS序列,探讨了基因组序列密码子的使用模式和特性,并进行了病毒间的聚类分析。结果表明:流感病毒的G+C含量均低于A+U含量,偏向使用以A、U结尾的密码子的程度比使用以G、C结尾的较高,CUG、UCA、AGU、AGC、AGA、AGG、GUG、CCA、ACA、GGA、GCA、AUU、UGA、CAU、CAA、AAU、AAA、GAA等18个密码子为流感病毒共有的偏好性密码子,且以A结尾的居多,尤其偏爱AGA、GGA。聚类结果表明首先亚洲流感病毒H2N2和香港流感病毒H2N2聚为一类,亚洲流感病毒H1N1和俄罗斯流感病毒H1N1聚为一类,1997年和2003年~2004年发生的人禽流感聚为一类,说明它们的密码子使用的偏好性相似;而2009年爆发的甲型H1N1流感和任何一个流感的距离都比较远,说明甲型H1N1流感病毒是一种新型的病毒,不同于以往任何一种流感病毒。     

Inhibition of influenza A virus sialidase activity by sulfatide

Sulfatide, which binds to influenza A viruses and prevents the viral infection, was found to inhibit the sialidase activities of influenza A viruses in a pH-dependent manner. The kinetic parameters of the effect of sulfatide on the sialidase activities of human influenza A viruses using fluorometric assay indicated that sulfatide was a powerful and non-competitive type inhibitor in low-pH conditions.     

A型流感病毒NS1与突触后密度蛋白-95的相互作用

目的 A型流感病毒NS1蛋白是一种多功能的致病因子,能够与被感染细胞中的多种蛋白相互结合,影响并干扰宿主细胞内的信号转导、蛋白质合成及抗病毒反应。突触后密度蛋白(Postsynaptic density protein95,PSD-95)主要存在于神经元及SH-SY-5Y等神经来源的细胞株中。假设NS1能够与PSD-95结合,则更有利于了解A型流感病毒对神经元及相关细胞的作用机制。方法通过酵母双杂交,GST-pull down及免疫荧光技术分别从体外和体内两方面检测NS1与PSD-95的相互作用。结果酵母双杂交表明,仅转染PGAD-NS51/PGBK-PSD-95的QDO有菌落生长,且α-半乳糖苷酶活性显著高于阳性对照;而转染PGAD-NS32/PGBK-PSD-95的QDO无菌落生长;GST-pull down表明仅NS51与PSD-95孵育后,能够被Western-blot检测到;免疫荧光表明NS51与PSD-95可能存在共定位,而NS32与PSD-95则不存在共定位。结论 H5N1(A/chicken/Guangdong/1/2005)的NS1能够与PSD-95结合;反之,H3N2(A/Shantou/602/06)的NS1则不能。