甲型流感病毒血凝素基因变异与抗原变异关系研究

目的 了解甲型流感病毒流行株的血凝素基因变异和抗原变异及两者间的关系,结合流行病学资料分析基因变异和抗原变异的吻合情况.方法 从2005-2007年上海市季节性流感样病例分离到的甲型流感病毒株中,选取H1N1、H3N2两个亚型中部分有代表性的病毒株,并同WHO北半球流感疫苗推荐株一起,进行血凝素全基因序列测定后做基因进化树分析.同时用灭活的全病毒抗原免疫金黄地鼠,通过血凝抑制试验测定病毒的血凝效价,对血凝抑制结果进行聚类分析,绘制病毒的抗原变异图.结果 H3N2分离株与WHO北半球疫苗推荐株A/Sydney/5/97、A/Fujian/411/2002处于不同的基因进化分枝上,时间间隔越久,进化距离越远,同样的结果也出现在抗原变异分析中.而H1N1分离株的基因变异情况和抗原变异情况则不一致,在基因变异中,与疫苗推荐株A/New Caledonia/20/1999的距离远近受到分离时间的影响,抗原变异还与病毒是否从散发病例或聚集性病例分离有关.结论 流感病毒血凝素的基因变异和抗原变异的结果基本吻合,采用流行株免疫血清的血凝抑制试验能更好地反映病毒的变异和进化情况.     

甲型流感病毒血凝素基因变异与抗原变异关系研究

目的 了解甲型流感病毒流行株的血凝素基因变异和抗原变异及两者间的关系,结合流行病学资料分析基因变异和抗原变异的吻合情况.方法 从2005-2007年上海市季节性流感样病例分离到的甲型流感病毒株中,选取H1N1、H3N2两个亚型中部分有代表性的病毒株,并同WHO北半球流感疫苗推荐株一起,进行血凝素全基因序列测定后做基因进化树分析.同时用灭活的全病毒抗原免疫金黄地鼠,通过血凝抑制试验测定病毒的血凝效价,对血凝抑制结果进行聚类分析,绘制病毒的抗原变异图.结果 H3N2分离株与WHO北半球疫苗推荐株A/Sydney/5/97、A/Fujian/411/2002处于不同的基因进化分枝上,时间间隔越久,进化距离越远,同样的结果也出现在抗原变异分析中.而H1N1分离株的基因变异情况和抗原变异情况则不一致,在基因变异中,与疫苗推荐株A/New Caledonia/20/1999的距离远近受到分离时间的影响,抗原变异还与病毒是否从散发病例或聚集性病例分离有关.结论 流感病毒血凝素的基因变异和抗原变异的结果基本吻合,采用流行株免疫血清的血凝抑制试验能更好地反映病毒的变异和进化情况.     

Antiviral agents active against influenza A viruses

The recent outbreaks of avian influenza A (H5N1) virus, its expanding geographic distribution and its ability to transfer to humans and cause severe infection have raised serious concerns about the measures available to control an avian or human pandemic of influenza A. In anticipation of such a pandemic, several preventive and therapeutic strategies have been proposed, including the stockpiling of antiviral drugs, in particular the neuraminidase inhibitors oseltamivir (Tamiflu; Roche) and zanamivir (Relenza; GlaxoSmithKline). This article reviews agents that have been shown to have activity against influenza A viruses and discusses their therapeutic potential, and also describes emerging strategies for targeting these viruses.     

Pyrosequencing as a tool to detect molecular markers of resistance to neuraminidase inhibitors in seasonal influenza A viruses

Pyrosequencing has been successfully used to monitor resistance in influenza A viruses to the first class of anti-influenza drugs, M2 blockers (adamantanes). In contrast to M2 blockers, resistance to neuraminidase (NA) inhibitors (NAIs) is subtype- and drug-specific. Here, we designed a pyrosequencing assay for detection of the most commonly reported mutations associated with resistance to NAIs, a newer class of anti-influenza drugs. These common mutations occur at residues: H274 (N1), E119 (N2), R292 (N2), and N294 (N2) in seasonal influenza A viruses. Additionally, we designed primers to detect substitutions at D151 in NAs of N1 and N2 subtypes. This assay allows detection of mutations associated with resistance not only in grown viruses but also in clinical specimens, thus reducing the time needed for testing and providing an advantage for disease outbreak investigation and management. The pyrosequencing approach also allows the detection of mixed populations of virus variants at positions of interest. Analysis of viruses in the original clinical specimens reduces the potential for introducing genetic variance in the virus population due to selection by cell culture. Our results showed that, in at least one instance, a D151E change seen in N1NA after virus propagation in cell culture was not detected in the original clinical specimen. Although the pyrosequencing assay allows high throughput screening for established genetic markers of antiviral resistance, it is not a replacement for the NA inhibition assays due to insufficient knowledge of the molecular mechanisms of the NAI-resistance.     

Photodynamic inactivation of influenza and herpes viruses by hematoporphyrin

Hematoporphyrin (HP), at concentrations as low as 0.5 microgram/ml, was found to inhibit the in vitro replication of influenza A and herpes simplex viruses, but not of several other viruses. The effect required exposure of the viruses or cells to visible light and was demonstrable when HP was administered shortly before virus inoculation or during the infection. In studies on the mechanism of action of HP, we found that in the presence of light, HP caused decomposition of GMP but not of various other nucleosides. It caused breakdown of yeast tRNA and inhibited polymerization of RNA and DNA by influenza virus and HSV-1-specific polymerases as well as some other polymerases isolated from bacterial and mammalian sources. Protective effects of HP and light were demonstrable in embryonated eggs infected with the WSN and PR8 strains of influenza A virus and in mice infected with the WSN strain. HSV-1-induced keratitis in rabbits and HSV-2-induced dermatitis in mice were not responsive to HP treatment.     

细胞基因工程重组人α_1干扰素对7种流感病毒的抗病毒作用

目的:研究细胞基因工程人α干扰素(rhIFN-α_1)对体外7种流感病毒感染的MDCK细胞以及对甲型流感病毒鼠肺适应株PR_8引起的小鼠肺炎的对抗作用。方法:接种7种病毒液(H_1N_1,H_2N_2,H_3N_3,B型,C型,A_1,B分离株)于MDCK细胞中,小鼠用PR_8病毒液滴鼻,观察rhIFN-α_1的抗病毒作用。结果:rhIFN-α_1对7种病毒液的最小有效浓度分别为12.5,25,50,25,12.5,25和12.5kU·L~(-1);在MDCK细胞上对7种病毒液的感染治疗指数为8×10~3,4×10~3,2×10~3,4×10~3,8×10~3,4×10~3和8×10~3;抑制指数为3.6,4.7,3.5,3.3,3.9,4.6和3.5 ;rhIFN-α_1能有效地抑制流感病毒的细胞内复制,却不能直接杀伤病毒;rhIFN-α_1对小鼠病毒肺炎有抑制作用,使肺组织炎症,纤维间质增生明显改善,降低病毒滴度,延长生命率为94.2％-132.7％,肺指数抑制率为14.8％-37.4％。结论:rhIFN-α_1对流感病毒的增殖有抑制作用,并且能够改善流感病毒引起的小鼠肺炎症状。     

细胞基因工程重组人α1干扰素对7种流感病毒的抗病毒作用

目的：研究细胞基因工程人α干扰素（ｒｈＩＦＮ－α１）对体外７种流感病毒感染的ＭＤＣＫ细胞以及对甲型流感病毒鼠肺适应株ＰＲ８引起的小鼠肺炎的对抗作用。方法：接种７种病毒液（Ｈ１Ｎ１、Ｈ２Ｎ２,Ｈ３Ｎ３,Ｂ型,Ｃ型,Ａ１,Ｂ分离株）于ＭＤＣＫ细胞中,小鼠用ＰＲ８病毒液滴鼻,观察ｒｈＩＦＮ－α１的抗病毒作用。结果：ｒｈＩＦＮ－α１对７种病毒液的最小有效深度分别１２．５、２５,５０,１２。５,２５和１２。     

L-nucleosides: antiviral activity and molecular mechanism

Drug discovery for antiviral chemotherapy has provided the effective treatment of numerous viral diseases. Among antiviral agents used in therapy, nucleoside analogues have been particularly useful. In fact, almost twenty nucleosides are currently used in antiviral therapy, seven of which are for the treatment of HIV infection. In the search for new and effective agents within this class, the focus has recently expanded on L-analogues, characterized by opposite configuration compared to the natural D-nucleosides. The interest in L-nucleosides has risen since the discovery of 3TC, one of the most important drugs used in the treatment of AIDS and hepatitis B infection. This review will discuss the latest advances in L-nucleosides as antiviral agents with a particular focus on the synthesis and molecular mechanism as well as metabolic differences between L- and D-nucleosides.     

Polycation-induced alterations of the envelope surface of influenza and parainfluenza viruses

Natural macromolecular polycations (protamine, histones H1, H2A, H2B and H3) caused the clustering of influenza and parainfluenza viruses into aggregates heterogeneous in size, as well as a decrease in their hemagglutinating capacity. Polycation-induced virus alterations consisted in surface pyknosis and an increase most efficient as regards the interaction with viruses.     

Beta-cyclodextrin inclusion complexes: effect of the host substitution on molecular recognition

The stability of inclusion complexes (i.e. the measure of molecular recognition between a guest and beta-cyclodextrin) is highly influenced by the fit of the guest into the cavity of the host and by the secondary bonds among the functional groups getting in close connection. The strength of these interactions depend on the size, shape and functional groups of the guests as well as on the facts that both the size of the cavity and the reactivity of beta-cyclodextrins are altered when the hydroxy groups are substituted. As best models, the interactions among hydroxypropylated cyclodextrins of different average degree (and pattern) of substitution and phenolphthalein (as a model for "large" guests) and p-nitrophenol-p-nitrophenolate couple (as for "small" ones) have been studied. The formation constants of phenolphthalein hydroxypropyl-beta-cyclodextrin complexes are continuously decreasing, while those of p-nitrophenol/phenolate ones show a maximum with increasing degree of substitution. The pattern of substitution has also a significant effect on the quality of the interactions: the substitution on O(6) position alters the type of the interactions most, and a series of different findings prove that this change is the basis in the chiral selectivity of different cyclodextrin derivatives, too.     

Mixed influenza A and B infections complicate the detection of influenza viruses with altered sensitivities to neuraminidase inhibitors

Previously, three influenza A(H3N2) isolates with a reduced susceptibility to the neuraminidase inhibitors (NAIs) zanamivir and oseltamivir were identified during screening by the Neuraminidase Inhibitor Susceptibility Network (NISN). The isolates were from untreated patients from the first three years post licensure of the NAIs. We plaque-purified progeny from each of these isolates and determined the NAI sensitivity of each plaqued population. Sequencing and serology for each population revealed that the isolates contained a mix of wild type influenza A(H3N2) and influenza B. The NAI susceptibility reductions that had originally been reported were a consequence of influenza B neuraminidases that have lower relative NAI sensitivities, rather than being due to resistant influenza A(H3N2) viruses. Our study highlights the need to check for mixed influenza infections when isolates with potentially lower sensitivities to NAIs are identified.