Stimulation of tumor necrosis factor secretion by purified influenza virus neuraminidase

We showed that purified neuraminidase (NA) of influenza virus, but not hemagglutinin (HA), possessed the potential to increase in vitro and in vivo the interleukin 1 (IL 1) activity of mouse peritoneal macrophages. In this study, we report the effect of NA and HA on the secretion of tumor necrosis factor (TNF) activity by murine peritoneal macrophages. TNF being a cytokine sharing many related and overlapping biological functions with IL 1. The two glycoproteins of the strain A/USSR/90/77 (H1N1) were purified electrophoretically and were tested in vitro at doses ranging from 0.5 to 5.0 micrograms using the adherent peritoneal macrophages of C3H/HeN mice elicited with thioglycolate. The TNF activity of culture supernatants, collected 24 hr after stimulation with viral protein, was evaluated by the standard cytolytic assay using L929 and WEHI.164 cells. No increase of the TNF activity was observed at 0.5 micrograms of NA (4.8 Units (U)/ml in the L929 assay and 20.4 U/ml in the WEHI assay) but further increase of NA to 1.0 microgram had a significant effect on the TNF activity (39.7 and 88.8 U/ml, respectively). Higher concentrations of NA (2.0 and 5.0 micrograms) did not improve the TNF activity. The addition of a rabbit anti-TNF-alpha serum to the assay system reduced the lysis of L929 cells by 85%, suggesting that the observed activity was due to TNF. In parallel, the enhancement of IL 1 activity due to NA was reverified using D10.G4.1 cells instead of the C3H/HeJ thymocytes assay used previously. NA augmented the IL 1 activity up to 1.0 micrograms (25.8 U/ml). The addition of monoclonal anti-IL 1 antibodies (100 neutralizing units) to the supernatants reduced the incorporation of [3H]-thymidine by 90 to 95%, suggesting that the observed activity was due to IL 1. Comparative results of NA and HA showed that only NA stimulated the TNF and IL 1 activities of murine macrophages.     

Pancreatic cholesterol esterases. 2. Purification and characterization of human pancreatic fatty acid ethyl ester synthase

Human pancreatic fatty acid ethyl ester synthase has been isolated and purified 1200-fold to homogeneity, and its activities, binding properties, and N-terminal amino acid sequence indicate that it is a member of the lipase family. This 52-kDa monomeric protein is present at 0.6-1.2 mg/g of pancreas, and it catalyzes the synthesis and hydrolysis of ethyl oleate at rates of 2400 nmol mg-1 h-1 and 30 nmol mg-1 h-1, respectively. Kinetic analyses reveal a pronounced substrate specificity for unsaturated octadecanoic fatty acids, with ethyl ester synthetic rates of 2400 nmol mg-1 h-1 (linoleic), 2400 nmol mg-1 h-1 (oleic), 400 nmol mg-1 h-1 (arachidonic), 300 nmol mg-1 h-1 (palmitic), and 100 nmol mg-1 h-1 (stearic). Like cholesterol esterase, the enzyme binds to immobilized heparin, and this property was critical for its purification to homogeneity. Its N-terminal amino acid sequence is virtually identical with that reported for human triglyceride lipase, NH2-X-Glu-Val-Cys-5Tyr-Glu-Arg-Leu-Gly-10Cys-Phe-Ser-Asp- Asp-15Ser-Pro-Trp-Ser-Gly-20Ile, and it differs by only four residues from that reported for porcine pancreatic lipase. The synthase purified here also cleaves triglycerides, hydrolyzing triolein at a rate of 30 nmol mg-1 h-1, and this activity is stimulated by colipase and inhibited by sodium chloride. Conversely, commercially available porcine triglyceride lipase exhibits fatty acid ethyl ester synthase activity (1530 nmol mg-1 h-1) and hydrolyzes triolein at a rate of 23 nmol mg-1 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purified glycoproteins of influenza virus stimulate cell-mediated cytotoxicity in vivo

Previously, we reported that purified surface influenza viral glycoproteins can induce cell-mediated cytotoxicity (CMC) in vitro. Both neuraminidase (NA) and hemagglutinin (HA) were equally good stimulators, on an equimolar basis. In order to broaden the scope of these observations, we examined whether these glycoproteins stimulate natural killer (NK) activity in vivo. Biologically active preparations of glycoproteins NA and HA were purified from virus A/USSR/90/77 (H1N1) and recombinant virus A/USSR/92/77 (H1) x A/Prague/1/56 (N7), respectively. The studies were carried out using the optimal doses of NA and HA. In a 4-hour NK assay, using NK-sensitive YAC-1 cells as targets, both viral glycoproteins stimulated the NK activity of splenocytes of BALB/c and C3H mice. This stimulation was independent of the route of administration (intravenous or intraperitoneal) of the antigen. The observed NK activity was viral antigen-specific and could be modulated to levels comparable to those observed with the standard stimulator, polyinosinic acid-polycytidylic acid, by the use of an appropriate synthetic adjuvant, stearyl tyrosinate. Direct and indirect evidences suggest that the enhanced CMC is due to NK cells. These observations imply that enhancement of NK activity is the intrinsic property of influenza NA and HA.     

Pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice

The Spanish influenza pandemic of 1918 to 1919 swept the globe and resulted in the deaths of at least 20 million people. The basis of the pulmonary damage and high lethality caused by the 1918 H1N1 influenza virus remains largely unknown. Recombinant influenza viruses bearing the 1918 influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins were rescued in the genetic background of the human A/Texas/36/91 (H1N1) (1918 HA/NA:Tx/91) virus. Pathogenesis experiments revealed that the 1918 HA/NA:Tx/91 virus was lethal for BALB/c mice without the prior adaptation that is usually required for human influenza A H1N1 viruses. The increased mortality of 1918 HA/NA:Tx/91-infected mice was accompanied by (i) increased (>200-fold) viral replication, (ii) greater influx of neutrophils into the lung, (iii) increased numbers of alveolar macrophages (AMs), and (iv) increased protein expression of cytokines and chemokines in lung tissues compared with the levels seen for control Tx/91 virus-infected mice. Because pathological changes in AMs and neutrophil migration correlated with lung inflammation, we assessed the role of these cells in the pathogenesis associated with 1918 HA/NA:Tx/91 virus infection. Neutrophil and/or AM depletion initiated 3 or 5 days after infection did not have a significant effect on the disease outcome following a lethal 1918 HA/NA:Tx/91 virus infection. By contrast, depletion of these cells before a sublethal infection with 1918 HA/NA:Tx/91 virus resulted in uncontrolled virus growth and mortality in mice. In addition, neutrophil and/or AM depletion was associated with decreased expression of cytokines and chemokines. These results indicate that a human influenza H1N1 virus possessing the 1918 HA and NA glycoproteins can induce severe lung inflammation consisting of AMs and neutrophils, which play a role in controlling the replication and spread of 1918 HA/NA:Tx/91 virus after intranasal infection of mice.     

The structure of oligosaccharide fragments of glycoproteins from influenza virus A/Krasnodar/101/59/(H2N2)--heavy and light chains of hemagglutinin and neuraminidase

The structure and heterogeneity of carbohydrate chains of hemagglutinin (HA) and neuraminidase (NA), the surface glycoproteins of influenza virus A/Krasnodar/101/59 (H2N2), were investigated. Hemagglutinin was reduced with beta-mercaptoethanol and its heavy (HA1) and light (HA2) chains were separated by gel chromatography. Amino acid and sugar composition of HA1, HA2 and NA was elucidated. The carbohydrate chains of the glycoproteins were cleaved off by the alkaline LiBH4 treatment and oligosaccharides were reduced with NaB[3H]4. They were fractionated by subsequent two-step HPLC on Ultrasphere-C8 and Zorbax-NH2 columns with simultaneous identification using nonlabelled oligosaccharides of known structures. Some of the major oligosaccharides isolated from HA1, HA2 and NA were thus identified as high mannose chains, containing 5-9 mannose residues, and complex chains, first of all biantennary chains having or not having bisecting N-acetylglucosamine and/or fucose residues. The approach which has been developed enables one to study the structure and heterogeneity of carbohydrate chains starting from one nmole of a desialylated N-glycoprotein.     

Plasma Membrane H+-ATPase Activity in Spores, Germ Tubes, and Haustoria of the Rust Fungus Uromyces viciae-fabae

Using plasma membrane-enriched vesicles, the properties of the H+-ATPase (EC 3.6.1.35) from the rust fungus Uromyces viciae-fabae were studied. The enzyme is strictly Mg2+-dependent and is inhibited by vanadate. The pH-optimum is at 6.7. By Western blot analysis using a monoclonal antibody against corn plasma membrane H+-ATPase a polypeptide of approximately 104 kDa could be detected. The vanadate-sensitive H+-ATPase activity of microsomal vesicles obtained from different stages of rust development was determined. Uredospores had only a very low enzyme activity (1.9 μmol Pi x mg-1 protein x h-1). In germ tubes the ATPase activity was about twofold higher (4.0 μmol Pi x mg-1 protein x h-1). An eightfold higher ATPase activity (16.1 μmol Pi x mg-1 protein x h-1) was found in microsomal vesicles from haustoria which had been isolated from rust-infected Vicia faba leaves. These results suggest, that the electrochemical gradient generated by the H+-ATPase of haustoria plays an important role for their function, possibly by promoting nutrient uptake from host cells.     

Choline and acetylcholine metabolism in rat neostriatal slices

Choline (Ch) uptake and release and acetylcholine (ACh) synthesis and release have been studied by gas chromatography mass spectrometry (GCMS) in slices of rat neostriatum in vitro to assess the effects of depolarization by 25 mM K+ and the influence of elevated concentrations of Ch in the incubation medium. During the first 60 min after preparation, 25 mM K+ increased ACh release by 182% and reduced ACh levels by 40%. The rate of ACh synthesis was unchanged. After a 1-h equilibration period, the rate of ACh synthesis was considerably less (2.41 nmol mg-1 h-1, compared to 9.78 nmol mg-1 h-1). Exposure to 25 mM K+ during the second hour increased the rate to 6.47 nmol mg-1 h-1. During the first 10 min of exposure to 25 mM K+, ACh synthesis was reduced, regardless of incubation. Increasing concentrations of external [2H4]Ch apparently favored initial rates of net ACh synthesis, since the rank order of initial net ACh synthesis rates is the same as the rank order of external [2H4] Ch concentration under both normal and depolarized conditions. However, the only significant effect of external [2H4]Ch on ACh metabolism was that it increased ACh release during the initial 10 min, when the preparation was depolarized with K+. The efflux of endogenous [2H0]Ch was increased initially (10 min) and slowed over a 60-min period by 25 mM K+, and increased when [2H4]Ch in the medium was increased. Changes in ACh synthesis and release were dependent upon the time exposure of slices to high K+, and the results suggest that Ch favors initial rates of ACh synthesis, but that Ch influences ACh release primarily under conditions of stress (i.e., depolarization).     

一株鹅源H5N2亚型高致病性禽流感病毒的分离鉴定及生物学特性分析

分离到一株鹅源 H5N2亚型高致病性禽流感病毒,SPF鸡静脉接种致病指数为2.99,但鸭子对该病毒不敏感．病毒感染小鼠后不致病,但能够在肺内有效复制,表明其具有感染哺乳动物的潜在风险．血凝素(hemagglutinin, HA)蛋白裂解位点上插入有多个连续的碱性氨基酸(-RRRKKR-),从分子上证实这是一株高致病性禽流感病毒．核酸序列比较分析表明,分离的流感病毒HA基因与A/chicken/Hubei/489/2004 (H5N1)同源率达到99.4%,神经氨酸酶(neuraminidase, NA)基因与A/chicken/Jilin/53/01(H9N2)同源率达到99.8%;氨基酸水平上,HA与2004年分离到的A/chicken/Hubei/489/2004(H5N1)、A/swan/Guangxi/307/2004(H5N1)、A/wildduck/Guangdong/314/　2004(H5N1)和A/chicken/Henan/210/2004(H5N1)同源率均为99.3%,NA 与A/chicken/Jilin/53/01(H9N2)同源率为99.6%．进化树分析结果表明,该流感病毒分离株可能是由H5N1和H9N2两个亚型病毒重排而来．     

Correlation between strong adjuvanticity of Klebsiella O3 lipopolysaccharide and its ability to induce lnterleukin-1 secretion

Adjuvant activity of Klebsiella O3 lipopolysaccharide (KO3 LPS) in augmenting antibody response and delayed-type hypersensitivity to protein antigens in SMA mice was much stronger than that of LPS from Escherichia coli O55 and O127 (EO55 LPS and EO127 LPS). Relationship between strength of the adjuvant activity and that of the ability to induce interleukin-1 (IL-1) secretion by peritoneal macrophages from C3H/HeN or SMA mice was investigated using these three kinds of LPS. When supernatant samples of macrophages cultured at 37 °C for 24 hr in the presence of 5 μg/ml LPS were assayed by their mitogenic effect on thymocytes from C3H/HeJ mice, KO3 LPS induced the secretion of about four to six times greater amounts of IL-1 activity than did EO127 LPS. When concentration of LPS used for stimulation of macrophages was varied from 0.1 to 50 μg/ml, KO3 LPS induced the secretion of definitely greater amounts of IL-1 activity than did EO55 LPS and EO127 LPS throughout the LPS concentrations tested. Nearly the same amount of IL-1 activity as that produced by 10 μg/ml EO55 LPS or 50 μg/ml EO127 LPS could be produced by 1.0 μg/ml or lower concentrations of KO3 LPS.     

Presence of a Ca2+-sensitive CDPdiglyceride-inositol transferase in canine cardiac sarcoplasmic reticulum

Sarcoplasmic reticulum (SR) and plasma membranes from canine left ventricle were used to evaluate the presence of the enzyme CDPdiglyceride-inositol transferase in these membranes. (K+,-Ca2+)-ATPase activity, a marker for SR, was 79.2 +/- 5.0 (SE) and 11.2 +/- 2.0 mumol.mg-1.h-1 in SR and plasma membrane preparations, respectively, and (Na+,K+)-ATPase activity, a marker for plasma membranes, was 5.6 +/- 1.2 and 99.2 +/- 8.0 mumol.mg-1.h-1, respectively. Contamination of SR and plasma membrane preparations by mitochondria was estimated to be 2% and 8%, respectively, and by Golgi membranes, 0.9% and 1.8%, respectively. Transferase activity, measured at pH 6.8, was 1.32 +/- 0.04 (SE) and 0.28 +/- 0.04 nmol of [3H]phosphatidylinositol ([3H]PtdIns).mg-1.min-1 in three SR and plasma membrane preparations, respectively. The transferase activity detected in the plasma membrane preparation could be accounted for largely, but not entirely, by contaminating SR membranes. The pH optimum for the SR transferase activity was between 8.0 and 9.0; little or no activity was detectable at pH 6.3 and 5.5, the lowest pH tested. Ca2+ inhibited the enzyme, half-maximal inhibition occurring at about 10 microM Ca2+; removal of the Ca2+ by addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid restored activity. No loss of [3H]PtdIns could be detected when membranes were incubated in the presence or absence of Ca2+. The Ca2+ inhibition of the transferase was noncompetitive with respect to CDP-dipalmitin while that with respect to myo-inositol was slightly noncompetitive at low [Ca2+] and became uncompetitive at higher [Ca2+].(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylene Reduction by Symbiosomes and Free Bacteroids from Broad Bean (Vicia faba L.) Nodules (Role of Oxalate)

We report the presence of oxalate in the organic acid fraction of broad bean (Vicia faba L.) nodule cytosol. Using both high-performance liquid chromatography and enzymic assays, high levels of oxalate were detected (70.4 [plus or minus] 2.4 mM). To study the potential role of oxalate as an energy-yielding substrate for nitrogenase activity, free bacteroids were isolated from nodules and found to oxidize oxalate in support of C2H2 reduction under O2 tensions that were lower than those required to oxidize succinate, another dicarboxylate commonly detected in legume nodules. Symbiosomes of broad bean, isolated for the first time from amide-producing nodules, were provided with [14C]oxalate and found to have uptake kinetics with a lower affinity [Km(oxalate) = 330 [mu]M] than that for free bacteroids [Km(oxalate) = 130 [mu]M]. In anaerobic preparations of symbiosomes supplied with purified oxyleghemoglobin, O2 consumption was stimulated by oxalate from 20.2 [plus or minus] 0.8 nmol O2 min-1mg-1 protein to 24.5 [plus or minus] 1.1 nmol O2 min-1 mg-1 protein but always remained lower than the rate of O2 consumption in free bacteroids (32.2 [plus or minus] 1.4 nmol O2 min-1 mg-1 protein). Under these conditions, C2H2 reduction activity was 9.7 [plus or minus] 0.8 and 15.1 [plus or minus] 0.9 nmol C2H4 min-1 mg-1 protein for symbiosomes and bacteroids, respectively. These data support the suggestion that oxalate may play a role as a carbon substrate in support of N2 fixation in broad bean nodules.     

Endotoxin-induced tumor necrosis factor (TNF): selective triggering of TNF and interleukin-1 production by distinct glucosamine-derived lipids

The isolated lipid A of Bordetella pertussis endotoxin (LipA) has been found to induce in vitro release of tumor necrosis factor (TNF) by murine macrophages, albeit much less efficiently than does the intact lipopolysaccharide. Synthetic analogs (monosaccharides M4 and M6) of both glucosamine units present in the LipA backbone induced production of TNF by peritoneal macrophages of Swiss mice. Macrophages from A/J mice gave higher responses than those from Swiss mice, while those of C3H/HeJ mice were unresponsive. Enhancement of TNF secretion was observed for all cells if they were pretreated with a calcium ionophore, and no otherwise inactive substance became active with cells thus treated. For synthetic monosaccharide derivatives, a phosphate group on O-4 was not required for, and a phosphate group on O-1 abolished, the TNF-inducing activity. Synthetic monosaccharides, chemically closely related to substructures recognized to be present in isolated lipid A preparations, could induce either TNF or interleukin-1 (IL-1) production, but not both simultaneously: the monosaccharides M4 and M6 were active TNF inducers, but did not initiate IL-1 production, while the monosaccharides M9 and lipid X efficiently elicited IL-1 production, but did not trigger TNF secretion. It should be noted, however, that the active synthetic compounds are considerably less efficient TNF inducers as is the intact B. pertussis endotoxin.     

The structure of carbohydrate chains of hemagglutinin and neuraminidase of influenza virus B/Leningrad/179/86

The main surface glycoprotein, hemagglutinin (HA), was obtained by treatment of influenza virus B/Leningrad/179/86 with bromelain. Amino acid and monosaccharide compositions of HA and neuraminidase (NA, earlier isolated from the same virus) were determined, thus showing HA and NA to contain 8-10 and 2 carbohydrate chains, respectively. The carbohydrate fragments were cleaved off by the alkaline LiBH4 treatment, the oligosaccharides released were reduced with NaB3H4 and fractionated by two-step HPLC on Ultrasphere-C18 and Zorbax-NH2 columns. Some higher mannose and complex oligosaccharides were identified in both cases by comparison with nonlabelled oligosaccharides of the known structure. The data obtained show that surface glycoproteins of influenza virus A and B are rather similar with regard to structure and heterogeneity of their carbohydrate chains.     

In vitro enhancement of human natural cell-mediated cytotoxicity by purified influenza virus glycoproteins.

The role of the glycoproteins of influenza virus, hemagglutinin (HA), and neuraminidase (NA) in the in vitro stimulation of natural cell-mediated cytotoxicity (NCMC) or natural killer activity of human peripheral blood lymphocytes was evaluated with radiolabeled K562 cells as target cells in an overnight chromium release assay. Three different approaches were used. (i) Purified viral proteins were obtained by extraction with Nonidet P-40, separation on a sucrose gradient, and further purification by affinity chromatography. Ficoll-Hypaque-purified peripheral blood lymphocytes exposed to HA or NA individually or to a mixture of both significantly increased NCMC (32 to 50%). (ii) Treatment of HA and NA with their respective homologous antisera or F(ab')2 antibody abrogated the stimulation of NCMC by these glycoproteins. (iii) Virions treated with proteolytic enzymes resulted in viral cores lacking either HA or NA or both activities. Compared to whole virions, viral cores devoid of HA activity only induced a 50% increase in NCMC, whereas viral cores lacking HA activity and with traces of NA activity stimulated only 10% of the NCMC. These results suggest that influenza virus-induced cell-mediated cytotoxicity is largely due to its glycoproteins.     

The effect of seasonality on oxidative metabolism in Nacella (Patinigera) magellanica

We studied the seasonal variation on aerobic metabolism and the response of oxidative stress parameters in the digestive glands of the subpolar limpet Nacella (P.) magellanica. Sampling was carried out from July (winter) 2002 to July 2003 in Beagle Channel, Tierra del Fuego, Argentina. Whole animal respiration rates increased in early spring as the animals spawned and remained elevated throughout summer and fall (winter: 0.09+/-0.02 micromol O2 h-1 g-1; summer: 0.31+/-0.06 micromol O2 h-1 g-1). Oxidative stress was assessed at the hydrophilic level as the ascorbyl radical content/ascorbate content ratio (A./AH-). The A./AH- ratio showed minimum values in winter (3.7+/-0.2 10(-5)AU) and increased in summer (18+/-5 10(-5) AU). A similar pattern was observed for lipid radical content (122+/-29 pmol mg-1 fresh mass [FW] in winter and 314+/-45 pmol mg-1 FW in summer), iron content (0.99+/-0.07 and 2.7+/-0.6 nmol mg-1 FW in winter and summer, respectively) and catalase activity (2.9+/-0.2 and 7+/-1 U mg-1 FW in winter and summer, respectively). Since nitrogen derived radicals are thought to be critically involved in oxidative metabolism in cells, nitric oxide content was measured and a significant difference in the content of the Fe-MGD-NO adduct in digestive glands from winter and summer animals was observed. Together, the data indicate that both oxygen and nitrogen radical generation rates in N. (P.) magellanica are strongly dependent on season.     

NA Proteins of Influenza A Viruses H1N1/2009, H5N1, and H9N2 Show Differential Effects on Infection Initiation,Virus Release,and Cell-Cell Fusion

Two surface glycoproteins of influenza virus, haemagglutinin (HA) and neuraminidase (NA), play opposite roles in terms of their interaction with host sialic acid receptors. HA attaches to sialic acid on host cell surface receptors to initiate virus infection while NA removes these sialic acids to facilitate release of progeny virions. This functional opposition requires a balance. To explore what might happen when NA of an influenza virus was replaced by one from another isolate or subtype, in this study, we generated three recombinant influenza A viruses in the background of A/PR/8/34 (PR8) (H1N1) and with NA genes obtained respectively from the 2009 pandemic H1N1 virus, a highly pathogenic avian H5N1 virus, and a lowly pathogenic avian H9N2 virus. These recombinant viruses, rPR8-H1N1NA, rPR8-H5N1NA, and rPR8-H9N2NA, were shown to have similar growth kinetics in cells and pathogenicity in mice. However, much more rPR8-H5N1NA and PR8-wt virions were released from chicken erythrocytes than virions of rPR8-H1N1NA and rPR8-H9N2NA after 1 h. In addition, in MDCK cells, rPR8-H5N1NA and rPR8-H9N2NA infected a higher percentage of cells, and induced cell-cell fusion faster and more extensively than PR8-wt and rPR8-H1N1NA did in the early phase of infection. In conclusion, NA replacement in this study did not affect virus replication kinetics but had different effects on infection initiation, virus release and fusion of infected cells. These phenomena might be partially due to NA proteins’ different specificity to α2-3/2-6-sialylated carbohydrate chains, but the exact mechanism remains to be explored.     

Influenza A viruses possessing type B hemagglutinin and neuraminidase: potential as vaccine components

A licensed live attenuated influenza vaccine is available as a trivalent mixture of types A (H1N1 and H3N2) and B vaccine viruses. Thus, interference among these viruses could restrict their replication, affecting vaccine efficacy. One approach to overcoming this potential problem is to use a chimeric virus possessing type B hemagglutinin (HA) and neuraminidase (NA) in a type A vaccine virus background. We previously generated a type A virus possessing a chimeric HA in which the entire ectodomain of the type A HA molecule was replaced with that of the type B HA, and showed that this virus protected mice from challenge by a wild-type B virus. In the study described here, we generated type A/B chimeric viruses carrying not only the chimeric (A/B) HA, but also the full-length type B NA instead of the type A NA, resulting in (A/B) HA/NA chimeric viruses possessing type B HA and NA ectodomains in the background of a type A virus. These (A/B) HA/NA chimeric viruses were attenuated in both cell culture and mice as compared with the wild-type A virus. Our findings may allow an effective live influenza vaccine to be produced from a single master strain, providing a model for the design of future live influenza vaccines.     

Characterization of a C21 neutral steroid hormone transforming enzyme, 21-dehydroxylase, in crude cell extracts of Eubacterium lentum.

A strain of the obligate anaerobe, Eubacterium lentum, isolated from human feces, catalyzes the 21-dehydroxylation of 11-deoxycorticosterone to progesterone. A quantitative radiochromatographic assay was developed to measure 21-dehydroxylase activity in cell extracts. Maximum enzyme activity in cell extracts required both a reduced pyridine nucleotide and an oxidized flavin coenzyme. However, photochemically reduced flavin (FMNH2) could replace the requirement for NAD(P)H plus oxidized flavin. NAD(P)H : flavin (either FMN or FAD) oxidoreductase activity was detected spectrophotometrically in cell extracts assayed under anaerobic conditions. 21-Dehydroxylase was active from pH 5.4 to 8.5 with an apparent optimum between 6.4 and 6.8 using mixtures of NADH plus FMN as coenzymes. The substrate concentration at half-maximal reaction velocity was 8.0 microM and a specific acitivity of 5.8 nmol [3H]progesterone formed . h-1 . mg-1 protein was determined using [3th]deoxycorticosterone as substrate. Atabrine, rotenone, acriflavin, and 2,4-dinitrophenol (all at 1 mM) inhibited 21-dehydroxylase activity in cell extracts by 25, 24, 35 and 84%, respectively. These results suggest that 21-dehydrogenase may be coupled to a NAD(P)H : flavin oxidoreductase system in E. lentum.     

Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus

To understand more fully the molecular events associated with highly virulent or attenuated influenza virus infections, we have studied the effects of expression of the 1918 hemagglutinin (HA) and neuraminidase (NA) genes during viral infection in mice under biosafety level 3 (agricultural) conditions. Using histopathology and cDNA microarrays, we examined the consequences of expression of the HA and NA genes of the 1918 pandemic virus in a recombinant influenza A/WSN/33 virus compared to parental A/WSN/33 virus and to an attenuated virus expressing the HA and NA genes from A/New Caledonia/20/99. The 1918 HA/NA:WSN and WSN recombinant viruses were highly lethal for mice and displayed severe lung pathology in comparison to the nonlethal New Caledonia HA/NA:WSN recombinant virus. Expression microarray analysis performed on lung tissues isolated from the infected animals showed activation of many genes involved in the inflammatory response, including cytokine, apoptosis, and lymphocyte genes that were common to all three infection groups. However, consistent with the histopathology studies, the WSN and 1918 HA/NA:WSN recombinant viruses showed increased up-regulation of genes associated with activated T cells and macrophages, as well as genes involved in apoptosis, tissue injury, and oxidative damage that were not observed in the New Caledonia HA/NA:WSN recombinant virus-infected mice. These studies document clear differences in gene expression profiles that were correlated with pulmonary disease pathology induced by virulent and attenuated influenza virus infections.     

The Evolutionary Pattern of Glycosylation Sites in Influenza Virus (H5N1) Hemagglutinin and Neuraminidase

Two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the surface of influenza viruses play crucial roles in transfaunation, membrane fusion and the release of progeny virions. To explore the distribution of N-glycosylation sites (glycosites) in these two glycoproteins, we collected and aligned the amino acid sequences of all the HA and NA subtypes. Two glycosites were located at HA0 cleavage sites and fusion peptides and were strikingly conserved in all HA subtypes, while the remaining glycosites were unique to their subtypes. Two to four conserved glycosites were found in the stalk domain of NA, but these are affected by the deletion of specific stalk domain sequences. Another highly conserved glycosite appeared at the top center of tetrameric global domain, while the others glycosites were distributed around the global domain. Here we present a detailed investigation of the distribution and the evolutionary pattern of the glycosites in the envelope glycoproteins of IVs, and further focus on the H5N1 virus and conclude that the glycosites in H5N1 have become more complicated in HA and less influential in NA in the last five years.