Arbidol and Other Low-Molecular-Weight Drugs That Inhibit Lassa and Ebola Viruses.

Antiviral therapies that impede virus entry are attractive because they act on the first phase of the infectious cycle. Drugs that target pathways common to multiple viruses are particularly desirable when laboratory-based viral identification may be challenging, e.g., in an outbreak setting. We are interested in identifying drugs that block both Ebola virus (EBOV) and Lassa virus (LASV), two unrelated but highly pathogenic hemorrhagic fever viruses that have caused outbreaks in similar regions in Africa and share features of virus entry: use of cell surface attachment factors, macropinocytosis, endosomal receptors, and low pH to trigger fusion in late endosomes. Toward this goal, we directly compared the potency of eight drugs known to block EBOV entry with their potency as inhibitors of LASV entry. Five drugs (amodiaquine, apilimod, arbidol, niclosamide, and zoniporide) showed roughly equivalent degrees of inhibition of LASV and EBOV glycoprotein (GP)-bearing pseudoviruses; three (clomiphene, sertraline, and toremifene) were more potent against EBOV. We then focused on arbidol, which is licensed abroad as an anti-influenza drug and exhibits activity against a diverse array of clinically relevant viruses. We found that arbidol inhibits infection by authentic LASV, inhibits LASV GP-mediated cell-cell fusion and virus-cell fusion, and, reminiscent of its activity on influenza virus hemagglutinin, stabilizes LASV GP to low-pH exposure. Our findings suggest that arbidol inhibits LASV fusion, which may partly involve blocking conformational changes in LASV GP. We discuss our findings in terms of the potential to develop a drug cocktail that could inhibit both LASV and EBOV.IMPORTANCE Lassa and Ebola viruses continue to cause severe outbreaks in humans, yet there are only limited therapeutic options to treat the deadly hemorrhagic fever diseases they cause. Because of overlapping geographic occurrences and similarities in mode of entry into cells, we seek a practical drug or drug cocktail that could be used to treat infections by both viruses. Toward this goal, we directly compared eight drugs, approved or in clinical testing, for the ability to block entry mediated by the glycoproteins of both viruses. We identified five drugs with approximately equal potencies against both. Among these, we investigated the modes of action of arbidol, a drug licensed abroad to treat influenza infections. We found, as shown for influenza virus, that arbidol blocks fusion mediated by the Lassa virus glycoprotein. Our findings encourage the development of a combination of approved drugs to treat both Lassa and Ebola virus diseases.

Sensitivity of influenza viruses to zanamivir and oseltamivir: a study performed on viruses circulating in France prior to the introduction of neuraminidase inhibitors in clinical practice.

Influenza virus neuraminidase inhibitors (NAIs) were introduced in clinical practice in various parts of the world since 1999 but were only scarcely distributed in France. Prior to the generalization of zanamivir and oseltamivir utilization in our country, we decided to test a large panel of influenza strains to establish the baseline sensitivity of these viruses to anti-neuraminidase drugs, based upon a fluorometric neuraminidase enzymatic test. Our study was performed on clinical samples collected by practitioners of the GROG network (Groupe Régional d'Observation de la Grippe) in the south of France during the 2002-2003 influenza season. Out of 355 isolates tested in the fluorometric neuraminidase activity assay, 267 isolates could be included in inhibition assay against anti-neuraminidase drugs. Differences in IC50 range were found according to the subtype and the anti-neuraminidase drug. Influenza B and A/H1N1 viruses appeared to be more sensitive to zanamivir than to oseltamivir (mean B IC50 values: 4.19 nM versus 13 nM; mean H1N1 IC50 values: 0.92 nM versus 1.34 nM), while A/H1N2 and A/H3N2 viruses were more sensitive to oseltamivir than to zanamivir (mean H3N2 IC50 values: 0.67 nM versus 2.28 nM; mean H1N2 IC50 values: 0.9 nM versus 3.09 nM). Out of 128 N2 carrying isolates, 10 isolates had zanamivir or oseltamivir IC50 values in upper limits compared to their respective data range. Sequencing of the neuraminidase of these outliers N2 highlighted several mutations, but none of them were associated with resistance to neuraminidase inhibitors.

Monoclonal antibody recognition of cholesterol monohydrate crystal faces.

BACKGROUND: The immune system can elicit antibodies against a wide variety of antigens. We have proposed that crystal surfaces may also operate as antigens, binding specific antibodies. Here we exploit the crystal surfaces of cholesterol monohydrate to investigate antibody-surface recognition at the molecular level. RESULTS: Four monoclonal antibodies were selected. Two specifically interact with cholesterol monohydrate crystals, and one with 1,4-dinitrobenzene crystals. The fourth interacts nonselectively with various solid substrates. The relative reactivities of the four antibodies to the different surfaces of cholesterol monohydrate and to other surfaces were compared. The nonspecific antibody adsorbs mainly at imperfections. Of the two specific antibodies, one shows a clear preference for one set of faces, relative to others, the second adsorbs selectively at one face of cholesterol monohydrate crystals. CONCLUSIONS: Monoclonal antibodies can be selected that specifically bind to the crystal surfaces of cholesterol monohydrate. The binding sites of such antibodies appear to recognize a number of molecular moieties, exposed at the surface in a specific structural organization. Different antibodies recognize different structural organizations with varying degrees of selectivity. Antibody-crystal surface interactions may serve as convenient models for studies aimed at an understanding of the molecular bases of antibody recognition.

A sensitive and specific ELISA immunocapture assay for rapid quantitation of influenza A/H3N2 neuraminidase protein.

Both HA and NA proteins elicit antibodies which have been shown to be capable of altering the course of infection. Nevertheless, while influenza virus vaccine standardization involves hemagglutinin (HA) and neuraminidase (NA) in terms of antigenic characterization, only HA protein quantitation is undertaken. An immunocapture ELISA (EIA) is described for N2 NA quantitation, based on the use of a highly specific monoclonal antibody (MAb) for capturing NA and an anti-NA antiserum for antigen detection. The amounts of NA in samples were deduced from the standard curve established by using purified NA. The NA-EIA is specific and detects as a little as 7 ng/ml. The capture and detector antibodies directed against A/Beijing/32/92 NA were shown to react with H3N2 prototype strains used in current influenza vaccines, provided that an antigenically matched reference NA is used as standard.

Simultaneous purification of influenza haemagglutinin and neuraminidase proteins by immunochromatography.

A new and rapid method for co-purification of haemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/H3N2 viruses is described. Surface glycoproteins were first solubilized using a non-ionic detergent under high ionic strength conditions, then they were separated by chromatography on sepharose previously bound to monoclonal antibodies (MAbs) directed either against HA (IaH-chromatography) or against NA (IaN-chromatography). Depending on the protein specificity of the MAb immobilized on the column, HA or NA was bound to sepharose and the counterpart protein was free in the flow-through volume. IaH-chromatography and IaN-chromatography proved equally efficient in term of recoveries (> 75%) and purity (> or = 99%) of both HA and NA but differences appeared when considering functional and antigenic properties of pure proteins. Those properties were highly retained in IaH- and IaN-derived HA as well as in IaH-derived NA while IaN-NA was partially degraded. IaH-chromatography allowed the co-purification of HA and NA proteins in heterologous antigen-antibody system with a 50% rate of cross reactivity. IaH-HA and IaH-NA may be suitable for immunity studies, standardization of influenza vaccine and for diagnostic purposes.

Initial study of using a laminar fluid diffusion interface for sample preparation in high-performance liquid chromatography.

This report describes a new microfluidic device called the H Filter for sample preparation prior to HPLC. The H Filters make possible a diffusional transfer of an analyte from a sample stream into a stream of a "receiver" fluid. Existing mathematical models can be used for optimizing experimental conditions. The authors have selected the extraction of the antibiotic cephradine from blood to demonstrate the utility of the new device. The extracts of blood samples spiked with cephradine levels between 0.2 and 100 microg/ml were analyzed using a C8 reversed-phase column and UV detection at 260 nm. The HPLC results were in good agreement with theory. The recovery of 32.2+/-2.8% was uniform over the entire range of cephradine concentrations. The new method completely avoids the use of centrifuges, that is otherwise typical for most current methodologies for the preparation of blood samples prior to HPLC analysis.

Neuraminidase assays.

Anti-neuraminidase (NA) antibodies (Ab) play a role in protection against influenza and in combination with anti-HA Ab they increase the protection in mice. To control the NA content of vaccines, which should improve vaccine standardisation and may benefit vaccine efficacy, a series of questions must be addressed: 1) The antigenic characterization of NA in vaccine strains and seed lots is based on the measurement of the enzymatic (E) activity using fetuin as substrate. The antigenic profile is established by inhibiting the E activity with post infectious ferret antisera. Overnight incubation ensures sensitivity, and fetuin substrate gives specificity by detection of variant specific antibodies. Several difficulties have to be overcome, such as the low level of E activity in MDCK grown viruses, and the lability of N1. 2) The NA protein content of the vaccines (in bulk or final product) can be measured by an ELISA capture test but the lability of the NA proteins at 4 degrees C must be checked. 3) The anti NA Ab response can be measured using a neuraminidase inhibition test. --The steric hindrance by HI antibodies does not exceed a titre of 20 in human sera. --Triton treatment of viruses reduces the steric hindrance in polyclonal sera and monoclonal antibodies but unmasks epitopes. 4) The correlations between neuraminidase inhibition, neutralization and protection, has been established in the mouse model, but remains to be shown in humans. 5) The use of a small fluorescent (MUN) or chemiluminescent (NA-STAR) substrate can be used for the rapid differentiation of N1 from N2 and NB, but not for the titration of protective NI antibodies.

[Acute appendicitis: to image or not to image?]. / Imagerie de l'appendicite: échographie, scanner ou rien du tout?

Acute appendicitis is frequently clinically suspected. However, about 50% of emergency room patients with such a diagnosis do not have acute appendicitis and between 20-25% of patients undergoing appendectomy based on clinical diagnosis have a normal appendix. On the other hand, if left untreated acute appendicitis may result in peritonitis. The purpose of this article is to review the indications for imaging patients with clinical suspicion of acute appendicitis, to describe the US and CT features of acute appendicitis, to review the advantages and limitations of US and CT, and to present the differential diagnosis to be considered in patients with right lower quadrant pain.

[Role of antineuraminidase antibodies in protection against influenza]. / Place des anticorps antineuraminidase dans la protection contre la grippe.

For improving the anti-influenza vaccination efficacy, the choice of strains carrying up dated neuraminidase antigen (NA) and the introduction of the optimal amount of NA antigen in the vaccine are critical. Monoclonal antibodies prepared against the neuraminidase N2 of A/Beijing/32/92 showed NA inhibition (NI) and neutralized (Nt) the cells infection by influenza virus either at an early stage (group 2 antibodies inhibit virus binding to cells) or at a late stage of infection (group 1 antibodies inhibit virus release). The specificity of the neutralization test is restricted to the homologous variant whereas the NI specificity is much broader. When both group 1 and group 2 antibodies are tested together, their neutralizing activity is significantly increased. The emergence in 1997 of an avian strain H5N1 in humans influenza infections at Hong Kong (Strain A/Hong-Kong/156/97) rose the threat of pandemic. The H5N1 strain carried H5 HA which is not recognized by the human immune system, but N1 might be related to other N1 antigens belonging to avian, swine and human strains. So we 1) characterized the N1 antigen from H5N1 in comparison with other known antigens, 2) we looked for anti N1 (H5N1) antibodies in humans according to the age and the vaccination status, 3) we checked the neutralizing activity of anti N1 antibodies. The N1 antigen (H5N1) appeared closely related to N1 from swine strains: Sw/31 correlated itself to the pandemic spanish virus (1918-19), and more recent swine isolates from 1982 and 1989. The anti N1 (H5N1) antibodies were present in sera collected from 75+ years old persons and these N1 antibodies were neutralizing H5N1 cells infection. Consequently, 75+ years old persons do not represent a priority group for vaccination in the case of H5N1 pandemic conditions.

Metabolite fingerprinting of barley whole seeds, endosperms, and embryos during industrial malting.

Samples of whole seeds, isolated endosperms including the aleurone layer and isolated embryos with attached scutellum from an industrial scale barley malting process (variety Braemar) were analysed for their water soluble metabolites by gas chromatography-mass spectrometry (GC-MS). 73 known metabolites and about 350 unknown signals were detected. Principal component analysis (PCA) showed a time dependent shift of sample profiles. Whole seeds and endosperm samples showed very similar patterns with nearly all compounds rising until the end of germination. In the embryos a maximum concentration of compounds was reached after 72-96 h of malting. Most concentrations decreased afterwards. The kilning step, namely the drying and roasting of germinated seeds, induced variable effects of increases, stability or decreases of metabolites and thereby separated kilned samples from germinated seeds in the PCA. A second barley cultivar (Quench) underwent the same malting and analysis procedures and gave nearly identical results. Fructose, malate, myo-inositol and raffinose exhibited the potential to serve as markers for specific developmental stages of seeds in both varieties. Biological markers represent targets for industrial process control. Their potential application would meet the maltsters' demand to flatten variances in germination properties and to produce equal composed malt by directed malting management.

Biochemical properties of paramyxovirus Duck/Mississippi/75 neuraminidase.

The neuraminidase activity of two strains of Duck/Mississippi/75 virus:DK/Mississippi/320 and DK/Mississippi/334 was studied. These neuraminidases hydrolyse the alpha 2 leads to 3 and alpha 2 leads to 8 ketosidic bonds of different substrates such as fetuin, N-acetyl neuramine lactose and colominic acid, but do not hydrolyse the alpha 2 leads to 6 bonds of mucin type I and type II. The kinetic values of the neuraminidases, Michaelis constant, maximal and initial velocities and the effect of pH, temperature and detergents were also evaluated. The isolates differ mainly in the optimal pH and temperature conditions of activity. As with other paramyxovirus neuraminidases, the enzyme of DK/Mississippi/75 was destroyed by ionic but not non-ionic detergents.

Study of a new strain of paramyxoviruses isolated from wild ducks: structural polypeptides.

By polyacrylamide gel electrophoresis, Duck/Mississippi/75 virus was shown to contain five different types of polypeptides of mol. wt. ranging from 76 X 1O(3) to 43 X 10(3), two of which were glycosylated (mol. wt. 76 X 10(3) and 57 X 10(3). A comparison of this data with similar results obtained using Yucaipa and Newcastle disease virus (NDV) revealed a similarity with NDV, concerning the number, position and mol. wt. of the polypeptides. Haemagglutinating and neuraminidase properties are associated with surface glycoproteins which represent at least 40% of the virus protein. After KCl and Triton X-100 treatment and centrifugation on linear sucrose density gradients (10 to 25%, w/w) it was possible to isolate glycoprotein VP1 of mol. wt 76 X 10(3) with which haemagglutinating and neuraminidase activities were associated.

Contribution of monoclonal antibodies to the study of parainfluenza virus antigens.

Splenic lymphocytes from BALB/c mice immunized with Parainfluenza 1 virus (PIV-1), HA2 strain, were fused to the non secreting 653 and secreting P3 X 63Ag8 myeloma cell lines. Fusion products were screened for antibody synthesis using an enzyme-linked immunosorbent assay (ELISA) with purified PIV-1. Antibodies were characterized by their biologic specificity and immune fluorescence staining--6 monoclones (2HI and 4 non HI) were used to further analysis by cross reactivity panels using different PIV strains. None of the anti PIV1 monoclones showed any cross reactivity with PIV2 and PIV3 whereas with HI and non HI monoclones antigenic relationships were observed between viruses belonging to serotype 1. Monoclonal antibodies were also applied to the study of host cell related changes in PIV1 proteins.

[Rapid serodiagnosis of influenza by a modified radial haemolysis test: immune response (author's transl)]. / Diagnostic sérologique rapide de la grippe par la méthode d'hémolyse radiale modifiée et évolution des anticorps.

A modification of the single radial haemolysis test by treatment with Cl3Cr of the virus coated red blood cells is proposed for a rapid, more sensitive and reproducible serodiagnosis of Influenza infections. The reagents "red blood cells-virus-Cl3Cr" is stable at 4 degrees C for at least 1 month: the modified test gives results within 3 hours, in measuring the H-anti-H specific reactions. The anti-influenza A immune response was studied in patients of different age, bled at different time after exposure. The IgM do not diffuse in the agarose and do not haemolyse. A clear haemolytic area is produced by IgG, partially haemolysed areas occur when there is a competition between specific IgM and IgG and in the case of reactions involving cross-reacting antigenic determinants.

Study of a new strain of paramyxoviruses isolated from wild ducks: antigenic and biological properties.

Among the different strains of avian paramyxoviruses isolated from migrating feral ducks, two were identified as Newcastle disease viruses (NDV) and the five others would correspond to a new serotype for which we suggest the name of Duck/Mississippi/75 virus. The characteristics of this new serotype are as follows: (1) Duck/Mississippi/75 virus is able to grow as well in allantoic as in amniotic cavities of embryonated hen's eggs; (2) the haemagglutinin and haemolytic activities can be detected with hen red blood cells; (3) the neuraminidase hydrolyses the alpha 2 leads to 3 bonds of the fetuin substrate and its pH activity could be species specific. Antigenically, this serotype is different from all human and animal paramyxoviruses, in spite of an antigenic relationship with NDV.

Difficulties in standardizing the neuraminidase content of influenza vaccines.

To achieve better standardization of influenza vaccines, an ELISA immunocapture assay was developed for N2 neuraminidase quantification. This sensitive and highly specific assay was successfully applied to vaccine preparations produced in embryonated hens' eggs from 1992 to 1997 and to antigenically related viral suspensions produced in MDCK cells. A study of the neuraminidase activity of prototype A/H3N2 strains stored at 4 degrees C showed the gradual development of enzymatic instability from 1994 onwards, accompanied by antigenic modifications of the antigen. As the phenomenon was also more pronounced with the recombinant, the question arose of the standard of immunity provided when such viruses are used for vaccination. The antibodies inhibiting neuraminidase activity in vaccinated subjects were monitored in parallel using both complete virus and purified N2 NA. The study revealed the existence of an interference phenomenon which resulted in the titre of the N1 antibodies being overestimated. The interference was due to anti-HA antibodies impeding access to the substrate at the enzymatic site by steric hindrance.

Contribution to the study of the parainfluenza antigens.

From epidemiological data it appeared that an epidemic of parainfluenza type 1 occurred in Lyons in October-November 1967. Thereafter a few sporadic cases were detected. Then in 1970 strains were isolated which showed a change in the hemagglutinin antigen. The persistence of antibodies in the population is shorter than that of the other parainfluenza viruses (i. e. parainfluenza 3); it is not known if reinfections with parainfluenza 1 occur since there are cross-reactions by CF test between parainfluenza 1 and parainfluenza 3 which are known to cause reinfections. The study of antigens of parainfluenza strains was made by HI, Nt, CF and immunodiffusion tests with various antisera prepared in various animal species. This study showed antigenic differences and similarities between parainfluenza 1 and parainfluenza 3.

Biological properties and carbohydrate composition of human parainfluenza virus type 1 in two host systems.

Human parainfluenza virus type 1 (HA2 virus) grown either in embryonated hens' eggs or in M. rhesus monkey kidney cells showed differences in size, biological properties and carbohydrate composition. Egg-grown virus showed a larger size (233 nm versus 167 nm), a higher neuraminidase activity (specific activity and initial and maximum velocity) and a higher haemolytic activity than monkey kidney cell-grown virus. The haemagglutinin titre was identical for the HA2 strain grown in both host systems when tested with human O Rh+, guinea-pig and hen red blood cells, but reduced by more than 100-fold when tested with grivet monkey red blood cells. In addition, the carbohydrate content (mainly neutral sugars) was higher in egg-grown virus (9.2%) than in virus grown in MK cells (5.7%), and the amino to neutral sugar ratio was lower (1.2 versus 2.1). The sugars were identified as fucose, mannose, galactose, glucose, glucosamine and galactosamine. The prominent neutral monosaccharide was glucose in egg-grown virus and fucose in MK cell-grown virus. HA2 virus infection of MK cells increased fucose and glucose, and decreased mannose and galactose levels.

Catalytic properties of the A/H3N2 influenza neuraminidases: influence of antigenic variations.

Antigenic variation of the neuraminidase of A/H3N2 influenza viruses may be associated with modifications of the catalytic activity of this enzyme. We observed this phenomenon when studying two prototype strains: A/Hong Kong/1/68 (X31K) and A/Bangkok/2/79. For the neuraminidases of these strains, we determined their substrate specificity, initial velocity, optimum pH, optimum temperature, heat inactivation and Michaelis constants and their inactivation by chemical group-specific reagents. In order to examine the relationship between antigenic variation and enzyme activity of the influenza neuraminidases, three X31K monoclonal variants were selected using anti-neuraminidase monoclonal antibodies. Two of these (X31/NC92 and X31/NC56) were modified at a single neuraminidase epitope, and the third one (X31/NC92/NC56) at two epitopes. The neuraminidase activity of the monoclonal variants was analysed and compared to that of the prototype strains. Compared to A/Hong Kong/1/68, the A/Bangkok/2/79 strain neuraminidase was more susceptible to inactivation by physical (pH, temperature) and chemical agents [urea, dithiothreitol, 1-ethyl-3-(3-dimethylaminopropyl carbodiimide), iodoacetamide, acetic anhydride, 2,3-butanedione] and showed a twofold lower substrate affinity for N-acetylneuraminlactose. The neuraminidase activity of the monoclonal variants of X31K became more susceptible to inactivation by both physical and chemical agents than the original strain and exhibited various substrate affinities. Therefore, we conclude that the enzymic properties of the structurally conserved active sites of the neuraminidase molecule may be influenced by antigenic modifications that affect the variable areas of the neuraminidase and that the degree of this enzymic variation is related to the nature and number of the modified epitope(s). A local conformational change in the neuraminidase molecule reflected as antigenic variation could be involved in modification of enzyme activity.