In vitro activity of expanded-spectrum pyridazinyl oxime ethers related to pirodavir: novel capsid-binding inhibitors with potent antipicornavirus activity.

Picornaviruses (PV) include human rhinovirus (HRV), the primary cause of the common cold, and the enteroviruses (EV), which cause serious diseases such as poliomyelitis, meningoencephalitis, and systemic neonatal disease. Although no compounds for PV infections have been approved in the United States, pirodavir was one of the most promising capsid-binding compounds to show efficacy in human clinical trials for chemoprophylaxis of the common cold. Susceptibility to hydrolysis precluded its use as an oral agent. We have developed orally bioavailable pyridazinyl oxime ethers that are as potent as pirodavir. Compounds BTA39 and BTA188 inhibited a total of 56 HRV laboratory strains and three clinical isolates as determined by neutral red uptake assay. At concentrations of <100 nM, BTA39 inhibited 69% of the HRV serotypes and isolates evaluated, BTA188 inhibited 75%, and pirodavir inhibited 59% of the serotypes and isolates. The 50% inhibitory concentrations (IC(50)s) for the two compounds ranged from 0.5 nM to 6,701 nM. The compounds also inhibited EV, including coxsackie A and B viruses (IC(50) = 773 to 3,608 nM) and echoviruses (IC(50) = 193 to 5,155 nM). BTA39 only inhibited poliovirus strain WM-1 at 204 nM, and BTA188 only inhibited poliovirus strain Chat at 82 nM. EV 71 was inhibited by BTA39 and BTA188, with IC(50)s of 1 and 82 nM, respectively. Both compounds were relatively nontoxic in actively growing cells (50% cytotoxic doses, >/=4,588 nM). These data suggest that these oxime ethers warrant further investigation as potential agents for treating selected PV infections.

Development of sampling and analytical methods for concerted determination of commonly used chloroacetanilide, chlorotriazine, and 2,4-D herbicides in hand-wash, dermal-patch, and air samples.

Sampling and analytical methods were developed for commonly used chloroacetanilide, chlorotriazine, and 2,4-D herbicides in hand washes, on dermal patches, and in air. Eight herbicides selected for study were alachlor, atrazine, cyanazine, 2,4-dichlorophenoxyacetic acid (2,4-D), metolachlor, simazine, and two esters of 2,4-D, the 2-butoxyethyl ester (2,4-D, BE) and the 2-ethylhexyl ester (2,4-D, EH). The hand-wash method consisted of shaking the worker's hand in 150 mL of isopropanol in a polyethylene bag for 30 seconds. The dermal-patch method entailed attaching a 10-cm x 10-cm x 0.6-cm polyurethane foam (PUF) patch to the worker for exposure; recovery of the herbicides was achieved by extraction with 40 mL of isopropanol. The air method involved sampling with an OVS-2 tube (which contained an 11-mm quartz fiber filter and two beds of XAD-2 resin) and recovery with 2 mL of 10:90 methanol:methyl t-butyl ether. Analysis of each of the three sample types was performed by gas chromatography with an electron-capture detector. Diazomethane in solution was employed to convert 2,4-D as the free acid to the methyl ester in each of the three methods for ease of gas chromatography. Silicic acid was added to sample solutions to quench excess diazomethane. Limits of detection for all eight herbicides were matrix-dependent and, generally, less than 1 microgram per sample for each matrix. Sampling and analytical methods met NIOSH evaluation criteria for all herbicides in hand-wash samples, for seven herbicides in air samples (all herbicides except cyanazine), and for six herbicides in dermal-patch samples (all herbicides except cyanazine and 2,4-D). Speciation of 2,4-D esters and simultaneous determination of 2,4-D acid were possible without losses of the esters or of other herbicides (acetanilides and triazines) being determined.

Distributions and determinants of pre-emergent herbicide exposures among custom applicators.

Custom applicators intensively apply herbicides to corn and soybean fields each spring. The primary objective of this study was to characterize the exposure distributions of the herbicides alachlor, atrazine, 2,4-D 2-ethylhexyl ester (2,4-D EH), and metolachlor among a group of applicators during the spring pre-emergent spray season. A secondary objective was to evaluate determinants of exposure and to estimate within- and between-worker variance components. Fifteen applicators were sampled using a systematic design that included spray and non-spray days and multiple measurements (five to seven) on each applicator. Air, patch, and handwash samples were collected on 89 applicator-days. Applicator-days were classified into three categories: target herbicide sprayed, non-target herbicide sprayed, and no herbicide sprayed. Mixed-model regression analysis was used. For all exposure metrics, adjusted mean herbicide exposures were significantly higher on days when target herbicides were sprayed as compared to non-spray days. For 2,4-D EH only, adjusted mean exposures on non-target herbicide spray days were significantly higher than on non-spray days. Wearing gloves significantly reduced adjusted mean hand exposure for all herbicides (4-20 fold) and adjusted mean thigh exposure for three herbicides (8-53 fold) on days the herbicides were sprayed; however, wearing gloves significantly increased adjusted mean atrazine hand and thigh exposures (9 and 7 fold, respectively) on days that non-atrazine herbicides were sprayed. Few of the other covariates were consistent determinants of exposure. For all exposure metrics, the within-worker variability (GSD(W) 2.1-5.6) was greater than the between-worker variability (GSD(B) 1.2-2.7).

Determination of capsaicin and dihydrocapsaicin in air in a pickle and pepper processing plant.

A sampling and analytical method has been developed for measurement of capsaicin and dihydrocapsaicin in air. This method is applicable to measurement of the capsaicinoids in air in pepper processing plants and involves air sampling with a 13-mm glass fiber filter, recovery of the sample with 2 mL of acetonitrile, filtration of the solution, and analysis by high performance liquid chromatography with fluorescence detection. Excitation and emission wavelengths of the detector were 281 and 312 nm, respectively. Average recoveries were 98 to 104% after fortification of glass fiber filters with 0.13- to 2.9-microg quantities of capsaicin. Average recoveries of dihydrocapsaicin were 93 to 99% after fortification of glass fiber filters with 0.11- to 3.0-microg quantities. Detection limits of capsaicin and dihydrocapsaicin were 0.015 and 0.02 microg per sample, respectively. This method was used successfully for determining air concentrations of capsaicin and dihydrocapsaicin in a health hazard evaluation at a large pickle and pepper processing plant. An interesting phenomenon was the fact that the ratio of capsaicin to dihydrocapsaicin in each of the largest air samples was in the range of 0.3:1 to 0.5:1. Generally, capsaicin is the capsaicinoid that occurs in Capsicum fruit in the greatest relative abundance.

A flu optical immunoassay (ThermoBioStar's FLU OIA): a diagnostic tool for improved influenza management.

ThermoBioStar's and Biota's flu optical immunoassay (FLU OIA) is a rapid test designed to diagnose influenza A and B infection using a variety of specimen types. The assay uses highly sensitive thin-film detection methods, coupled with specific monoclonal antibodies to the nucleoprotein. The test is simple to perform, requires no instrumentation and is intended to provide a result within 15 min of test initiation in the 'point-of-care' environment. In initial clinical studies, the assay was demonstrated to be equivalent to culture in identifying infected individuals. Subsequent independent studies using a variety of sample types have demonstrated sensitivity ranging from 48 to 100% and specificities ranging from 93 to 97%. In addition to detecting human strains, this assay has been demonstrated to be capable of detecting a variety of avian and non-human mammalian influenza viruses. The FLU OIA test has been used in large-scale surveillance schemes intended to provide rapid epidemiological data during normal influenza seasons and has demonstrated the potential for fulfilling a similar role for multispecies surveillance in, for example, conditions that offer challenges for conventional virus isolation methods. Conceivably, such use should facilitate the timely recognition of influenza outbreaks and prioritization of positive specimens for more conventional, laboratory characterization, leading to improved interpandemic surveillance and rapid reaction in the face of the next pandemic.

The core of the respiratory syncytial virus fusion protein is a trimeric coiled coil.

Entry into the host cell by enveloped viruses is mediated by fusion (F) or transmembrane glycoproteins. Many of these proteins share a fold comprising a trimer of antiparallel coiled-coil heterodimers, where the heterodimers are formed by two discontinuous heptad repeat motifs within the proteolytically processed chain. The F protein of human respiratory syncytial virus (RSV; the major cause of lower respiratory tract infections in infants) contains two corresponding regions that are predicted to form coiled coils (HR1 and HR2), together with a third predicted heptad repeat (HR3) located in a nonhomologous position. In order to probe the structures of these three domains and ascertain the nature of the interactions between them, we have studied the isolated HR1, HR2, and HR3 domains of RSV F by using a range of biophysical techniques, including circular dichroism, nuclear magnetic resonance spectroscopy, and sedimentation equilibrium. HR1 forms a symmetrical, trimeric coiled coil in solution (K(3) approximately 2.2 x 10(11) M(-2)) which interacts with HR2 to form a 3:3 hexamer. The HR1-HR2 interaction domains have been mapped using limited proteolysis, reversed-phase high-performance liquid chromatography, and electrospray-mass spectrometry. HR2 in isolation exists as a largely unstructured monomer, although it exhibits a tendency to form aggregates with beta-sheet-like characteristics. Only a small increase in alpha-helical content was observed upon the formation of the hexamer. This suggests that the RSV F glycoprotein contains a domain that closely resembles the core structure of the simian parainfluenza virus 5 fusion protein (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309-319, 1999). Finally, HR3 forms weak alpha-helical homodimers that do not appear to interact with HR1, HR2, or the HR1-HR2 complex. The results of these studies support the idea that viral fusion proteins have a common core architecture.

An approach to area sampling and analysis for total isocyanates in workplace air.

An approach to sampling and analysis for total isocyanates (monomer plus any associated oligomers of a given isocyanate) in workplace air has been developed and evaluated. Based on a method developed by the Occupational Health Laboratory, Ontario Ministry of Labour, Ontario, Canada, isocyanates present in air are derivatized with a fluorescent reagent, tryptamine, in an impinger and subsequently analyzed via high-performance liquid chromatography (HPLC) with fluorescence detection. Excitation and emission wavelengths are set at 275 and 320 nm, respectively. A modification to the Ontario method was made in the replacement of the recommended impinger solvents (acetonitrile and 2,2,4-trimethylpentane) with dimethyl sulfoxide (DMSO). DMSO has the advantages of being compatible with reversedphase HPLC and not evaporating during sampling, as do the more volatile solvents used in the Ontario method. DMSO also may dissolve aerosol particles more efficiently during sampling than relatively nonpolar solvents. Several formulations containing diisocyanate prepolymers have been tested with this method in the laboratory. This method has been issued as National Institute for Occupational Safety and Health (NIOSH) Method 5522 in the first supplement to the fourth edition of the NIOSH Manual of Analytical Methods. This method is recommended for area sampling only due to possible hazards from contact with DMSO solutions containing isocyanate derivatives. The limits of detection are 0.1 microgram/sample for 2,4-toluene diisocyanate, 0.2 microgram/sample for 2,6-toluene diisocyanate, 0.3 microgram/sample for methylene bisphenyl diisocyanate, and 0.2 microgram/sample for 1,6-hexamethylene diisocyanate.

Estimate of the frequency of human immunodeficiency virus type 1 protease inhibitor resistance within unselected virus populations in vitro.

The frequency of drug-resistant human immunodeficiency virus type 1 (HIV-1) variants in virus populations not previously exposed to drug was determined in vitro by using HIV-1RF and the protease inhibitor SC-55389A. Two variants with single mutations responsible for drug resistance (V82A and N88S) were quantifiably isolated after only one round of replication, yielding a crude frequency estimate of at least 1 SC-55389A-resistant variant per 3.5 x 10(5) wild-type infectious units.

A mutation in human immunodeficiency virus type 1 protease at position 88, located outside the active site, confers resistance to the hydroxyethylurea inhibitor SC-55389A.

The hydroxyethylurea human immunodeficiency virus type 1 (HIV-1) protease inhibitors SC-55389A and SC-52151 were used to select drug-resistant variants in vitro. One clinical HIV-1 strain (89-959) and one laboratory HIV-1 strain (LAI) were passaged in peripheral blood mononuclear cells or CEMT4 cells in the presence of SC-55389A. Resistant isolates from both strains consistently had a mutation to serine for asparagine at amino acid 88 (N88S) in the protease gene either alone or in combination with a change to phenylalanine at position 10. The N88S mutation, recreated by oligonucleotide-mediated site-directed mutagenesis in HXB2, was sufficient to confer resistance to SC-55389A. In contrast, SC-52151-resistant variants selected from the monocytotropic strain SF162 had multiple substitutions in the protease gene (I11V, M461, F53L, A71V, and N88D), and the N88D mutation, re-created by oligonucleotide-mediated site-directed mutagenesis in HXB2, did not confer resistance to SC-52151. The potencies of L735,524 and Ro31-8959 were not reduced when these compounds were assayed against variants with either the N88S or N88D substitution. Position 88 is in a helix that lies behind the substrate binding pocket and may indirectly influence inhibitor binding through interactions with the amino acid at position 31. The selected mutations were persistent in the viral populations after more than 20 passages in the absence of drugs. Passaging of virus first in SC-55389A alone and then in combination with SC-52151 resulted in the accumulation of more mutations in the protease gene (L10F, D35E, D37M, I47V, 154L, A71V, V82I, and S88D) and in the selection of a variant that was cross-resistant to multiple protease inhibitors. These results indicate that a mutation in the HIV-1 protease at a position that is located outside of the substrate binding pocket confers resistance to a protease inhibitor and that mutations in the protease gene accumulate with increasing selection pressure and can persist in the absence of selection pressure.

Oligomerization of the hydrophobic heptad repeat of gp41.

The transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a leucine zipper-like (hydrophobic heptad) repeat which has been predicted to form an amphipathic alpha helix. To evaluate the potential of the hydrophobic heptad repeat to induce protein oligomerization, this region of gp41 has been cloned into the bacterial expression vector pRIT2T. The resulting plasmid, pRIT3, expresses a fusion protein consisting of the Fc binding domain of monomeric protein A, a bacterial protein, and amino acids 538 to 593 of HIV-1 gp41. Gel filtration chromatography demonstrated the presence of oligomeric forms of the fusion protein, and analytical centrifugation studies confirmed that the chimeric protein formed a higher-order multimer that was greater than a dimer. Thus, we have identified a region of HIV-1 gp41 which is capable of directing the oligomerization of a fusion protein containing monomeric protein A. Point mutations, previously shown to inhibit the biological activity of the HIV-1 envelope glycoprotein, have been engineered into the segment of gp41 contained in the fusion protein, and expressed mutant proteins were purified and analyzed via fast protein liquid chromatography. A point mutation in the heptad repeat, which changed the central isoleucine to an alanine, caused a significant (> 60%) decrease in oligomerization, whereas changing the central isoleucine to aspartate or proline resulted in almost a complete loss of oligomerization. Deletions of one, two, or three amino acids following the first isoleucine also resulted in a profound decrease in oligomerization. The inhibitory effects of the mutations on oligomer formation correlated with the effects of the same mutations on envelope glycoprotein-mediated fusion. A possible role of the leucine zipper-like region in the fusion process and in an oligomerization event distinct from assembly of the envelope glycoprotein complex is discussed.

Human immunodeficiency virus type 1 envelope glycoprotein is modified by O-linked oligosaccharides.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been shown to be extensively modified by N-linked glycosylation; however, the presence of O-linked carbohydrates on the glycoprotein has not been firmly established. We have found that enzymatic deglycosylation of the HIV-1 envelope glycoprotein with neuraminidase and O-glycosidase results in a decrease in the apparent molecular weight of the envelope glycoprotein. This result was observed in both vaccinia virus recombinant-derived envelope glycoproteins and glycoproteins derived from the IIIB, SG3, and HXB2, strains of HIV-1. The decrease in molecular weight was also observed when the envelope glycoprotein had been deglycosylated with N-glycanase F after treatment with neuraminidase and O-glycosidase, indicating that the decrease in apparent molecular weight was not attributable to the removal of N-linked carbohydrate. Treatment with neuraminidase, O-glycosidase, and N-glycanase F was found to be necessary to remove all radiolabel from [3H]glucosamine-labelled envelope glycoprotein, a result seen for both recombinant and HIV-1-derived envelope glycoprotein. [3H]glucosamine-labelled carbohydrates liberated by O-glycosidase treatment were separated by paper chromatography and were found to be of a size consistent with O-linked oligosaccharides. We, therefore, conclude that the HIV-1 envelope glycoprotein is modified by the addition of O-linked carbohydrates.

Determination of pentamidine isethionate in air.

A concern currently exists regarding the potential for exposure of health care workers to pentamidine isethionate, a drug used for prevention and treatment of Pneumocystis carinii pneumonia in immunocompromised patients, including those with human immunodeficiency virus infection. In order to evaluate worker exposures, a sampling and analytical method for pentamidine isethionate in air has been developed. This method involves sampling with a 37-mm PVC membrane filter at 1 to 2 L/min, recovery with 3 mL of 50:50 ethanol:water with 0.085% phosphoric acid and 0.04% tetramethylammonium chloride in an ultrasonic bath for 10 min, and analysis by high performance liquid chromatography with fluorescence detection. The limit of detection is about 18 ng per sample, and the lower limit of quantitation is 50 ng per sample. Recoveries of pentamidine isethionate from PVC filters were 0.76 to 0.91 at 50 to 8820-ng levels of fortification. Samples were stable on PVC filters during 27 days of storage at room temperature. Because patients who are treated with pentamidine isethionate are at increased risk of contracting tuberculosis (TB), safety precautions for handling samples contaminated with TB were included in the sampling and analytical method.

Vectorial release of poliovirus from polarized human intestinal epithelial cells.

Polarized epithelial cells represent the primary barrier to virus infection of the host, which must also be traversed prior to virus dissemination from the infected organism. Although there is considerable information available concerning the release of enveloped viruses from such cells, relatively little is known about the processes involved in the dissemination of nonenveloped viruses. We have used two polarized epithelial cell lines, Vero C1008 (African green monkey kidney epithelial cells) and Caco-2 (human intestinal epithelial cells), infected with poliovirus and investigated the process of virus release. Release of poliovirus was observed to occur almost exclusively from the apical cell surface in Caco-2 cells, whereas infected Vero C1008 cells exhibited nondirectional release. Structures consistent with the vectorial transport of virus contained within vesicles or viral aggregates were observed by electron microscopy. Treatment with monensin or ammonium chloride partially inhibited virus release from Caco-2 cells. No significant cell lysis was observed at the times postinfection when extracellular virus was initially detected, and transepithelial resistance and vital dye uptake measurements showed only a moderate decrease. Brefeldin A was found to significantly and specifically inhibit poliovirus biosynthetic processes by an as yet uncharacterized mechanism. The vectorial release of poliovirus from the apical (or luminal) surface of human intestinal epithelial cells has significant implications for viral pathogenesis in the human gut.

Secretion of a truncated form of the human immunodeficiency virus type 1 envelope glycoprotein.

We have characterized a truncated secreted form of the HIV-1 envelope glycoprotein gene. Expression via a recombinant vaccinia virus resulted in a glycoprotein product of approximately 140 kDa (gp160t) and a minor cleavage product of 120 kDa (gp120). Pulse-chase analysis revealed that the majority of gp160t remained cell-associated and underwent degradation within 10-20 hr of synthesis. A secreted form (gp160t/sec) and gp120 were detected in the media 2-4 hr postsynthesis and were not significantly degraded within a period of 20 hr. Most of the cell-associated gp160t remained sensitive to digestion with endoglycosidase H, whereas gp160t/sec and gp120 were largely resistant. Gp160t, gp160t/sec, and gp120 formed oligomers which were stabilized by intermolecular disulfide bonds and/or noncovalent interactions and were also found to bind to soluble CD4. Both wild type gp160 and wild type gp160t were observed to undergo a post-translational modification 4-5 hr postsynthesis, resulting in glycoproteins with a slightly increased electrophoretic mobility. These differences in electrophoretic mobility remained following treatment with N-glycosidase F, indicating that they are not a consequence of N-linked oligosaccharide processing, but may represent an additional modification of the envelope glycoprotein.

Bidirectional entry of poliovirus into polarized epithelial cells.

The interactions of viruses with polarized epithelial cells are of some significance to the pathogenesis of disease because these cell types comprise the primary barrier to many virus infections and also serve as the sites for virus release from the host. Poliovirus-epithelial cell interactions are of particular interest since this virus is an important enteric pathogen and the host cell receptor has been identified. In this study, poliovirus was observed to adsorb to both the apical and basolateral surfaces of polarized monkey kidney (Vero C1008) and human intestinal (Caco-2) epithelial cells but exhibited preferential binding to the basolateral surfaces of both cell types. Localization of the poliovirus receptor by a receptor-specific monoclonal antibody (D171) revealed a similar distribution predominantly on basolateral membranes, and treatment of cells with antibody D171 inhibited virus adsorption to both membrane surfaces. Poliovirus was able to initiate infection with similar efficiency following adsorption to either surface, and infection was blocked at both surfaces by D171, indicating that functional receptor molecules are expressed on both surfaces at sufficient density to mediate efficient infection at the apical and basolateral plasma membranes. Poliovirus infection resulted in a decrease in transepithelial resistance which was inhibited by prior treatment with monoclonal antibody D171 and occurred prior to other visible cytopathic effects. These results have interesting implications for viral pathogenesis in the human gut.

Migration of polarized epithelial cells through permeable membrane substrates of defined pore size.

We have observed that cells of various epithelial lines exhibit the ability to migrate through permeable membrane substrates containing 3.0 microns pores. Scanning and transmission electron microscopic observations of Vero C1008 and Caco-2 cell lines grown on polycarbonate membranes containing 3.0 microns pores revealed extensive penetration of the filter and the establishment of virtually complete monolayers on the opposing surface. The migration of MDCK cells was also observed to occur under the same conditions; however, the extent of MDCK cell growth on the opposing surface was significantly less than observed for Vero C1008 and Caco-2 cells. Morphological differences were apparent between cells growing on the upper and lower faces of the filter membrane, although cells growing on both surfaces exhibited a polarized phenotype. The cells which invaded the filter were collected and maintained by serial passage. The passaged cells exhibited morphological differences and an altered rate of differentiation in comparison to the parental cell type, suggesting that the invasive cells represent a variant of the parental cell population. Studies using filters of different pore sizes indicated that cellular migration also occurs through pores of 2.0 microns diameter, but not through 1.0 micron (or smaller) pores. These observations have significant implications for studies involving the growth of epithelial cells on permeable membrane substrates containing large pores.

A 585-bp deletion found in the spleen focus-forming virus (SFFV) env gene is responsible for the defective intracellular transport of SFFV gp52.

The Friend spleen focus-forming virus (F-SFFV) codes for a transport defective, leukemogenic envelope glycoprotein designated as gp52. Gp52 closely resembles the envelope glycoproteins (gp70-p15E) encoded by the mink cell focus-forming viruses (MCFV). The major differences between SFFV and MCFV include a 585-bp deletion and a frame-shift mutation near the 3' end of the SFFV env gene. We have constructed a mutant MCFV env gene, which contains a 585-bp deletion like that found in the SFFV env gene, and expressed this gene using recombinant vaccinia vectors or retroviral vectors. The mutant MCFV env gene expressed a truncated, transport defective glycoprotein (gp57). Only a small proportion of gp57 underwent further oligosaccharide processing. Intracellular gp57 remained predominantly monomeric and only a small proportion of gp57 (and its processed forms) formed disulfide-linked dimers and trimers which resembled those formed by SFFV gp52. Processed forms of gp57 were found on the cell surfaces and in culture fluids. The extracellular forms had a faster electrophoretic mobility than the intracellular-processed forms of gp57. These results indicate that the 585-bp deletion found in SFFV env gene is responsible for the folding, transport, and secretion of gp52. Retroviral vectors carrying the mutant MCFV env gene were nonpathogenic (or weakly pathogenic) in adult mice. The results indicate that the 585-bp deletion, although essential, is not the sole determinant of SFFV-induced disease in adult mice.