RESUMEN

Rapid and precise detection of influenza viruses in a point of care setting is critical for applying appropriate countermeasures. Current methods such as nucleic acid or antibody based techniques are expensive or suffer from low sensitivity, respectively. We have developed an assay that uses glucose test strips and a handheld potentiostat to detect the influenza virus with high specificity. Influenza surface glycoprotein neuraminidase (NA), but not bacterial NA, cleaved galactose bearing substrates, 4,7di-OMe N-acetylneuraminic acid attached to the 3 or 6 position of galactose, to release galactose. In contrast, viral and bacterial NA cleaved the natural substrate, N-acetylneuraminic acid attached to the 3 or 6 position of galactose. The released galactose was detected amperometrically using a handheld potentiostat and dehydrogenase bearing glucose test strips. The specificity for influenza was confirmed using influenza strains and different respiratory pathogens that include Streptococcus pneumoniae and Haemophilus influenzae; bacteria do not cleave these molecules. The assay was also used to detect co-infections caused by influenza and bacterial NA. Viral drug susceptibility and testing with human clinical samples was successful in 15 minutes, indicating that this assay could be used to rapidly detect influenza viruses at primary care or resource poor settings using ubiquitous glucose meters.

RESUMEN

An electrochemical assay has been designed to rapidly diagnose influenza viruses. Exposure of a glucose-bearing substrate to influenza viruses or its enzyme, neuraminidase (NA), releases glucose, which was detected amperometrically. Two methods were used to detect released glucose. First, we used a standard glucose blood meter to detect two viral NAs and three influenza strains. We also demonstrated drug susceptibility of two antivirals, Zanamivir and Oseltamivir, using the assay. Finally, we used disposable test strips to detect nineteen H1N1 and H3N2 influenza strains using this assay in one hour. The limit and range of detection of this first generation assay is 10(2) and 10(2)-10(8) plaque forming units (pfu), respectively. Current user-friendly glucose meters can be repurposed to detect influenza viruses.

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RESUMEN

We have developed a panel of synthetic glycans as receptor mimics for the specific capture of influenza viruses. The glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. The microarray was evaluated for its ability to capture three different strains of influenza A virus, two H1N1, A/Brisbane/59/2007 and A/Solomon Islands/3/2006 and one H3N2, A/Aichi/2/1968. We observed an excellent detection ability with some compounds exhibiting clinically relevant (10(1) plaque forming units) limit of detection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using this microarray and could determine antiviral resistance for these strains.

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Adenovirus empty capsids are immature intermediates that lack DNA and viral core proteins. Highly purified preparations of empty and full capsids were generated by subjecting purified adenovirus preparations to repeated cesium chloride gradient separations. PAGE results revealed that empty capsids contain at least five bands that correspond to proteins absent from the mature virus proteome. Peptide mapping by matrix-assisted laser desorption/ionization time-of-flight MS revealed that three of these bands correspond to varying forms of L1 52/55kDa, a protein involved in the encapsidation of the viral DNA. One band at around 31kDa was found to include precursors to proteins VI and VIII. These precursors correspond to proteins that have not been cleaved by the adenovirus-encoded protease and are not present in the mature full capsids. The precursor to protein VIII (pVIII), a capsid cement protein, is used in this study as a marker in reverse-phased HPLC (RP-HPLC) analyses of adenovirus for the quantitation of empty capsids. A novel calculation method applied to the integration of RP-HPLC chromatograms allowed for the generation of a percentage empty capsid value in a given adenovirus preparation. The percentage empty capsid values generated to date by this method show a high degree of precision and good agreement with a cesium chloride gradient/SDS-PAGE quantitation method of empty capsids. The advantage of this method lies in the accurate, precise, and rapid generation of the percentage of empty capsids in a given purified virus preparation without relying on tedious and time-consuming cesium chloride gradient separations and extractions.

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RESUMEN

A simple, accurate, and precise method to determine adenovirus particle concentration using 260-nm absorbance was developed as an enhancement to the method of Maizel et al. (1968, Virology 36, 115-125). This modified method ensures complete disruption of virus particles and viral DNA prior to absorbance measurements, therefore eliminating absorbance measurement errors due to hyperchromic shift and thus providing an extinction coefficient at 260 nm that is directly related to protein concentration as measured by the method of Lowry et al. (1951, J. Biol. Chem. 193, 265-275). Application of this modified method will reduce interlaboratory variability in determining adenovirus particle concentrations, as current practices reflected in the literature utilize varying sample preparation procedures and calculation methods which cause underestimation of adenovirus concentration by almost twofold. The sample pretreatment conditions used in this modified method entail incubation in 1% sodium dodecyl sulfate at 100 degrees C for 4 min and result in an adenovirus 260-nm absorptivity of 1.8 x 10(12) viral particles per milliliter per absorbance unit in a 1-cm-pathlength cell.

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