We have designed an electrochemical assay 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. We used two methods 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. Second, we used disposable test strips to detect nineteen H1N1 and H3N2 influenza strains using this assay in 1 hour. The limit and range of detection of this first generation assay is 102 and 102–108 plaque forming units (pfu), respectively. Existing, ubiquitous, user friendly glucose meters can be repurposed to detect influenza viruses.

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Glycoprotein D (gD) plays an essential role in cell entry of many simplexviruses. B virus (Macacine herpesvirus 1) is closely related to herpes simplex virus 1 (HSV-1) and encodes gD, which shares more than 70% amino acid similarity with HSV-1 gD. Previously, we have demonstrated that B virus gD polyclonal antibodies were unable to neutralize B virus infectivity on epithelial cell lines, suggesting gD is not required for B virus entry into these cells. In the present study, we confirmed this finding by producing a B virus mutant, BV-⌬gDZ, in which the gD gene was replaced with a lacZ expression cassette. Recombinant plaques were selected on complementing VD60 cells expressing HSV-1 gD. Virions lacking gD were produced in Vero cells infected with BV⌬gDZ. In contrast to HSV-1, B virus lacking gD was able to infect and form plaques on noncomplementing cell lines, including Vero, HEp-2, LLC-MK2, primary human and macaque dermal fibroblasts, and U373 human glioblastoma cells. The gD-negative BV-⌬gDZ also failed to enter entry-resistant murine B78H1 cells bearing a single gD receptor, human nectin-1, but gained the ability to enter when phenotypically supplemented with HSV-1 gD. Cell attachment and penetration rates, as well as the replication characteristics of BV-⌬gDZ in Vero cells, were almost identical to those of wild-type (wt) B virus. These observations indicate that B virus can utilize gD-independent cell entry and transmission mechanisms, in addition to generally used gD-dependent mechanisms. A lphaherpesviruses share a strategy to enter host cells (1-3). Initial cell attachment of free virions is mediated by glycoprotein C (gC) and/or gB binding to cell surface heparan sulfate (4). This interaction facilitates specific binding of gD to one of several cellular receptors. To date, five gD receptors have been identified, including herpesvirus entry mediator (HVEM, or HveA), nectin-1 (HveC), nectin-2 (HveB), poliovirus receptor (PVR, or HveD), and 3-O-sulfated heparin sulfate (5-8). Receptor binding induces a conformational change in gD and subsequent transition into an active state. Activated gD then induces gB and gH-gL conformational changes, which trigger fusion between viral and cellular membranes (9). A key role of gD homologs in cell entry was established for all known alphaherpesviruses expressing the protein, including herpes simplex virus 1 (HSV-1), pseudorabies virus (PRV), bovine herpesvirus 1 (BHV-1), and equine herpesvirus 1 (EHV-1). Investigations of deletion mutants of these viruses showed that gD is essential for virus penetration into target cells (10)(11)(12)(13)(14). Numerous studies showing complete inhibition of virus cell entry by monoclonal gD antibodies, soluble recombinant gD protein, or soluble gD receptors further confirmed the crucial role of gD in in vitro infectivity of alphaherpesviruses (15)(16)(17)(18). Experiments demonstrating that vaginal infection of experimental animals with HSV-1 and HSV-2 could be prevented by pretreatment of a virus inoculum with gD-specific anti...

We have designed an electrochemical assay 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. We used two methods 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. Second, we used disposable test strips to detect nineteen H1N1 and H3N2 influenza strains using this assay in 1 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. Existing, ubiquitous, user friendly glucose meters can be repurposed to detect influenza viruses.

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