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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B virus (Macacine herpesvirus 1), a simplex virus endemic in macaques, causes encephalitis, encephalomyelitis, and death in 80% of untreated zoonotically infected humans with delayed or no treatment. Here we report a significant difference in PI3K/Akt-dependent apoptosis between B virus infected human and macaque dermal fibroblasts. Our data show that B virus infection in either human or macaque fibroblasts results in activation of Akt via PI3K and this activation does not require viral de novo protein synthesis. Inhibition of PI3K with LY294002 results in a significant reduction of viral titers in B virus infected macaque and human fibroblasts with only a modest difference in the reduction of virus titers between the two cell types. We, therefore, tested the hypothesis that B virus results in the phosphorylation of Akt (S473), which prevents apoptosis, enhancing virus replication in B virus infected macaque dermal fibroblasts. We observed markers of intrinsic apoptosis when PI3K activation of Akt was inhibited in B virus infected macaque cells, while, these apoptotic markers were absent in B virus infected human fibroblasts under the same conditions. From these data we suggest that PI3K activates Akt in B virus infected macaque and human fibroblasts, but this enhances virus replication in macaque fibroblast cells by blocking apoptosis.

Sleep disturbance has been found to trigger a stress response with a subsequent activation of the psychoneuroimmunological (PNI) pathway associated with adverse health outcomes. This study aimed to assess the association among selected PNI biomarkers, sleep disturbances, and adverse health outcomes (depressive symptoms, physical symptoms). A stratified, quota sample (14 poor sleepers and 15 good sleepers) was drawn from a pool of healthy college women from a larger scale of study. The participants reported their sleep, stress, depressive, and physical symptoms. Wrist actigraphy was used to collect objective sleep data, and the Enzyme-Linked ImmunoSorbent Assay was used to assess PNI biomarkers. Poor sleep quality, higher stress perception, elevated serum serotonin, and lower serum interleukin-10 explained 75.3% of the variances for the depressive symptoms. Poor sleep quality along with delayed peak activity rhythms accounted 31.4% of the physical symptoms. High serotonin and tumor necrosis factor-α were the significant predictors for poor sleep efficiency, and serotonin was the single significant predictor for poor daytime functioning. Stress and sleep disturbances negatively impact the health of college women and should be as part of regular check-ups on campus. PNI effects on health outcomes should be further explored. Educational materials in the areas of sleep hygiene, health impacts from sleep disturbances, and strategies to maintain synchronized circadian rhythms should be mandatorily included in the college curriculum.

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