The virulent influenza virus clone 7a produced a greater level of apoptosis in MDCK cells compared with the attenuated strain A/Fiji. In both cases, apoptosis could be partially blocked by treatment with three anti-neuraminidase compounds [4-amino-(GR121158A) and 4-guanidino-(GG167 ; Zanamivir) 2,3-dehydro-N-acetylneuraminic acid and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA)] when they were given to cells during the virus attachment/entry phase, but not subsequent to this phase. In contrast, GG167, which does not enter cells, did not affect the numbers of infected cells and, in addition, acted late in the infection cycle to inhibit virus yields. Clone 7a neuraminidase was more active than A/Fiji neuraminidase when fetuin was used as the substrate. Similar differences in activity between the two viruses were seen when α-2,6 sialyl lactose was used as a substrate, but not with α-2,3 sialyl lactose. No sequence differences in the enzyme active site of the two neuraminidases were observed, indicating that differences in neuraminidase specificity and activity may be dictated by other residues. These results suggest that neuraminidase plays some role in the induction of apoptosis and that it acts prior to or during virus entry. However, apoptosis was considerably reduced when UV-irradiated virus, which retains 75 % of its neuraminidase activity, was used. In addition, ammonium chloride, used to prevent virus entry, reduced virus-induced apoptosis. Amantadine, which inhibits virus uncoating, also inhibited apoptosis induced by the amantadine-sensitive strain A/Udorn/307/72 (H3N2), but not the amantadine-resistant clone 7a. Hence, one or more intracellular processes are also involved in influenza virus-induced apoptosis.
Zanamivir is a highly selective neuraminidase (NA) inhibitor with demonstrated clinical efficacy against influenza A and B virus infections. In phase II clinical efficacy trials (NAIB2005 and NAIB2008), virological substudies showed mean reductions in virus shedding after 24 h of treatment of 1.5 to 2.0 log 10 50% tissue culture infective doses compared to a placebo, with no reemergence of virus after the completion of therapy. Paired isolates (n ؍ 41) obtained before and during therapy with zanamivir demonstrated no shifts in susceptibility to zanamivir when measured by NA assays, although for a few isolates NA activity was too low to evaluate. In plaque reduction assays in MDCK cells, the susceptibility of isolates to zanamivir was extremely variable even at baseline and did not correlate with the speed of resolution of virus shedding. Isolates with apparent limited susceptibility to zanamivir by plaque reduction proved highly susceptible in vivo in the ferret model. Further sequence analysis of paired isolates revealed no changes in the hemagglutinin and NA genes in the majority of isolates. The few changes observed were all natural variants. No amino acid changes that had previously been identified in vitro as being involved with reduced susceptibility to zanamivir were observed. These studies highlighted problems associated with monitoring susceptibility to NA inhibitors in the clinic, in that no reliable cell-based assay is available. At present the NA assay is the best available predictor of susceptibility to NA inhibitors in vivo, as measured in the validated ferret model of infection.
We describe the in vitro selection and characterisation of virus derived from B/Beijing/1/87 passaged in the presence of zanamivir. During zanamivir passage, the phenotype of virus isolates was either drug dependent or drug resistant in plaque reduction assays. The susceptibility of the neuraminidase of the drug-dependent isolates was unchanged from that of the wild-type enzyme. The drug-dependent isolates contained two mutations in the viral haemagglutinin: V90A, close to the proposed secondary sialic acid-binding site, and L240Q, close to the primary sialic acid-binding site. Virus isolates that were drug resistant contained the same mutations in the haemagglutinin but also contained the mutation E116G in the neuraminidase. For the drug-dependent viruses, zanamivir susceptibility could not be measured because plaque numbers increased with increasing drug concentration. The in vitro zanamivir susceptibility of drug-resistant viruses was lower than that of the wild-type virus by a factor of 275- to >2532-fold. Neuraminidase containing the E116G mutation has a 33-fold lower affinity for zanamivir than the wild-type enzyme. The finding that the same haemagglutinin mutations are found in both drug-dependent and drug-resistant viruses confirms that the same changes to the receptor binding function can contribute to both phenotypes. This observation demonstrates the interplay between the influenza virus haemagglutinin and neuraminidase in escape from zanamivir inhibition in vitro.
Interviews were conducted with 67 Black and non-Hispanic White adolescents to gain detailed descriptions of personal and ethnic identities, perceptions of drugs and drug use norms, and reports of their own drug use behavior. In addition, this study examined ways in which ethnic identity and perceptions of cultural norms were linked to adolescent drug use attitudes. Findings imply that perceptions of culturally specific norms may be related to adolescent drug use attitudes and behavior. The authors argue for increased health campaign prevention efforts directed at erroneous perceptions of ethnic cultural norms.
We describe the detection characteristics of a device the Resonant Coil Magnetometer (RCM) to quantify paramagnetic particles (PMPs) in immunochromatographic (lateral flow) assays. Lateral flow assays were developed using PMPs for the measurement of total prostate specific antigen (PSA) in serum samples. A detection limit of 0.8 ng/mL was achieved for total PSA using the RCM and is at clinically significant concentrations. Comparison of data obtained in a pilot study from the analysis of serum samples with commercially available immunoassays shows good agreement. The development of a quantitative magneto-immunoassay in lateral flow format for total PSA suggests the potential of the RCM to operate with many immunoassay formats. The RCM has the potential to be modified to quantify multiple analytes in this format. This research shows promise for the development of an inexpensive device capable of quantifying multiple analytes at the point-of-care using a magneto-immunoassay in lateral flow format.
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