Maryam Saleh

Autophagy induction regulates influenza virus replication in a time-dependent manner

Purpose. Autophagy plays a key role in host defence responses against microbial infections by promoting degradation of pathogens and participating in acquired immunity. The interaction between autophagy and viruses is complex, and this pathway is hijacked by several viruses. Influenza virus (IV) interferes with autophagy through its replication and increases the accumulation of autophagosomes by blocking lysosome fusion. Thus, autophagy could be an effective area for antiviral research. Methodology. In this study, we evaluated the effect of autophagy on IV replication. Two cell lines were transfected with Beclin‐1 expression plasmid before (prophylactic approach) and after (therapeutic approach) IV inoculation. Results/Key findings. Beclin‐1 overexpression in the cells infected by virus induced autophagy to 26%. The log10haemagglutinin titre and TCID50 (tissue culture infective dose giving 50% infection) of replicating virus were measured at 24 and 48 h post‐infection. In the prophylactic approach, the virus titre was enhanced significantly at 24 h post‐infection (P≤0.01), but it was not significantly different from the control at 48 h post‐infection. In contrast, the therapeutic approach of autophagy induction inhibited the virus replication at 24 and 48 h post‐infection. Additionally, we showed that inhibition of autophagy using 3‐methyladenine reduced viral replication. Conclusion. This study revealed that the virus (H1N1) titre was controlled in a time‐dependent manner following autophagy induction in host cells. Manipulation of autophagy during the IV life cycle can be targeted both for antiviral aims and for increasing viral yield for virus production.

Biotransformation of alcohols to aldehydes by immobilized cells of Saccharomyces cerevisiae PTCC5080

Saccharomyces cerevisiae was immobilized in polyacrylamide gel beads. The effect of each gel constituents on whole cell alcohol dehydrogenase activity of yeast were studied. Prior to entrapment the cells were permeabilized by cetyltrimethylammonium bromide (CTAB) where 27% increase in activity was obtained. The permeabilized entrapped cells were treated with different detergents. CTAB was found to increase the gel permeability. The optimum concentration of nicotinamide adenine dinucleotide (NAD) was determined to be 30 μM. The optima concentration of ethanol for permeabilized cells, permeabilized and entrapped yeast cells were 3 and 2 M, respectively. Entrapped yeast cells converted almost 32.4 and 45% ethanol and 2-butanol to their respective aldehydes.

Expression of the influenza M2 protein in three different eukaryotic cell lines.

Current influenza virus vaccines provide protection in part by antibodies induced to the two surface glycoproteins, the hemagglutinin and the neuraminidase. As a result of the continuous antigenic drift of these glycoproteins, a frequent update of the composition of influenza vaccines is required. The search for more conserved viral epitopes which would induce protective immunity against seasonal influenza viruses and eventually also to novel pandemic influenza viruses has a long history. The ectodomain of the Influenza A Virus M2 Protein has been identified as a possible candidate immunization against influenza. The present study describes the expression of cloned M2 gene in MDCK, HeLa, and COS-7 cells, i.e., in three established eukaryotic cell lines. The expression efficiency was demonstrated by immunofluorescent staining of transfected cells by ELISA, by SDS-PAGE-, and by Western blot-analysis. High level of expression was observed in COS-7 cells. Expression in HeLa and MDCK cells was less efficient. The plasmids constructed in this study may, after modifications, be used for the production of a DNA vaccine. Alternatively the expression product could be refined and used as a purified antigen for the vaccine. Thus, the M2 recombinant protein provides an ideal product for further antigenic, biochemical, structural and functional characterization of the protein and for evaluating its potential for immunodiagnosis and in vaccine studies.

Subsite mapping of purified glucoamylase I, II, III produced by Arthrobotrys amerospora ATCC 34468

Arthrobotrys amerospora ATCC 34468 produced glucoamylase in a medium containing maize starch as carbon source. On native PAGE, crude glucoamylase showed three isoenzymes which were designated as Glu I, Glu II, Glu III according to their electrophoretic mobility. These were purified by column chromatography techniques. The energy of binding for each glucoamylase was calculated using Hiromis kinetic based calculation. At subsite 1, the binding energies for Glu I, II and III were found to be negative.

Comparison between MDCK and MDCK-SIAT1 cell lines as preferred host for cell culture-based influenza vaccine production

ObjectivesTo evaluate MDCK and MDCK-SIAT1 cell lines for their ability to produce the yield of influenza virus in different Multiplicities of Infection.ResultsYields obtained for influenza virus H1N1 grown in MDCK-SIAT1 cell was almost the same as MDCK; however, H3N2 virus grown in MDCK-SIAT1 had lower viral titers in comparison with MDCK cells. The optimized MOIs to infect the cells on plates and microcarrier were selected 0.01 and 0.1 for H1N1 and 0.001 and 0.01 for H3N2, respectively.ConclusionsMDCK-SIAT1 cells may be considered as an alternative mean to manufacture cell-based flu vaccine, especially for the human strains (H1N1), due to its antigenic stability and high titer of influenza virus production.

Influenza virus hemagglutinin: a model for protein N-glycosylation in recombinant Escherichia coli.

Background: The hemagglutinin molecule of influenza virus is considered as an ideal model to study biological processes as well as the effect of glycosylation on the function of glycoproteins. Objectives: The large subunit of the influenza virus A/New Caledonia/20/99 (H1N1) hemagglutinin (HA1) was expressed in recombinant Escherichia coli containing the glycosylation system of Campylobacter jejuni. This viral glycoprotein contains glycosylation motifs recognized by prokaryotic and eukaryotic oligosaccharyltransferases. Methods: In order to express the hemagglutinin large subunit gene, the gene was amplified using reverse transcription polymerase chain reaction (RT-PCR), and it was cloned in pET22b for periplasmic expression. Results: Western blotting and lectin blotting bands confirmed glycosylation of the HA1 in recombinant E. coli. Conclusion: Such a successful accomplishment of hemagglutinin expression in recombinant E. coli can be used to construct carbohydrates in hemagglutinin molecules of different strains in order to produce effective antigens for vaccine and rapid diagnostic kits against new emerging viruses.

A novel chimeric influenza virosome containing Vesicular stomatitis G protein as a more efficient gene delivery system.

ObjectivesTo enhance the efficiency of influenza virosome-mediated gene delivery by engineering this virosome.ResultsA novel chimeric influenza virosome was constructed containing the glycoprotein of Vesicular stomatitis virus (VSV-G), along with its own hemagglutinin protein. To optimize the transfection efficiency of both chimeric and influenza cationic virosomes, HEK cells were transfected with plasmid DNA and virosomes and the transfection efficiency was assessed by FACS analysis. The chimeric virosome was significantly more efficient in mediating transfection for all amounts of DNA and virosomes compared to the influenza virosome.ConclusionsChimeric influenza virosome, including VSV-G, is superior to the conventional influenza virosome for gene delivery.