Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells

<p>Abstract</p> <p>Background</p> <p>Recent incidents where highly pathogenic influenza A H5N1 viruses have spread from avian species into humans have prompted the development of cell-based production of influenza vaccines as an alternative to or replacement of current egg-based production. Madin-Darby canine kidney (MDCK) cells are the primary cell-substrate candidate for influenza virus production but an efficient system for the direct rescue of influenza virus from cloned influenza cDNAs in MDCK cells did not exist. The objective of this study was to develop a highly efficient method for direct rescue of influenza virus in MDCK cells.</p> <p>Results</p> <p>The eight-plasmid DNA transfection system for the rescue of influenza virus from cloned influenza cDNAs was adapted such that virus can be generated directly from MDCK cells. This was accomplished by cloning the canine RNA polymerase I (pol I) promoter from MDCK cells and exchanging it for the human RNA pol I promoter in the eight plasmid rescue system. The adapted system retains bi-directional transcription of the viral cDNA template into both RNA pol I transcribed negative-sense viral RNA and RNA pol II transcribed positive-sense viral mRNA. The utility of this system was demonstrated by rescue in MDCK cells of 6:2 genetic reassortants composed of the six internal gene segments (PB1, PB2, PA, NP, M and NS) from either the cold-adapted (<it>ca</it>) influenza A vaccine strain (<it>ca </it>A/Ann Arbor/1/60) or the <it>ca </it>influenza B vaccine strain (<it>ca </it>B/Ann Arbor/1/66) and HA and NA gene segments from wild type influenza A and B strains. Representative 6:2 reassortants were generated for influenza A (H1N1, H3N2, H5N1, H6N1, H7N3 and H9N2) and for both the Victoria and Yamagata lineages of influenza B. The yield of infectious virus in the supernatant of transfected MDCK cells was 10⁶to 10⁷plaque forming units per ml by 5 to 7 days post-transfection.</p> <p>Conclusion</p> <p>This rescue system will enable efficient production of both influenza A and influenza B vaccines exclusively in MDCK cells and therefore provides a tool for influenza pandemic preparedness.</p

Human cytomegalovirus uracil DNA glycosylase associates with ppUL44 and accelerates the accumulation of viral DNA

BACKGROUND: Human cytomegalovirus UL114 encodes a uracil-DNA glycosylase homolog that is highly conserved in all characterized herpesviruses that infect mammals. Previous studies demonstrated that the deletion of this nonessential gene delays significantly the onset of viral DNA synthesis and results in a prolonged replication cycle. The gene product, pUL114, also appears to be important in late phase DNA synthesis presumably by introducing single stranded breaks. RESULTS: A series of experiments was performed to formally assign the observed phenotype to pUL114 and to characterize the function of the protein in viral replication. A cell line expressing pUL114 complemented the observed phenotype of a UL114 deletion virus in trans, confirming that the observed defects were the result of a deficiency in this gene product. Stocks of recombinant viruses without elevated levels of uracil were produced in the complementing cells; however they retained the phenotype of poor growth in normal fibroblasts suggesting that poor replication was unrelated to uracil content of input genomes. Recombinant viruses expressing epitope tagged versions of this gene demonstrated that pUL114 was expressed at early times and that it localized to viral replication compartments. This protein also coprecipitated with the DNA polymerase processivity factor, ppUL44 suggesting that these proteins associate in infected cells. This apparent interaction did not appear to require other viral proteins since ppUL44 could recruit pUL114 to the nucleus in uninfected cells. An analysis of DNA replication kinetics revealed that the initial rate of DNA synthesis and the accumulation of progeny viral genomes were significantly reduced compared to the parent virus. CONCLUSION: These data suggest that pUL114 associates with ppUL44 and that it functions as part of the viral DNA replication complex to increase the efficiency of both early and late phase viral DNA synthesis

Classroom climate – an affirmation of interculturalism

Pod utjecajem intenziviranja migracijskih tokova, kao i upotrebe komunikacijskih tehnologija, kontakti između pripadnika različitih kultura više nisu iznimka, već pravilo svakodnevnog života. Unatoč trendu globalnog povezivanja brojni su primjeri iskazivanja ksenofobije, socijalne distance, rasizma i drugih oblika društveno neprihvatljiva ponašanja i među školskom populacijom. Škola više nego ikoja druga socijalna institucija ima društveno-moralnu obvezu djelovati preventivno u smjeru osposobljavanja učenika za suočavanje s različitostima. Stoga, nastavnici imaju zadaću implementirati interkulturalna načela u školsko i razredno-nastavno ozračje.Predmet ovog rada jest promišljanje odnosa razredno-nastavnog ozračja i interkulturalizma. Polazeći od shvaćanja interkulturalnog odgoja i obrazovanja prvenstveno kao programa usmjerenih na zajednicu s ciljem unapređenja međuljudskih odnosa, ističe se zahtjev za nastavnikovom refleksijom interkulturalnog aspekta odgojno-obrazovnog ozračja. U funkciji navedenog, razlažu se smjernice u okviru različitih dimenzija razredno-nastavnog ozračja kojemogu poslužiti kao polazište za vrednovanje i unapređivanje interkulturalnog dijaloga.As a result of the expansion of migration flows and communication technology, contacts among members of different cultures have become the norm of everyday life. Nevertheless, a troublesome level of explicit xenophobia, social distance, racism and other forms of socially unacceptable behaviors still exists among the school population. More than any other social institution, the school has a social-moral obligation to act preventively through training pupils for encounteringdiversity. Thus, teachers gain a new role as mediators of intercultural principles in order to create a school and classroom climate which includes an intercultural dimension.The subject of this paper is the systematic analysis of the relationship of the classroom climate and interculturalism. Starting from an understanding of intercultural education as a community oriented program which aims to enhance human relations, the demand for the teacher\u27s reflection of the intercultural aspect of the educational climate is highlighted. In that function, guidelines within the different dimensions of the classroom atmosphere are elaborated, which can be utilized as a starting point for evaluating and improving the intercultural dialogue.</p

Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells-5

<p><b>Copyright information:</b></p><p>Taken from "Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells"</p><p>http://www.virologyj.com/content/4/1/102</p><p>Virology Journal 2007;4():102-102.</p><p>Published online 23 Oct 2007</p><p>PMCID:PMC2241602.</p><p></p>ed into an artificial vRNA-CAT reporter construct. These constructs were individually combined with expression plasmids for PB1, PB2, PA and NP proteins and transfected into MDCK cells. At 44 hrs after transfection, cell lysates were analyzed for CAT expression by a colorimetric ELISA assay. In the plasmid pHW72-CAT, the human RNA pol I promoter directs transcription of a negative sense CAT gene

Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells-8

<p><b>Copyright information:</b></p><p>Taken from "Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells"</p><p>http://www.virologyj.com/content/4/1/102</p><p>Virology Journal 2007;4():102-102.</p><p>Published online 23 Oct 2007</p><p>PMCID:PMC2241602.</p><p></p>ed by arrows

Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells-10

<p><b>Copyright information:</b></p><p>Taken from "Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells"</p><p>http://www.virologyj.com/content/4/1/102</p><p>Virology Journal 2007;4():102-102.</p><p>Published online 23 Oct 2007</p><p>PMCID:PMC2241602.</p><p></p>ere aligned with MDCK sequences flanking the transcription initiation site mapped by primer extension. The first base of the predominate RNA transcript of the indicated species is labeled +1. Conserved residues are indicated blue, red and green lettering. For all the indicated species, the -1 position is a T (red) and the +1 position a purine residue (green). The sequences from +2 to +9 (blue) are conserved in the indicated mammalian species. Based on the aligned sequences, the G residue (arrow) in the MDCK sequences was predicted to be the RNA pol I promoter transcription initiation site

Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells-9

<p><b>Copyright information:</b></p><p>Taken from "Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells"</p><p>http://www.virologyj.com/content/4/1/102</p><p>Virology Journal 2007;4():102-102.</p><p>Published online 23 Oct 2007</p><p>PMCID:PMC2241602.</p><p></p>oducts and P ΦX174 size marker DNAs (lane M) were subjected to electrophoresis on a 6% polyacrylamide, 7 M urea gel followed by detection of the radioisotope in the gel with a BioRad Molecular Imager Fx. The maximum length of the observed products were approximately 370 bases and 220 bases, respectively, for the reactions using PrimEx1 (lane 1) and PrimEx2 (lane 2). (B) The products from the PrimEx2 reaction were subjected to electrophoresis adjacent to a M13 sequencing ladder on a 6% polyacrylamide, 7 M urea sequencing gel in order to more accurately determine the maximum length of the products synthesized. (C) The MDCK DNA sequences adjacent to the positions where the largest PrimEx2 products terminated

Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells-7

<p><b>Copyright information:</b></p><p>Taken from "Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells"</p><p>http://www.virologyj.com/content/4/1/102</p><p>Virology Journal 2007;4():102-102.</p><p>Published online 23 Oct 2007</p><p>PMCID:PMC2241602.</p><p></p>ion of the MDCK 7.1 kb EcoR I fragment which hybridized to the 18S rRNA gene probe. (B) Southern hybridization of MDCK DNA. Left panel: Single restriction enzyme digestions of MDCK DNA were subjected to electrophoresis on a 0.7% agarose gel and detected by ethidium bromide staining. M: 1 kb ladder (Invitrogen); Lanes 1–8: Avr II, BamH I, EcoR I, Hind III, Sac I, Spe I, Sph I, Xba I; C: 18S rRNA gene probe. Right panel: Southern blot of gel in left panel after hybridization to a psoralen-biotin labeled 18S rRNA gene probe (0.5 kb) and detection by chemilumiscense. The 7.1 kb EcoR I fragment (arrow) was cloned and analyzed for RNA pol I promoter activity. (C) Comparison of the size of selected restriction fragments predicted by the genomic sequence to hybridize to the 18S rRNA gene probe and those restriction fragments from MDCK DNA which were observed to hybridize

Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells-13

<p><b>Copyright information:</b></p><p>Taken from "Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells"</p><p>http://www.virologyj.com/content/4/1/102</p><p>Virology Journal 2007;4():102-102.</p><p>Published online 23 Oct 2007</p><p>PMCID:PMC2241602.</p><p></p>ectional vector, by replacement of the human pol I promoter sequence in pAD3000 with the MDCK 469 bp sequence upstream of the transcription initiation site. In addition, the two BI restriction sites in pAD3000, which are used for cloning sequences between the two promoters, were changed to I sites because the former restriction site occurs in the MDCK pol I containing fragment