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A Normann

Publications and source records attributed to A Normann.

16 recordsLinked to original sources
PubMed2003

Microdevices for separation, accumulation, and analysis of biological micro- and nanoparticles.

Microfabrication and performance of a novel microsystem for separation, accumulation and analysis of biological micro- and nanoparticles is reported. Versatile chip functions based on dielectrophoresis and microfluidics were integrated to isolate particles from complex sample solutions such as serum. A bead-based assay for virus detection is proposed. Separation of micro- and sub-mum beads employing dielectrophoretic deflector and bandpass structures is demonstrated. Individual antibody coated beads with hepatitis A virus bound to their surface were trapped by negative dielectrophoresis in a field cage and analysed by fluorescence microscopy.

Journal Article↗
PubMed2001

Genetic relatedness of Cuban HAV wild-type isolates.

Knowledge of the nucleotide sequence in the region of the putative VP1/2A junction of the Hepatitis A virus (HAV) genome has enabled differentiation of HAV strains and their classification into seven genotypes, in some of which sub-genotypes A and B can be defined. A 168 base segment encompassing the putative VP1/2A junction of 27 clinical wild-type isolates of HAV from Cuba was amplified by reverse transcriptase-polymerase chain reaction and then sequenced. The Cuban isolates are clustered within sub-genotype IA. A single amino acid substitution, which does not correspond with any of the reported changes for other strains, was observed. A new sub-genotype variant is therefore postulated. This study provides new data on the genetic relatedness of HAVs from Cuba and on the distribution of sub-genotype IA in the Caribbean area.

Adult↗
PubMed1997

Influence of the 5' noncoding region of hepatitis A virus strain GBM on its growth in different cell lines.

Previous sequence analysis of consecutive passages of the hepatitis A virus (HAV) strain GBM/WT in human embryonic kidney cells (HEK cells), human embryonic lung fibroblasts (HFS cells) and in FRhK-4 cells (foetal rhesus monkey kidney cells) pointed to a host cell dependent cell culture adaptation of GBM/WT in HFS cells involving mutations in the 5' noncoding region (5'NCR). Multiple nucleotide changes occurred in the 5'NCR of the GBM genome after the cell line used for virus passage was changed from HEK cells to HFS cells. In contrast, no mutations in the 5'NCR occurred during the first 20 passages of GBM/WT in FRhK-4 cells. In order to analyse the influence of the 5'NCR on host cell specific adaptation of HAV strain GBM in different cell cultures, GBM/HM175 chimeras were constructed which contained 5'NCRs from different GBM variants by replacing the 5'NCR of the infectious clone pHAV/7. Parallel transfection assays in FRhK-4 and HFS cells, performed with transcripts from the chimeric GBM/HM175 constructs, showed that the 5'NCR of the GBM variant GBM/HFS is essential for virus growth in HFS cells. The GBM/HM175 chimeric RNA, which contained the 5'NCR of GBM/HFS, exclusively, was able to produce infectious virus after transfection of HFS cells. The growth of the different GBM/HM175 chimeras in FRhK-4 cells, in contrast, did not seem to be strongly influenced by a specific sequence of the 5'NCR.

Animals↗
PubMed1995

Molecular epidemiology of an outbreak of hepatitis A in Italy.

The relationship of hepatitis A virus (HAV) isolates associated with an outbreak in Genoa, Italy, in 1993 was examined using direct sequencing of amplicons derived by antigen capture PCR (AC/PCR) from faecal samples of the infected persons. Forty samples recovered from 38 primary and two secondary cases were examined. The latter were household contacts of the primary cases. In addition, faecal material of 2 unrelated persons infected simultaneously with hepatitis A in Genoa were tested. The PCR products derived from the P1/P2 junction of the HAV genome were analysed. A 100% nucleotide identity was detected between the viral isolates originating from the primary as well as the secondary cases. The viral isolates recovered from the faecal samples of the two unrelated cases differed from the virus causing the outbreak as well as from each other. These results indicate that a single HAV strain caused the outbreak. The virus might have been transmitted by ingestion of contaminated food or water since all hepatitis A infected employees of the factory had eaten in the same canteen. Definitions of HAV genotypes are based on numerous genetic comparisons of different strains. The sequence comparison of the investigated isolates with published HAV sequences of the P1/P2 genome region revealed that the virus associated with the outbreak belongs to HAV subgenotype IA, whereas the strains recovered from the viral isolates of the unrelated cases belong to subgenotype IB.

Antigens, Viral↗
PubMed1995

Hepatitis A virus and polymerase chain reaction amplification: methodology and results.

Hepatitis A infection among patients receiving solvent/detergent inactivated factor VIII preparations in various locations in Europe have been documented recently. In investigations in Italy, Germany and Ireland, polymerase chain reaction (PCR) amplification was used to detect hepatitis A virus in frozen plasma pools, purified factor VIII, patient sera and samples from animal transmission studies; nucleic acid sequencing was used to clarify and identify the virus responsible based upon genotype analysis. Unique virus strains were found among the cases in Italy and Germany, and identical virus sequences were also found in some factor VIII lots. However, with the exception of the Italian investigation, lack of appropriate samples have precluded the identification of virus in these outbreaks. In addition, animal infectivity studies have not been successful in demonstrating infectivity under laboratory conditions. We discuss the limitations of PCR amplification with respect to detecting virus within these situations, and the necessity for the corresponding epidemiologic investigations.

Animals↗
PubMed1994

Mutational events in consecutive passages of hepatitis A virus strain GBM during cell culture adaptation.

In order to study the adaptation of hepatitis A virus (HAV) in cell culture, we examined the mutational events of the genome in early passages of HAV strain GBM propagated either in FRhK-4 cells (fetal rhesus monkey kidney-derived) or in human embryonic kidney (HEK) and human embryonic fibroblast cells (HFS) in relation to their growth characteristics. Sequence analysis of the nucleotide region encoding 2B, 2C, and the beginning of 3A as well as the nucleotide region encompassing the 5' noncoding region (5'NCR) of the genome was performed on consecutive virus passages after amplification of the viral RNA from the cell culture supernatant by antigen-capture PCR. By the 2nd passage of the GBM variants cultured in FRhK-4 or in HEK cells we found a mutation at nucleotide position 3889 (2B coding region) which results in an amino acid substitution from alanine to valine. Further mutations present in the 2B/2C region of the cell culture-adapted GBM variants differ from each other and occur after the 10th or even the 40th virus passage. Another early change, an in frame deletion of nine nucleotides in the 3A region, appeared in the 5th virus passage only in GBM cultured on FRhK-4 cells. This genome region showed different mutations in the virus passages on HEK and HFS cells. The 5'NCR of the cell culture-adapted GBM variants, in contrast, did not show any mutations before the 8th virus passage. The faster and more efficient growth of the HAV strain GBM during successive propagation on cell cultures seems to correlate with the appearance of mutations in the investigated genome regions.

Adaptation, Physiological↗
PubMed1994

Nucleotide sequence of wild-type hepatitis A virus GBM in comparison with two cell culture-adapted variants.

In order to study cell tropism and attenuation of hepatitis A virus (HAV), the genome of HAV wild-type GBM and two cell culture-adapted variants, GBM/FRhK and GBM/HFS, were cloned and sequenced after amplification by reverse transcriptase-PCR. During virus cultivation, the HAV variant GBM/FRhK had a strict host range for FRhK-4 cells, in contrast to GBM/HFS, which can be grown in HFS and FRhK-4 cells. The HAV variant GBM/HFS was shown to be attenuated when inoculated into chimpanzees (B. Flehmig, R. F. Mauler, G. Noll, E. Weinmann, and J. P. Gregerson, p. 87-90, in A. Zuckerman, ed., Viral Hepatitis and Liver Disease, 1988). On the basis of this biological background, the comparison of the nucleotide sequences of these three HAV GBM variants should elucidate differences which may be of importance for cell tropism and attenuation. The comparison of the genome between the GBM wild type and HAV wild types HM175 (J. I. Cohen, J. R. Ticehurst, R. H. Purcell, A. Buckler-White, and B. M. Baroudy, J. Virol. 61:50-59, 1987) and HAV-LA (R. Najarian, O. Caput, W. Gee, S. J. Potter, A. Renard, J. Merryweather, G. Van Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985) showed a 92 to 96.3% identity, whereas the identity was 99.3 to 99.6% between the GBM variants. Nucleotide differences between the wild-type and the cell culture-adapted variants, which were identical in both cell culture-adapted GBM variants, were localized in the 5' noncoding region; in 2B, 3B, and 3D; and in the 3' noncoding region. Our result concerning the 2B/2C region confirms a mutation at position 3889 (C-->T, alanine to valine), which had been shown to be of importance for cell culture adaptation (S. U. Emerson, C. McRill, B. Rosenblum, S. M. Feinstone, and R. H. Purcell, J. Virol. 65:4882-4886, 1991; S. U. Emerson, Y. K. Huang, C. McRill, M. Lewis, and R. H. Purcell, J. Virol. 66:650-654, 1992), whereas other mutations differ from published HAV sequence data and may be cell specific. Further comparison of the two cell culture-adapted GBM variants showed cell-specific mutations resulting in deletions of six amino acids in the VP1 region and three amino acids in the 3A region of the GBM variant GBM/FRhK.

Adaptation, Biological↗
PubMed1994

Processing of truncated mouse or human rRNA transcribed from ribosomal minigenes transfected into mouse cells.

The processing of pre-rRNA in eukaryotic cells involves a complex pattern of nucleolytic reactions taking place in preribosomes with the participation of several nonribosomal proteins and small nuclear RNAs. The mechanism of these reactions remains largely unknown, mainly because of the absence of faithful in vitro assays for most processing steps. We have developed a pre-rRNA processing system using the transient expression of ribosomal minigenes transfected into cultured mouse cells. Truncated mouse or human rRNA genes are faithfully transcribed under the control of mouse promoter and terminator signals. The fate of these transcripts is analyzed by the use of reporter sequences flanking the rRNA gene inserts. Both mouse and human transcripts, containing the 3' end of 18S rRNA-encoding DNA (rDNA), internal transcribed spacer (ITS) 1, 5.8S rDNA, ITS 2, and the 5' end of 28S rDNA, are processed predominantly to molecules coterminal with the natural mature rRNAs plus minor products corresponding to cleavages within ITS 1 and ITS 2. To delineate cis-acting signals in pre-rRNA processing, we studied series of more truncated human-mouse minigenes. A faithful processing at the 18S rRNA/ITS 1 junction can be observed with transcripts containing only the 60 3'-terminal nucleotides of 18S rRNA and the 533 proximal nucleotides of ITS 1. However, further truncation of 18S rRNA (to 8 nucleotides) or of ITS 1 (to 48 nucleotides) abolishes the cleavage of the transcript. Processing at the ITS 2/28S rRNA junction is observed with truncated transcripts lacking the 5.8S rRNA plus a major part of ITS 2 and containing only 502 nucleotides of 28S rRNA. However, further truncation of the 28S rRNA segment to 217 nucleotides abolishes processing. Minigene transcripts containing most internal sequences of either ITS 1 or ITS 2, but devoid of ITS/mature rRNA junctions, are not processed, suggesting that the cleavages in vivo within either ITS segment are dependent on the presence in cis of mature rRNA sequences. These results show that the major cis signals for pre-rRNA processing at the 18S rRNA/ITS 1 or the ITS2/28S rRNA junction involve solely a limited critical length of the respective mature rRNA and adjacent spacer sequences.

3T3 Cells↗
PubMed1994

Sequence variability of hepatitis A virus and factor VIII associated hepatitis A infections in hemophilia patients in Europe. An update.

Outbreaks and sporadic cases of hepatitis A have been observed in 4 European countries in hemophilia patients receiving factor VIII preparations. PCR amplification of potential hepatitis A virus (HAV) nucleic acid present in plasma pools, purified factor VIII and acute-phase sera from infected individuals has been performed and the nucleic acid sequence determined for those samples that resulted in a positive PCR product. HAV sequences were detected in the serum of 2 German patients, but not in the factor VIII lots administered to these individuals. Screening of plasma pools and the corresponding 5 lots of factor VIII associated with the outbreak in Ireland did not reveal any HAV sequences. In contrast, a study of samples from Italy detected HAV sequences in 5 of 12 lots and in 2 hemophilia patients who developed hepatitis A. These data suggest that implicated factor VIII preparations might have been involved in the outbreaks of HAV infection among Italian hemophiliacs. However, no molecular evidence was obtained for a similar association in Germany or Ireland. The preliminary data from these two investigations must be verified by animal inoculation studies and supported by epidemiologic analysis.

Base Sequence↗
PubMed1994

Detection of hepatitis A virus in a factor VIII preparation by antigen capture/PCR.

The antigen capture/PCR (AC/PCR) has been applied in the analysis of various factor VIII preparations, which were suspected to be contaminated with hepatitis A virus (HAV). AC/PCR involves capturing the antigen, i.e. the intact virus particles, by binding to the HAV monoclonal antibody mAb 7e7, reverse transcription of the viral RNA and amplification of the cDNA with HAV-specific primer pairs. The PCR analysis of one factor VIII concentrate yielded an HAV-specific DNA product, which could be confirmed by Southern blot analysis. The HAV strain recovered by AC/PCR from this factor VIII concentrate could be classified into genotype III after solid-phase sequencing of the product and comparison with the consensus sequences for the known HAV genotypes. Analysis of the sera from 3 haemophiliacs treated with this batch has not resulted in a reliable product. However, the results obtained indicate that HAV can be detected in purified factor VIII preparations by AC/PCR.

Base Sequence↗
PubMed1988

The mouse ribosomal gene terminator consists of three functionally separable sequence elements.

The structural requirements for 3' end formation of mouse pre-rRNA have been studied. Three sequence elements are shown to be required for accurate and efficient transcription termination by RNA polymerase I (pol I) assayed both in a cell-free transcription system and in vivo after transfection of rDNA minigene constructs into 3T6 cells. The essential termination signal is the previously identified 18-bp conserved element (AGGTCGACCAGATTANTCCG) that contains a SalI restriction site. This sequence motif (the 'Sal box') interacts with a specific nuclear protein that directs transcription termination. Here we demonstrate that the 'Sal box' sequence motif is sufficient for termination of pol I transcripts and the release of the nascent RNA chains from the template. However, in addition to this termination signal, pyrimidine-rich sequences flanking the box at the 5' and 3' side play a role in the efficient and correct formation of authentic pre-rRNA termini. Downstream sequences contribute to the efficiency of the termination reaction, whereas the position of 3' end formation (i.e. 21 bp upstream of the 'Sal box') is affected by 5' flanking regions. These flanking regions are recognized by at least two different nuclear factors which specifically bind to DNA sequences located upstream and downstream of the 'Sal box'.

Animals↗
PubMed1986

The core promoter of mouse rDNA consists of two functionally distinct domains.

We have determined the sequences constituting the minimal promoter of mouse rDNA. A very small region immediately upstream of the transcription start site (from -1 to -39) is sufficient to direct correct transcription initiation. Sequences immediately downstream of the transcription start site (+1 to +11) increase the efficiency of transcription initiation. Point mutations within the core promoter have been generated and assayed for their effects on template activity and on interaction with the pol I specific transcription factor TIF-IB. The core promoter element appears to consist of two functionally different domains. The distal sequence motif from position -22 to -16 is recognized by factor TIF-IB. Mutations within this region lead to similar changes of both template activity and binding of TIF-IB. Two point mutations within the proximal sequence motif from -15 to -1 do not affect TIF-IB binding although they severely impair transcription initiation. It is suggested, that this proximal region plays a role in the assembly of functional transcription initiation complexes rather than in the primary binding of TIF-IB.

Animals↗