Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i) mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii) mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.The Hepatitis C virus (HCV) is a leading cause of chronic liver disease (37). Most infections are inapparent or initially associated with mild symptoms, but the virus persists in ϳ80% of all patients, leading to a high risk of developing severe liver damage such as liver cirrhosis or hepatocellular carcinoma.HCV is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae (33). The genome of HCV encompasses a single ϳ9,600-nucleotide (nt) RNA molecule carrying one large open reading frame that is flanked by nontranslated regions (NTRs). The 5Ј NTR contains an internal ribosome entry site (IRES) that directs translation of the open reading frame (43, 45). In addition, the 5Ј NTR is required for RNA replication as is the case with the 3Ј NTR (11, 12, 27, 47). All HCV proteins are generated from a polyprotein precursor that is cleaved by cellular and viral proteases into at least 10 different products (for reviews, see references 2 and 38). The structural proteins core, E1, and E2 are located in the amino terminus of the polyprotein (19), followed by p7, a hydrophobic pepti...
The proposed technique provides a reproducible and sensitive index. It is hoped that its independence from the partial volume effect will improve consistency in quantitative measurements between centres with different imaging devices and analysis software.
A series of donor‐functionalized pyrylium salts have been prepared by classical condensation reactions which were further converted into the corresponding thienyl‐ and pyridyl‐substituted polydentate λ3‐phosphinines by reaction with P(SiMe3)3. Further chemical modification of these phosphorus heterocycles with Hg(OAc)2 in the presence of methanol resulted in the formation of λ5‐phosphinines. The photophysical properties of a selected series of thienyl‐ and pyridyl‐functionalized pyrylium salts, λ3‐ and λ5‐phosphinines, were investigated and the results compared and supported by theoretical calculations on the DFT level. Significant fluorescence was observed for the pyrylium salts and λ5‐phosphinines. In contrast, the heteroaromatic substituted λ3‐phosphinines show very little emission which is consistent with the low oscillator strength predicted by DFT calculations for this π→π* transition. Furthermore, all three classes of compounds show readily observable phosphorescence in solution, which was determined by time‐gated detection at low temperature.
The first Danish cases of Shewanella alga bacteremia in two patients with chronic lower leg ulcers are reported. Both patients were admitted to the hospital during the same month of a very warm summer and had been exposed to the same marine environment, thereby suggesting the same source of infection. Both patients survived; however, one of them had extensive myonecrosis, while the other patient had an uncomplicated course. The strains were initially believed to be Shewanella putrefaciens on the basis of key characteristics and results of the API 20NE identification system (bioMérieux, Marcy l'Etoile, France), but further genetic and physiological analyses identified them as Shewanella alga.
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