[show abstract][hide abstract] ABSTRACT: DNA methylation regulates gene expression in a cell-type specific way. Although peripheral blood mononuclear cells (PBMCs) comprise a heterogeneous cell population, most studies of DNA methylation in blood are performed on total mononuclear cells. In this study, we investigated high resolution methylation profiles of 58 CpG sites dispersed over eight immune response genes in multiple purified blood cells from healthy adults and newborns. Adjacent CpG sites showed methylation levels that were increasingly correlated in adult blood vs. cord blood. Thus, while interindividual variability increases from newborn to adult blood, the underlying methylation changes may not be merely stochastic, but seem to be orchestrated as clusters of adjacent CpG sites. Multiple linear regression analysis showed that interindividual methylation variability was influenced by distance of average methylation levels to the closest border (0 or 100%), presence of transcription factor binding sites, CpG conservation across species and age. Furthermore, CD4+ and CD14+ cell types were negative predictors of methylation variability. Concerns that PBMC methylation differences may be confounded by variations in blood cell composition were justified for CpG sites with large methylation differences across cell types, such as in the IFN-γ gene promoter. Taken together, our data suggest that unsorted mononuclear cells are reasonable surrogates of CD8+ and, to a lesser extent, CD4+ T cell methylation in adult peripheral, but not in neonatal, cord blood.
Epigenetics: official journal of the DNA Methylation Society 11/2012; 7(12). · 4.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: High-pathogenic avian influenza viruses (HPAIVs) evolve from low-pathogenic precursors specifying the HA serotypes H5 or H7 by acquisition of a polybasic HA cleavage site. As the reason for this serotype restriction has remained unclear, we aimed to distinguish between compatibility of a polybasic cleavage site with H5/H7 HA only and unique predisposition of these two serotypes for insertion mutations. To this end, we introduced a polybasic cleavage site into the HA of several low-pathogenic avian strains with serotypes H1, H2, H3, H4, H6, H8, H10, H11, H14, or H15, and rescued HA reassortants after cotransfection with the genes from either a low-pathogenic H9N2 or high-pathogenic H5N1 strain. Oculonasal inoculation with those reassortants resulted in varying pathogenicity in chicken. Recombinants containing the engineered H2, H4, H8, or H14 in the HPAIV background were lethal and exhibited i.v. pathogenicity indices of 2.79, 2.37, 2.85, and 2.61, respectively, equivalent to naturally occurring H5 or H7 HPAIV. Moreover, the H2, H4, and H8 reassortants were transmitted to some contact chickens. The H2 reassortant gained two mutations in the M2 proton channel gate region, which is affected in some HPAIVs of various origins. Taken together, in the presence of a polybasic HA cleavage site, non-H5/H7 HA can support a highly pathogenic phenotype in the appropriate viral background, indicating requirement for further adaptation. Therefore, the restriction of natural HPAIV to serotypes H5 and H7 is likely a result of their unique predisposition for acquisition of a polybasic HA cleavage site.
Proceedings of the National Academy of Sciences 02/2012; 109(7):2579-84. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: From infection studies with cultured chicken cells and experimental mammalian hosts, it is well known that influenza viruses use the nonstructural protein 1 (NS1) to suppress the synthesis of interferon (IFN). However, our current knowledge regarding the in vivo role of virus-encoded NS1 in chickens is much more limited. Here, we report that highly pathogenic avian influenza viruses of subtypes H5N1 and H7N7 lacking fully functional NS1 genes were attenuated in 5-week-old chickens. Surprisingly, in diseased birds infected with NS1 mutants, the IFN levels were not higher than in diseased birds infected with wild-type virus, suggesting that NS1 cannot suppress IFN gene expression in at least one cell population of infected chickens that produces large amounts of the cytokine in vivo. To address the question of why influenza viruses are highly pathogenic in chickens although they strongly activate the innate immune system, we determined whether recombinant chicken alpha interferon (IFN-α) can inhibit the growth of highly pathogenic avian influenza viruses in cultured chicken cells and whether it can ameliorate virus-induced disease in 5-week-old birds. We found that IFN treatment failed to confer substantial protection against challenge with highly pathogenic viruses, although it was effective against viruses with low pathogenic potential. Taken together, our data demonstrate that preventing the synthesis of IFN is not the primary role of the viral NS1 protein during infection of chickens. Our results further suggest that virus-induced IFN does not contribute substantially to resistance of chickens against highly pathogenic influenza viruses.
Journal of Virology 05/2011; 85(15):7730-41. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the field, highly pathogenic avian influenza viruses (HPAIV) originate from low-pathogenic strains of the haemagglutinin (HA) serotypes H5 and H7 that have acquired a polybasic HA cleavage site. This observation suggests the presence of a cryptic virulence potential of H5 and H7 low-pathogenic avian influenza viruses (LPAIV). Among all other LPAIV, the H9N2 strains are of particular relevance as they have become widespread across many countries in several avian species and have been transmitted to humans. To assess the potential of these strains to transform into an HPAIV, we introduced a polybasic cleavage site into the HA of a contemporary H9N2 isolate. Whereas the engineered polybasic HA cleavage site mutant remained a low-pathogenic strain like its parent virus, a reassortant expressing the modified H9 HA with engineered polybasic cleavage site and all the other genes from an H5N1 HPAIV became highly pathogenic in chicken with an intravenous pathogenicity index of 1.23. These results suggest that an HPAIV with a subtype other than H5 or H7 would only emerge under conditions where the HA gene could acquire a polybasic cleavage site and the other viral genes carry additional virulence determinants.
Journal of General Virology 04/2011; 92(Pt 8):1843-53. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: The prime virulence determinant of highly pathogenic avian influenza viruses (HPAIVs) is the polybasic haemagglutinin (HA) cleavage site. However, engineering of a polybasic cleavage site into an avian influenza virus of low pathogenicity does not result in transformation into an HPAIV, indicating the importance of other adaptations. Here, the influence of amino acids adjacent to the HA cleavage site on virulence was studied. Most HPAIVs of subtype H5 carry serine or threonine at position 346 (corresponding to position 323 according to H3 numbering), whereas almost all low-pathogenic H5 viruses have valine. Moreover, all H5 low-pathogenic strains carry threonine at position 351 (corresponding to position 328 according to H3 numbering), suggesting that acquisition of a polybasic cleavage site involves several steps. This study generated a virus mutant derived from HPAIV A/Swan/Germany/R65/06 H5N1 (R65) with a monobasic cleavage site, R65(mono)-S-ER, and the following additional mutants: R65(mono)-V-ER with serine changed to valine at position 346, and R65(mono)-S-ETR and R65(mono)-V-ETR with threonine inserted at position 351. Moreover, in the R65 HA, serine was replaced with valine at position 346 (R65-V). Infection of chickens with R65(mono)-S-ETR or R65(mono)-S-ER led to slight transient respiratory symptoms, whereas R65-infected animals died within 2 days. However, chickens infected with R65-V survived longer than R65-infected animals, indicating that serine 346 in R65 HA contributes to virulence. These data suggest that evolution of H5 HPAIVs from low-pathogenic precursors, besides acquisition of a polybasic cleavage site, involves adaptation of neighbouring regions.
Journal of General Virology 09/2010; 92(Pt 1):51-9. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Highly pathogenic avian influenza viruses (HPAIV) originate from avirulent precursors but differ from all other influenza viruses by the presence of a polybasic cleavage site in their hemagglutinins (HA) of subtype H5 or H7. In this study, we investigated the ability of a low-pathogenic avian H5N1 strain to transform into an HPAIV. Using reverse genetics, we replaced the monobasic HA cleavage site of the low-pathogenic strain A/Teal/Germany/Wv632/2005 (H5N1) (TG05) by a polybasic motif from an HPAIV (TG05(poly)). To elucidate the virulence potential of all viral genes of HPAIV, we generated two reassortants carrying the HA from the HPAIV A/Swan/Germany/R65/06 (H5N1) (R65) plus the remaining genes from TG05 (TG05-HA(R65)) or in reversed composition the mutated TG05 HA plus the R65 genes (R65-HA(TG05poly)). In vitro, TG05(poly) and both reassortants were able to replicate without the addition of trypsin, which is characteristic for HPAIV. Moreover, in contrast to avirulent TG05, the variants TG05(poly), TG05-HA(R65), and R65-HA(TG05poly) are pathogenic in chicken to an increasing degree. Whereas the HA cleavage site mutant TG05(poly) led to temporary non-lethal disease in all animals, the reassortant TG05-HA(R65) caused death in 3 of 10 animals. Furthermore, the reassortant R65-HA(TG05poly) displayed the highest lethality as 8 of 10 chickens died, resembling "natural" HPAIV strains. Taken together, acquisition of a polybasic HA cleavage site is only one necessary step for evolution of low-pathogenic H5N1 strains into HPAIV. However, these low-pathogenic strains may already have cryptic virulence potential. Moreover, besides the polybasic cleavage site, the additional virulence determinants of H5N1 HPAIV are located within the HA itself and in other viral proteins.
PLoS ONE 01/2010; 5(7):e11826. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Reverse genetics has become pivotal in influenza virus research relying on rapid generation of tailored recombinant influenza viruses. They are rescued from transfected plasmids encoding the eight influenza virus gene segments, which have been cloned using restriction endonucleases and DNA ligation. However, suitable restriction cleavage sites often are not available. Here, we describe a cloning method universal for any influenza A virus strain which is independent of restriction sites. It is based on target-primed plasmid amplification in which the insert provides two megaprimers and contains termini homologous to plasmid regions adjacent to the insertion site. For improved efficiency, a cloning vector was designed containing the negative selection marker ccdB flanked by the highly conserved influenza A virus gene termini. Using this method, we generated complete sets of functional gene segments from seven influenza A strains and three haemagglutinin genes from different serotypes amounting to 59 cloned influenza genes. These results demonstrate that this approach allows rapid and reliable cloning of any segment from any influenza A strain without any information about restriction sites. In case the PCR amplicon ends are homologous to the plasmid annealing sites only, this method is suitable for cloning of any insert with conserved termini.
Nucleic Acids Research 11/2008; 36(21):e139. · 8.28 Impact Factor