Overview of JC virus NCCR rearrangements (<i>i.e.</i> deletions and insertions) identified by Sanger sequencing and 454 sequencing.

<p>Comparison of the consensus NCCR as determined by Sanger sequencing (n = 61) revealed four distinct groups of samples: 1) archetype sequences with no DNA rearrangements compared to the reference NCCR (CY isolate), 2) samples harboring polymorphic deletions, 3) samples carrying polymorphic insertions and 4) sample harboring both a deletion and an insertion. 454 sequencing was further applied (n = 54) to identify JCV quasispecies. Healthy subject (HS) samples in which quasispecies were identified, the nature of the minority sequences contributing to the quasispecies and the percentage of viral NCCR sequences harboring the minority sequences are indicated. The sequence blocks (a to f) affected by the NCCR rearrangements are indicated between brackets. Of note, HS11 carries a 1 bp deletion at position 165 that in ∼1.5% of the viral population is part of a more extensive deletion. The numbering scheme is based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070950#pone.0070950-Frisque1" target="_blank">[12]</a>.</p

Validation of JC virus quasispecies by nested PCR.

<p>(A) A nested PCR approach was developed to validate the presence of the 28 bps deletion identified in ∼1.5% of the viral DNA sequences in HS11. (B) The nested PCR approach was applied on viral DNA extracted from urine from HS11. Urine aliquots donated at different time points were included: T0, T1, T2 and T3. Viral DNA extracted from HS6 T1 was included as a negative control, together with a no template control (NTC). The upper panel shows successful amplification of the consensus DNA fragment in all samples. In contrast, only in HS11 T1 a PCR fragment could be amplified when using the reverse primer spanning the deletion instead of the consensus reverse primer, confirming the presence of this deletion. L = 25 base pair DNA ladder (Invitrogen). (C) A similar nested PCR approach was used to demonstrate the existence of quasispecies in HS53. DNA fragments were generated when a primer set designed to amplify a consensus sequence from both HS53 and HS26 (lanes HS53 con and HS26 con) were used. When the reverse primer was replaced by a primer specifically targeting the sequence deleted in the quasispecies succesfull amplification was only detected in HS53 (HS53 del), but not in HS26 (HS26 del). NTC: no template control. L = 25 base pair DNA ladder (Invitrogen).</p

Phylogenetic analysis based on the VP1 coding sequence from healthy subjects.

<p>The full length VP1 coding sequence (1065 bp) was obtained for all healthy subjects for which also the non-coding control region (NCCR) was Sanger sequenced (n = 61). The relatedness of the HS samples to defined JC virus genotypes is illustrated in a phylogenetic tree. For each JCV genotype the following reference VP1 coding sequences were used (NCBI accession number between brackets; reference sequences taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070950#pone.0070950-Agostini1" target="_blank">[26]</a>): genotype 1A (AF015526), genotype 1B (AF015527), genotype 2A (AF015529), genotype 2B (AF015533), genotype 2C (AF015535), genotype 2D (AF015536), genotype 2E (AF281606), genotype 3A (U73500), genotype 3B (U73501), genotype 4 (AF015528), genotype 6 (AF015537), genotype 7 (U61771). Healthy subjects (HS) in which deletions or insertions were identified in the non-coding control region via Sanger sequencing are indicated by * (deletion) or + (insertion). HSs in which JCV quasispecies were identified by 454 sequencing are circled.</p

Additional file 1: of Virology analysis in HCV genotype 1-infected patients treated with the combination of simeprevir and TMC647055/ritonavir, with and without ribavirin, and JNJ-56914845

Study design. HCV NS3/4A, NS5B and NS5A sequence analysis. (DOCX 32 kb

HCMV Displays a Unique Transcriptome of Immunomodulatory Genes in Primary Monocyte-Derived Cell Types

<div><p>Human cytomegalovirus (HCMV) is a betaherpesvirus which rarely presents problems in healthy individuals, yet may result in severe morbidity in immunocompromised patients and in immune-naïve neonates. HCMV has a large 235 kb genome with a coding capacity of at least 165 open reading frames (ORFs). This large genome allows complex gene regulation resulting in different sets of transcripts during lytic and latent infection. While latent virus mainly resides within monocytes and CD34⁺ progenitor cells, reactivation to lytic infection is driven by differentiation towards terminally differentiated myeloid dendritic cells and macrophages. Consequently, it has been suggested that macrophages and dendritic cells contribute to viral spread <i>in vivo</i>. Thus far only limited knowledge is available on the expression of HCMV genes in terminally differentiated myeloid primary cells and whether or not the virus exhibits a different set of lytic genes in primary cells compared with lytic infection in NHDF fibroblasts. To address these questions, we used Illumina next generation sequencing to determine the HCMV transcriptome in macrophages and dendritic cells during lytic infection and compared it to the transcriptome in NHDF fibroblasts. Here, we demonstrate unique expression profiles in macrophages and dendritic cells which significantly differ from the transcriptome in fibroblasts mainly by modulating the expression of viral transcripts involved in immune modulation, cell tropism and viral spread. In a head to head comparison between macrophages and dendritic cells, we observed that factors involved in viral spread and virion composition are differentially regulated suggesting that the plasticity of the virion facilitates the infection of surrounding cells. Taken together, this study provides the full transcript expression analysis of lytic HCMV genes in monocyte-derived type 1 and type 2 macrophages as well as in monocyte-derived dendritic cells. Thereby underlining the potential of HCMV to adapt to or influence different cellular environments to promote its own survival.</p></div

Expression levels of the HCMV genes transcriptome in fibroblasts (MOI 0.16).

<p>Log(10) of the RPKM values per gene were binned in six categories and mapped on the reference genome of TB40/E.</p

Percentage of mapped reads to the HCMV transcriptome in primary cell types.

<p>Six different blood donors (A-F) were processed to obtain monocytes which were differentiated to DCs, MΦ1 and MΦ2. Subsequently, these primary cell types were infected with TB40/E at MOI5. As a comparison, we infected NHDF cells (n = 3) at MOI 0.16, MOI 0.5 and MOI 5. Given in the top panel are the percentages of mapped reads on the HCMV genome for each cell type.</p

Enrichment of functional groups in DCs, MΦ1 and MΦ2 compared to NHDF.

<p>Genes involved in cell tropism, viral spread and immunomodulation are shown to be the most enriched groups in DCs, MΦ2 and MΦ1. Given are the p-vales of each individual gene.</p

Enrichment of functional groups when comparing DCs with both types of macrophages and when comparing MΦ1 with MΦ2 macrophages.

<p>The HCMV was annotated and a mathematical model was used to determine which functional groups were significantly enriched in the differentially regulated genes. Based on this algorithm, genes involved in cell tropism, viral spread and immunomodulation are the most enriched groups in DCs, MΦ2 and MΦ1.</p

Differential regulation of HCMV gene expression in monocyte-derived DCs, MΦ1 and MΦ2 (MOI5) compared to lytic infection in NHDF fibroblasts (MOI0.16).

<p>On the graph is the fold increase or decrease (reflecting genes which are expressed higher/lower in fibroblasts than in primary cell types). Red bars represent significant changes in gene expression between the indicated cell type and NHDF fibroblasts (adjusted p<0.05), black bars indicate modulated genes which did not reach the significance threshold (adjusted p≥0.05).</p