CpG Distribution and Methylation Pattern in Porcine Parvovirus

<div><p>Based on GC content and the observed/expected CpG ratio (oCpGr), we found three major groups among the members of subfamily <i>Parvovirinae</i>: Group I parvoviruses with low GC content and low oCpGr values, Group II with low GC content and high oCpGr values and Group III with high GC content and high oCpGr values. Porcine parvovirus belongs to Group I and it features an ascendant CpG distribution by position in its coding regions similarly to the majority of the parvoviruses. The entire PPV genome remains hypomethylated during the viral lifecycle independently from the tissue of origin. <i>In vitro</i> CpG methylation of the genome has a modest inhibitory effect on PPV replication. The <i>in vitro</i> hypermethylation disappears from the replicating PPV genome suggesting that beside the maintenance DNMT1 the <i>de novo</i> DNMT3a and DNMT3b DNA methyltransferases can’t methylate replicating PPV DNA effectively either, despite that the PPV infection does not seem to influence the expression, translation or localization of the DNA methylases. SNP analysis revealed high mutability of the CpG sites in the PPV genome, while introduction of 29 extra CpG sites into the genome has no significant biological effects on PPV replication <i>in vitro</i>. These experiments raise the possibility that beyond natural selection mutational pressure may also significantly contribute to the low level of the CpG sites in the PPV genome.</p> </div

Titration of the mutant PPVs with extra CpGs.

<p>A, Schematic representation of the novel CpG sites in the mutant viruses. Red vertical bars symbolize the CpGs in the genomes. B, Titer of the mutant stocks on PT and Cos7 cells. Infected cells were detected by immunofluorescence labeling at 20 and 48 hours PI fixation, respectively. C, Titer of the mutant stocks after 10 additional passages on PT cells. Quantification was carried out by qPCR. Vertical bars indicate the standard deviation in each case.</p

GC content and observed/expected CpG ratio in parvoviral genomes.

<p>Values are shown in percentage; viruses are listed by increasing GC content. Group I, Group II and Group III parvoviruses are indicated by red, violet and green colors respectively.</p

SNP frequency of the CpG and GC sites in the PPV genome.

<p>The frequency was calculated by dividing the rate of SNPs in the CpG and GC sites with the rate of SNPs in the total number of Gs and Cs in the genome.</p

Deep sequencing of the bisulfite treated PPV genome.

<p>Vertical bars label the position of the CpGs in the PPV genome. Height of the bars represents the frequency of unmethylated CpGs. Coverage of the CpG sites having more than 8% methylation rate was between 47 and 13694. </p

CpG distribution in the coding positions of different organisms.

<p>A, Total number of CpGs in the main coding ORFs (NS and VP) of parvoviral genomes. B, Number of CpGs in the different positions of the mRNAs of the human and drosophila proteomes. Numbers on the X axis indicate distance from the start codon of the proteins; Number of CGX (Arg codons) are indicated in 1,4,7,10… positions, numbers of XCG codons (Ser, Pro, Thr, Ala) are indicated in 2,5,8,11… positions and numbers of CGs in consecutive codons XXC GXX are indicated in 3,6,9,12… positions.</p

Initiation of the replication by differentially methylated PPV DNAs.

<div><p>A, and B, PT cells on 96 well plate were transfected by 0.2 µg differentially methylated DNA and PPV infected cells were detected by immunofluorescence 24 hour post-transfection. </p> <p>The fluorescent nuclei count (FNC) in one well is indicated by columns. C, Number of viral genomes in the supernatant of differentially methylated DNA transfected cells 24 and 48 hours post-transfection. Vertical bars indicate standard deviation in each case.</p></div

Diagrammatic presentation of the occurrence of lipase-, protease- and pseudohyphae-producer strains in the <i>C. parapsilosis sensu lato</i> complex.

<p>n: number of isolates, upper number in fraction: number of pseudohypha-producer strains, lower number in fraction: number of pseudohypha negative strains.</p

Interactions of different <i>C. parapsilosis sensu lato</i> isolates (see Table S1.) with primary human monocyte-derived macrophages.

<p>(A) Killing efficiency of <i>C. parapsilosis sensu lato</i> species based on CFU-determinations (Cp, <i>C. parapsilosis sensu stricto</i>; Co, <i>C</i><i>. orthopsilosis</i>; Cm, <i>C</i><i>. metapsilosis</i>), (B) killing efficiency of lipase producer vs. non-producer and pseudohypha positive vs. negative strains in the <i>C. parapsilosis sensu lato</i> group [lip+, lipase positive (regardless of pseudohypha production); lip-, lipase negative; psh+, pseudohyphae positive (regardless of lipase production); psh-, pseudohyphae negative], (C) killing efficiency of lipase or pseudohyphae positive vs. negative isolates of <i>C. parapsilosis sensu stricto</i>, (D) killing efficiency of lipase or pseudohyphae producer vs. non-producer strains of <i>C</i><i>. orthopsilosis</i>, (E) host-cell damaging capacity of <i>C. parapsilosis sensu lato</i> species based on the release of LDH (lactate dehydrogenase), (F) host-cell damaging capacity of lipase or pseudohyphae producer vs. non-producer strains in the <i>C. parapsilosis sensu lato</i> group, (G) host-cell damaging capacity of lipase or pseudohyphae positive vs. negative isolates of <i>C. parapsilosis sensu stricto</i>, (H) host-cell damaging capacity of lipase or pseudohyphae producer vs. non-producer strains of <i>C</i><i>. orthopsilosis</i>. Cp, <i>C. parapsilosis sensu stricto</i>; Co, <i>C</i><i>. orthopsilosis</i>; Cm, <i>C</i><i>. metapsilosis</i>. Data points on graphs represent individual strains. Experiments were performed in triplicates. Data were analyzed by the Kruskal-Wallis test (A, E), the Mann-Whitney test (B, D, F, G, H) or the Wilcoxon rank sum test (C). * p<0.05, ** p<0.01, *** p<0.001.</p

Phagocytosis of one representative isolate each of <i>C. parapsilosis sensu stricto</i>, <i>C</i><i>. orthopsilosis</i> and <i>C</i><i>. metapsilosis</i> by J774.2 macrophages.

<p>(A) Light microscopic picures of J774.2 macrophages phagocytosing <i>C. parapsilosis sensu lato</i> species, (B) phagocytosis of <i>C. parapsilosis sensu stricto</i>, <i>C</i><i>. orthopsilosis</i> and <i>C</i><i>. metapsilosis</i> by J774.2 macrophages assessed by quantitative imaging, R2: phagocytosing macrophage population discriminated by the presence of red fluorescence due to ingestion of Alexa Fluor 647-labeled yeast cells, (C) numbers of ingested yeast cell/macrophage in the R2 population in case of <i>C. parapsilosis sensu stricto</i>, <i>C</i><i>. orthopsilosis</i> and <i>C</i><i>. metapsilosis</i> determined by the spot-counting feature of IDEAS software.</p