Pre-Existing Intrarenal Parvovirus B19 Infection May Relate to Antibody-Mediated Rejection in Pediatric Kidney Transplant Patients

Viral infections can lead to transplant dysfunction, and their possible role in rejection is described. In total, 218 protocol biopsies performed in 106 children at 6, 12 and 24 months after transplantation were analyzed according to Banff ’15. RT-PCR for cytomegalovirus, Epstein-Barr virus, BK virus and Parvovirus B19 was performed on blood and bioptic samples at the time of transplant and each protocol biopsy. The prevalence of intrarenal viral infection increases between 6 and 12 months after transplantation (24% vs. 44%, p = 0.007). Intrarenal Parvovirus B19 infection is also associated with antibody-mediated rejection (ABMR) (50% ABMR vs. 19% T-cell-mediated rejection, p = 0.04). Moreover, Parvovirus infection is higher at 12 months of follow-up and it decreases at 48 months (40.4% vs. 14%, p = 0.02), while in 24% of grafts, Parvovirus is already detectable at the moment of transplantation. Intrarenal Parvovirus B19 infection seems to be related to ABMR in pediatric kidney recipients. The graft itself may be the way of transmission for Parvovirus, so performance of a PCR test for Parvovirus B19 should be considered to identify high-risk patients. Intrarenal Parvovirus infection presents mainly during the first-year post-transplantation; thus, we recommend an active surveillance of donor-specific antibodies (DSA) in patients with intrarenal Parvovirus B19 infection during this period. Indeed, it should be considered a treatment with intravenous immunoglobulins in patients with intrarenal Parvovirus B19 infection and DSA positivity, even in the absence of ABMR criteria for kidney biopsy

Next Generation Sequencing for the Analysis of Parvovirus B19 Genomic Diversity

Parvovirus B19 (B19V) is a ssDNA human virus, responsible for an ample range of clinical manifestations. Sequencing of B19V DNA from clinical samples is frequently reported in the literature to assign genotype (genotypes 1-3) and for finer molecular epidemiological tracing. The increasing availability of Next Generation Sequencing (NGS) with its depth of coverage potentially yields information on intrinsic sequence heterogeneity; however, integration of this information in analysis of sequence variation is not routinely obtained. The present work investigated genomic sequence heterogeneity within and between B19V isolates by application of NGS techniques, and by the development of a novel dedicated bioinformatic tool and analysis pipeline, yielding information on two newly defined parameters. The first, alpha-diversity, is a measure of the amount and distribution of position-specific, normalised Shannon Entropy, as a measure of intra-sample sequence heterogeneity. The second, sigma-diversity, is a measure of the amount of inter-sample sequence heterogeneity, also incorporating information on alpha-diversity. Based on these indexes, further cluster analysis can be performed. A set of 24 high-titre viraemic samples was investigated. Of these, 23 samples were genotype 1 and one sample was genotype 2. Genotype 1 isolates showed low alpha-diversity values, with only a few samples showing distinct position-specific polymorphisms; a few genetically related clusters emerged when analysing inter-sample distances, correlated to the year of isolation; the single genotype 2 isolate showed the highest alpha-diversity, even if not presenting polymorphisms, and was an evident outlier when analysing inter-sample distance. In conclusion, NGS analysis and the bioinformatic tool and pipeline developed and used in the present work can be considered effective tools for investigating sequence diversity, an observable parameter that can be incorporated into the quasispecies theory framework to yield a better insight into viral evolution dynamics

Co-localization of two different viral genomes in the same sample by double-chemiluminescence in situ hybridization

A double-chemiluminescence in situ hybridization has been developed that combines the advantages of chemiluminescence with the detection of two different viral DNAs, i.e., herpes simplex virus (HSV..

Genetic Characterization of Cancer of Unknown Primary Using Liquid Biopsy Approaches

Cancers of unknown primary (CUPs) comprise a heterogeneous group of rare metastatic tumors whose primary site cannot be identified after extensive clinical\u2013pathological investigations. CUP patients are generally treated with empirical chemotherapy and have dismal prognosis. As recently reported, CUP genome presents potentially druggable alterations for which targeted therapies could be proposed. The paucity of tumor tissue, as well as the difficult DNA testing and the lack of dedicated panels for target gene sequencing are further relevant limitations. Here, we propose that circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) could be used to identify actionable mutations in CUP patients. Blood was longitudinally collected from two CUP patients. CTCs were isolated with CELLSEARCH\uae and DEPArrayTM NxT and Parsortix systems, immunophenotypically characterized and used for single-cell genomic characterization with Ampli1TM kits. Circulating cell-free DNA (ccfDNA), purified from plasma at different time points, was tested for tumor mutations with a CUP-dedicated, 92-gene custom panel using SureSelect Target Enrichment technology. In parallel, FFPE tumor tissue was analyzed with three different assays: FoundationOne CDx assay, DEPArray LibPrep and OncoSeek Panel, and the SureSelect custom panel. These approaches identified the same mutations, when the gene was covered by the panel, with the exception of an insertion in APC gene. which was detected by OncoSeek and SureSelect panels but not FoundationOne. FGFR2 and CCNE1 gene amplifications were detected in single CTCs, tumor tissue, and ccfDNAs in one patient. A somatic variant in ARID1A gene (p.R1276 17) was detected in the tumor tissue and ccfDNAs. The alterations were validated by Droplet Digital PCR in all ccfDNA samples collected during tumor evolution. CTCs from a second patient presented a pattern of recurrent amplifications in ASPM and SEPT9 genes and loss of FANCC. The 92-gene custom panel identified 16 non-synonymous somatic alterations in ccfDNA, including a deletion (I1485Rfs 1719) and a somatic mutation (p. A1487V) in ARID1A gene and a point mutation in FGFR2 gene (p.G384R). Our results support the feasibility of non-invasive liquid biopsy testing in CUP cases, either using ctDNA or CTCs, to identify CUP genetic alterations with broad NGS panels covering the most frequently mutated genes

Parvovirus B19 DNA CpG Dinucleotide Methylation and Epigenetic Regulation of Viral Expression

CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression

A flow-FISH assay for the quantitative analysis of parvovirus B19 infected cells

Human parvovirus B19 (B19V) replication is a process highly dependent on the cellular environment, therefore methodologies allowing for analysis at single cell level could represent effective tools to understand cell-to cell differences in the replication process and to investigate cell-virus interactions. Fluorescence in situ hybridization (FISH) can be combined with flow cytometry (flow-FISH) to enable the detection of target nucleic acid sequences in thousands of individual cells in a short amount of time. In the present study, a flow-FISH assay based on the use of a digoxigenin-labeled genomic probe has been developed to discriminate B19V infected cells following in vitro infection of UT7/EpoS1 cell line and EPCs (erythroid progenitor cells) generated from peripheral blood mononuclear cells. In B19V infected UT7/EpoS1 and EPCs, viral nucleic acids were detected by the flow-FISH assay starting from 24hpi up to 48hpi. The method, used together with quantitative PCR techniques, can be very useful to describe the kinetics of B19V infection within a heterogeneous cell population