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, α-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, σ-diversity, is a measure of the amount of inter-sample sequence heterogeneity, also incorporating information on α-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 α-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 α-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.

Parvovirus B19 (B19V) is a human ssDNA virus with ample pathogenic potential. It is characterized by a selective tropism for erythroid progenitor cells (EPC), exerting a cytotoxic effect with blockade of erythropoiesis. In our work, we investigated both viral and cellular expression profile in the course of infection of EPCs cultures via mRNA high throughput sequencing technology (HTS) and a dedicated bioinformatic pipeline, reconstructing both the viral and cellular transcriptome and their variations. A productive infection was confirmed as restricted to EPCs expressing mature differentiation markers and the specific receptor for virus VP1u region. mRNA HTS reconstructed the viral transcriptome in terms of localization and abundance of the different mRNA species, detailing the differential expression profile of B19V among early or late times in the course of infection. Analysis of cellular transcriptome indicated that variation was mainly driven by the cellular differentiation process, with the virus impacting to a lesser level, but still clearly separating infected vs. non-infected profiles. At early times post-infection, variations were typical of cellular sensing of viral infection and aimed at the induction of an antiviral state. At later times in the course of infection, the cellular population showed induction of an inflammatory response, related to TNF and IL-10, and a transition to adaptive immunity with evidence of upregulation of genes involved in MHC-II presentation. This dual-transcriptome analysis on infected EPCs population can lay the ground for future research aimed at a better definition of the pathogenetic mechanisms of B19V.

Parvovirus B19 (B19V) is a human pathogenic virus of clinical relevance, characterized by a selective tropism for erythroid progenitor cells in bone marrow. Relevant information on viral characteristics and lifecycle can be obtained from experiments involving engineered genetic systems in appropriate in vitro cellular models. Previously, a B19V genome of defined consensus sequence was designed, synthesized and cloned in a complete and functional form, able to replicate and produce infectious viral particles in a producer/amplifier cell system. Based on such a system, we have now designed and produced a derived B19V minigenome, reduced to a replicon unit. The genome terminal regions were maintained in a form able to sustain viral replication, while the internal region was clipped to include only the left-side genetic set, containing the coding sequence for the functional NS1 protein. Following transfection in UT7/EpoS1 cells, this minigenome still proved competent for replication, transcription and production of NS1 protein. Further, the B19V minigenome was able to complement B19-derived, NS1-defective genomes, restoring their ability to express viral capsid proteins. The B19V genome was thus engineered to yield a two-component system, with complementing functions, providing a valuable tool for studying viral expression and genetics, suitable to further engineering for purposes of translational research.