Keeping Pace with Parvovirus B19 Genetic Variability: a Multiplex Genotype-Specific Quantitative PCR Assay

Abstract

Three genotypes have been identified within the parvovirus B19 species (B19V), and such genetic diversity may have significant implications for the development of molecular detection assays. In the present study, B19V genetic variability has been examined on a subset of genomic sequences available in the NCBI nucleotide database, and a quantitative PCR (qPCR) assay able to detect, differentiate, and quantify all viral variants has been established. The designed primers and probes have been used for the development of alternative detection formats, based on a combined use of intercalating dye and genotype-specific hydrolysis probes. The qPCR assay analytical performances have been determined on the 1st WHO International Reference Panel for Parvovirus B19 Genotypes. The developed qPCR protocols allow for the detection of genotypes 1 to 3 with equal accuracy, and with a limit of detection (LOD) of 200 IU/ml. A comparison of routine performance was carried out with respect to a previously established assay specifically validated on B19V genotype 1. For 130 clinical samples analyzed, 126 showed concordant results (31 positive and 97 negative), while 4 showed discordant results. Overall, the genotype-specific qPCR assay showed a sensitivity of 93.94% and a specificity of 97.94%, with an agreement rate of 96.92%. The proposed qPCR assay and the alternative protocols developed, each with robust performance, may allow choice with respect to operational systems and diagnostic requirements and might contribute to provide a more reliable diagnostic service and epidemiological surveillance of B19 virus. H uman parvovirus B19 (B19V) is a widespread human-pathogenic virus and a member of the Erythrovirus genus in theParvoviridae family (1). In the last decade, due to the introduction of innovative molecular techniques, the taxonomy in the family has grown in complexity to accommodate a still-increasing number of newly recognized viruses (2). Specifically, B19 viruses are now formally subdivided into three genotypes, the prototype genotype 1 and the two variant genotypes 2 and 3, and further subtypes (3, 4). In particular, within genotype 1, the majority of isolates are referred to as genotype 1a, while a few isolates from Asia show separate clustering and are referred to as genotype 1b (5). Within genotype 3, two distinct subtypes are usually referred to as genotypes 3a and 3b (6). All B19V genotypes appear to cocirculate, but their relative frequencies are strikingly different: genotype 1a is ubiquitous worldwide, genotype 2 is rare and sporadically detected in different geographic settings, including Europe, and genotypes 3a and 3b are detected at higher frequencies in western Africa and at lower frequencies in other geographic areas (7-10).The three B19V genotypes are assumed to have similar biological properties, pathogenic capacities, and transmission routes and pose a similar diagnostic challenge in the clinical setting (11). Of relevance, all genotype variants can be contaminants of blood and blood-derived products, so ...

Parvovirus B19 Replication and Expression in Differentiating Erythroid Progenitor Cells

“…A B19V viremic serum sample, identified in our laboratory in the course of routine diagnostic analysis and available for research purposes according to Italian privacy law, was used as source of virus for the infection experiments. The viremic serum contained 10 12 B19V (genotype 1) genome copies (geq)/mL, as determined by quantitative PCR analysis, and resulted negative by routine diagnostic assays to other viruses [ 17 ].…”

Human Parvoviruses

“…They were later replaced by more sensitive qualitative PCRs (542,1104,(1104)(1105)(1106) and qPCRs (1107,1108). There are many in-house and commercial qPCR assays for genotype-specific as well as pan-B19V amplification for diagnostic and blood screening purposes (242,1042,(1109)(1110)(1111)(1112)(1113)(1114). Many PCR methods show poor sensitivity or fail to detect all three genotypes.…”

Serological and molecular analysis of parvovirus B19 infection in Mayan women with systemic lupus erythematosus in Mexico.

“…Primers and probes used in amplification reactions were described by Bonvicini et al . 41 , (B19 primer forward 5'-CGCCTGGAACASTGAAACCC-3', B19 primer reverse 5´-TCAACCCCWACTAACAGTTC-3', and genotype 1 probe 6FAMGTTGTAGCTGCATCGTGGGAAGAMGBNFQ). They were designed by Applied Biosystems and directed against the non-structural protein 1 (NS1, 616-2631 nucleotides) of PV-B19 genotype 1.…”