Premature ovarian failure (POF), also termed as primary ovarian insufficiency (POI), is a highly heterogenous condition affecting 0.5-3.0% of women in childbearing age. These young women comprise quite a formidable group with unique physical and psychological needs that require special attention. Premature ovarian senescence (POS) in all of its forms evolves insidiously as a basically asymptomatic process, leading to complete loss of ovarian function, and POI/POF diagnoses are currently made at relatively late stages. Well-known and well-documented risk factors exist, and the presence or suspicion of autoimmune disorder should be regarded as an important one. Premature ovarian failure is to some degree predictable in its occurrence and should be considered while encountering young women with loss of menstrual regularity, especially when there is a concomitant dysfunction in the immune system.
SUMMARYThe variability of the Apple stem pitting virus (ASPV) coat protein (CP) gene was investigated. The CP gene of ten virus isolates from apple and pear trees was sequenced. Comparison of all sequenced virus isolates revealed high diversity of the CP gene (70.7-93.5 % at the nucleotide level and 77.8-98.7 % at the amino acid level). Additionally, one or two deletions in the N-terminal part of the coat protein gene of the studied virus isolates were identified. The ratio of nonsynonymous to synonymous polymorphic sites indicated that purifying selection has acted to eliminate deleterious mutations in coding sites. Moreover, the evidences for recombination in analyzed sequences were provided. It is likely that recombination, along with selection, enhances the speed of elimination of deleterious mutations in ASPV, following the mutational deterministic hypothesis of Kondrashov.
Although the significance of molecular diagnostics in routine plant virus detection is rapidly growing, the preferred methods are still antibody-based enzyme immunoassays. In the past decade, aptamers have been demonstrated to be viable alternatives of antibodies in many applications. We set out to select apple stem pitting virus (ASPV)-specific aptamers and to apply them as antibody substitutes in various immunoassay methods. The applied systematic evolution of ligands by exponential enrichment (SELEX) procedure resulted in highly discriminative aptamers selectively binding to the target virus coat protein even in complex protein matrixes. We developed protocols for exploitation of aptamers in diverse plant virus diagnosis methods, such as dot and Western blot analyses and enzyme-linked oligonucleotide assay (ELONA). Our selected aptamers proved to be superior to the available antibody in all aspects. In contrast to the antibody, the aptamers decorate both native and denaturated proteins, and ELONA produces higher signal intensity than traditional enzyme-linked immunosorbent assay (ELISA) with virus-infected plant extract. Summarily, our results present the selection and practical utilization of first plant virus-specific aptamers. Most important, the first application of ELONA for virus detection is demonstrated, which proposes a novel, more flexible, and cost-effective means of virus diagnostics.
In this study, 267 coat protein gene (CP) sequences from 48 Polish isolates of Apple stem pitting virus (ASPV) were determined. The genetic structure of the virus population was analysed and possible mechanisms of molecular evolution explored. We found evidence of recombination within the ASPV population and the presence of 17 ASPV molecular variants that differ in the length, number and arrangement of deletions in the CP. Population genetic analyses showed significant variation among isolates from pear and apple trees, between isolates from the same host species and, more interestingly, within isolates, supporting the existence of significant levels of variability within individual hosts, as expected by a quasispecies population structure. In addition, different tests support that selection might have been an important force driving diversification within isolates: positive selection was found acting upon certain amino acids. Phylogenetic analyses also showed that isolates did not classify according to the host species (pear or apple trees) but according to the pattern of deletions, suggesting a possible role for deletions during clade diversification.
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