Polyvalent degenerate oligonucleotides reverse transcription-polymerase chain reaction: a polyvalent detection and characterization tool for trichoviruses, capilloviruses, and foveaviruses.

ABSTRACT A polyvalent nested reverse transcription-polymerase chain reaction (RT-PCR) test using degenerate primers containing inosine (polyvalent degenerate oligonucleotides [PDO]) was developed for filamentous fruit tree viruses belonging to the genera Trichovirus, Capillovirus, and Foveavirus. The 362-bp product was amplified from nucleic acid extracts obtained from Prunus and Malus leaf samples. All the viruses targeted were detected, demonstrating the polyvalence of the test. The variability of a collection of Apple chlorotic leaf spot virus isolates was analyzed using the sequence of the PDO RT-PCR amplified cDNAs. The technique was also used to screen stone fruit materials infected with known agents or with virus-like graft-transmissible diseases of unknown etiology. The results obtained further validated the broad specificity of the assay, with positive amplification obtained for uncharacterized or partially characterized viruses associated with cherry and peach disorders. Sequencing the amplified PCR products either directly or after cloning allowed the identification of variants of known agents and the tentative identification of two new agents, a Trichovirus and a Foveavirus. In addition, sequence comparisons demonstrated that the sequence of the targeted region is phylogenetically informative and of predictive taxonomic value.

Fatigue and cognitive symptoms in patients with diabetes: relationship with disease phenotype and insulin treatment.

Neurobehavioral symptoms are frequently reported in patients with diabetes. Nevertheless, the characterization of the specific symptom dimensions that develop in diabetic patients with respect to disease phenotype and treatment status remains obscure. This study comparatively assessed fatigue symptoms and cognitive performance using a dimensional approach in 21 patients with insulin-treated type 1 diabetes, 24 type 2 diabetic patients either insulin-free or undergoing insulin treatment for at least six months, and 15 healthy subjects. Specific dimensions of fatigue were assessed using the Multidimensional-Fatigue-Inventory (MFI). Cognitive performance on tests of choice reaction time, pattern recognition memory and spatial planning was evaluated using the Cambridge-Neuropsychological-Automated-Battery (CANTAB). Body mass index (BMI) and glycated-hemoglobin (HbA1C) concentrations were collected, as well as information on diabetes complications and disease duration. Patients with type 2 diabetes, regardless of insulin treatment status, exhibited higher scores of fatigue, primarily in the dimensions of general and physical fatigue as well as reduced activity. Cognitive alterations, in the form of longer reaction times and impaired spatial planning, were also detected in type 2 diabetic patients treated with insulin. These alterations were overall unrelated to glucose control, as reflected in HbA1C levels, and were not explained by complications and duration of diabetes. No specific alteration was measured in type 1 diabetic patients who exhibited fatigue scores and cognitive performance comparable to healthy participants. While associated with fatigue, increased BMI did not significantly account for the relationship of type 2 diabetes with general fatigue and physical fatigue. BMI, however, modulated the association of type 2 diabetes with reduced activity and the association of insulin-treated type 2 diabetes with psychomotor slowing. These findings reveal specific fatigue and cognitive symptoms in patients with type 2 diabetes and suggest the involvement of differential pathophysiological processes.

Host species-dependent population structure of a pollen-borne plant virus, Cherry leaf roll virus.

Cherry leaf roll virus (CLRV) belongs to the Nepovirus genus within the family Comoviridae. It has a host range which includes a number of wild tree and shrub species. The serological and molecular diversity of CLRV was assessed using a collection of isolates and samples recovered from woody and herbaceous host plants from different geographical origins. Molecular diversity was assessed by sequencing a short (375-bp) region of the 3' noncoding region (NCR) of the genomic RNAs while serological diversity was assessed using a panel of seven monoclonal antibodies raised initially against a walnut isolate of CLRV. The genomic region analyzed was shown to exhibit a significant degree of molecular variability with an average pairwise divergence of 8.5% (nucleotide identity). Similarly, serological variability proved to be high, with no single monoclonal antibody being able to recognize all isolates analyzed. Serological and molecular phylogenetic reconstructions showed a strong correlation. Remarkably, the diversity of CLRV populations is to a large extent defined by the host plant from which the viral samples are originally obtained. There are relatively few reports of plant viruses for which the genetic diversity is structured by the host plant. In the case of CLRV, we hypothesize that this situation may reflect the exclusive mode of transmission in natural plant populations by pollen and by seeds. These modes of transmission are likely to impose barriers to host change by the virus, leading to rapid biological and genetic separation of CLRV variants coevolving with different plant host species.