Use of a new RNA next generation sequencing approach for the specific detection of virus infection in cells.

The utilization of the current combination of in vitro, in vivo and PCR assays for the identification of adventitious viruses in production cells has a limited range of detection. While Next Generation Sequencing (NGS) has a broader breadth of detection, it is unable to differentiate sequences from replicating viruses versus background inert sequences. In order to improve NGS specificity, we have designed a new NGS approach which targets subsets of viral RNAs only synthesized during cell infection. In order to evaluate the performance of this approach for detecting low levels of adventitious viruses, we selected two difficult virus/cell systems. This included B95-8 cells persistently infected by Human herpesvirus 4 (HHV-4) and serially diluted into HHV-4 negative Ramos cells and Madin-Darby bovine kidney cells with an early infection produced via a low dose of Bovine viral diarrhea virus. We demonstrated that the sensitivity of our RNA NGS approach was equivalent to targeted PCR with an increased specificity for the detection of viral infection. We were also able to identify a previously undetected Murine Leukemia Virus contaminant in Ramos cells. Based on these results, we conclude that this new RNA NGS approach is suitable for conducting viral safety evaluations of cells.

Vitamin B status in patients with type 2 diabetes mellitus with and without incipient nephropathy.

AIM: To investigate the vitamin B status, with particular focus on vitamin B6, in adults with and without incipient nephropathy secondary to type 2 diabetes mellitus. METHODS: Plasma and/or urine concentrations of vitamins B6, B1, B12, related vitamers and biomarkers (including total homocysteine, methylmalonic acid) were measured in 120 adults with type 2 diabetes (including 46 patients with microalbuminuria) and 52 non-diabetic control subjects. RESULTS: Plasma concentrations of pyridoxal 5'-phosphate (PLP) were significantly lower in patients with type 2 diabetes than in control subjects (median: 22.7 nmol/L, diabetes with microalbuminuria; 26.8 nmol/L, diabetes without microalbuminuria; 39.5 nmol/L, non-diabetic control; p<0.0001). The prevalence of low PLP (<30 nmol/L) was 63%, 58%, and 25% in the diabetes groups with and without microalbuminuria and the control group, respectively. Plasma levels of pyridoxine and pyridoxal were also lower (p<0.0001), but levels of pyridoxamine, pyridoxamine 5'-phosphate, and pyridoxic acid were higher in both groups with diabetes compared to the control group (p<0.001). Thiamine deficiency was highly prevalent in all groups, whereas low vitamin B12 and elevated methylmalonic acid were rare. Increased levels of C-reactive protein and soluble vascular cell adhesion molecule-1 were observed in the groups with diabetes (p<0.05, versus healthy control). CONCLUSIONS: Deficiency of vitamin B6 (PLP, pyridoxine, pyridoxal) and vitamin B1 (thiamine) was prevalent in type 2 diabetes. Incipient nephropathy was associated with more pronounced alterations in vitamin B6 metabolism and stronger indications of endothelial dysfunction and inflammation.

Genotyping of human platelet antigen-1 by gene amplification and labelling in one system and automated fluorescence correlation spectroscopy.

Genotyping of human platelet antigen-1 (HPA-1) is required for the diagnosis and appropriate therapy of alloimmunization. Recently, the HPA-1 polymorphism has been identified as an inherited risk factor for thrombosis. Most currently used methods for HPA-1 genotyping have the disadvantage of time-consuming post-polymerase chain reaction (PCR) processes such as ligation (oligonucleotide ligation assay), restriction enzyme digestion (allele-specific restriction enzyme analysis) and electrophoresis (single-strand conformation polymorphism). We present a novel method for HPA-1 genotyping based on a homogeneous PCR strategy (GALIOS, gene amplification and labelling in one system) combined with automated fluorescence correlation spectroscopy (FCS). The PCR uses one pair of gene-specific amplification primers and two allele-specific, semi-nested labelling primers. The allele-specific labelling primers differ in a single nucleotide (T for HPA-1a/1a, C for HPA-1b/1b) and are coupled to different fluorescent dyes. The quantities of generated fluorescent PCR products are analysed by FCS at 543 nm and 633 nm excitation wavelength respectively. The genotypes determined using this method were in 100% concordance with the results obtained by allele-specific restriction analysis (n = 380 samples). The assay was validated for specificity, reliability and the dynamic range. This innovative method of rapid HPA-1 genotyping offers a specific and robust system, which is applicable for routine HPA-1 genotyping.