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We describe the development of a nested polymerase chain reaction (PCR) technique used to detect human parvovirus B19 DNA. It was performed in a two-step reaction, first with a pair of outer primers, then with a pair of inner (ie, nested) primers. Primer sequences were selected in the VP1 gene, corresponding to the capsid protein, of human parvovirus B19. To establish the nested PCR assay, the plasmid pGEM-1 containing almost the entire coding sequence of a human parvovirus B19 isolate was used. The nested PCR could detect up to 1.8 x 10(-3) ag of B19 DNA, equivalent to 10(-4) genomes, by electrophoresis. No amplification product was detected by gel electrophoresis when the reaction mixture contained human placental DNA, cytomegalovirus DNA, and sterile distilled water as templates. We used this assay to evaluate four cases of maternal B19 infection that were diagnosed by determination of the presence of anti-B19 immunoglobulin-M in maternal serum. The advantages of our nested PCR for detecting B19 DNA are plating simplicity, safety, sensitivity, and specificity. Our results suggest that this method may have general applicability in the evaluation of nonimmune hydrops fetalis and in the documentation of the natural history of fetal infection with B19 when applied to specimens of amniotic fluid or fetal blood.

In rare cases, measles virus (MV) in children leads to fatal neurological complications such as primary measles encephalitis, post-acute measles encephalitis, subacute sclerosing panencephalitis and measles inclusion-body encephalitis. To investigate the pathogenesis of MV-induced encephalitis, rodent brain-adapted MV strains CAM/RB and CAMR40 were generated. These strains acquired mutations to adapt to the rodent brain during 40 passages in rat brain. However, it is still unknown which genes confer the neurovirulence of MV. We previously established a rescue system for recombinant MVs possessing the backbone of wild-type strain HL, an avirulent strain in mice. In the present study, to identify the genes in CAMR40 that elicit neurovirulence, we generated chimeric recombinant MVs based on strain HL. As a result, recombinant wild-type MV in which the haemagglutinin (H) gene was substituted with that of CAMR40 caused a non-lethal mild disease in mice, while additional substitution of the HL phosphoprotein (P) gene with that of strain CAMR40 caused lethal severe neurological signs comparable to those of CAMR40. These results clearly indicated that, in addition to the H gene, the P gene is required for the neurovirulence of MV CAMR40.

ObjectiveThe tumour-to-normal ratio (T/N) is a representative index reflecting brain tumour activity by 18F-fluorodeoxyglucose (FDG) and 11C-methionine (MET) PET. We proposed a new automated method of calculating the normal reference value (N-value) for use as the denomination of T/N. This method uses voxel-based analysis of FDG- and MET-PET images. We compared the results of this method with those of the standard region-of-interest (ROI) method.MethodsData sets were obtained from 32 patients with newly diagnosed glioma and 13 patients with recurrent brain tumour. Our methods were as follows: (1) FDG-PET and MET-PET images were co-registered. (2) The areas where the FDG uptake was higher than a set threshold were selected. (3) For the corresponding areas of MET-PET images, mode and mean voxel values were calculated as tentative MET N-values. (4) Applying the same coordinates to FDG-PET, the voxel values were averaged and used as tentative FDG N-values. (5) The threshold of FDG-PET and whether to use the mode or the mean voxel values were computationally optimized using learning data sets. (6) Applying the optimal threshold and either the mode or mean, N-values of FDG and MET were finally determined.ResultsN-values determined by our automated method showed excellent agreement with those determined by a manual ROI method (ICC(2,1) > 0.78). These values were significantly correlated with mean manual N-values (p < 0.001).ConclusionsOur new method shows sufficiently good agreement with the standard method and can provide a more objective metabolic index.Electronic supplementary materialThe online version of this article (doi:10.1007/s12149-017-1153-8) contains supplementary material, which is available to authorized users.

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