The discovery of human metapneumovirus and its implications for respiratory tract disease have emphasized the need for a sensitive, specific, and rapid assay to detect this virus in a clinical setting. It recently became clear that human metapneumovirus can be grouped into at least four genetic lineages. Previously described assays for the detection of human metapneumovirus were developed by using limited sequence information and failed to detect viruses from all four genetic lineages with comparable sensitivities. Here we describe the development and evaluation of a real-time reverse transcriptase PCR assay that detects human metapneumovirus from the four known genetic lineages with equal specificities and sensitivities.The isolation and characterization of a novel paramyxovirus, human metapneumovirus (hMPV), were recently described (13,14). hMPV was first isolated from children suffering from acute respiratory tract disease (13); however, infections in all other age groups were later identified as well (2,6,12). The virus has been circulating in humans for at least 50 years. The clinical signs associated with hMPV infection appear to be similar to those caused by human respiratory syncytial virus (RSV), which is the most common cause of respiratory tract disease in children (3,4,15,17). hMPV appears to be responsible for about 7 to 10% (3,5,8,9,11,12,15) of cases of acute respiratory tract infections in infants. This relatively high incidence of hMPV infections and the fact that hMPV-associated disease may be severe have emphasized the need for a reliable, sensitive, and rapid diagnostic test for the detection of this virus.In a diagnostic setting, PCR is generally accepted for the detection of viral infections, particularly for viruses that are difficult to isolate in cell cultures, such as hMPV. To date, four distinct genetic lineages of hMPV have been described (16). For the detection of hMPV, a single assay that is equally sensitive for all four hMPV genetic lineages is preferred. Here we describe the development and evaluation of a new and sensitive real-time reverse transcriptase (RT) PCR (RT-PCR) assay which meets these requirements and a comparison of this assay with previously described assays. MATERIALS AND METHODSClinical samples and virus isolates. hMPV-positive specimens were obtained from nasopharyngeal samples collected from patients with symptoms of respiratory tract disease (13, 15). Viruses were grown on tertiary monkey kidney (tMK) cells, and the isolates were stored at Ϫ70°C. For each of the four genetic lineages of hMPV, a single virus isolate was selected as the prototype isolate (16). RNA from these prototype virus isolates was used to test the designed Taqman primers and probes. The nucleoprotein sequence of lineage A1, strain NL/1/00 can be obtained from the GenBank database (accession number AF371337) (see below for the accession numbers of the sequences submitted for this study).For validation of the Taqman RT-PCR assay, we used nasopharyngeal samples that were collected in the 2000-...

hMPV was detected in a substantial number of children with URIs and concomitant AOM.

Nitrate redudase (NR) is the first enzyme in nitrate assimilation, a critica1 process for plant survival. l h e regulation of NR gene expression is complex, involving both interna1 and externa1 factors. O f these, nitrate indudion of NR gene expression has been studied most extensively and is well conserved among baderia, fungi, and higher plants. We are interested in understanding the mechanism of nitrate indudion of higher plant NR genes. Here we describe promoter analyses of the 5' flanking regions of the Arabidopsis NR genes, NR1 and NRZ, with resped to nitrate indudion of gene expression. To facilitate these analyses, a nitrate indudion procedure using 1, transgenic tobacco plants was established. Approximately 1.5-kb 5' flanking regions of the two Arabidopsis NR genes (NR1 and NRZ) were fused to a reporter gene and its expression in transgenic plants was analyzed. Deletion analyses of these regions show that 238-and 188-bp 5' flanking regions of the NR1 and NRZ, respedively, contain sequences responsive to nitrate indudion.

Accreditation standards in counseling allow triadic supervision, one supervisor meeting with two supervisees simultaneously, as a substitute for individual supervision. Research, however, has indicated that triadic sessions may complement individual and group supervision by offering unique learning opportunities not present in other supervision modalities. We describe a peer supervision approach that capitalizes on these learning opportunities while addressing some of the challenges in conducting triadic supervision (e.g., helping supervisees give constructive feedback, keeping both engaged). We include structured peer review forms used with practicum and internship supervisees and describe different supervisor roles during triadic supervision with these two groups.

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