No abstract

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.

No abstract

Nucleotide sequence comparisons were performed on a highly heterogeneous region of three human cytomegalovirus strains, Toledo, Towne, and AD169. The low-passage, virulent Toledo genome contained a DNA segment of approximately 13 kbp that was not found in the Towne genome and a segment of approximately 15 kbp that was not found in the AD169 genome. The Towne strain contained approximately 4.7 kbp of DNA that was absent from the AD169 genome, and only about half of this segment was present, arranged in an inverted orientation, in the Toledo genome. These additional sequences were located at the unique long (U L)/b (IR L) boundary within the L component of the viral genome. A region representing nucleotides 175082 to 178221 of the AD169 genome was conserved in all three strains; however, substantial reduction in the size of the adjacent b sequence was found. The additional DNA segment within the Toledo genome contained 19 open reading frames not present in the AD169 genome. The additional DNA segment within the Towne genome contained four new open reading frames, only one of which shared homology with the Toledo genome. This comparison was extended to five additional clinical isolates, and the additional Toledo sequence was conserved in all. These findings reveal a dramatic level of genome sequence complexity that may explain the differences that these strains exhibit in virulence and tissue tropism. Although the additional sequences have not altered the predicted size of the viral genome (230 to 235 kbp), a total of 22 new open reading frames (denoted UL133 to UL154), many of which have sequence characteristics of glycoproteins, are now defined as cytomegalovirus specific. Our work suggests that wild-type virus carries more than 220 genes, some of which are lost by large-scale deletion and rearrangement of the U L /b region during laboratory passage.