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.

Background Human metapneumovirus (HMPV) is a leading cause of acute respiratory illness (ARI) in children. Population-based incidence rates and comprehensive clinical characterizations of disease have not been established. Methods We conducted population-based prospective surveillance for HMPV in two U.S. counties among children <5 years hospitalized with ARI or fever for two years. Nasal/throat swabs were tested for HMPV by real-time RT-PCR and genotyped. Results Forty-two of 1104 (3.8%) children tested positive for HMPV. The overall annual rate of HMPV-associated hospitalizations per 1000 children <5 years was 1.2 (95%CI 0.9–1.6). This rate was highest in infants 0–5 months (4.9/1000 [95%CI 2.9–7.2]), followed by children 6–11 months (2.9/1000 [95%CI 1.4–4.7]). The annual rate of HMPV-associated hospitalizations was less than respiratory syncytial virus, but similar to influenza and parainfluenzavirus (PIV) 3 in all age groups. The mean age of children hospitalized with HMPV was 6 months. Bronchiolitis, pneumonia and asthma were the most common diagnoses in children with HMPV. All four HMPV subgroups were detected during both years at both sites. HPMV was most prominent from March through May. Conclusions HMPV was detected in 3.8% of children hospitalized with ARI or fever, with a population incidence similar to that of influenza and PIV3.

Background: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in children. We examined the diversity and molecular evolution of HMPV using 85 full-length F (fusion) gene sequences collected over a 20-year period.

Background Human metapneumovirus (HMPV) is an important cause of acute respiratory illnesses in children. HMPV encodes two major surface glycoproteins, fusion (F) and glycoprotein (G). The function of G has not been fully established, though it is dispensable for in vitro and in vivo replication. Methods We analyzed 87 full-length HMPV G sequences from isolates collected over 20 years. Results The G sequences fell into four subgroups with a mean 63% amino acid identity (minimum 29%). The length of G varied from 217 to 241 residues. Structural features such as proline content and N- and O-glycosylation sites were present in all strains but quite variable between subgroups. There was minimal drift within the subgroups over 20 years. The estimated time to the most recent common ancestor was 215 years. Conclusions HMPV G was conserved within lineages over 20 years, suggesting functional constraints on diversity. However, G was poorly conserved between subgroups, pointing to potentially distinct roles for G among different viral lineages.