Real-Time Reverse Transcriptase PCR Assay for Detection of Human Metapneumoviruses from All Known Genetic Lineages

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-...

Association of human metapneumovirus with acute otitis media

“…First, the NW and MEF specimens we tested had been thawed and frozen several times for previous studies, which may have decreased sensitivity, although we used a sensitive RT-PCR method that is capable of detecting all four genetic lineages of hMPV at 50 copies or less [11]. In a previous study, we retested freeze-thawed samples for hMPV and other viruses and did not find a significant loss of sensitivity [5].…”

“…Metapneumovirus testing was performed by a realtime RT-PCR assay on a Smart Cycler (Cepheid, Sunnyvale, CA) using primers and probe for the N gene [11] with the Quantitect RT-PCR kit (Qiagen, Valencia, CA). The probe was altered slightly, with BHQ-3 (Invitrogen, Carlsbad, CA) substituted for TAMRA as the 3′ fluorescent quencher.…”

Human Metapneumovirus and Human Bocavirus in Children

“…Because of the genetic variability of RNA viruses in general, and hMPV specifically, care must be taken to select regions that are more stable for PCR primers. One such region is the N gene, for which a real-time PCR assay that detects all four subgroups was described in 2004 and is commonly used (43). Until recently, respiratory viral PCR was done only using "home-brew" assays in research or reference laboratories.…”

Multi-year study of human metapneumovirus infection at a large US Midwestern Medical Referral Center

“…Since hMPV was first reported (van den Hoogen et al, 2001) in 2001, much progress has been made in its detection Ebihara et al, 2003Ebihara et al, , 2004Hamelin and Boivin, 2005;Leung et al, 2005;Mackay et al, 2003;Maertzdorf et al, 2004), in understanding its genetic diversity Falsey et al, 2003;Ludewick et al, 2005;Mackay et al, 2003) and in documenting its varied clinical presentation (Falsey et al, 2003;Principi et al, 2006). By linking demographic and clinical data to a large, 3-year, viral culture specimen repository, we sought to describe hMPV infections among patients evaluated by a large US Midwestern referral laboratory.…”