Human metapneumovirus as a cause of community-acquired respiratory illness

Emerging Infectious Diseases, Sept, 2002 by Joanne Stockton, Iain Stephenson, Douglas Fleming, Maria Zambon

Human metapneumovirus (HMPV) is a recently identified Paramyxovirus first isolated from hospitalized children with acute respiratory tract infections (ARTI). We sought evidence of HMPV infection in patients who had visited general practitioners, had influenzalike illnesses (ILI), and had negative tests for influenza and Human respiratory syncytial virus (HRSV). As part of national virologic surveillance, sentinel general practices in England and Wales collected samples from patients of all ages with ILI during winter 2000-01. Reverse transcriptase-polymerase chain reaction (PCR) for HMPV, influenza A (H1 and H3), influenza B, and HRSV (A and B) was used to screen combined nose and throat swabs. PCR products from the HMPV-positive samples were sequenced to confirm identity and construct phylogenetic trees. Of 711 swabs submitted, 408 (57.3%) were negative for influenza and HRSV; HMPV was identified in 9 (2.2%) patients. HMPV appears to be associated with community-acquired ARTI. The extent of illness and possible complications related to this new human virus need to be clarified.

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Despite control of many infectious diseases in the industrialized world, acute viral respiratory tract infections (ARTI) remain a leading cause of illness. Although usually self-limiting in healthy adults, these infections are responsible for a substantial loss of productive time and are important factors in the illness and death of the elderly population. Various genetically diverse viruses, often with multiple types, may cause respiratory illness; of these, influenza receives the greatest attention (1). Human respiratory syncytial virus (HRSV) is also increasingly implicated as an important pathogen (2).

The association between the incidence of ARTI and excess winter deaths in the United Kingdom is well recognized (1). Regression modeling associates excess winter deaths with influenza and HRSV but also suggests that other pathogens may be involved (3).

Studies of the impact of respiratory virus infections are limited by difficulty in distinguishing respiratory pathogens clinically and in the laboratory (4,5). Despite improved sensitivity with diagnostic techniques such as reverse transcriptase-polymerase chain reaction (RT-PCR), approximately 40% of specimens from patients with community-acquired respiratory illnesses during peak winter months contain no identified viral pathogen (2,5,6).

A new pneumovirus, Human Metapneumovirus (HMPV), has recently been isolated in the Netherlands (7). The Pneumovirinae subfamily is classified into Pneumovirus, containing HRSV, and Metapneumovirus genera. In 2001, Van den Hoogen et al. (7) reported the detection of HMPV in nasopharyngeal aspirates taken in a 10-year period from 28 hospitalized children and infants with respiratory tract infections who had signs and symptoms similar to those of HRSV infection.

Establishing sensitive methods for virus detection helps to clarify the relative contribution of different pathogens to the extent of illness in the community. This information is important for future development of specific antiviral therapies and vaccines. We examined specimens submitted from patients seen in general practice with influenzalike illnesses (ILI) during winter 2000-01 to detect HMPV as a possible cause of influenza- and HRSV-negative ILI.

Materials and Methods

Sentinel General Practice Networks

Clinical episodes of ILI are recorded by continuous monitoring in approximately 75 sentinel practices in England and Wales, covering a population of 700,000. New episodes of illness are noted and weekly returns submitted to the Royal College of General Practitioners research unit. ILI was defined as symptoms of fever, cough, and muscle pains with duration of [less than or equal to] 5 days (8,2). Virologic surveillance of ILI is performed by a subset of the sentinel practices. Combined nose and throat swabs are taken from persons diagnosed with ILI at the time they see a clinician, which is often several days after onset of illness. Swabs are mailed in virus transport medium to the Central Public Health Laboratory for analysis. Samples are divided into aliquots, labeled, and then frozen at -80[degrees]C on receipt.

HRSV and Influenza Detection

Detection of HRSV and influenza was performed prospectively on receipt of samples by using multiplex PCR as previously described (2,8).

HMPV Detection

Stored nucleic acid from 348 samples negative for influenza and HRSV was analyzed by using a PCR for HMPV (7). No optimization of the PCR detection was undertaken. Detection of HPMV was based on primers located in the L gene. For a further 60 (14.8%) samples from which the nucleic acid stored was insufficient, an aliquot of the original clinical material was re-extracted by using a Magnapure (Roche Diagnostics GmbH, Mannheim, Germany) automated extraction machine according to the manufacturer's instruction; RT was performed as described previously (8). Twenty microliters of eDNA was added to 80 [micro]L of reaction mix, which yielded a final concentration of 20 mM Tris-HCl pH8.4, 10 mM Mg[Cl.sub.2], 50 mM KCl, and 3 U Taq polymerase and 50 pmol of each primer. Amplification, using a DNA Engine thermocycler (MJ Research, Essex, England), consisted of 1 cycle at 94[degrees]C for 2 min, followed by 35 cycles of 94[degrees]C 1 min, 58[degrees]C 1 min, and 72[degrees]C for 1 min. Amplicons of the expected size (171 bp) were visualized by agarose gel electrophoresis. PCR sensitivity was determined by cloning of the PCR product into TOPO PCR4 vector (Invitrogen Corp., San Diego, CA) according to the manufacturer's instructions. Plasmid preparations were performed by using a Qiagen minprep kit (QIAGEN, Inc., Valencia, CA), according to the manufacturer's instructions. DNA quantitation followed by PCR, using the original primers and conditions (as above) with the addition of 0.1 mM of each deoxynucleoside triphosphate per reaction, was undertaken using the plasmid plus insert as template. Total RNA at the time of extraction was estimated by spectrophotometry at [A.sub.260].