A newly discovered human pneumovirus isolated from young children with respiratory tract disease

Abstract

From 28 young children in the Netherlands, we isolated a paramyxovirus that was identified as a tentative new member of the Metapneumovirus genus based on virological data, sequence homology and gene constellation. Previously, avian pneumovirus was the sole member of this recently assigned genus, hence the provisional name for the newly discovered virus: human metapneumovirus. The clinical symptoms of the children from whom the virus was isolated were similar to those caused by human respiratory syncytial virus infection, ranging from upper respiratory tract disease to severe bronchiolitis and pneumonia. Serological studies showed that by the age of five years, virtually all children in the Netherlands have been exposed to human metapneumovirus and that the virus has been circulating in humans for at least 50 years.

Figure 1. Electron micrograph of hMPV particles.

Virus concentrated from infected tMK–cell culture supernatants were visualized by negative contrast electron microscopy after PTA staining. Magnification, ×92,000.

Figure 2. Schematic representation of genomic fragments obtained from hMPV.

A schematic diagram of APV (3′ to 5′ end, left to right) is shown above the fragments obtained with RAP-PCR and RT-PCR on virus isolate 00-1. Fragments 1–10 were obtained using RAP-PCR. Fragment A was obtained with a primer in RAP-PCR fragments 1 and 2 and a primer designed on basis of alignment of leader and trailer sequences of APV and RSV (ref. 7). Fragment B was obtained with primers designed in RAP-PCR fragments 1 and 2 and RAP-PCR fragment 3. Fragment C was obtained with primers designed in RAP-PCR fragment 3 and RAP-PCR fragments 4–7.

Figure 3. Nucleotide and deduced amino-acid sequences for selected regions of the hMPV genome.

a and b, The nucleotide sequences for the 3′ end of the viral genome and the intergenic region between M and F ORFs. Note that the underlined sequence in a refers to the primer used for PCR amplification, and therefore does not necessarily reflect the actual hMPV leader sequence. c–f Comparison of the amino acid sequences of the putative N (panel c), P (panel d), M (panel e) and F (panel f) ORFs of hMPV, aligned with those of APV and RSV. Residues that differ between isolate 00-1 and the other viruses are shown, identical amino acids are represented by periods, gaps are represented by dashes.

Figure 4. Phylogenetic analysis of ORFs of hMPV and selected pneumoviruses.

a–d, The F (panel a), N (panel b), M (panel c), and P (panel d) ORFs of hMPV isolate 00-1 were aligned with those of other members of the genus Pneumovirinae, and phylogenetic trees were generated by maximum likelihood analyses using 100 bootstraps and 3 jumbles. The scale representing the number of nucleotide changes is shown for each tree. Bootstrap values are based on the consensus trees.

Figure 5. Phylogenetic analysis of 9 hMPV isolates.

a–d, Parts of the F (panel a), N (panel b), M (panel c) and L (panel d) ORFs of the indicated hMPV isolates were amplified by PCR and sequenced directly (fragments of 142, 71, 143, and 102 nucleotides, respectively). Phylogenetic trees were generated as described in Fig. 4. The first 2 numbers in the isolate names refer to the year from which the isolate originated. We were unable to amplify the F fragment for isolate 93-4.