Genetic variability of the G glycoprotein gene of human metapneumovirus

Abstract

Human metapneumovirus (hMPV) has been associated with respiratory illnesses like those caused by human respiratory syncytial virus (HRSV) infection. Similar to other pneumoviruses, genetic diversity has been reported for hMPV. Little information is currently available on the genetic variability of the G glycoprotein (G), which is the most variable gene in RSV and avian pneumovirus. The complete nucleotide sequences of the G open reading frame (ORF) of 24 Canadian hMPV isolates were determined. Phylogenetic analysis showed the existence of two major groups or clusters (1 and 2). All but one of the hMPV isolates that we examined belonged to cluster 1. Additional genetic variability was observed in cluster 1, which separated into two genetic subclusters. Within cluster 1 the nucleotide sequence identity for the G ORF was 74.2 to 100%, and the identity for the predicted amino acid sequence was 61.4 to 100%. The G genes of cluster 1 isolates were more divergent from the cluster 2 isolates, with 45.6 to 50.5% and 34.2 to 37.2% identity levels for the nucleotide and amino acid sequences, respectively. Sequence analysis also revealed changes in stop codon usage, resulting in G proteins of different lengths (217, 219, 228, and 236 residues). Western blot analysis with the use of hMPV-specific polyclonal antisera to each hMPV cluster showed significant antigenic divergence between the G proteins of clusters 1 and 2. These results suggest that the G protein of hMPV is continuously evolving and that the genetic diversity observed for the hMPV genes is reflected in the antigenic variability, similar to HRSV.

FIG. 1.

Phylogenetic analysis of hMPV isolates. Nucleotide sequences were determined for the G ORF. The corresponding ORF sequences from previously reported hMPV Canadian (CAN97-83 and CAN98-75) and Dutch (NDL-01) isolates were also analyzed. Phylogenetic analysis was performed using the neighbor-joining method of the MEGA program. Bootstrap proportions were plotted at the main internal branches of the phylogram to show support values. The year in which the isolates was collected is indicated by the first (for example, CAN99-81) or last (for example, 40-02) two numerals in the isolate name.

FIG. 2.

(a) Alignment of the predicted amino acid sequences of the hMPV G protein of the hMPV Canadian isolates with the Dutch isolate NDL-01. Only residues that differ from isolate NDL-01 are shown; identical amino acids are represented by periods; gaps are represented by dashes. Potential N-linked glycosylation sites are shaded, and the conserved cysteine residue at position 27 is marked by a dot. (b) Predicted hydrophilicity profile of CAN99-81 (cluster 1) and CAN99-80 (cluster 2) isolates.

FIG. 2.

(a) Alignment of the predicted amino acid sequences of the hMPV G protein of the hMPV Canadian isolates with the Dutch isolate NDL-01. Only residues that differ from isolate NDL-01 are shown; identical amino acids are represented by periods; gaps are represented by dashes. Potential N-linked glycosylation sites are shaded, and the conserved cysteine residue at position 27 is marked by a dot. (b) Predicted hydrophilicity profile of CAN99-81 (cluster 1) and CAN99-80 (cluster 2) isolates.

FIG. 3.

Antigenic analysis of CAN99-80 (cluster 2) and CAN99-81 (cluster 1) G proteins by Western blotting with hMPV-specific polyclonal antisera raised against CAN99-81 (a) and CAN99-80 (b). Whole-cell lysates of recombinant baculovirus-infected SF21 cells expressing the G protein were analyzed. Mock-infected SF21 cells were used as a negative control. Numbers at left of each panel are molecular masses in kilodaltons.

FIG. 4.

Immunoprecipitation of the G glycoprotein of CAN99-80 and CAN99-81 with hMPV-specific polyclonal antisera raised against CAN99-80 and CAN99-81. The immunoprecipitated G proteins were analyzed on an SDS-10% polyacrylamide gel followed by fluorography. Numbers at left are molecular masses in kilodaltons.