Genetic variability of human metapneumovirus amongst an all ages population in Cambodia between 2007 and 2009

Abstract

First identified in 2001, human metapneumovirus (HMPV) is a novel pathogen and causative agent of acute respiratory tract infection. Re-infection with HMPV is common, and currently there is no available vaccine against HMPV infection. Two genotypes of HMPV have been identified, A and B, both of which can be divided further into at least two distinct sub-genotypes. Here we report the results of the first study to investigate the genetic variability of HMPV strains circulating within Cambodia. The overall incidence of HMPV infection amongst an all-ages population of patients hospitalised with ALRI in Cambodia during 3 consecutive years, between 2007 and 2009, was 1.7%. The incidence of HMPV infection was highest amongst children less than 5 years of age, with pneumonia or bronchopneumonia the most frequent clinical diagnoses across all age groups. The incidence of HMPV infection varied annually. As anticipated, genetic diversity was low amongst the conserved F gene sequences but very high amongst G gene sequences, some strains sharing as little as 56.3% and 34.2% homology at the nucleotide and amino acid levels, respectively. Simultaneous co-circulation of strains belonging to the HMPV sub-genotypes B1, B2 and lineage A2b, amongst patients recruited at 2 geographically distinct provincial hospitals, was detected. Sub-genotype B2 strains were responsible for the majority of the infections detected, and a significant (p=0.013) association between infection with lineage A2b strains and disease severity was observed.

Fig. 1

(A) Seasonal distribution of HMPV positive samples. Data represent the incidence of HMPV infections amongst the total number of specimens tested from patients hospitalised for acute lower respiratory infections between June 2007 and December 2009. (B) Average monthly rainfall in Kampong Cham and Takeo provinces. Data represent the combined average monthly rainfall (mm) in Kampong Cham and Takeo provinces, for 2001 and 2002.

Fig. 2

Phylogenetic analysis of partial F gene sequences from Cambodian and reference HMPV strains. Phylogeny was constructed using the neighbour-joining method with 1000 bootstrap replicates. Only bootstrap values  > 70% are shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Cambodian strains are indicated by ‘Cam’ followed by the year of collection. Cambodian strains isolated from Takeo province are indicated by ●; strains collected in Kampong Cham province by ▴. Lineages are indicated. An avian metapneumovirus (AMPV) strain was used as an outgroup.

Fig. 3

Phylogenetic analysis of partial G ORF sequences from Cambodian and reference HMPV strains. Phylogeny was constructed using the neighbour-joining method with 1000 bootstrap replicates. Only bootstrap values  > 70% are shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. All positions containing gaps and missing data were eliminated. Cambodian strains are indicated by ‘Cam’ followed by the year of collection. Cambodian strains isolated from Takeo province are indicated by ●; strains collected in Kampong Cham province by ▴. Lineages are indicated. An avian metapneumovirus (AMPV) strain was used as an outgroup.

Fig. 4

Circulation of HMPV lineages in Cambodia from 2007 to 2009. Relative percentage of HMPV lineages detected following analysis of F and G gene sequences obtained during the study period by year and overall.