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Dispatch
Global Genetic Diversity of
Human Metapneumovirus Fusion Gene
Guy Boivin,*
Ian Mackay,† Theo P. Sloots,† Shabir Madhi,‡ François Freymuth,§ Dana
Wolf,¶ Yonat Shemer-Avni,# Herbert Ludewick,‡ Gregory C. Gray,** and Éric
LeBlanc*
*Centre Hospitalier Universitaire de Québec and Laval University, Québec
City, Canada; †Royal Children’s Hospital, Herston, Queensland, Australia;
‡University of Witwatersrand, Bertsham, South Africa; §Centre Hospitalier
Universitaire de Caen, Caen, France; ¶Hadassah University Hospital, Jerusalem,
Israel; #Soroka Medical Center, Beer Sheva, Israel; and **University of
Iowa, Iowa City, Iowa, USA
Suggested citation
for this article:
Boivin G, Mackay I, Sloots TP, Madhi S, Freymuth F, Wolf D, et al. Global
genetic diversity of human metapneumovirus fusion gene. Emerg Infect
Dis [serial on the Internet]. 2004 Jun [date cited]. Available
from: http://www.cdc.gov/ncidod/EID/vol10no6/03-1097.htm
We analyzed 64 human
metapneumovirus strains from eight countries. Phylogenetic analysis
identified two groups (A and B, amino acid identity 93%–96%) and four
subgroups. Although group A strains predominated, accounting for 69%
of all strains, as many B as A strains were found in persons >3
years of age.
Studies from various parts of the world have identified human metapneumovirus
(HMPV) as one of the leading causes of hospitalization for acute respiratory
tract infections in young children (1–5). Severe respiratory
infections associated with HMPV have also been reported in elderly and
immunocompromised persons (6,7). Studies from our group
(6,8) and others (2,5) have identified
two major lineages of HMPV, with some studies indicating the splitting
of those groups into subgroups. Recently, the complete genomic sequence
of a representative strain from each of the two groups was determined
(9). In this study, our objective was to analyze the
fusion (F) gene sequences of a large set of HMPV strains collected from
various countries over several years and to identify sequence signatures
in different HMPV subtypes.
The Study
HMPV sequences included were from isolates grown in LLC-MK2 (monkey kidney)
cells or polymerase chain reaction (PCR)-amplified products from nasopharyngeal
aspirates (NPA) (1,3,5,6,10). Viral RNA was extracted
from 200 µL of cell culture supernatants or NPA specimens by using
the QIAamp viral RNA Mini Kit (QIAGEN, Inc., Mississauga, ON, Canada).
For phylogenetic studies, nucleotide sequences were obtained from amplified
HMPV F-gene products as previously described (6). The
sequence region comprising nucleotides 60-708 of the F gene was entered
into a multiple alignment generated by Clustal-W and corrected through
final visual inspection with the SeqLab application (Wisconsin package
version 10.3, Accelrys Inc., San Diego, CA). Phylogenetic analysis was
performed by using distance methods with the PAUP 4.0b10 program (Sinauer
Associates, Inc., Sunderland, MA) for the Macintosh. The parameters for
the distance method were Kimura 2-parameters, using the neighbor-joining
algorithm. Five hundred additional bootstrap analyses were performed on
those phylogenetic trees.
A total of 64 HMPV sequences were analyzed, 34 from Canada (years 1993–2002)
and 30 from various other countries (years 2000–2002): Peru (n = 2), the
United States (n = 2), France (n = 6), Israel (n = 5), Republic of South
Africa (n = 7), Australia (n = 7), and the prototype strain 001 (GenBank
accession no. AF371337) from the Netherlands. Phylogenetic analysis of
the HMPV F gene showed the existence of two main groups (A and B) that
could be further subdivided into two subgroups (1 and 2; Figure
1). Bootstrap analysis strongly supported dividing HMPV into the A
and B clusters (100% of bootstrap replicas) as well as subdividing them
into B1, B2, and A2 (99%–100% of bootstrap replicas). However, the topology
of the tree regarding the A1 subgroup was supported by only 71% of the
bootstrap replicas. This result may be explained by the strain AUS-2001-4,
which could not be firmly assigned to any of the two A subgroups. In addition,
even though the strain CAN-1993-1 was strongly supported as a member of
the A1 group by 91% of the bootstrap replicas, it was clearly the most
divergent, as can be expected from an older strain.
Identical F-gene sequences (648 nt) were found for some strains isolated
in the same year from the same country (CAN-1998-5/7, CAN-2000-11/14,
CAN-2001-18/19/20) but also for strains isolated in different years from
different countries (ISRAEL-2002-4 and CAN-1999-9, FRA-2001-5 and CAN-2002-23/25/27/28/29/32/33/34).
Topology of the phylogenetic tree was also supported by analysis of homology
between sequences. Nucleotide identity between groups A and B was 81.5%–85.3%,
whereas it was 91.6%–95.3% and 92.0%–94.1% between subgroups A1–A2 and
B1–B2, respectively. The sequences within the three subgroups A2, B1,
and B2 shared a nucleotide identity of 96.3%–99.0%, 96.0%–99.9%, and 97.2%–99.4%,
respectively. The A1 group was the most divergent; sequences shared 94.4%–99.9%
of nucleotide identity, which was consistent with phylogenetic data. AUS-2001-4
and CAN-1993-1 strains shared only 94.2% and 96.5% of nucleotide identity
with other members of the A1 subgroup. At the amino acid level, the identity
was 93.1%–96.3% between groups A and B, whereas it was 96.3%–99.1% and
98.1%–99.1% between subgroups A1–A2 and B1–B2, respectively. Within subgroups,
amino acid identity was 98.2%–100%, 96.8%–100%, 99.5%–100%, and 99.1%–100%
for subgroups A1, A2, B1, and B2, respectively. Based on phylogenetic
analysis, the distance between the two HMPV groups was slightly smaller
than between prototypes from the two respiratory syncytial virus (RSV)
groups (14.5 versus 16.5 substitutions/100 residues, data not shown).
An amino acid alignment of all distinct HMPV F sequences (representing
amino acids 20–233 of the prototype strain NETH-001), as well as those
of other metapneumoviruses and pneumoviruses, is shown in Figure
2. Cysteine residues were conserved at positions 28, 60, and 182 in
all HMPV strains. Analysis of this multiple sequence alignment showed
six amino acid substitutions found at positions 61, 122, 135, 167, 175,
and 233 that may be used as signature sequences to differentiate group
A and B strains. In addition, we also identified an amino acid substitution
at position 185 that may allow us to differentiate the A1 subtype from
others, whereas substitutions specific to B2 subtypes were located at
positions 143 and 179. Some of these substitutions were located in potential
functional domains, such as the fusion domain (codon 122) and the heptad
repeat A (HRA) region (codons 135, 143, and 167). Two potential N-glycosylation
sites were found in all HMPV sequences.
We investigated associations between HMPV genotypes and demographic or
clinical data, although the small number of non-Canadian strains limited
analyses. Based on phylogenetic analysis, a total of 44 (68.8%) HMPV strains
were classified in group A (9 [14.1%] A1 and 35 [54.7%] A2), whereas 20
(31.2%) belonged to group B (15 [23.4%] B1 and 5 [7.8%] B2). Group B strains
accounted for 38.2% and 23.3% of Canadian and non-Canadian strains, respectively
(p = 0.20). No B strains were recovered from France, Australia, and Israel
as part of this study. Co-circulation of both A and B strains was found
in Canada during 2000, 2001, and 2002 as well as in South Africa during
2000. Among the 26 HMPV strains recovered before 2001, 57.7% were from
group A compared to 42.3% from group B, whereas the ratios were 76.3%
and 23.7% for the 38 strains analyzed in 2001 and 2002 (p = 0.12). When
analysis was restricted to Canadian strains, group A strains accounted
for 46.7% of all HMPV recovered before 2001 and 73.7% of HMPV recovered
after 2001 (p = 0.11). All but one of the recent (year 2001 or later)
A strains were from the A2 subgroup.
Subsequent analyses were performed without the prototype strain NETH-001,
for which no detailed information was available. Most strains from the
Northern Hemisphere (41 [87.2%] of 47) and the Southern Hemisphere (11
[68.8%] of 16) were recovered over the typical respiratory virus season,
spanning a 5-month period during winter and spring. Most (69.8%) of the
HMPV strains were recovered from young children <3 years, whereas
only 25.6% were from adults >18 years of age, although this
finding may only reflect more intense investigation into viral cases in
children. Group B strains accounted for 25% of total HMPV strains from
study participants <3 years of age compared to 47.4% of those
from patients >3 years of age (p = 0.08). The ratios of group A/group
B strains were 62.5%/37.5% for the 32 male and 74.2%/25.8% for the 31
female patients (p = 0.32). Groups A and B strains were associated with
a similar proportion of cases of pneumonitis (12 [27.9%] of 43 vs. 7 [35%]
of 20, p = 0.57). Finally, although group B strains were found in approximately
half of the positive LLC-MK2 cultures (14 [53.8%] of 26), such strains
only represented 16.2% of total HMPV strains detected by reverse transcription–PCR
from NPA samples (p = 0.002).
Conclusions
Our results, based on a large dataset of viral strains collected over
several years from the Northern and Southern Hemispheres, confirm two
main HMPV groups and at least four minor subgroups. One strain (AUS-2001-4)
could not be ascribed to one of the two A subtypes and may constitute
a third subtype. We have further demonstrated that both HMPV genotypes
could circulate in a single year during a typical respiratory virus season,
i.e., over the winter and early spring months in countries in both hemispheres.
Our phylogenetic data were based on analysis of the HMPV F protein, one
of two viral glycoproteins considered the major antigenic determinant
in RSV (11). Our data confirm that HMPV is more closely
related to avian pneumovirus type C than to RSV, which is classified in
a separate genus (Pneumovirus) within the same subfamily.
Overall, the amino acid sequence identity of the two HMPV F groups was
slightly higher than that calculated for the two RSV groups (93%–96%
vs. 89%) (11). Although only the first half of the F
gene was sequenced in this study, our data indicate that all A and B strains
can be easily differentiated on the basis of unique amino acid changes,
some of which are located in functional regions of the protein, e.g.,
the putative fusion domain and the HRA region implicated in paramyxovirus
fusion to their cell receptors. Some of the latter amino acid changes
were nonconservative. Of note, the sequence signatures reported here were
also present in two other recent Canadian isolates representing the two
major HMPV groups (12). Additional studies will be required
to evaluate the impact of such changes on pathogenesis and immune response.
Close to 70% of all HMPV strains belonged to group A with a possible
shift towards more A2 strains in recent years. However, more young children
were evaluated in our study, and the group A genotype infects three times
as many children <3 years of age. In addition, group B strains, which
occurred more frequently in adults, could have been underestimated, as
suggested by the detection of fewer group B strains in clinical samples
(16%) than in infected LLC-MK2 cells (54%). In that regard, most HMPV
PCR primers designed so far have been selected from the sequence of strain
NETH-001 (which belongs to group A2 in this study), and consequently,
they may not have been optimal to detect group B strains. Alternatively,
isolating HMPV group A strains may be less efficient in LLC-MK2 cells.
Additional studies are required to validate these hypotheses. In RSV,
group A strains are often thought to be associated with more severe disease
than are group B strains (13). However, we found no
differences in severity between the two HMPV genotypes when we used pneumonitis
as the clinical endpoint in this small, retrospective study.
Our study has some limitations. We analyzed relatively few non-Canadian
strains over a short period of time, consistent with the recent description
of this viral pathogen in 2001. Also, much more genetic variability could
have been observed by sequencing the gene encoding for the attachment
glycoprotein (G gene) as suggested by RSV and limited HMPV sequences (14,15).
Using different PCR primers to initially identify HMPV and studying different
populations in the various centers may have also introduced bias in interpreting
our results. Nevertheless, our study confirms the worldwide distribution
of HMPV and provides initial insights into the epidemiology of the two
main viral genotypes.
Dr. Boivin is an
infectious disease specialist and virologist at the Centre Hospitalier
Universitaire de Québec. He is also an associate professor of microbiology
at Laval University, Québec City, Québec, Canada. His main research
interests include the characterization of old and emerging respiratory
viruses and the mechanisms of antiviral drug resistance for herpesviruses.
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