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BRIEF REPORT
Human metapneumovirus strains circulating in Latin America
Josefina Garcia • Merly Sovero • Tadeusz Kochel • V. Alberto Laguna-Torres •
Maria Ester Gamero • Jorge Gomez • Felix Sanchez • Ana E. Arango •
Sergio Jaramillo • Eric S. Halsey
Received: 20 August 2011 / Accepted: 21 November 2011
� Springer-Verlag (outside the USA) 2011
Abstract The human metapneumovirus (HMPV) is
responsible for acute respiratory tract infections in young
children, elderly patients, and immunocompromised hosts.
In this study, we genetically analyzed the circulating
HMPV in Central and South America from July 2008 to
June 2009 and characterized the strains present in this
region. Samples were collected during an international
collaborative influenza like illness surveillance study and
then sequenced with specific primers for the HMPV G
gene. Our results show that two distinct clusters of HMPV
circulated in Central and South America, subtypes A2 and
B2 being the predominant strains.
Introduction
The human metapneumovirus (HMPV) is a relatively
recently discovered member of the family Paramyxoviri-
dae responsible for acute respiratory tract infections in
young children, elderly patients, and immunocompromised
hosts [5, 31]. It was first isolated in 2001 from nasopha-
ryngeal aspirates obtained from young children in the
Netherlands [32]. The virus has been detected in samples
from throughout the world, including Canada [7], Austra-
lia, Norway [11], Tunisia, France, Italy [8], Hong Kong
[22], Japan, the United States of America [36], South
Africa, Thailand, and Israel [6, 26].
HMPV is genetically related to the human respiratory
syncytial virus (HRSV). The clinical manifestations of
both infections in young children are indistinguishable
[6]. Studies have shown that HMPV is associated with
the common cold (complicated by otitis media in about
one-third of cases) and with lower respiratory tract ill-
nesses such as bronchiolitis, pneumonia, croup, and
exacerbation of reactive airways disease [20, 27]. Since
its discovery, this virus has also been detected in spec-
imens from adults, elderly, and immunocompromised
patients suffering from acute respiratory infections
[31]. Features of HMPV infections include tachypnea,
fever, cough, hypoxia, and changes on chest radiographs
such as infiltrates, hyperinflation, and peribronchial
cuffing [6].
Based on genomic sequencing and phylogenetic analy-
sis, there are two major genotypes of HMPV, designated A
and B [9, 13]. These analyses are based on sequencing and
comparison of the N, M, F, G, or L genes and genotype
grouping is concordant regardless of which gene is studied
[10]. Whether these two genotypes represent distinct ser-
otypes remains controversial. Each genotype appears to
The views expressed in this article are those of the authors and do not
necessarily reflect the official policy or position of the Department of
the Navy, Department of Defense, nor the U.S. Government.
J. Garcia (&) � M. Sovero � T. Kochel �V. A. Laguna-Torres � M. E. Gamero � E. S. Halsey
United States Naval Medical Research Unit 6, Lima, Peru
e-mail: josefina.garcia@med.navy.mil
J. Gomez
Direccion General de Epidemiologıa, Ministerio de Salud,
Lima, Peru
F. Sanchez
Hospital Infantil Manuel de Jesus Rivera, Managua, Nicaragua
A. E. Arango
Grupo Inmunovirologıa, Universidad de Antioquia,
Medellın, Colombia
S. Jaramillo
Hospital Pablo Tobon Uribe, Medellın, Colombia
123
Arch Virol
DOI 10.1007/s00705-011-1204-8
have at least two distinct subgroups, named A1 and A2,
and B1 and B2 [2, 19, 27, 33].
The genomic organization of the two viral genotypes is
identical. The major differences between the A and B
genotypes are nucleotide polymorphisms and the G and SH
proteins contain the highest concentration of these. The G
gene of HMPV displays significant strain-to-strain vari-
ability [3, 17, 23, 24, 27]. The G protein possesses 32 to
37% amino acid identity between the A and B genotypes of
HMPV [1, 2, 23].
Although many reports describe HMPV infection in
Latin America [12, 14, 16, 18, 21, 25, 28, 30], only a
handful characterize the genotypes of HMPV circulating in
the region [12, 16, 21, 25]. In this study, we focus on the
circulating strains in Latin America from July 2008 to July
2009, the period preceding the influenza A-panH1N1
appearance and dissemination in the region.
Material and methods
Ethics
The study protocol was approved by the Naval Medical
Research Center Institutional Review Board (Protocol
NMRCD.2002.0019) in compliance with all applicable
Federal regulations governing the protection of human
subjects. All participants underwent a verbal consent pro-
cess. No informed consent document was prepared since
samples were obtained through procedures considered less
than minimal risk by the mentioned IRB.
Specimen collection and identification of HMPV
positive samples
We collected 7,196 pharyngeal throat swab specimens at
hospitals throughout Central and South America from
patients, regardless of age, who presented with influenza
like illness (ILI), defined as fever (C to 38�C) plus either
cough or sore throat. In keeping with each hospital’s pre-
existing sample collection method, oropharyngeal swabs
were collected at all sites except in Nicaragua where
nasopharyngeal swabs were collected. Swabs were trans-
ported in viral transport media at -70�C to the Naval
Medical and Research Unit 6-Lima (NAMRU-6,
previously known as NMRCD) in Lima, Peru. Virus iso-
lation was carried out by inoculation into four cell lines for
virus isolation: Madin-Darby canine kidney (MDCK),
African green monkey kidney (Vero76 and VeroE6), and
Rhesus monkey kidney (LLCMK2) cells. Upon the
appearance of a cytopathic effect in any of these cell lines,
an immunofluorescence assay was performed to identify
the virus. The Respiratory Virus Screening and Identifica-
tion Kit (D3 DFA Respiratory Virus Diagnostic Hybrids;
Athens, OH) was utilized for the identification of adeno-
viruses, influenza A virus, influenza B virus, parainfluenza
viruses (types 1, 2, and 3), HRSV and HMPV. HMPV-
positive isolates obtained by isolation and immunofluo-
rescence were required for strain characterization. The
same data collection form was employed at all sites. This
form collected patient demographic data as well as signs
and symptoms of the acute illness.
RNA extraction and RT-PCR
Nucleic acid was extracted with the use of viral RNA kit
(QIAamp, Qiagen�; Valencia, CA) from 140 ll of the
naso/oro-pharyngeal swab solution and was amplified in a
reverse transcriptase PCR (RT-PCR). RT-PCR was per-
formed by using a SuperScript III One-Step RT-PCR
System (Invitrogen; San Diego, CA). The primers used
were specific to the G gene segments of HMPV:
hMPVG1F (ATG GAG GTG AAA GTG GAG AAC AT)
[2] and hMPVG1R (GTG GAT TCA TTG AGA GGA
TCC AT). For further verification, the isolates underwent
also a PCR with primers specific for the N gene: hmpv1
(CCC TTT GTT TCA GGC CAA) and hmpv2 (GCA GCT
TCA ACA GTA GCT). Amplification was carried out in a
thermocycler 7700 (Applied Biosystems; Foster City, CA).
Phylogenetic analysis
Phylogenetic trees were constructed on the basis of the G
and N gene sequences of 15 random samples. For direct
sequencing of viral nucleic acids from clinical specimens,
gene fragments were amplified and sequenced with the use
of Big Dye terminator cycle sequencing kit (version 3.1,
Applied Biosystems; Foster City, CA) on an ABI 3130
DNA Sequencer (Applied Biosystems; Foster City, CA).
Nucleotide sequences of PCR products were analyzed by
sequencing using Sequencher and BioEdit (version 7.0.0 -
Isis Pharmaceuticals, Inc.) software, and then aligned with
the CLUSTAL X version 2.0.1 software to compare with
HMPV sequences from the GenBank database.
Phylogenetic trees were constructed by the neighbor-
joining method in MEGA software (version 4). The statis-
tical significance of the tree topology was tested by boot-
strapping (1,000 replicas). Pairwise distances between and
within the genotypes at the nucleotide level were calculated
with Kimura 2 parameters and with Poisson correction at the
amino acid level with MEGA software. Sequences are
available on GenBank #JN604816-JN604830.
J. Garcia et al.
123
Results and discussion
This study used a convenience sample from an ILI sur-
veillance network in Central and South America to char-
acterize the strains of HMPV circulating in the region. We
detected HMPV in 32 of the 7,196 naso/oro-pharyngeal
samples collected in ten countries in Latin America
(Fig. 1A). Of the 32 samples identified, 17 were isolated
only on the LLCMK2 cell line, 4 were isolated only on the
Vero E6 cell line, and 10 were isolated on both.
Despite obtaining samples from a surveillance network
spanning ten countries, the 32 HMPV-positive samples
detected came from only three countries: Peru, Nicaragua,
and Colombia (Fig. 1A). Half came from male participants
(n = 16) and half from female participants (n = 16). The
average age was 18.4 years (with a minimum of one month
and a maximum of 72 years). Figure 1B shows that
patients of all ages were infected with HMPV. Eight of the
HMPV-positive patients had another virus identified,
mostly adenovirus (n = 3) and influenza B (n = 3) virus,
but also influenza A (n = 1) and enterovirus (n = 1). Of
the three countries where we detected HMPV, only Peru is
completely located in the Southern Hemisphere, and we
observed that the months of May (n = 3), June (n = 11),
and July (n = 1) accounted for more than half of the 26
isolations from this country.
Not surprisingly the most common signs and symptoms
were those of our inclusion criteria: fever (n = 32), cough
(n = 30) and a sore or swollen throat (n = 30). Frequent
non-inclusion criteria manifestations included malaise
(n = 23), headache (n = 19), tearing (n = 14), and eye
pain (n = 12). Gastrointestinal manifestations–including
abdominal pain (n = 6), vomiting (n = 6), and diarrhea
(n = 4)–affected a significant minority of participants.
Phylogenetic trees for the G and N proteins were
constructed and Fig. 2 shows the result obtained by ana-
lyzing a portion of the G protein. It illustrates a very clear
division of our samples into two subtypes, A2 and B2.
The phylogenetic tree constructed with the portion of the
N protein produced the same division of subtypes as those
found with the G protein, confirming both our G protein
findings and the close linkage between these two proteins
described in previous studies [24]. These results, together
with other reports from the region (Peru [16], Uruguay
[25], and Chile [12]), show a change in the circulating
strains between 2000-2003, when all subtypes of HMPV
were detected, and 2006-2009, when the A2 and B2
subtypes predominated.
Finally, we analyzed the amino acid sequences of the
extracellular region of the G protein and compared them
with sequences obtained from Peru in previous years (2002
and 2003) to detect any possible mutations. Compared to
its nucleotide variability, the HMPV G protein exhibits a
great amino acid of diversity, suggesting selective pressure,
although the reasons for this remain unknown [24]. Fig-
ure 3 shows sequences for all four genotypes with signifi-
cant differences between samples, even in what has been
proposed as the most conserved region (framed) [17]. In
respect to the B2 genotype, one of the viruses possessed the
mutation Q/R which had been detected in samples from
previous years in Peru and also in the BJ1816 sample from
Beijing-China. For the A2 genotype, there were two sam-
ples with mutations at the conserved region but at least five
other mutations were found (in red) that were present in
most of the isolates. We were not able to compare to
previous years’ sequences as we did not isolate this
genotype before in Peru. These alignments show a clear
variability of the G protein even at the conserved region of
the extracellular domain. The unique cysteine residue of
the extracellular domain is conserved throughout the B
genotype isolates and is not present on the A genotype
isolates consistent with prior reports [24]. Nevertheless, the
significance of such alterations in the extracellular portion
of the G protein remains speculative, as replication is not
hindered even in the absence of this region [4] and no
A
B
Country Number of Samples HMPV-positive
Argentina 350Bolivia 157Colombia 343 2Ecuador 546El Salvador 127Honduras 362Nicaragua 684 4Paraguay 277Peru 4110 26Venezuela 240
Total 7196 32
5
7
2
5
3
0
4
2
4
0
3
6
9
Nu
mb
er o
f H
MP
V-p
osi
tive
Age (years)
--
---
-
-
Fig. 1 HMPV-positive samples. This figure shows the number of
HMPV-positive samples (A) by country of collection and (B) by age
of participant
HMPV in Latin America
123
difference in disease severity has been noted between
viruses heterologous at this site [1].
A major limitation of our study was the method of
detection used. We used exclusively viral cell culture, and
although no ‘‘gold standard’’ exists for HMPV detection,
direct PCR is often the preferred method for identification
[27]. For this reason, frequencies and prevalences are not
addressed in this manuscript; we have focused solely on the
viruses detected by the ILI surveillance network already in
place.
Nevertheless, our culture-based detection techniques
allowed for a side-by-side comparison between two culture
media, LLCMK2 and Vero E6 cells. A higher number of
isolates were identified in LLCMK2 cells, a finding
reported by others [29]. Our use of culture may also have
impacted our co-infection results. Whereas previous stud-
ies have noted a predilection for HRSV to be found as a co-
infecting virus with HMPV [8], we did not discover a
single case of concomitant infection with these two viruses.
This could be attributable to the fact that HRSV, like
HMPV, may be hard to identify using culture methods
alone [34].
Although our data was collected over only one year and
should be interpreted with caution, a similar non-summer
seasonal predilection has been found in many studies from
the Southern [14, 15] and Northern Hemispheres [8, 35]
whereas only a handful of reports describe a summer pre-
dominance [22, 28].
FLA5055 Peru Dec2008
FLA5834 Peru Jan2009
FLA4032 Peru Nov2008
FLA4816 Peru Nov2008
FLA4882 Colombia Jul2009
FLA8694 Colombia Oct2008
FLA5066 Peru Dec2008
FLA4574 Peru Oct2008
FLA6964 Peru May2009
FLA3859 Colombia May2008
CAN97-83 / AY297749FLA9903 Peru Jun2009
NL/00/17 / FJ168779
NL/1/00 / AF371337
CAN99-81 / AY574224
Peru1-2002 / DQ393715
FLA4362 Peru Jul2008
BJ1816 / DQ843658
CAN99-80 / AY574247
NL/1/94 / AY296040
FLA8902 Nicaragua Jun2009
FLA9019 Nicaragua Jun2009
FLA9218 Peru Jun2009
Arg/1/02 / DQ362958
NL/1/99 / AY525843
JPS-194 / AY530094
Peru5-2003 / DQ393719
Peru4-2003 / DQ393718
0.1
A
B
A2
A1
96
88
87
83
B1
B2
99
92
100
Fig. 2 HMPV phylogenetictree. 601 nucleotides of the G
protein gene were amplified,
sequenced, and compared to
published sequences from
GenBank. We have labeled the
samples according to the
following format: ‘‘Sample code
/ Country of collection / Month-
Year of collection.’’ The
comparison sequences are
complete genome sequences
from GenBank labeled ‘‘Sample
code / Accession number.’’
Nucleotide sequences were
aligned by using Clustal X.
Phylogenetic analyses were
performed using the Kimura
two-parameter model as a
model of nucleotide substitution
and using the neighbor-joining
method to reconstruct
phylogenetic trees (MEGA
version 2.1)
J. Garcia et al.
123
We found a wide variety of clinical manifestations in
our patients infected with HMPV, highlighting the over-
lapping clinical syndromes associated with HMPV and
other respiratory viruses. An interesting finding was the
high prevalence of eye complaints such as tearing and pain,
symptoms described only infrequently in other reports [6].
On the other hand, our low level of gastrointestinal com-
plaints has been a common finding in other studies [11, 22,
36].
In summary, our results demonstrate the clinical and
molecular characteristics of HMPV isolates collected from
a large respiratory surveillance network in South and
Central America. In addition, our findings note the pres-
ence of HMPV in Nicaragua and Colombia and indicate a
possible shift in dominant HMPV subtypes in Peru from B1
and B2 to A2 and B2.
Acknowledgments We are grateful to Mrs. Victoria Espejo and
Mrs. Ada Romero for technical support. This study was funded by the
US Department of Defense Global Emerging Infections Surveil-
lance and Response System (DoD-GEIS), a division of the Armed
Forces Health Surveillance Center, WORK UNIT NUMBER:
847705.82000.25GB.B0016.
Conflict of interest None of the authors has a financial or personal
conflict of interest related to this study. The corresponding author had
full access to all data in the study and final responsibility for the
decision to submit this publication.
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