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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