NEW MICROBIOLOGICA, 32, 297-301, 2009
Human metapneumovirus infection in a cohort
of young asymptomatic subjects
Rosalinda Bruno1, Stefania Marsico1, Chiara Minini2, Paola Apostoli2,
Simona Fiorentini2, Arnaldo Caruso2
1Section of Microbiology, Department of Pharmaco-Biology, University of Calabria, Rende (Cosenza), Italy;
2Section of Microbiology, Department of Experimental and Applied Medicine, University of Brescia, Italy
Section of Microbiology
Department of Experimental and Applied Medicine
University of Brescia
Piazzale Spedali Civili, 1 - 25123 Brescia, Italy
Human metapneumovirus (HMPV) was identi-
fied in 2001 in the Netherlands from nasopha-
ryngeal aspirate samples (Van den Hoogen et al.,
2001) of children with acute respiratory infec-
tions (ARI). HMPV has been classified into the
subfamily pneumovirinae within the family
paramyxoviridae (Van den Hoogen et al., 2001;
Galiano et al., 2006) genus metapneumovirus,
species human metapneumovirus. HMPV has
been identified worldwide (Kahn et al., 2006) and
longitudinal surveys have indicated that HMPV
has a seasonal distribution, mainly occurring dur-
ing winter to early spring (Van den Hoogen et al.,
2001; Bastien et al., 2003; Williams et al., 2006;
Caracciolo et al., 2008). Incidence of HMPV in-
fection can substantially vary from year to year,
being sometimes infrequent or rivaling respira-
tory syncitial virus (RSV) (Maggi et al., 2003;
Caracciolo et al., 2008).
Based on genomic sequencing and phylogenetic
analysis, there are two major genotypes of HMPV,
designated A and B (Biacchesi et al., 2003; Kahn,
2006) and each genotype appears to have at least
two distinct subgroups (MacKay et al., 2004). It
has been shown that concurrent annual circula-
tion of all four subgroups in the same geograph-
ical area is common, with a single, usually dif-
ferent, HMPV subgroup predominating each year
(Kahn 2006; Caracciolo
Seroprevalence studies in the Netherlands (Van
den Hoogen et al., 2001), Israel (Wolf et al., 2003)
and Japan (Ebihara et al., 2003) have shown that
by the age of 5 to 10 years, seropositivity reach-
es virtually 100%. Moreover, reinfections have
been observed in all age groups.
Since its initial description, HMPV has been con-
sidered a leading cause of serious lower respira-
tory diseases in infants and young children (Van
den Hoogen et al., 2001; Hamelin et al., 2004).
Belonging to the same subfamily, HMPV is com-
pared frequently with RSV because they share
many biologic properties and cause a similar, but
not identical, disease (Regev et al., 2006; Wolf et
al., 2006; Caracciolo et al., 2008).
The importance of HMPV infection in high risk
adults, especially among the elderly and im-
Human metapneumovirus (HMPV) is a recently described cause of respiratory infection. The purpose of this study was to
to further the available information on the circulation of HMPV among young asymptomatic subjects. The epidemiology of
HMPV was examined in a cohort of 73 university students during a winter season by using polymerase chain reaction.
HPMV was detected in 3/73 (4.1%) asymptomatic subjects. Phylogenetic analysis has shown that circulating viruses be-
longed to different HMPV sublineages. Our findings indicate that asymptomatic carriage of HMPV might be a neglected source
of viral transmission in the community.
KEY WORDS: Human metapneumovirus; molecular epidemiology
Received March 03, 2009
Accepted March 15, 2009
munocompromised hosts, has also been increas-
ingly recognized (Falsey et al., 2006) even if the re-
al pathogenic role of HMPV in some of these cat-
egories of patients remains uncertain (Debiaggi et
al., 2007). In symptomatic adults the clinical char-
acteristics of HMPV do not appear to be distinc-
tive from those of illness due to other winter res-
piratory viruses even if HMPV infection may be
somewhat less severe than RSV infection and in-
fluenza. Colds, asthma exacerbations, and in-
fluenza-like illness have been documented in old-
er children and young adults (Falsey et al., 2003;
Hamelin et al., 2004). On the other hand, HMPV
infections in younger healthy adults generally
have not been appreciated being often an asymp-
tomatic infection. Cases of reinfection in adult
subjects suggest that, despite universal infection
in childhood, new infections can occur through-
out life due to incompletely protective immune
responses and/or acquisition of new genotype.
At present, consistent data on the presence of
HMPV in asymptomatic persons in the commu-
nity are still not available.
The purpose of this study was to determine the
prevalence of HMPV in specimens obtained from
young students at Calabria University that were
randomly sampled. Students who were in good
general health were eligible for the study.
Exclusion criteria included current lower respi-
ratory symptoms, cardiovascular or other serious
disease (i.e., immunodeficiencies, cystic fibrosis)
or a history of chronic respiratory diseases.
A nasal swab specimen was collected from each
subject between January 15th and January 26th
2007 by gently rubbing the nasal turbinates with
a moistened cotton tip swab and placed in 2 ml
of transport medium (ViralPack, Biomedics s.r.l.,
Madrid, Spain). Specimens collected were main-
tained at 4°C and transferred within 4 hours af-
ter sampling to the Microbiology Laboratory at
the University of Calabria where they were im-
mediately frozen at -70°C. Specimens were
screened for the presence of HMPV, RSV, in-
fluenza A and B viruses, adenoviruses, and
parainfluenza viruses (serotypes 1-3) by PCR as-
says as previously described (Caracciolo et al.,
2008). Briefly, nucleic acids were extracted from
a 200 µl aliquot of samples with the RNeasy Kit
(Qiagen, Milan, Italy), according to the manu-
facturer’s instructions and cDNAs were synthe-
sized from 20 µl of eluted RNA using the cDNA
Archive mini kit (Applied Biosystems, Monza,
Italy). Primers used for the amplification of the
HMPV fusion (F) gene were as follows: F-f (5′-
GTY AGC TTC AGT CAA TTC AAC AGA AG-3′);
F-r (5′-CCT GTG CTG ACT TTG CAT GGG-3′)
(Huck et al., 2006). To confirm HMPV infection
samples were also amplified with HMPV N gene
primers (N-f: 5′-CCY TCA GCA CCA GAC ACA
CC-3′; N-r: 5′-AGA TTC AGG RCC CAT TTC TC-
3′). Finally, the amplified products were analyzed
by electrophoresis on agarose gel after ethidium
bromide staining, and the size of the amplicons
were compared with standard molecular weight
markers. To validate the amplification process
and to exclude carryover contamination, positive
and negative controls were run in each PCR. The
PCR products of the F gene of HMPV (nt 3,624-
4,130) were purified with Wizard SV Gel and PCR
Clean-Up System (Promega, Milan, Italy) and se-
quences were performed at the CRIBI BMR
Genomics Sequence Facility (Padua, Italy).
Sequences were aligned with prototype HMPV
strains that are representative of all the known
HMPV different subgroups [NL/1/00 (GenBank
accession number AF371337) for A1,1 JPS 03-240
(GenBank accession number AY530095) for A2b,
AY145296) for A2a, NL/1/99 (GenBank accession
number AY304361) for B1 and CAN98/79
(GenBank accession number AY145293) for B2]
by using the CLUSTAL W algorithm of the
BioEdit software (http://www.mbio.ncsu.edu/
bioEdit/bioedit.htlm). Phylogenetic trees were
constructed by FastME algorythm of the DAMBE
asp). The F gene of the avian pneumovirus sub-
group C (GenBank accession number AF187152)
was chosen as outgroup to root this phylogenet-
ic tree. The significance of the grouping was eval-
uated by bootstrap parsimony analysis.
Nasal swabs were obtained from asymtomatic
young volunteers referring to the Calabria
University Health Centre for routine medical ex-
aminations. All the volunteers gave their in-
formed consent to the proposed study. There were
53 females and 20 males with a median age of
24.3 years (range 19 to 29 years). At least one
identifiable respiratory virus was detected in 10
(13.7%) cases, whereas none was detected in 63
(86.3%). HMPV was detected in specimens ob-
tained from 3 (30%) of the total virus-infected
R. Bruno, S. Marsico, C. Minini, P. Apostoli, S. Fiorentini, A. Caruso
young adults, all females. RSV was detected in 5
(50%) subjects, 4 (80%) males and 1 (20%) fe-
male. The occurrence of HMPV and RSV was
4.1% and 6.8% respectively, when virus detection
was reported to the total number of cases (n =73)
examined. Distribution and characteristics of the
cohort taken into consideration are summarized
in Table 1. None of the HMPV-infected subjects
was referred for respiratory symptoms, nor had
a background respiratory disease.
Moreover, within the HMPV-infected population,
this virus was the solely pathogen found among
the microorganisms routinely tested. On the oth-
er hand, a significant percentage of RSV-infected
subjects (60%) were symptomatic. In this study
we never observed the concomitant presence of
multiple viruses in the same subject. Co-infec-
tions are instead frequent events in infected chil-
dren with severe respiratory diseases (Caracciolo
et al., 2008). If the lack of co-infection was due to
the development of more severe symptoms,
falling into one of the exclusion criteria or not, is
a question that remains to be addressed. It is
worth noting that during the season of observa-
tion (winter 2006/2007) influenza virus circula-
tion was rare (Caracciolo et al., 2008). In this
study we did not observe the presence of in-
fluenza virus either in symptomatic or asympto-
matic subjects. The lack of circulation of impor-
tant pathogens has been hypothesized as an event
favoring the circulation of other respiratory
pathogens. The relatively high rate of circulation
of HMPV and RSV observed in this study seems
to strengthen this hypothesis.
The circulation incidence of HMPV observed in
this study is in agreement with other studies
where 4.1% of asymptomatic young persons were
found infected with HMPV infection (Falsey et
al., 2003) whereas RSV asymptomatic infection
was a relatively uncommon finding (Hall et al.,
Asymptomatic respiratory infections both com-
munity- and nosocomially-acquired are recog-
nized as an important source of virus spreading
to risk population. In fact, even asymptomatic in-
fections may be accompanied by the release of
appreciable quantity of virus, as already demon-
strated for RSV (Hall et al., 1979). In this context,
it is relevant that HMPV infection is more fre-
quent in young healthy persons that in elderly
people (Falsey et al., 2003). This is likely explained
by their extensive contact with children (Hashem
et al., 2003) but they may, in turn, become a
source of infection. Moreover, it is worth stress-
ing that infections in adult individuals, even if
subclinical, may also represent an unrecognized
risk, since the virus population is expanding in
the face of a previous immune response. This sit-
uation might increase the probability of antigenic
variants being selected from the expanded viral
As shown in Figure 1A, sequencing of a portion
of the conserved F gene showed that two of the
three HMPV isolates were genetically related
(Cs01/06-7 and Cs01/06-73) whereas the third
(Cs01/06-14) showed consistent mutations. This
result suggests that more than one HMPV strain
may co-circulate in a restricted and crowded en-
vironment such as the university community and
that HMPV circulation within this population
may be not exclusively due to a spread within this
Human metapneumovirus infection in a cohort of young asymptomatic subjects
TABLE 1 - Characteristics of the cohort examined.
Total HMPVRSV Other viruses
Subjects 73 (100)3 (4.1) 5 (6.8) 2 (2.7)
53 (72.6) 3 (100) 4 (80)1 (50)
20 (27.4) 0 (0)1 (20)1 (50)
Age (Range) 23.3 (19-29)23.3 (21-27)22.2 (19-25) 25.5 (23-28)
Upper respiratory symptoms (%) 13 (17.8) 0 (0)3 (60)1 (50)
aCalculated within the virus-positive population.
This was confirmed by phylogenetic analysis. In
fact, Cs01/06-7 and Cs01/06-73 were found to be-
long to the genotype A2a while Cs01/06-14 was
related to the HMPV subgroup A2b (Figure 1B).
No HMPV subgroup A1, B1 and B2 were identi-
fied during this study period. This result is in
agreement with other epidemiological studies
showing that a concurrent annual circulation of
different HMPV subtypes is common, with a sin-
gle HMPV subtype predominating in a given year
(Kahn et al., 2006). We recently demonstrated that
during the winter 2006 circulation of HMPV was
indeed frequent in Italy with the subtype A2a pre-
dominating over the others (Caracciolo et al.,
In summary, our data demonstrated that asymp-
tomatic young adults can harbor HMPV and
might represent a neglected source of transmis-
sion of HMPV to risk populations.
Additional studies will be needed to fully deter-
mine the rate of HMPV circulation in different
symptomatic and asymptomatic populations as
well as their potential role in the spread of infec-
R. Bruno, S. Marsico, C. Minini, P. Apostoli, S. Fiorentini, A. Caruso
FIGURE 1 - (A) Nucleotide sequence alignment of the HMPV isolates from a region of gene F of HMPV. Viruses iden-
tified among the asymptomatic university population were compared with the reference viruses NL-001 (A1 strain),
CAN97/83 (A2a strain), JPS03-240 (A2b strain), NL/01/99 (B1 strain) and CAN99/75 (B2 strain) representing the pro-
totype HMPV strains and with primary HMPV isolates representative of each strain derived from a symptomatic pe-
diatric population (Caracciolo et al., 2008). Viruses detected in the present study have the prefix Cs. Dots indicate iden-
tical residues. Alignment was performed with the Clustal W program. (B) Phylogenetic analysis of HMPV strains cir-
culating during the winter season 2006-2007 was performed using the same F gene fragment. The divergence bar is
shown at the bottom of the figure.
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Human metapneumovirus infection in a cohort of young asymptomatic subjects
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