Detection and Genetic Diversity of Human Metapneumovirus in
Hospitalized Children with Acute Respiratory Infections in Southwest
Cui Zhang, Li-Na Du, Zhi-Yong Zhang, Xian Qin, Xi Yang, Ping Liu, Xin Chen, Yao Zhao, En-Mei Liu, and Xiao-Dong Zhao
Division of Immunology, Children’s Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory
of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, China
lower respiratory tract infections (ALRTI) in the Netherlands
21, 24). Children ?5 years of age, elderly adults, and immuno-
compromised patients are at increased risk for severe hMPV in-
fection (23, 24). Morphologically, hMPV consists of a negative-
sense, single-stranded, and nonsegmented RNA that encodes at
least 9 distinct proteins (26). Among them, the two major trans-
duction of protective immune responses and therefore are anti-
(95% homology at the amino acid level between group A and B
homology between group A and B (3).
genetically and antigenically and have been classified into two
broad groups: group A and group B (4, 26), with each group di-
vided into genetic subgroups 1 and 2 (12). More recently, phylo-
genetic analysis showed a further bipartition of subgroup A2 into
two new genetic clusters designated A2a and A2b (3). Both anti-
genic group A and group B were noted to cocirculate in the same
city during the epidemic periods and had various patterns of pre-
Among all the sublineages of hMPV, the A2 sublineage shows
the greatest diversity. Antigenic variability is thought to contrib-
ute to reinfection throughout the life of the patient and may pose
a challenge to vaccine development. Future planning for vaccine
positions of the hMPV strains prevalent in target populations.
However, less information is available on the distribution pat-
uman metapneumovirus (hMPV) was first isolated in 2001
epidemiological study on hMPV in the Chongqing area from
April 2006 to March 2008, and genotype A2 was found to be the
most predominant during the study period (6). Thus, ongoing
epidemiological surveillance in a consecutive manner will help
assess the disease burden and genetic diversity of hMPV. The aim
of the present study was to evaluate the genetic diversity of the G
protein gene of hMPV in hospitalized infants and young children
with ALRTI in the past three epidemic periods in Chongqing.
be beneficial for the development of hMPV vaccines.
MATERIALS AND METHODS
Collection of specimens. From April 2008 to March 2011, NPAs were
obtained on 3 fixed days of each week from all children admitted to
Chongqing Children’s Hospital due to ALRTI. ALRTI was diagnosed ac-
cording to the criteria developed by the World Health Organization. The
specimens were immediately placed at 4°C in tubes containing 3 ml of
cold viral transport medium (phosphate-buffered saline [PBS], 100 U/?l
penicillin, and 100 ?g/ml streptomycin) and transported to the Depart-
ment of Virology within 4 to 6 h. Specimens were vortexed and centri-
nofluorescence assay (DFA) to detect seven common viral respiratory
Received 27 March 2012 Returned for modification 7 May 2012
Accepted 2 June 2012
Published ahead of print 12 June 2012
Address correspondence to Xiao-Dong Zhao, email@example.com.
C.Z., L.-N.D., and Z.-Y.Z. contributed equally to the article.
Supplemental material for this article may be found at http://jcm.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jcm.asm.org Journal of Clinical Microbiologyp. 2714–2719 August 2012 Volume 50 Number 8
pathogens (respiratory syncytial virus, parainfluenza virus 1, 2, and 3,
influenza virus A and B, and adenovirus) on the same day. The cell pellet
being adjusted to a suitable concentration. Subsequently, cells were fixed
was approved by the Ethics and Research Council of Chongqing Chil-
dren’s Hospital, and informed consent was obtained from the parents or
guardians of the children.
of frozen specimens with an RNeasy minikit (Qiagen, Germany) accord-
?l of DNase-free, RNase-free water. For cDNA synthesis, 100 ng of total
Real-time PCR assays. All NPAs were tested for hMPV by real-time
PCR. Real-time PCR primers and probes for the hMPV F gene were used
for the diagnosis of hMPV infection and designed based on the sequence
using real-time PCR with the Premix Ex Taq kit (Perfect Real Time; Ta-
KaRa, China) in a LightCycler instrument (Roche Diagnostics). Two mi-
croliters of cDNA was added to a 22-?l real-time PCR master mixture
cycling protocol was 94°C for 2 min, followed by 35 cycles of 94°C for 1
72°C. The positive control was defined at a cycle threshold (CT) of ?36.
The negative and positive controls were introduced in each real-time
PCR. The positive control was the cDNA from recombinant hMPV-in-
fected Vero E6 cells. The sequence of real-time PCR primers and probes
for the hMPV F gene was as follows: hMPV-F(F), 5=-CGTTTCTAACAT
GCCGACATCTG-3; hMPV-F(R), 5=-GCTCCCGTAGACCCCTATCA
G-3=; and the probe, 5=(FAM)-CCCTTTCTTCGCACCATCGCACGG(E
clipse)-3= (where FAM is 6-carboxyfluorescein). To verify the presence
hMPV F and G genes to ensure reproducibility. All PCR assays were ver-
ified to be able to amplify the F and G genes of the recombinant hMPV
PCR amplification of the full-length G protein gene. We selected 23
hMPV strains by using a random-digit table, including seven in the epi-
demic season of 2008 to 2009, seven in 2009 to 2010, and nine in 2010 to
2011. Primers used to amplify the full-length G protein were hMPVGF,
extension at 72°C for 10 min. Five microliters of PCR products were
subjected to 1.5% agarose gel electrophoresis, stained with GoldViewTM
nucleic acid stain (SBS Genetech), and visualized under UV light.
DNA sequencing. The PCR products were purified with a QIAquick
gel extraction kit (Qiagen, Germany) according to the manufacturer’s
instructions. The purified PCR products were sequenced in both direc-
tions using the same PCR primers for amplification on an ABI Prism 310
genetic analyzer (Applied Biosystems) by using an ABI Prism BigDye
terminator cycle sequencing ready reaction kit (Applied Biosystems).
A and B hMPV were compared independently with the available hMPV
sequences in the GenBank database using ClustalX version 1.81 and
manually edited with BioEdit version 7.0.1. Phylogenetic trees were
constructed by the neighbor-joining method in MEGA version 5. The
statistical significance of the tree topology was tested by bootstrapping
(1,000 replicas). The phylogenetic analysis included the hMPV sequences
in GenBank belonging to four different subgroups: A1, CHN03-
06(EF571502), FL-8-01(AY295989), CAN99-81(AY574224), and NL1-
00(AY296035); A2a, Arg-4-00(DQ362953) and CAN97-83(AY297749);
A2b, CHN07-06(EF571506), BJ1819(DQ270215), BJ1887(DQ843659),
00(AY296035), Arg-1-00(DQ362958), and NL-1-99(AY296034); and B2,
Nucleotide sequence accession numbers. The GenBank accession
numbers of the nucleotide sequences obtained in the present study are
JX082172 to JX082194.
from inpatients with ALRTI from April 2008 to March 2011, and
Positive rates of hMPV were 11.8% during 2008 to 2009, 7.31%
during 2009 to 2010, and 12.8% during 2010 to 2011. The preva-
lence of hMPV in Chongqing was high in April, May, and June of
2008 to 2009 and 2009 to 2010 while hMPV circulated predomi-
nantly during the late winter and spring of 2010 to 2011 (Fig. 1).
months). Most infected individuals were infants aged 0 to 6
months, accounting for 45.9% of subjects positive for hMPV in-
years accounted for 20.1%. (Table 1).
FIG 1 Seasonal distribution of hMPV infection in infants and children with ALRTI in Chongqing between April 2008 and March 2011.
Genetic Diversity of Human Metapneumovirus in China
August 2012 Volume 50 Number 8 jcm.asm.org 2715
All 144 hMPV-positive patients had clinical symptoms. The
(n ? 142; 99.1%), wheezing (n ? 102; 71.1%), and cyanosis (n ?
110; 85.0%). The hMPV-infected children were diagnosed with
bronchopneumonia (61.1%; 88/144), bronchiolitis (19.4%; 28/
144), bronchial asthma exacerbation (15.9%; 23/144), and acute
upper respiratory infection (3.4%; 5/144) (Table 2). Eighteen out
of 144 inpatients had comorbidities, including congenital heart
disease (15/144), tracheomalacia (3/144), and congenital laryn-
geal stridor (1/144). Eight patients developed respiratory failure
and one required mechanical ventilation. Among the eight pa-
tients with respiratory failure, congenital heart disease was iden-
virus type 3 (PIV3).
Other respiratory viruses coinfecting with hMPV. Addition-
ally, 29.2% (n ? 42) of hMPV-positive patients were found to have
RSV, 11 with PIV3, 6 with adenovirus, and 6 with influenza B virus.
Phylogenetic analysis and genotype distribution patterns.
Phylogenetic analysis of 23 hMPV strains in Chongqing con-
firmed two main genetic lineages, A and B. Interestingly, all the
strains in Chongqing were clustered as A2b, A1, and B1, and sub-
group B2 was not identified. During 2008 to 2009, strains from
subgroups A2b, A1, and B1 cocirculated, with 82% (n ? 9) be-
only subgroup A2b was identified in the next two epidemic sea-
sons (2009 to 2010 and 2010 to 2011). The results of phylogenetic
analysis of 23 hMPV strains are shown in Fig. 2.
Sequencing of the full length of the G protein gene revealed
homology ranging from 55.8 to 58.4% at the nucleotide level and
44.1 to 49.7% at the amino acid level between group A and B
totype strain NL/1/99 (94.9% at the nucleotide level; 91.9% at the
amino acid level), whereas subgroup A2b strains revealed homol-
ogy with the prototype strain CAN97-83 ranging from 89.6 to
99.8% at the nucleotide level and 82.3 to 84.9% at the amino acid
level. Subgroup A1 strains shared high homology with the proto-
type strain CAN99-81 (100% at the nucleotide level). In compar-
ison with the prototype A2 strains circulating from April 2006 to
March 2008 in Chongqing, 21 A2 strains prevalent from April
2008 to March 2011 revealed homology ranging from 95.7 to
97.7% at the nucleotide level and 94.3 to 96.2% at the amino acid
study shared high homology with the prototype B2 strains preva-
lent from April 2006 to March 2008 in Chongqing (92.8% at the
nucleotide level; 90.7% at the amino acid level).
Amino acid analysis. The comparisons of the deduced amino
acid sequence of the G protein gene of strains in Chongqing with
their prototype strains revealed that intracellular and transmem-
in the supplemental material). Most of the changes in amino acid
were observed in the extracellular domain due to nucleotide sub-
nt 694 (UGA), and nt 709 (UAA)], which corresponded to vari-
able lengths in polypeptides. Strains from subgroup A2b had two
(aa) (UAG) and 219 aa (UAG), whereas subgroup B1 strains ter-
The subgroup A1 strains terminated at the UAA stop codon and
exhibited protein with 236 aa.
Respiratory tract infections are the major cause of hospitalization of
sociated with respiratory infections include rhinoviruses, coronavi-
ruses, influenza viruses, parainfluenza viruses, RSV, and adenovi-
respiratory infections, hMPV, was described in the Netherlands.
hMPV is an important causative pathogen for ALRTI in children in
with a large population remains largely unknown due to the limita-
In the present study, 1,410 specimens were collected from pe-
diatric patients with ALRTI from April 2008 to March 2011 and
tested for hMPV by real-time RT-PCR. The enrolled patients ac-
counted for a significant proportion of total inpatients with
ALRTI in that period. Although this study was designed to enroll
were not necessarily free from selection bias because we collected
NPAs in the daytime only, and some of the admitted patients
refused to provide NPAs. The age and gender distributions and
diseases between the enrolled 1,410 patients in this study and the
total 9,231 inpatients in the same period were not statistically dif-
ferent, reflecting that the selected subjects may be relatively rep-
resentative. To detect hMPV, the F protein gene was chosen be-
cause it is highly conserved and has been used in previous studies
among hospitalized children with ALRTI in most studies (1, 18,
TABLE 1 Age distribution of 144 patients with hMPV infection
% of total
TABLE 2 Clinical features of 144 patients with hMPV infection
Clinical presentation/diagnostic disease
% of total
Bronchial asthma exacerbation
Acute upper respiratory infection
Zhang et al.
jcm.asm.orgJournal of Clinical Microbiology
22, 25) and 7.31% to 12.8% annually in this study by using real-
time PCR to detect the hMPV F protein gene. The prevalence of
46% hMPV infection varied from year to year, with the lowest in
hMPV infection was relatively high compared with that in some
other regions, probably due to a higher population density in this
seroprevalence studies have previously shown that 90 to 100% of
children are infected by the age of 5 to 10 years. Comparable to
other reports (4, 5, 20), the present study showed that 71.4% of
children with hMPV infection were ?2 years of age. The high
prevalence of hMPV infection in infants (?6 months) might be
attributed to weak immune function. The symptoms of hMPV
infection among children were similar to those described previ-
ously for respiratory syncytial viral infection (11, 21, 27). We
ated ALRTI were fever, cough, wheezing, and cyanosis. The main
diseases ranged from acute laryngotracheobronchitis, asthmatic
bronchitis, and bronchitis to bronchiolitis and pneumonia. We
erbations, and bronchiolitis as described previously (7, 8, 13).
Consistent with our findings, other studies have shown that
hMPV infection may lead to severe respiratory distress requiring
to an intensive care unit (2, 11, 14). Thus, hMPV infection is
associated with a substantial clinical and likely economic impact.
Although the winter-spring season is traditionally regarded as
for the comparison by selecting representatives of distinct clusters in previous studies and selecting isolates from GenBank that gave the best hits in BLAST
searches with each of the Chinese clusters. The genotype assignment is indicated by brackets on the right.
Genetic Diversity of Human Metapneumovirus in China
August 2012 Volume 50 Number 8jcm.asm.org 2717
the typical epidemic season of hMPV infection in the temperate
2010 and in the winter-spring season (November to February) of
2010 to 2011. The hMPV seasonality observed in this study was
comparable to that reported in Japan, which showed a biennial
hMPV infection, with a peak between November and March in
odd-numbered years and between March and June in the succes-
sive even-numbered years (19). The difference in the hMPV sea-
sonality between our study and other studies is unclear. Previous
studies have shown that climate can affect the seasonality of
hMPV infection (14, 18, 22). However, the climate records for
Chongqing fail to indicate significant variability in the same
months of different years. Apparently, more studies of a longer
duration are needed to elucidate the real pattern of hMPV circu-
lation in this region.
respiratory viruses, the potential for a coinfection likely existed.
Some studies have found a coinfection rate of 10% to 75% in
hMPV and other respiratory viruses (10, 15, 23). In this study,
coinfection of hMPV with other respiratory viruses was detected
in 29.2% of hMPV-infected patients, and the coinfected viruses
tion with RSV, 3 developed respiratory failure, indicating that
Phylogenetic analysis of G protein gene sequences in the
present study showed that both group A and B hMPV circu-
lated in Chongqing. Generally, group A hMPV is reported
more commonly than group B hMPV (9, 14, 16). In the present
study, among 23 hMPV strains, 22 strains were grouped as A
and 1 strain was grouped as B (subgroup B1). hMPV group A
was a likely predominant virus circulating in Chongqing,
which was consistent with other studies (8, 17, 20). Genotype
2008 to 2009, of which genotype A2b viruses were more com-
mon. In 2009 to 2010 and 2010 to 2011, only genotype A2b
in the present study. Indeed, circulation of hMPV of different
genotypes has been reported to vary annually with the replace-
ment of predominant genotypes every 1 to 3 years in a given
population. Such genotype replacement is believed to result
from adaptive immunity of a population to the predominant
dominant in the consecutive three epidemic seasons in
Chongqing, which was consistent with the findings in an epi-
demiological study from April 2006 to March 2008 (6). We
found that genotype A2b viruses prevalent in the study period
revealed high homology with those circulated from April 2006
to March 2008, which may be attributed to the low G gene
hMPV may be more pathogenic than group B hMPV (22), there
was no significant difference in the severity of ALRTI between
groups or subgroups because only one group B hMPV strain was
detected. Further studies are required to better understand the
clinical significance, seasonality, and molecular epidemiology of
The predicted G protein of different hMPV strains had differ-
ent lengths, which may be attributed to amino acid substitution
and insertion, deletion, and/or change in the stop codon. G pro-
circulating in China.
In summary, hMPV subgroups were observed among inpa-
seasons. Genotype A2b has become a dominant genotype in the
three epidemic seasons. This study thus contributes to a better
understanding of the molecular epidemiology of hMPV in main-
land China. Comprehensive information on the prevalence of
and thus may contribute to vaccine development.
This work was supported by the National Natural Science Foundation of
China (grant 30730098) and in part by the Chongqing Excellent Young
Scientists Fund (CSCT; grant 2008BA5040).
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