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Dispatch
Human Metapneumovirus in Severe
Respiratory Syncytial Virus Bronchiolitis
Julie Greensill,* Paul S. McNamara,* Winifred Dove,* Brian Flanagan,*
Rosalind L. Smyth,* and C. Anthony Hart*
*University of Liverpool, Liverpool, United Kingdom
Suggested citation for this article: Greensill
J, McNamara PS, Dove W, Flanagan B, Smyth RL, Hart CA. Human metapneumovirus
in severe respiratory syncytial virus bronchiolitis. Emerg Infect Dis
[serial online] 2003 Mar [date cited]. Available from: URL: http://www.cdc.gov/ncidod/EID/vol9no3/02-0289.htm
Reverse transcription-polymerase
chain reaction was used to detect segments of the M (matrix), N (nucleoprotein),
and F (fusion) genes of human metapneumovirus in bronchoalveolar fluid
from 30 infants with severe respiratory syncytial virus bronchiolitis.
Seventy percent of them were coinfected with metapneumovirus. Such coinfection
might be a factor influencing the severity of bronchiolitis.
Bronchiolitis is an important cause of illness and death in infants.
An estimated 25 in every 1,000 children will require hospitalization with
bronchiolitis; for 1% to 2% of these children, infection will be severe
enough to require ventilatory support (1). The etiologic
agents of bronchiolitis include adenovirus, influenza and parainfluenza
viruses, and Bordetella pertussis. However, most infections are
due to respiratory syncytial virus (RSV) (2). In June
2001, Osterhaus and colleagues reported the discovery of a “new” human
respiratory virus (Pneumovirinae subfamily, Paramyxoviridae
family) (3). RSV is in the genus Pneumovirus,
and the new virus, although related to RSV, is more closely related to
avian pneumovirus serotype C (previously known as turkey rhinotracheitis
virus) (4), the only other member of the Metapneumovirus
genus. The new virus, however, does not infect chickens or turkeys (3),
and they are unlikely to be a zoonotic source. Thus, the new virus was
termed human metapneumovirus (hMPV). Of 28 isolates, all isolated in the
winter, 13 came from infants <12 months of age who had disease patterns
resembling those seen in RSV infections. Subsequently, human metapneumovirus
has been detected in children in Australia (5) and Canada
(6).
The Study
As part of a larger study examining the immunopathogenesis of severe
bronchiolitis, nonbronchoscopic bronchoalveolar lavage samples were collected
from 30 infants ventilated with RSV bronchiolitis (determined by antigen
detection in nasopharyngeal aspirates) in 2000 and 2001. The sampling
was performed according to recent European Respiratory Society guidelines
(7). Briefly, a suction catheter was passed down the
endotracheal tube beyond the bifurcation of the bronchi. Two 1-mL/kg aliquots
of sterile normal saline were instilled separately down the suction catheter.
Lavage fluid was recovered with constant suction pressure into a mucous
trap. Samples were frozen at –80°C until analyzed. Control samples were
obtained in winter from children within the same age range who were being
intubated and ventilated for noninfection-related conditions. The local
research ethics committee approved the study, and informed consent was
obtained from parents.
Detection of the human metapneumovirus genome was performed by reverse
transcription-polymerase chain amplification (RT-PCR) of the matrix (M),
fusion (F), and nucleoprotein (N) genes. RNA was extracted from the specimens
by the guanidine/silica method (8), and 15 µL was added
to a 50-µL RT reaction with a polyT primer and 1.25 U AMV-RT (Gibco, Basingstoke,
U.K.) to produce cDNA. Each PCR reaction contained 10 µL of cDNA with
2.5 U Amplitaq Gold (Applied Biosystems, Warrington, U.K.), 2.0 mM MgCl2,
300 µM deoxynucleoside triphosphate, 0.4 µM of each primer, and the buffer
supplied. For amplification of the M gene, primers hMPV-MF1 (AAG TGA ATG
CAT CAG CCC AAG) and hMPV-MR1 (CAC AGA CTG TGA GTT TGT CAA A), which amplified
the region between nucleotides 212 and 331, were used to give an amplicon
of 120 bp. PCR cycling conditions were 95°C for 10 min followed by 45
cycles of 95°C for 1 min, 58°C for 1 min, 72°C for 1 min, and a final
extension step of 72°C for 10 min in a Perkin-Elmer (Warrington, Cheshire,
U.K.) 2400 model thermal cycler. Positive samples were confirmed by PCR
amplification for the F or N gene products, or by amplification of a longer
product of the M gene. The longer M product used primer hMPV-MR1 and a
new primer, hMPV-MF2 (ATG GAG TCC TAT CTA GTA GAC A), amplifying the region
between nucleotides 1 and 331 of the M gene, yielded a 331-bp product.
For the F PCR, primers hMPV-FF1 (GTG AGC TGT TCC ATT GGC AG) and hMPV-FR1
(CCC TCA ACT TTG CTT AGC TGA TA) amplified the region between nucleotides
1162 and 1295 of the F protein to give a 134-bp PCR product. PCR cycling
conditions were as for the M protein but with an annealing temperature
of 62°C. The N PCR used primers hMPV-NF1 (GTA TTA CAG AAG TTT GTT CAT
TGA G) and hMPV-NR1 (GAG AAC AAC ACT TGC AAA GTT GG), which amplified
the region between nucleotides 710 and 1034 of the N protein to give a
325-bp product. PCR cycling conditions were as for the F protein. In each
case, the primers were designed by aligning the M, N, and F gene sequences
of the original Dutch isolates (93.1, 93.2, 93.3, 94.1, 94.2, 99.1, 99.2,
and 00.1) (3) to define regions that were held in common.
The primers did not amplify sequences from either human RSV or avian pneumovirus.
Selected PCR products were cloned into a TA cloning vector (pGEM-T, Promega,
Southampton, Hampshire, U.K.), and the sequence was determined to confirm
the identity of the virus detected by the PCR reaction. Sequences from
the M, N, and F proteins of one virus (LIV08) have been deposited in EMBL,
with the accession numbers AJ439505, AJ439504, and AJ439503, respectively.
The phylogenetic relationship between isolates was examined. Human metapneumovirus
sequences were aligned with the CLUSTALX multiple alignment program (EMBL,
Heidelberg, Germany), and analyses were carried out by using the Phylogeny
Inference Package (PHYLIP version 3.5c, (J. Felenstein and the University
of Washington). DNA sequences were analyzed by using DNADIST followed
by neighbor joining; 100 replica samplings were analyzed. Detection of
RSV by RT-PCR and N gene typing was as described (9).
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Figure
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Click to view
enlarged image
Figure. The phylogenetic relationship
of human metapneumovirus from Liverpool (LIV08) to those from Holland
and to avian pneumovirus...
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M gene and at least one other of N, F, or long M amplicons of human metapneumovirus
were detected in bronchoscopic bronchoalveolar lavage fluids from 21 (70%)
of 30 infants ventilated for RSV bronchiolitis (Table).
Four of the M-gene amplicons were cloned and sequenced. Amplicons were
cut from agarose gels, eluted, and purified by using a gel purification
kit (QIAGEN, West Sussex, U.K.). The amplicons were cloned into pGEM-T
(Promega), and sequencing was conducted with MI3 primers by Lark Technologies
(Essex, U.K.). In each case, the amplicons were very similar (1 base different)
to each other and closely related to those reported for human metapneumovirus
(82% to 97%) (3). The Figure shows
a neighbor-joining tree that uses a 102-bp (nucleotide 208–309) region
of the M gene, demonstrating the relationship of a Liverpool (LIV08) strain
to those from Holland. RSV was detectable by RT-PCR in only 24 (80%) of
30 nonbronchoscopic bronchoalveolar lavage samples, despite RSV antigens
being detectable from all nasopharyngeal aspirates at the onset of illness.
Of these 24, 23 could be assigned an N genotype (18 NP-4, 4 NP-2, 1 NP-3).
Human metapneumovirus coinfection was detectable with each of the genotypes,
although only one (25%) of the four infants infected with NP-2 was coinfected
with human metapneumovirus, compared with 13 (76%) of 17 of those with
NP-4. Human metapneumovirus was detectable in three infants from whom
no RSV amplicon could be obtained. Neither RSV nor human metapneumovirus
amplicons were detectable in nonbronchoscopic bronchoalveolar lavage fluid
from 10 control patients ventilated for reasons unrelated to bronchiolitis.
Most infants have serologic evidence of infection with RSV by 2 years
of age (10), but other than host factors such as underlying
cardiac, respiratory, or genetic factors and prematurity, why severe disease
develops in some infants and mild disease in others is unclear. For example,
although 87% of children have antibodies to RSV by age 18 months (11),
most children have only upper respiratory tract symptoms such as rhinitis,
cough, and coryza (12). The age of the patient is also
important because lower respiratory tract signs develop in up to 40% of
infants <1 year, and 0.5% to 2% of all infants need to be hospitalized
(12). Coinfection with RSV and other pathogens has been
described previously. In studies in Northern Ireland (13)
and India (14), coinfection occurred in 1.1% (all with
rhinovirus) and 15% (with influenza and parainfluenza viruses) of patients
with RSV acute lower respiratory tract infection, respectively. However,
apart from in viral infections that might alter host immunity (15),
acute respiratory tract infections involving RSV alone were
not significantly different from those caused by RSV and another virus
(16). RSV and human metapneumovirus might have similar
seasonal patterns (3), so coinfection is possible. In
the present study, we detected coinfection with human metapneumovirus
in 70% of infants with RSV bronchiolitis sufficiently severe to require
admission to the pediatric intensive-care unit for ventilatory support.
Sequence analysis of a portion of the M gene from four of the detected
human metapneumoviruses indicated that they were closely related both
to each other and to the Dutch isolates. This high rate of coinfection
raises the possibility that coinfection with RSV and human metapneumovirus
might be another determinant of RSV disease severity. Indeed, of 10 studied
infants with severe RSV bronchiolitis and no other risk factors for severe
disease, 9 (90%) were coinfected with human metapneumovirus. Confirmation
of a role for coinfection will require longitudinal studies over several
bronchiolitis seasons, comparing disease severity with frequency of coinfection
across the full spectrum of RSV disease. Finally, this high RSV–human
metapneumovirus coinfection rate in severe disease means that previous
studies into RSV subtype/genotype, disease severity, and cytokine and
chemokine expression in RSV disease may need to be reevaluated.
Action Research
provided financial support for this study.
Dr. Greensill is
a postdoctoral fellow working on characterization of Kaposi’s sarcoma–like
herpesviruses in Old World monkeys. Her doctoral degree focused on investigating
the pathogenesis and molecular epidemiology of respiratory syncytial
virus.
References
- Stott EJ, Taylor G. Respiratory
syncytial virus: brief overview. Arch Virol 1985;84:1–52.
- McNamara PS, Smyth RL. The
pathogenesis of respiratory syncytial virus in childhood. Br Med
Bull 2002;61:13–28.
- van den Hoogen BG, deJong JC, Groen J, Kuiken T, de Groot R, Fouchier
RAM, et al. A
newly discovered human pneumovirus isolated from young children with
respiratory tract disease. Nat Med 2001;7:719–24.
- Collins MS, Gough RE. Characterization
of a virus associated with turkey rhinotracheitis. J Gen Virol 1988;69:909–16.
- Nissen MD, Siebert DJ, Mackay IM, Sloots TP, Withers SJ. Evidence
of human metapneumovirus in Australian children. Med J Aust 2002;176:188.
- Peret TCT, Boivin G, Li Y, Couillard M, Humphrey C, Osterhaus ADME,
et al. Characterization
of human metapneumoviruses isolated from patients in North America.
J Infect Dis 2002;185:1660–3.
- de Blic J, Midulla F, Barbato A, Clement A, Dab I, Eber E, et al.
Bronchoalveolar
lavage in children: ERS Task Force on bronchoalveolar lavage in children:
European Respiratory Society. Eur Respir J 2000;15:217–31.
- Fletcher JN, Smyth RL, Thomas HM, Ashby D, Hart CA. Respiratory
syncytial virus genotypes and disease severity among children in hospital.
Arch Dis Child 1997;77:508–11.
- Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van
der Noordaa J. Rapid
and simple method for purification of nucleic acids. J Clin Microbiol
1990;28:495–503.
- Kim HW, Arrobio JO, Brandt CD, Jeffries BC, Pyles G, Reid JL, et al.
Epidemiology
of respiratory syncytial virus infection in Washington, D.C. 1: importance
of the virus in different respiratory tract disease syndromes and temporal
distribution of infection. Am J Epidemiol 1973;98:216–25.
- Simoes EA. Respiratory
syncytial virus infection. Lancet 1999;354:847–52.
- Hall CB. Respiratory syncytial virus. In: Feigin RD, Cherry JD, editors.
Textbook of pediatric infectious diseases. Philadelphia: WB Saunders;
1998. p. 2084–111.
- Ong GM, Wyatt DE, O’Neill HJ, McCaughey C, Coyle PV. A
comparison of nested polymerase chain reaction and immunofluorescence
for the diagnosis of respiratory infections in children with bronchiolitis
and the implication for a cohorting strategy. J Hosp Infect 2001;49:122–8.
- Maitreyi RS, Broor S, Kabra SK, Ghosh M, Seth P, Dar L, et al. Rapid
detection of respiratory viruses by centrifugation enhanced culture
from children with acute respiratory tract infections. J Clin Virol
2000;16:41–7.
- Abughali N, Khiyami A, Birnkrant DJ, Kumar ML. Severe
respiratory syncytial virus pneumonia associated with primary Epstein-Barr
virus infection. Pediatr Pulmonol 2002;33:395–8.
- Ray CG, Minnich LL, Holberg CJ, Shehab ZM, Wright AL, Barton LL, et
al. Respiratory
syncytial virus associated lower respiratory illnesses: possible influence
of other agents. Pediatr Infect Dis J 1993;12:15–9.
| Table.
Characteristics of infants with severe bronchiolitisa |
|
| Pt. no. |
Age (weeks)
|
Weight (kg)
|
Predisposing factors
|
RSV
|
hMPV
|
Days of illness before BAL
|
|
|
1
|
3
|
3.5
|
None
|
NP-4
|
+
|
8
|
|
2b
|
48
|
6.6
|
Premature, BPD
|
–
|
+
|
6
|
|
3
|
18
|
7.5
|
None
|
NP-4
|
+
|
7
|
|
4
|
35
|
6.0
|
Cardiac, genetic
|
–
|
+
|
7
|
|
5
|
11
|
2.8
|
Premature
|
NP-4
|
+
|
6
|
|
6
|
21
|
5.5
|
Premature
|
NP-4
|
+
|
4
|
|
7
|
3
|
2.3
|
Premature
|
–
|
–
|
7
|
|
8
|
5
|
2.5
|
Premature
|
NP-4
|
+
|
3
|
|
9
|
18
|
5.1
|
Premature, cardiac
|
NP-2
|
–
|
6
|
|
10
|
11
|
5.7
|
None
|
NP-4
|
+
|
10
|
|
11
|
21
|
7.5
|
None
|
NP-2
|
+
|
9
|
|
12
|
9
|
2.0
|
Premature
|
NP-4
|
–
|
3
|
|
13b
|
16
|
4.4
|
Premature, cardiac
|
NP-2
|
–
|
3
|
|
14
|
7
|
2.6
|
Premature
|
NP-3
|
+
|
5
|
|
15
|
15
|
2.8
|
Premature
|
NP-4
|
+
|
5
|
|
16
|
4
|
4.0
|
None
|
NP-4
|
+
|
2
|
|
17
|
7
|
4.3
|
None
|
NP-4
|
+
|
5
|
|
18
|
12
|
3.2
|
Premature, cardiac
|
NP-4
|
–
|
6
|
|
19
|
12
|
5.4
|
None
|
–
|
+
|
13
|
|
20
|
5
|
3.2
|
None
|
NP-4
|
+
|
7
|
|
21
|
5
|
2.8
|
Premature
|
NP-2
|
–
|
5
|
|
22
|
9
|
4.7
|
Cardiac
|
NP-4
|
–
|
8
|
|
23b
|
22
|
5.7
|
Genetic
|
NP-4
|
+
|
4
|
|
24
|
7
|
3.4
|
Premature
|
+
|
+
|
11
|
|
25
|
19
|
4.8
|
Premature, BPD
|
NP-4
|
–
|
6
|
|
26
|
13
|
3.4
|
Premature
|
NP-4
|
+
|
6
|
|
27
|
8
|
2.5
|
Premature
|
NP-4
|
+
|
6
|
|
28
|
29
|
3.5
|
Premature, BPD
|
–
|
+
|
6
|
|
29
|
3
|
4.1
|
None
|
NP-4
|
–
|
5
|
|
30
|
5
|
4.1
|
None
|
–
|
+
|
5
|
|
| aAbbreviations
used: Pt., patient; RSV, respiratory syncytial virus; hMPV, human
metapneumovirus; BPD: bronchopulmonary dysplasia; BAL, bronchoalveolar
lavage; NP, nucleoprotein genotype. |
| bDied. |
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