ONLINE DEPOSITORY MATERIAL:
Serial viral infections in infants with recurrent respiratory illnesses
Tuomas Jartti1, Wai-Ming Lee1, Tressa Pappas1, Michael Evans2 ,Robert F. Lemanske Jr3,
James E. Gern1
Departments of 1Pediatrics, 2Biostatistics and Medical Informatics, and 2Medicine, University
of Wisconsin, Madison, WI.
Short title: Recurrent respiratory illnesses in infants
Correspondence to: Dr. Tuomas Jartti, Sirkkalankatu 4 C 59, FIN-20520 Turku, Finland.
Phone: +358 40 7270 284. Email: tuomas.jartti@utu.fi. Fax: +358 2 313 1460.
2
Online repository methods
Microbiology
Diagnostic virology was performed for all nasal samples in the first year of life whether a
child had symptoms or not. Results and methods for viral culture, immunofluorescent
antibody staining for RSV, and nested polymerase chain reaction (PCR) for first year were
previously described (1). All the samples of the 27 children with frequent respiratory
illnesses, but not the samples of the rest of the COAST cohort, were reanalyzed with a new
multiplex PCR-based assay (Respiratory Multicode Assay, EraGen Biosciences, Madison,
WI, USA) that uses 18 virus-specific primer sets to detect the following viruses: HRV, EV,
AdV (B, C, and E), Flu (A, B), PIV (1, 2, 3, 4a, 4b), CoV (SARS, OC48, 229, and NL63),
RSV (A, B) and hMPV (2). Each primer set detects as little as 20 copies of intended target
cDNA per sample (2).
Molecular typing of HRV was performed as described (3). Briefly, a 260-bp variable region at
the 5' noncoding region of HRV was amplified from the cDNA leftover of Respiratory
Multicode Assay by semi-nested PCR. This semi-nested PCR protocol requires as little as 10
copies of cDNA template per sample to produce sufficient product for cloning and does not
produce nonspecific product from original clinical specimens. The PCR fragment was cloned
and sequenced. The identity of the HRV was determined by comparing the resulting sequence
with the homologous sequences of the 101 known serotypes using phylogenetic tree
reconstruction and bootstrap analysis. A new isolate or detection was assigned the serotype to
which it clustered with in the phylogenetic tree with a significant bootstrap value (>50%). All
assigned serotypes in this study had 94-100% sequence identities with the respective
reference serotypes. In contrast, if the sequence of a new isolate or detection did not cluster
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with one of the 101 serotypes in the phylogenetic tree, and had >9% pairwise nucleotide
divergence from the nearest reference serotype, it was designated as a new strain (prefixed
with W).
Of the 115 HRV positive samples available for strain analysis, 62 HRV findings were
assigned to 41 serotypes with 94-100% identity with the respective reference serotypes (data
not shown). The remaining 53 HRV findings did not cluster with any of the 101 known
serotypes in phylogenetic tree reconstructions (data not shown). These 53 HRVs clustered
into 34 new unique strains with high degree of nucleotide divergence from the respective
nearest reference serotypes (range 12-35%).
Bacterial etiology was not studied. In moderate-to-severe respiratory viral infections in
children, the risk for concomitant bacterial infection is low in developed countries (4-6).
References for online repository methods
1. Lemanske RF Jr, Jackson DJ, Gangnon RE, et al. Rhinovirus illnesses during infancy
predict subsequent childhood wheezing. J Allergy Clin Immunol 2005;116:571-577.
2. Lee WM, Grindle K, Pappas T, et al. A High-Throughput, Sensitive and Accurate
Multiplex PCR-Microsphere Flow Cytometry System for Large-Scale Comprehensive
Detection of Respiratory Viruses. J Clin Microbiol 2007;45:2626-2634.
3. Lee WM, Kiesner C, Pappas T, et al. A diverse group of previously unrecognized
human rhinoviruses are common causes of respiratory illnesses in infants. PLoS ONE
2007;2:e966.
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4. Byington CL, Enriquez FR, Hoff C, et al. Serious bacterial infections in febrile infants
1 to 90 days old with and without viral infections. Pediatrics 2004;113:1662-1666.
5. Purcell K, Fergie J. Concurrent serious bacterial infections in 912 infants and children
hospitalized for treatment of respiratory syncytial virus lower respiratory tract
infection. Pediatr Infect Dis J 2004;23:267-269.
6. Lehtinen P, Jartti T, Virkki R, et al. Bacterial coinfections in children with viral
wheezing. Eur J Clin Microbiol Infect Dis 2006;25:463-469.
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Table E1. Respiratory symptom scorecard1
Symptom
Point score
Fever
1
Cough
Mild
1
Moderate
2
Severe
3
Rhinorrhea
Mild2
1
Moderate to severe3
2
Hoarseness
1
Duration of illness > 4 d
1
Apnea
3
Wheezing
5
Retractions
5
Tachypnea
5
Cyanosis
5
1
From: Copenhaver CC, Gern JE, Li Z, et al. Cytokine response patterns, exposure to viruses,
and respiratory infections in the first year of life. Am J Respir Crit Care Med 2004;170:17580.
2
Mild was defined as the requirement for suction from 0–4 times per day, or a wipe every 2 h
or less.
3
Moderate to severe was defined as the requirement for suction 5 or more times per day, or a
wipe 1 or more times per hour.
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Table E2. Viral etiology of wheezing in infants with recurrent MSIs
Factor
No. (%) of
No. (%) of
infants or episodes
infants or episodes
associated with virus
associated with virus
as sole agent
Infants
27
27
Wheezing infants
23
23
23 (100%)
20 (87%)
HRV1
18 (78%)
14 (61%)
RSV
16 (70%)
8 (35%)
PIV
9 (39%)
3 (13%)
CoV
6 (26%)
4 (17%)
hMPV
8 (35%)
3 (13%)
Flu
6 (26%)
1 (4%)
AdV
6 (26%)
0
0
0
Wheezed at least once with > 1 virus
EV
>2 viruses
14 (61%)
All episodes
157
157
Wheezing episodes
73
73
Wheezing episodes with nasal wash
68
68
60 (88%)
38 (56%)
32 (47%)
17 (25%)
Episodes with > 1 virus
> 1 HRV2, 3
7
> 1 RSV4
16 (24%)
7 (10%)
PIV
9 (13%)
3 (4%)
hMPV
8 (12%)
3 (4%)
CoV
6 (9%)
4 (6%)
Flu
6 (9%)
1 (1%)
AdV
6 (9%)
0
0
0
EV
>2 viruses
25 (37%)
Abbreviations: MSI, moderate-to-severe illness; HRV, rhinovirus; RSV, respiratory syncytial
virus; PIV, parainfluenza virus; CoV, coronavirus; hMPV, metapneumovirus; Flu, influenza
virus; AdV, adenovirus; EV, enteroviruses.
1
Number of infants who wheezed at least once with a particular virus.
2
Number of episodes positive for a particular virus.
3
Two episodes with dual rhinovirus infection (one case with HRV-33 and HRV-W20 and one
case with HRV-78 and HRV-W33) were classified as mixed infections.
4
One episode with dual RSV infection was classified as mixed infection.