M A J O R A R T I C L E
Viral Shedding and Immune Responses to
Respiratory Syncytial Virus Infection in Older
Edward E. Walsh,1,3Derick R. Peterson,2Aja E. Kalkanoglu,1Frances Eun-Hyung Lee,1,aand Ann R. Falsey1,3
1Department of Medicine and2Department of Biostatistics and Computational Biology, University of Rochester School of Medicine and Dentistry,
New York; and3Rochester General Hospital, New York
syncytial virus (RSV) infection in adults have not been performed.
Methods.Adults with RSV infection identified in both outpatient and inpatient settings were evaluated.
Upper and lower respiratory tract virus load, duration of virus shedding, select mucosal chemokine and cytokine
levels, humoral and mucosal immunoglobulin responses, and systemic T-cell responses were measured.
Results.A total of 111 RSV-infected adults (61 outpatients and 50 hospitalized patients) were evaluated. Hos-
pitalized subjects shed virus in nasal secretions at higher titers and for longer durations than less ill outpatients,
had greater mucosal interleukin 6 (IL-6) levels throughout infection, and had higher macrophage inflammatory
protein 1α (MIP-1α) levels early in infection. Persons >64 years old and those with more severe disease had a
higher frequency of activated T cells in the blood than younger, less ill subjects at infection. Multivariate analysis
found that the presence of underlying medical conditions, female sex, increased mucosal IL-6 level, and longer
duration of virus shedding were associated with severe disease. Older age and increased nasal MIP-1α levels were
of borderline statistical significance.
Conclusions.Multiple factors, but not older age, are independently associated with severe RSV infection in
adults. The presence of underlying medical conditions had the greatest influence on disease severity.
Comprehensive analyses of host, viral, and immune factors associated with severe respiratory
Keywords. respiratory syncytial virus; adults; pathogenesis; viral shedding; immune response.
Respiratory syncytial virus (RSV), a single-stranded
RNA virus of the family Paramyxoviridae, is the
leading cause of severe respiratory illness in infants
and young children [1, 2]. A hallmark of RSV infection
throughout life [3–5]. Although reinfection is generally
mild, certain adult populations can have severe illness
with lower respiratory tract symptoms, resulting in
hospitalization and death [3, 6–8]. Factors associated
with severe disease in infants are well described and
include underlying congenital cardiac or neurological
conditions, bronchopulmonary dysplasia, prematurity,
low RSV-specific serum antibody titers, and high virus
load [2, 9–11]. Additionally, it has been considered that
innate and adaptive T-cell responses, as well as cyto-
kine responses, such as interleukin 6 (IL-6) level,
strongly influence the pathogenesis of RSV disease in
infants [12–14]. Although less well studied, analogous
risk factors also appear to be operative in adults. Previ-
ously, we found that severe RSV disease in adults was
associated with underlying cardiopulmonary disease,
frailty, and low serum levels of RSV-specific antibody
[15, 16]. In addition, high virus load in hospitalized
persons has been associated withrespiratory failure .
Thus, as in infants, disease severity in adults is likely
multifactorial, as advancing age is associated with in-
creased frequency of underlying medical conditions
and global changes in immune function .
However, a comprehensive assessment of factors,
including involvement of the lower airways, virus load,
Received 26 August 2012; accepted 3 December 2012; electronically published
4 February 2013.
aPresent affiliation: Department of Medicine, Emory University, Atlanta, Georgia.
Correspondence: Edward E. Walsh, MD, Infectious Diseases Unit, Rochester
General Hospital, 1425 Portland Ave, Rochester, NY 14621 (edward.walsh@
The Journal of InfectiousDiseases2013;207:1424–32
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local and systemic cytokine responses, and T-lymphocyte
activation during infection, has not been performed in adults.
Therefore, we examined these factors in adults with a wide
spectrum of disease severity.
Patient Population and Illness Surveillance
The study spanned 3 winters, from 2005 through 2008, in
Rochester, New York. Two cohorts of adults were recruited
after they provided informed consent. The first included
persons ≥21 years of age living independently in the commu-
nity, some with underlying medical conditions. Subjects were
enrolled in the late summer and early fall and were followed
for development of RSV infection during subsequent winters.
They remained under surveillance for 1–3 winters and were
asked to call study personnel to report symptoms consistent
with respiratory illness (ie, new or worse cough, increase in
chronic sputum production, sore throat or nasal congestion,
and worsening dyspnea, with or without fever) when they
were evaluated as described below.
The second cohort included persons ≥21 years of age hos-
pitalized at Rochester General Hospital during the winter with
an admitting diagnosis consistent with an acute respiratory in-
fection (ie, community-acquired pneumonia, acute exacerba-
tion of chronic obstructive pulmonary disease, bronchitis,
congestive heart failure, respiratory failure, or acute viral syn-
drome). Exclusion criteria included residence in a long-term
care facility or presence of a significant immunocompromising
condition (ie, receipt of chemotherapy or radiation therapy
within 6 months of illness, human immunodeficiency virus
infection, or receipt of immunosuppressive medications in-
cluding >15 mg prednisone for >3 weeks).
At enrollment, demographic and medical history, baseline
functional score, resting saturated oxygen (SaO2) level, and
findings on a focused respiratory examination were recorded.
The study was approved by the institutional review boards of
the University of Rochester and Rochester General Hospital.
For the prospective cohort, most illness and daily assessments
were performed by a study nurse during home visits. For the
hospitalized cohort, initial evaluations occurred in the hospi-
tal, and visits after discharge took place at home. Symptoms
and signs of respiratory illness were recorded, and a nasal
swab specimen was obtained by gently swabbing one nostril
just below the inferior turbinate for 5 seconds. The nasal swab
specimen was placed in 3 mL of distilled water, kept on ice,
and processed immediately by reverse transcription polymer-
ase chain reaction (RT-PCR) for RSV RNA. Sputum samples
were diluted in an equal volume of distilled water and vor-
texed vigorously before processing as described elsewhere .
RSV-positive subjects were seen again as soon as feasible, gen-
erally within 48 hours, when a directed history was obtained,
a physical examination was performed, and the following
specimens were collected: repeat nasal swab specimen from 1
nostril and a sputum specimen for quantitative RT-PCR anal-
ysis, a nasal swab specimen from the opposite nostril for cyto-
kine measurements, a respiratory condensate obtained during
a 5-minute interval, 30 mL of heparinized blood for lympho-
cyte purification, and 10 mL of blood for serum IL-6 and RSV
antibody determinations. Heparinized blood was kept at room
temperature and transported to the laboratory for lymphocyte
purification within 12 hours. Other samples were processed
immediately and frozen at −80°C until assayed. Subjects were
reevaluated daily if possible for clinical signs and symptoms of
respiratory illness and collection of a nasal swab specimen and
sputum specimen for RSV RT-PCR. Assessment visits and
collection of nasal swab and sputum samples continued until
2 consecutive samples were RT-PCR negative for RSV. Nasal
swab and blood samples were also obtained between days 12
and 16 and days 25 and 32 after symptom onset for cytokine
measurements, peripheral blood mononuclear cell (PBMC)
purification, and serum antibody determinations.
Diagnosis of RSV Infection, Identification of RSV Group, and
Determination of RSV Titer
RSV infection, including virus group, was diagnosed using
real-time RT-PCR performed on initial nasal swab and
sputum specimens as described. Positive and subsequent
nasal swab and sputum specimens were assayed using a pub-
lished quantitative RSV RT-PCR . RSV titers were ex-
pressed as plaque-forming unit
milliliter of sample. Peak nasal and sputum titers were defined
as the highest titer on any day.
(PFU) equivalents per
Serum immunoglobulin G (IgG) titers to purified RSV F, Ga,
and Gb proteins and neutralizing antibody titers to RSV groups
A and B were determined as described elsewhere [16, 21]. Nasal
immunoglobulin A (IgA) titers to RSV F, Ga, and Gb proteins
was determined and corrected to a total protein concentration
of 100 µg/mL, according to published methods .
High-sensitivity enzyme immunoassays (Biosource Interna-
tional, Camarillo, CA) were used for measuring IL-6, IL-8,
and macrophage inflammatory protein 1α (MIP-1α) levels in
nasal swab and serum specimens. Nasal cytokine values were
corrected to a total protein concentration of 100 µg/mL.
Multicolor Flow Cytometry of T Cells
PBMCs were isolated within 12 hours of collection in sodium
heparin sulfate tubes, divided into aliquots of 1 million cells,
and frozen in liquid nitrogen, as described elsewhere .
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