JOURNAL OF VIROLOGY, Mar. 2008, p. 2040–2055
Vol. 82, No. 5
Viral and Host Factors in Human Respiratory Syncytial
Peter L. Collins1* and Barney S. Graham2
Laboratory of Infectious Diseases1and Vaccine Research Center,2National Institute of Allergy and
Infectious Diseases, Bethesda, Maryland 20892
Human respiratory syncytial virus (RSV) was first isolated in
1956 from a laboratory chimpanzee with upper respiratory
tract disease (for general reviews, see references 21, 57, 102,
and 145). RSV was quickly determined to be of human origin
and was shown to be the leading worldwide viral agent of
serious pediatric respiratory tract disease. In a 13-year pro-
spective study of infants and children in the United States,
RSV was detected in 43%, 25%, 11%, and 10% of pediatric
hospitalizations for bronchiolitis, pneumonia, bronchitis, and
croup, respectively (110). Approximately two-thirds of infants
are infected with RSV during the first year of life, and 90%
have been infected one or more times by 2 years of age. The
rate of hospitalization for primary infection is approximately
0.5% but can vary by situation and ethnic group and can be as
high as 25% (77).
RSV also is a significant cause of morbidity and mortality in
the elderly, with an impact approaching that of nonpandemic
influenza virus (39). RSV readily infects severely immunocom-
promised individuals, most notably allogeneic bone marrow
transplant recipients, causing high mortality. RSV also makes
a substantial contribution to upper respiratory tract disease in
individuals of all ages (59, 65). Globally, the World Health
Organization estimates that RSV causes 64 million infections
and 160,000 deaths annually (Initiative for Vaccine Research:
respiratory syncytial virus, World Health Organization [http:
accessed 5 December 2005]).
Although RSV has a single serotype, reinfection can occur
throughout life. RSV in yearly winter/early spring epidemics in
temperate regions; elsewhere, the timing of RSV activity can
vary widely with the locale. The RSV reservoir in the off-
season is unknown. Strains circulate quickly around the earth
(150). Neither a vaccine nor an effective antiviral therapy is
available, although there is active research in both areas (23,
78, 138). However, infants at high risk for serious disease can
receive passive immunoprophylaxis during the epidemic sea-
son by a monthly injection of a commercial RSV-neutralizing
monoclonal antibody, palivizumab (Synagis), which provides a
55% reduction in RSV-associated hospitalization (17).
RSV (family Paramyxoviridae, order Mononegavirales) is an
enveloped virus with a single-stranded negative-sense RNA
genome of 15.2 kb (21). There are animal versions of RSV,
including bovine RSV (BRSV) and pneumonia virus of mice
(PVM), suggesting that species jumping occurred during the
evolution of these viruses. However, there is no animal reser-
voir for human RSV.
Efficient infection by RSV of established cell lines in vitro
involves binding to cell-surface glycosaminoglycans (62). How-
ever, it is not known how closely this binding models attach-
ment in vivo or whether it is an initial interaction that is
followed by a second, higher-affinity step that remains to be
identified. The nucleocapsid gains entry to the cytoplasm by
membrane fusion; surprisingly, this may involve clathrin-medi-
ated endocytosis rather than surface fusion typical of
paramyxoviruses (85). Viral gene expression and RNA repli-
cation occur in the cytoplasm, and virions acquire a lipid en-
velope by budding through the plasmid membrane (Fig. 1).
Virions are pleomorphic and include spheres and long, fragile
filaments. Studies with RSV are impeded by modest viral yields
in cell culture and physical instability of the particle; interest-
ingly, this instability may reside in the glycoproteins (133).
The negative-sense RNA genome contains a short 3?-extra-
genic leader region, 10 viral genes in a linear array, and a
5?-trailer region (Fig. 1). Each gene is transcribed into a sep-
arate, capped, polyadenylated mRNA encoding a single viral
protein, except in the case of the M2 mRNA, which contains
two overlapping open reading frames that are expressed by a
ribosomal stop-restart mechanism into two distinct proteins,
M2-1 and M2-2 (52).
Five RSV proteins are involved in nucleocapsid structure
and/or RNA synthesis (21). The nucleocapsid N protein tightly
encapsidates genomic RNA as well as its positive-sense repli-
cative intermediate, called the antigenome. This provides pro-
tected, flexible templates and probably reduces detection of
these viral RNAs by host cell toll-like receptors (TLRs) and
intracellular RNA recognition helicases that initiate innate
immune responses through interferon (IFN) regulatory factors
and nuclear factor ?B (NF-?B) (3, 94). The large L protein is
the major polymerase subunit and contains the catalytic do-
mains. The P phosphoprotein is an essential cofactor in RNA
synthesis (31) and also is thought to associate with free N and
L to maintain them in soluble form for assembly of and inter-
action with nucleocapsids. The M2-1 and M2-2 proteins are
* Corresponding author. Mailing address: NIAID, NIH, 50 South
Drive, MSC 8007, Bethesda, MD 20892. Phone: (301) 594-1590. Fax:
(301) 496-8312. E-mail: email@example.com.
?Published ahead of print on 10 October 2007.
FIG. 1. RSV virion, RNA genome, and encoded proteins. (A) The negative-sense RNA genome (strain A2) is depicted 3? to 5? showing the
extragenic 3? leader (le) and 5? trailer (tr) regions and the intervening 10 viral genes (rectangles) that are each expressed as a separate mRNA (21). M2-1
and M2-2 are overlapping open reading frames of the M2 mRNA. The M2 and L genes overlap slightly, and L is expressed by polymerase backtracking
(25). (B) Electron photomicrographs showing an RSV virion budding through the plasma membrane of an infected cell (left) and a free virion (right).
Protein functions and amino acid lengths (in parentheses, unmodified form) are indicated. (C) Schematic diagrams of the F, G, and SH proteins, with
lengths approximately to scale (21). Filled rectangles indicate the hydrophobic cleaved signal sequence (sig.), transmembrane anchors (TM), and fusion
peptide (FP). Cross-hatched rectangles indicate heptad repeats (HR) that drive conformational changes involved in fusion. CT, cytoplasmic domain.
Potential acceptor sites for N-linked sugars are indicated as downward-facing stalks with an N. For the F protein, the locations and amino acid positions
of the two cleavage sites are indicated, as are the cleavage products (F2, p27, F1). For the G protein, the 25 potential acceptor sites for O-linked sugars
predicted to be the most likely to be utilized are indicated as downward-facing lollipops. The sequence and disulfide-bonding pattern (dotted lines) of
the central domain are shown with the CX3C fractalkine motif boxed and the highly conserved 13-amino-acid sequence of unknown significance
underlined. The M-48 translational start site for the secreted form and the mature secreted form is indicated.
VOL. 82, 2008 MINIREVIEW2041
factors involved, respectively, in transcription (42) and in mod-
ulating the balance between transcription and RNA replication
Four other RSV proteins associate with the lipid bilayer to
form the viral envelope (21). The matrix M protein lines the
inner envelope surface and is important in virion morphogen-
esis (147). The heavily glycosylated G, fusion F, and small
hydrophobic SH proteins are transmembrane surface glyco-
proteins (Fig. 1). G and F are the only virus neutralization
antigens and are the two major protective antigens (21).
The G glycoprotein plays a major but not exclusive role in
viral attachment (148). It contains several N-linked carbohy-
drate side chains and an estimated 24 to 25 O-linked side
chains. This increases the apparent molecular weight of the
polypeptide backbone from 32,500 to 90,000. Most of the
ectodomain is thought to have an extended, unfolded, heavily
glycosylated, mucin-like structure that is unique to RSV and its
close relatives and appears to be very distinct from the globular
attachment proteins of other paramyxoviruses. The signifi-
cance of the similarity to mucin is unknown, although it is
tempting to speculate that it might alter the physicochemical
properties of the virus so as to facilitate spread or evade trap-
ping by mucus. G is anchored in the membrane by a signal/
anchor sequence near the N terminus and also is expressed as
a secreted form. This secreted form arises from translational
initiation at the second methionine (codon 48) in the open
reading frame followed by proteolytic trimming to yield a final
form that lacks the N-terminal 65 amino acids, including the
entire signal/anchor (129). The G ectodomain contains a highly
conserved domain of 13 amino acids whose significance is
unknown (146). This conserved sequence overlaps a disulfide-
bonded tight turn that is called a cystine noose and contains a
CX3C motif that is discussed later.
The F protein directs viral penetration by membrane fusion
and also mediates fusion of infected cells with their neighbors
to form syncytia. F is synthesized as a precursor, F0, which is
activated by cleavage by furin-like intracellular host protease.
Unusual for a viral penetration protein, cleavage occurs at two
sites (amino acids 109/110 and 136/137) (Fig. 1) (51). This
yields, in amino-to-carboxy-terminal order, F2(109 amino ac-
ids), p27 (27 amino acids), and F1(438 amino acids). F2and F1
remain linked by a disulfide bond and represent the active
The remaining two RSV proteins, NS1 and NS2, are small
species that do not appear to be packaged significantly in the
virion. As described below, they are nonessential accessory
proteins involved in modulating the host response to infection.
Gene expression and RNA replication by RSV broadly fol-
low the mononegavirus model, although admittedly there are
substantial gaps in our understanding of these processes even
for prototypical mononegavirues (25). The polymerase enters
the genome at or near its 3? end, and the genes are transcribed
into individual mRNAs by sequential start-stop-restart synthe-
sis that is guided by short transcription signals flanking the
genes. There is a polar gradient of mRNA abundance due to
polymerase fall-off. RNA replication involves synthesis of the
full-length positive-sense antigenome that in turn is copied into
RSV adds some complexity of its own with the M2-1 and
M2-2 proteins, which are found only in close relatives of RSV.
With RSV, processive transcription depends on the M2-1 pro-
tein, which is essential for viral viability (42). In its absence,
transcription terminates nonspecifically within several hundred
nucleotides and results in (reduced) expression of NS1 and
NS2 alone (42). It is tempting to speculate that a reduction in
the level of M2-1 might facilitate persistent infection by down-
regulating the expression of most of the viral genes while
maintaining some expression of the NS1 and NS2 host defense
antagonists. The other product of the M2 gene, the M2-2
protein, is not essential but appears to downregulate transcrip-
tion in favor of RNA replication as infection progresses (9). It
is unclear why RSV needs these extra proteins while other
mononegaviruses, which seem to have a very similar RNA
synthetic program, do not. Interestingly, the M2-1 protein of
human metapneumovirus (HMPV) shares substantial se-
quence identity with that of RSV but is not essential for pro-
cessive transcription or viral viability (15). It may be that there
are other M2-1 functions that remain to be identified.
CLINICAL INFECTION AND DISEASE
Inoculation of the nose or eyes occurs by large particle
aerosol or direct contact and results in viral replication in the
nasopharynx, with an incubation period of 4 to 5 days, and can
be followed over the next several days by spread to the lower
respiratory tract (21, 57, 102). Rhinorrhea, cough, and low-
grade fever are common. Signs of lower airway infection are
common even in infants with mild disease. Clinical signs of
bronchiolitis include increased airway resistance, air trapping,
and wheezing. Pneumonia accounts for the hypoxia frequently
detected in RSV-infected infants.
Infection normally is highly restricted to the superficial cells
of the respiratory epithelium (72, 159). Ciliated cells of the
small bronchioles and type 1 pneumocytes in the alveoli are
major targets of infection in the lower airway. It is likely that
other cells, including nonciliated epithelium and intraepithelial
dendritic cells (DCs), are also infected (Fig. 2), but the basal
cells appear to be spared (72). Pathological findings include
necrosis of epithelial cells, occasional proliferation of the bron-
chiolar epithelium, infiltrates of monocytes and T cells cen-
tered on bronchiolar and pulmonary arterioles, and neutro-
phils between vascular structures and small airways. Infection
and tissue damage tends to be patchy rather than diffuse.
There are abundant signs of airway obstruction due to slough-
ing of epithelial cells, mucus secretion, and accumulated im-
mune cells. Syncytia are sometimes observed in the bronchio-
lar epithelium but are not common. However, syncytium
formation and giant-cell pneumonia are hallmarks of infection
in individuals with extreme T-cell deficiency.
Fifty percent or more of infants hospitalized with RSV lower
respiratory tract disease have subsequent episodes of wheezing
that in some cases can persist until 11 years of age or more (57,
140). It is of interest whether infection is a causal factor or
whether severe infection and wheezing are comarkers of an
underlying vulnerability. Evidence for causality in at least a
subset of individuals comes from a recent study in which suc-
cessful palivizumab prophylaxis of preterm infants was associ-
ated with reduced wheezing compared to untreated controls
when assessed at approximately 3.5 years of age (140). There
also is evidence that congenital vulnerability is involved and an
2042 MINIREVIEWJ. VIROL.
indication that there is considerable host-dependent heteroge-
neity in long-term effects of infection (98, 99). Some studies
also have linked severe early RSV infection with allergic sen-
sitization leading to asthma, but other researchers have dis-
puted this link (36, 139). In a small study, antibody prophylaxis
against RSV was associated with a higher incidence of normal
lung function and lower incidence of atopy compared with
controls when evaluated 7 to 10 years later (160).
There presently is renewed interest in whether RSV can
establish persistent infection in vivo, which might contribute to
pathogenesis and also help maintain the virus in the popula-
tion. In mice previously infected with, and apparently cleared
of, RSV, suppression of T cells several months later resulted in
the emergence of infectious virus in some animals (136). An
analysis of patients with chronic obstructive pulmonary disease
provided evidence of long-term infection in one study (162);
however, a second study with a comparable patient group in-
dicated that RSV was present in acute rather than persistent
The following sections will outline host risk factors for se-
vere RSV disease, the protective immune response and possi-
ble deficiencies, the contribution of host immunity to patho-
genesis, and viral factors in pathogenesis. Figure 3 depicts a
model of the relative contributions of host versus viral factors
to pathogenesis versus protection.
HOST RISK FACTORS
The risk of severe RSV disease is increased by factors that
compromise the ability to control and withstand a respiratory
tract infection: young age (?6 months), premature birth (?35
weeks of gestation), bronchopulmonary dysplasia, congenital
heart disease, immunodeficiency or immunosuppression, the
first or second RSV infection in life, unusually narrow airways,
low birth weight, male gender, a low titer of RSV-specific
serum antibodies, and frail old age (157). Although prematu-
rity and underlying disease play important roles in pediatric
RSV disease, more than two-thirds of pediatric hospitaliza-
tions involve previously healthy infants. Environmental factors,
including ones that affect lung function (e.g., household to-
bacco use) or that increase exposure to infection (e.g., day
care, hospitalization, multiple siblings), also play a role.
A role for genetic predisposition in severe RSV disease is
indicated by (i) association of susceptibility with a family his-
tory of asthma or severe infant lower respiratory tract disease
and (ii) differences in susceptibility between ethnic, racial, and
gender groups (69). More recently, studies have provided ev-
idence associating increased incidence of severe pediatric RSV
disease with genetic polymorphisms in a number of genes se-
lected for analysis, although these studies seem preliminary
and sometimes are inconsistent. These associations include
genes encoding cytokines and chemokines, including interleu-
kin-4 (IL-4), IL-8/CXCL8, IL-10, IL-13, and RANTES/CCL5,
or encoding proteins involved in surface interactions or intra-
cellular signaling, such as TLR4, CD14, IL-4R, CX3CR1,
CCR5, and surfactant protein A (SP-A), SP-B, SP-C, and SP-D
(4, 6, 69, 71, 115, 125–127, 144). Some of these polymorphisms
have been associated with functional effects that give clues to
possible beneficial or pathological roles of host factors in RSV
infection, some of which are noted below.
PROTECTIVE AND PATHOGENIC FEATURES OF THE
Protective immunity. As with other acute respiratory viruses,
RSV infection usually is completely resolved by innate and
adaptive immunity (21, 57, 102, 145). As with many viruses,
RSV infection or uptake by respiratory epithelial cells and
resident macrophages results in widespread changes in cellular
gene expression and upregulates expression of a variety of
factors, including surfactants, cytokines, chemokines, and cell-
surface molecules. Some of these factors have direct antiviral
properties; others stimulate the influx and activation of natural
killer (NK) cells, granulocytes, monocytes, macrophages, den-
dritric cells, and T lymphocytes that provide direct antiviral
activities and initiate an effective adaptive immune response.
Virus-neutralizing antibodies in the respiratory tract likely
FIG. 2. Immunohistochemistry of autopsy lung specimens from a
15-month-old patient with an untreated RSV infection (72). Immuno-
staining for RSV antigens (a) revealed infection of the bronchiolar
epithelium in a near circumferential pattern sparing the basal cells.
The basal cells can restore the epithelium, but in the process may lead
to mucus metaplasia and remodeling of airways. In addition to infec-
tion of the polarized ciliated epithelium, it appears that other cell
types, potentially including intraepithelial DCs, can be infected (ar-
rows point to the projections of irregularly shaped RSV-infected cells
that are thought to be DCs, based on morphology). Immunostaining
for CD1a?DCs (b) showed that they are not prominent in the lung
parenchyma during infection, but when present can have a distinctive
morphology with multiple projections that can extend beyond the
apical or basal boundary of the columnar epithelium. CD69?mono-
cytes were the major cell type present in peribronchiolar infiltrates.
However, CD3?T cells also were abundant and, while most of them
appeared to be double negative for CD4 and CD8, many were positive
for CD8 immunostaining (c). In panels a to c, L indicates airway
lumen; in panel c, the lower lumen is occluded with debris and immune
VOL. 82, 2008 MINIREVIEW2043
contribute to viral clearance and certainly play an important
role in protection against reinfection (21). They include secre-
tory immunoglobulin A (IgA) and transudated, serum-derived
IgG. Secretory IgA is particularly important in protecting the
upper respiratory tract, which is accessed only very inefficiently
by serum IgG (124, 137). The IgA response is short-lived fol-
lowing primary infection but can increase in duration following
reinfection (109). Serum IgG antibodies are somewhat more
efficient in accessing the lower respiratory tract and can pro-
vide substantial protection in that compartment. In RSV-naı ¨ve
infants, the maternal serum antibody titer is positively corre-
lated with a reduced level of severe RSV disease. The clinical
experience with palivizumab also shows that serum antibodies
alone can provide substantial protection from severe disease.
However, protection from passive antibodies quickly wanes,
because they decay with a half-life of approximately 21 to 24
days. CD8?T lymphocytes are important for clearing virus-
infected cells as well as for contributing cytokines, notably
gamma IFN (IFN-?), that promote a protective Th1 response
Protective immunity to RSV induced by natural infection is
generally described in the literature as weak and short-lived.
This is based mainly on the frequent incidence of reinfection of
humans in nature and under experimental conditions. How-
ever, as discussed later, viral immune evasion strategies also
may contribute to reinfection. Typical RSV-neutralizing serum
antibody titers in adults are quite high (mean reciprocal titer of
1,450 in a 50%-infected-well-reduction assay), and following
natural infection, they were increased fourfold or more in 64%
of young adult and 79% of frail elderly patients, suggestive of
good responses (41). While postinfection increases in antibody
titers wane in most individuals within a year, this decay might
not be unique to RSV (40), and the residual titers remain quite
high. Brisk serum antibody responses also have been noted in
children with primary and secondary infections (66), and even
young infants of 2 months of age can have substantial neutral-
izing serum antibody responses when the titer of immunosup-
pressive maternal antibody is low (R. A. Karron, personal
communication). Primary RSV infection of seronegative ex-
perimental animals, including the chimpanzee, results in ro-
bust protective immune responses, at least in the short term
(23). In addition, in clinical studies, experimental live RSV
vaccines do not seem to be obviously reduced in immunoge-
nicity compared to live human parainfluenza virus type 3
(HPIV3) and influenza A virus vaccines, although these studies
were not designed for virus-to-virus comparisons (R. A. Kar-
ron, personal communication). There are reports of effects on
cellular immunity. RSV-specific T-helper cell responses, as
measured by in vitro lymphoproliferation, appeared to be de-
ficient during reinfection in infancy (12). Increased apoptosis
of CD4?and CD8?lymphocytes resulting in lymphopenia also
has been described for RSV-infected infants compared to un-
infected controls, with the effect being greater with younger
age and more severe illness (130). Mitogen-induced prolifera-
tion of peripheral blood lymphocytes in vitro was inhibited by
contact with RSV-infected cell monolayers, an effect that did
not prevent the expression of T-cell activation markers but
impeded the cell cycle (135). This effect appeared to be medi-
ated by the viral F protein and was augmented by G. Studies of
mice suggested that the pulmonary CD8?CTL response to
RSV was less functional and shorter lived than that to influ-
enza virus (18). However, this difference between viruses has
not been confirmed and, as noted later, functional impairment
to the pulmonary CTL response might be a feature of the
FIG. 3. Estimated contributions of host and viral factors to RSV pathogenesis in the overall pediatric population, as discussed in the text.
Factors are placed in the vertical dimension approximately according to the extent to which they are determined by the host (top) or virus (bottom)
or a combination (in between). Placement in the horizontal dimension indicates the extent to which they are pathogenic (left) or protective (right).
The size of the symbol represents speculated aggregate impact.
tissue rather than the virus (153). In summary, it remains
unclear to what extent frequent reinfection by RSV reflects
inadequate or inappropriate responses by the host versus viral
immune evasion strategies.
Contribution of the host response to pathogenesis. The most
dramatic demonstration of the potential of host immunity to
contribute to disease associated with an RSV infection was the
experience with a formalin-inactivated RSV (FI-RSV) vaccine
that was administered intramuscularly to infants and children
in the 1960s. This vaccine was poorly protective and, in RSV-
naı ¨ve individuals, it primed for enhanced disease upon subse-
quent natural infection with RSV, with up to 80% of vaccinees
hospitalized with RSV-like disease, resulting in two deaths (21,
123). Retrospective studies showed that FI-RSV induced se-
rum antibodies that bound efficiently to viral antigen but did
not efficiently neutralize infectivity, contributing to the poor
vaccine efficacy (111). This atypical antibody response proba-
bly reflected denaturation of the antigen as well as a possible
deficiency in antibody affinity maturation. An analysis of lung
tissue from the fatal vaccine cases and from experimental an-
imal models of enchanced disease provided evidence of anti-
body-antigen complex deposition and complement activation
in the lung occurring during subsequent RSV infection (120).
In addition, peripheral blood lymphocytes from FI-RSV vac-
cinees exhibited an exaggerated proliferative response to RSV
antigens in vitro compared to lymphocytes isolated following
natural infection (82). Subsequent studies with experimental
animals confirmed that, compared to natural infection, FI-
RSV induced a heightened response of virus-specific CD4?T
lymphocytes biased toward the Th2 subset (54). Th1 and Th2
CD4?T lymphocytes play important roles in immune regula-
tion and function and to some extent are self-stimulatory and
reciprocally inhibitory. Th1 responses (signature cytokines
IFN-? and IL-12) tend to promote cell-mediated immunity
important for protection against intracellular pathogens, such
as viruses. Th2 responses (signature cytokines IL-4, IL-5, IL-
10, and IL-13) can be associated with eosinophilia, goblet cell
hyperplasia, mucus overproduction, IgE production, and air-
way hypersensitivity. The Th2-biased response to FI-RSV ap-
pears to be a consequence of inefficient induction of IFN-?-
secreting NK cells and CD8?
nonreplicating vaccine (70, 74). In FI-RSV-immunized ani-
mals, depletion studies confirmed that the Th2 cells and cyto-
kines were important in vaccine-enhanced pathology (24). This
experience amply demonstrated immune-mediated (particu-
larly Th2-mediated) pathology associated with an inactivated
vaccine and subsequent RSV infection. However, comparable
disease enhancement does not occur with natural RSV infec-
tions and reinfections, and the relevance of FI-RSV-associated
pathogenesis to natural infection is unclear.
As often is the case for acute infections, host immunity
appears to contribute to pathogenesis during natural RSV
infection, although not as dramatically as with FI-RSV. How-
ever, the relative contributions to RSV pathogenesis of direct
viral cytopathology versus the host immune response, and the
host factors that are responsible, remain controversial.
Several observations suggest a substantial contribution of
host immunity to RSV disease. For example, clinical observa-
tions (as noted above) and in vitro studies (described later)
showed that RSV is not highly cytopathic or invasive. In infants
T lymphocytes by this
coinfected with human immunodeficiency virus type 1, pro-
longed clinical shedding occurred for more than 199 days with-
out substantial disease (83). When cotton rats with an estab-
lished RSV infection were administered a neutralizing
antibody that reduced pulmonary virus replication more than
1,000-fold, there was little effect on pulmonary pathology; the
addition of anti-inflammatory glucocorticoid therapy was nec-
essary to reduce pathology (122). A similar lack of clinical
improvement was observed for intubated children with an es-
tablished infection, for whom antibody therapy reduced viral
shedding 30-fold compared to controls (97). The inability to
block disease progression by sharply reducing virus replication
is suggestive of immunopathology rather than direct viral cy-
topathology. Finally, genetic polymorphisms that increase ex-
pression of the IL-4, IL-8, and (tentatively) CCR5 genes were
associated with an increased frequency of severe pediatric
RSV disease, suggesting that these host factors can contribute
to pathogenesis (69).
Conversely, other observations indicate a contribution of
viral cytopathology to RSV disease. RSV disease often is more
frequent and more severe in highly immunosuppressed or im-
munocompromised individuals, which seems inconsistent with
disease being primarily immune mediated (43). In humans,
immunity to RSV due to maternal antibodies or prior infection
ameliorates rather than enhances disease upon reinfection.
While it is not as cytopathic as influenza virus, RSV perturbs
ciliary action and leads to cell shedding, effects that would
contribute to airway obstruction. There generally is a positive
correlation between the magnitude of virus replication and
clinical disease in natural and experimental infections with
wild-type or attenuated RSV (30, 79), although findings to the
contrary also have been reported (163). However, this does not
argue solely for viral cytopathology, since diminishing the viral
load will reduce the antigenic stimulation driving immunopa-
thology. In experimental infections of chimpanzees and human
adults, children, and infants with wild-type or attenuated RSV,
symptoms began 1 to 4 days following the onset of viral shed-
ding and ended either coincident with the cessation of shed-
ding or continued for several days, depending on the individual
(8, 75, 79, 81, 128). This result suggests that both viral cytopa-
thology and host immunity contribute to disease and that there
is variation among different individuals. A recent evaluation of
lung specimens from young infants with rapidly fatal, untreated
RSV infection provided evidence of extensive viral replication
with few CD8?T lymphocytes or NK cells and minimal lym-
phocyte-derived cytokines (159). This suggested that these
cases of fatal disease involved an inadequate rather than ex-
cessive adaptive immune response. However, another recent
study involving a case of RSV disease in a 15-month-old pa-
tient, in which death was due to a vehicular accident rather
than the virus, provided evidence of substantial immune infil-
trate, including monocytes, T lymphocytes, and neutrophils
(72). The controversy over the contribution of immunopathol-
ogy versus viral cytopathology continues because the natural
human host is not amenable to pathogenesis studies and ani-
mal models are poor surrogates.
Excessive T-lymphocyte cytotoxicity is one potential mech-
anism of immune-mediated pathogenesis. Studies of mice
showed that the T-cell response helps resolve RSV infection
but can make a substantial contribution to disease. For exam-
VOL. 82, 2008MINIREVIEW 2045
ple, depletion of CD4?or CD8?T cells reduced disease, and
depletion of both resulted in long-term infection without ill-
ness (53). In a patient with severe combined immunodeficiency
and a high level of persistent RSV shedding, T-cell reconsti-
tution dramatically reduced viral shedding but also resulted in
a dramatic increase in pulmonary disease (35). Conversely,
there also is evidence arguing against a prominent role of
T-lymphocyte cytotoxicity in RSV pathogenesis. Among in-
fants with immunodeficiencies, those with cell-mediated defi-
cits have more difficulty controlling the virus and have more
severe outcomes, suggesting that the net effect of T cells is
protective rather than pathogenic (43). As already noted, one
study of lung autopsy specimens provided evidence of a defi-
cient rather than overly robust CD8?T-cell response associ-
ated with fatal RSV disease (159), although a substantial re-
sponse was observed in a second study (72). In infants
hospitalized for RSV bronchiolitis, RSV-specific CTLs were
not detected until at least 6 days later (20) and therefore did
not correlate temporally with severe disease. Thus, T cells are
important for clearing RSV infection, but their contribution to
pathogenesis may depend on the situation.
A role for Th2-biased responses in RSV pathogenesis was
suggested by (i) the Th2-mediated disease associated with FI-
RSV discussed above, (ii) the Th2 bias of the young infant
(discussed later), in whom severe disease is more frequent, and
(iii) the association of Th2 responses with asthma, which in-
volves small airway constriction, mucus plugging, and wheezing
similar to patterns seen with RSV disease. A number of clinical
studies have documented elevated ratios of Th2/Th1 cytokines
or their mRNAs, measured in nasal secretions or in stimulated
or nonstimulated peripheral blood mononuclear cells, in asso-
ciation with severe pediatric RSV disease (80, 92, 93, 131). In
some cases, the overall Th response actually was decreased,
but the relative Th2 component was increased. In addition, two
studies demonstrated a positive association between RSV dis-
ease and a genetic polymorphism in the Th2 cytokine IL-4
gene that increases gene expression (69). Conversely, other
groups have reported a Th1-biased response or mixed re-
sponses associated with severe pediatric RSV disease (13, 47,
107). Thus, there is suggestive but inconsistent evidence of a
link between an increased Th2/Th1 ratio and RSV pathogen-
esis. In some studies, patients with severe disease fell into
subgroups with respect to Th responses, suggestive of substan-
tial host variability (80, 107).
The Th2 cytokines IL-4 and IL-13 promote isotype switching
to IgE. IgE is bound by receptors on mast cells and basophils
and, upon contact with antigen, induces cell activation and the
release of mediators, including histamine and leukotrienes.
These can mediate neural, vascular, and muscular responses,
including rhinorrhoea, cough, and wheeze, which are disease
signs associated with RSV. Indeed, some studies have found
persistent increased levels of free RSV-specific IgE and hista-
mine in secretions of infants experiencing an RSV infection
with wheezing (158). However, other studies have not con-
firmed these findings (28), and the association of IgE to RSV
disease remains unclear.
Eosinophils can be involved in either Th2-mediated or in-
flammatory responses (see below) and have been suspected to
have a role in RSV pathogenesis based on several observa-
tions. Increased numbers of eosinophils were reported in au-
topsy lung tissues from two infants who died of enhanced RSV
disease subsequent to vaccination with FI-RSV, although a
reanalysis of these specimens indicated that they were a minor
population (123). Pulmonary eosinophilia is observed in con-
nection with Th2 responses to RSV antigens in BALB/c mice,
as already noted. Also, increased levels of eosinophil degran-
ulation proteins were found in respiratory secretions from pa-
tients with severe cases of RSV disease (46, 64). The promi-
nent association of eosinophils in asthma also makes them of
particular interest. Eosinophils are recruited by Th2 or inflam-
matory chemoattractants, including IL-5, eotaxin/CCL11, and
RANTES, and are activated to release cytotoxic proteins and
antiviral RNase as well as Th2 cytokines and inflammatory
chemokines and cytokines. However, the prevalence of eosin-
ophils among airway leukocytes from RSV-infected infants, 1
to 3%, was low and approximately the same as for influenza
virus, and thus does not seem to represent a prominent or
unique feature of RSV pathogenesis (101, 143). Furthermore,
the net effects of eosinophils are not necessarily pathogenic:
hypereosinophilia in a transgenic mouse that overexpresses
IL-5 was associated with protective and disease-sparing effects
against RSV rather than increased disease (118). Thus, while
eosinophilia sometimes is prominent in animal models of RSV
pathogenesis, its contribution to authentic human disease is
Overly robust inflammatory responses also have been sug-
gested to contribute to RSV pathogenesis. These initiate when
the virus interacts with respiratory epithelial cells and macro-
phages and is detected by TLRs and other pattern-recognition
proteins, leading to upregulation of the expression of inflam-
matory factors, such as chemokines and surface proteins in-
volved in cell interactions. The further activation of NF-?B by
the RSV F and NS2 proteins (described later) likely augments
the response (90, 141). This leads to the influx and activation of
leukocytes, which add to the production of inflammatory fac-
tors and can help resolve infection but also contribute to
pathogenesis through tissue damage and other effects. Clinical
studies have documented increased expression of inflamma-
tory mediators or their mRNAs in respiratory secretions of
infants and children hospitalized for RSV disease compared
to controls, including IL-6, tumor necrosis factor ?, IL-8,
RANTES, macrophage inflammatory protein 1?/CCL3, eotaxin,
and monocyte chemotactic protein 1/CCL2, among others (47,
64, 100). As is typical for acute inflammation, neutrophils are
the predominant airway leukocyte in infants with RSV bron-
chiolitis, accounting for 84% or more of the cells, compared to
66% for influenza virus (101, 143). IL-8 is the major chemoat-
tractant for neutrophils. Its concentration in respiratory secre-
tions is elevated in infants with severe RSV disease (1, 64) and
in some reports appeared to be somewhat increased for RSV
versus other respiratory virus infections (48, 50). Activation of
neutrophils occurs in response to inflammatory mediators and
possibly to RSV itself and results in the release of cytotoxic
enzymes in addition to inflammatory cytokines and chemo-
kines (1). Neutrophils presumably can have antiviral activity
due to the destruction of RSV-infected cells, to which they may
be attracted by the virus-induced expression of surface adhe-
sion molecules. However, the extent to which RSV neutro-
philia and other aspects of the inflammatory response are
2046 MINIREVIEWJ. VIROL.
protective versus pathogenic, and whether this is particular to
RSV compared to other respiratory viruses, is unclear.
As noted, genetic polymorphisms that increase IL-8 and
CCR5 expression have been associated with increased RSV
disease, consistent with a role of inflammatory chemokines in
RSV disease (69). However, a strong inflammatory response
does not seem to be essential for the development of viral
respiratory tract disease: in one study, the level of inflamma-
tory cytokines and cytokines in nasal wash samples from in-
fected infants was substantially lower for HMPV than for RSV
despite similar disease signs (91). In most clinical studies, anti-
inflammatory therapy (oral or inhaled corticosteroid) has not
provided significant improvement of short- or long-term out-
comes of RSV infection (14).
Thus, a number of host immune factors involved in restrict-
ing and clearing the virus likely also contribute to pathogene-
sis, at least under some conditions. However, it is unclear
whether one or more factors are particularly responsible for
RSV disease, and whether this is different for RSV than for
other respiratory viruses.
PATHOGENIC FACTORS DETERMINED MAINLY
BY THE VIRUS
High infectivity. RSV is one of the most contagious human
pathogens, comparable to measles virus. In prospective stud-
ies, the natural introduction of RSV into a day-care setting
resulted in infection of more than 90% of infants and children
(75). RSV is readily introduced and spreads with ease in hos-
pitals, nursing homes, families, and other close-contact settings
(61). High infectivity contributes to yearly epidemics and to the
high frequency of reinfection.
RSV is not highly cytopathic or invasive. In an in vitro model
of a polarized, mucociliary airway epithelium, RSV preferen-
tially infected and was shed from the ciliated cells of the apical
surface (165). This is consistent with clinical histopathology
findings, as already noted. Ciliary action was disrupted, and
infected cells were shed and replaced over several weeks with-
out gross histological effect despite the ongoing infection. No
cell-to-cell fusion was detected, probably because the F protein
was expressed on the apical surface and had minimal contact
with neighboring cells. In contrast, control cultures infected
with influenza A virus were rapidly destroyed within 2 days,
and all cell types within this complex tissue appeared to be
susceptible to infection. Thus, RSV appears to be inherently
less cytopathic and less invasive within the epithelium than
influenza A virus and, instead, is more similar to HPIV3 (164).
The viral replication cycle in vitro is relatively long (30 to 48 h).
Total cellular DNA, RNA, and protein synthesis are reduced
only modestly and only late in the cycle, probably through
Limited antigenic and strain diversity. RSV has a single
serotype with two major antigenic subgroups (A and B) (21).
Some studies have described subgroup A strains as being as-
sociated with increased disease severity, but other studies have
not confirmed this difference in virulence. Recently, a sub-
group A strain called line 19 was shown to induce increased
airway hypersensitivity and goblet cell expansion in mice com-
pared to the related A2 prototype strain, providing the first
clear example (albeit in mice) of a strain-specific difference in
RSV illness (96). So far, however, strain differences do not
seem to play an important role in disease variability in humans.
Between the two subgroups, G is the most divergent protein,
with 53% amino acid sequence identity and 1% to 7% anti-
genic relatedness between subgroups (21). However, because
the other neutralization antigen, F, is less divergent (90%
amino acid sequence identity and 50% antigenic identity), the
two subgroups exhibit only a three- to fourfold difference in
reciprocal cross-neutralization in vitro. The subgroup differ-
ence has been estimated to confer a fourfold advantage in
reinfection (161). Epidemics typically involve multiple circu-
lating strains in which subgroup A and B may alternate in
predominance every 1 or 2 years. Yearly variation in the virus
population in any locale mainly involves changes in the mixture
of circulating strains rather than progressive antigenic drift.
The two antigenic subgroups are estimated to have diverged
350 years ago (167) and, as already noted, still retain a high
level of antigenic relatedness.
Infection very early in life. RSV infects patients earlier in
life with greater consequences than other respiratory viruses.
Rhinoviruses, influenza viruses, HPIVs, and HMPV commonly
infect children ?6 months of age (55), but RSV causes more
frequent and severe infections at an earlier age. Rhinovirus
infection in particular is common during the first year of life
and is associated with wheezing, much like RSV, but has a
much lower incidence in infants ?6 months of age (105). In
contrast, the peak of hospitalization for RSV disease is at the
remarkably young age of 2 months.
The ability to infect infants very early in life increases the
impact of RSV. Very young infants are less tolerant of severe
infection than older individuals, in part because they have
airways that are narrower in diameter and thus more suscep-
tible to obstruction, a prominent feature of RSV disease. Im-
mune responses in general are lower in magnitude and pre-
sumably less effective in young infants than in older children or
adults. For example, in a clinical trial of a live-attenuated
intranasal RSV vaccine, 100% of seronegative vaccinees ages 6
months or more achieved a fourfold-or-greater increase in
RSV-specific serum antibodies after one vaccine dose com-
pared to 44% of infants ages 2 to 4 months after two doses
(78). Comparable differences are seen for natural infections
with wild-type virus. RSV-specific cytotoxic cellular responses
to natural infection were detected in less than 40% of in-
fants under 5 months of age compared to 65% of children 6
months to 2 years of age (20). Protective responses against
respiratory viruses in infancy also are not long lasting (66,
The reduced immune responses are due to two phenomena:
immunosuppression by RSV-specific maternal serum antibod-
ies (26) and immunologic immaturity (2, 87). Antibody-medi-
ated immunosuppression affects mainly the humoral response
to RSV (26), and its mechanism is poorly understood. Immu-
nologic immaturity has multiple aspects and affects innate,
humoral, and cell-mediated immunity (2, 87). Compared to
adults, young infants have a smaller number of lymphocytes in
peripheral lymphoid tissues. Neonatal DCs have been shown
to be less efficient than adult cells in supporting T-cell prolif-
eration in response to antigen stimulation in vitro and to se-
crete fewer cytokines in response to RSV (87). They also are
deficient in the production of IL-12 and in inducing IFN-?. In
VOL. 82, 2008 MINIREVIEW2047
addition, infants exhibit a reduced frequency of somatic mu-
tation in the development of their antibody response and a
diminished capacity for class switching (155, 156). While the
impact of antibody-mediated immunosuppression on RSV-
specific antibody responses is well documented and is substan-
tial (26), the impact of immunologic immaturity is less well
There is a lingering Th2 bias in responses bias during the
neonatal period; during pregnancy, this helps block Th1-me-
diated immune rejection of the fetus (2, 121). Infection with
RSV during the first 3 months of life has been shown to induce
a Th2-biased response compared to infection of older infants,
based on an analysis of cytokines in respiratory secretions (88).
Interestingly, this also was true of infants infected with HPIV
or influenza virus and thus may be more related to host age
rather than virus (88), but the overall impact of this effect is
likely to be greater for RSV, since it infects patients with a
greater frequency at this young age. Infection in a Th2-biased
environment has the potential to affect the quality of the pri-
mary and recall responses. Infection of neonatal mice with
RSV was associated with reduced and delayed expression of
IFN-? during primary infection and increased disease and Th2-
associated cytokines upon reinfection compared with older
animals (27). This result indicates that the age at first infection
can have consequences on the host primary and recall re-
sponses (27). The majority of human infants do not have se-
vere disease or lingering clinical manifestations of RSV infec-
tion, and there is no evidence of enhanced disease upon
reinfection. However, there may be subtle effects of immuno-
logical imprinting of early infection in genetically susceptible
individuals. The Th2 environment also has the potential to
promote sensitization to bystander environmental antigens, as
has been demonstrated for mice (116). Conversely, the “hy-
giene hypothesis” postulates that (nonsevere) infections early
in life by RSV and other pathogens can stimulate the maturing
immune system toward adult-like Th1 responses, but whether
this occurs with respiratory viruses in general and with RSV in
particular remains a topic of investigation (44).
In addition to immune maturation, infancy is a time of rapid
lung maturation involving pulmonary alveolar expansion and
airway remodeling to accommodate growth (49). Whether vi-
rus-induced damage and overexpression of cellular factors dur-
ing this period can have long-term effects on lung function or
the immune response in some individuals is largely unknown.
This might depend on infection occurring in a genetically sus-
ceptible infant at a critical time in the development of the
immune system or the lung (49, 98). As one example, RSV
infection of rats was shown to increase the pulmonary expres-
sion of nerve growth factor (NGF) and its receptors, an effect
that was substantially greater (and well beyond normal levels)
in weanling versus older animals (68). NGF upregulates the
expression of the tachykinin substance P and its receptor,
which augment inflammation and airway hypersensitivity. The
age-specific aspect of this effect might contribute to greater
disease in the young. Overexpression of these neurogenic fac-
tors at critical times might potentially have long-term conse-
quences. Increases in NGF and another neurotropin also have
been found in cells recovered from the lower airways of infants
with severe RSV disease versus controls (149).
Reinfection. RSV is able to reinfect throughout life, and
even in the same epidemic season, despite limited antigenic
variation (59). A 10-year prospective day-care study found that
the majority of children were infected in each of the first 3
years of life (66). Frequent reinfection also is seen in adults
that are exposed to the virus: during a typical RSV season, 25
to 50% of health-care workers are reinfected, and family mem-
bers of sick children are readily reinfected. The annual inci-
dence of natural RSV infection in healthy adults not selected
for high exposure to the virus is estimated at 5 to 10% (39, 59).
Experimental infection studies have documented considerable
variability in the susceptibility of adults to reinfection (60).
Higher RSV-specific serum and nasal wash antibody titers tend
to correlate with greater resistance to reinfection, although a
substantial proportion of subjects with high titers can be rein-
fected (60, 106).
RSV reinfection usually is associated with reduced disease
compared with primary infection, although even in healthy
adults, 84% of infections in a prospective study were symptom-
atic and 26% had lower respiratory tract involvement (59).
Reinfection in infants, in the elderly, and in severely immuno-
suppressed individuals can result in severe disease. Impor-
tantly, the ability of RSV to reinfect maintains its presence in
the population and increases its access to susceptible individ-
uals of all ages. The ability of RSV to reinfect without consid-
erable antigenic change is in sharp contrast with the ability of
influenza A virus to reinfect, which is strongly dependent on
antigenic drift or shift. However, reinfection is not unique to
RSV among respiratory viruses: the HPIVs also can reinfect
without antigenic variation, although this does not occur as
frequently as with RSV (57, 112).
Tissue tropism. The tropism of RSV for the superficial cells
of the respiratory tract reduces the effectiveness of host immu-
nity. As noted, local IgA responses can be short-lived. Serum
antibodies (maternal, prophylactic, or postinfection) gain ac-
cess to the respiratory lumen by the inefficient process of tran-
sudation, which results in an estimated 80- to 160-fold gradient
between serum and the respiratory lumen (124). A further
10-fold increase in serum antibodies is required for protection
in the nasal cavity compared to the lungs, which likely reflects
a difference in the efficiency of transudation into these com-
partments (137). Thus, a high serum antibody titer is required
to confer protection. These factors leave the upper respiratory
tract particularly vulnerable to infection. However, that should
be the case for any respiratory virus and thus does not alone
account for the unusually high infectibility and reinfectibility of
It was recently shown that virus-specific CD8?cytotoxic T
lymphocytes (CTLs) became functionally impaired in the pro-
duction of antiviral cytokines and in cytolytic capacity after
recruitment to the lungs in virus-infected mice, an effect that
was observed for RSV, influenza virus, and simian virus 5
(153). Thus, this impairment was tissue specific rather than
virus specific, and it did not require CTL contact with a specific
viral antigen. This may be a host mechanism to prevent exces-
sive pulmonary inflammation and damage, but it has the po-
tential to inappropriately reduce CTL activity against respira-
Tropism to the superficial luminal cells of the airway, low
invasiveness, and inhibition of apoptosis (by the NS1, NS2, and
2048 MINIREVIEW J. VIROL.
SH proteins; see below) might each contribute to delay the
presentation of RSV antigens to the immune system. Lack of
virus-mediated destruction of the full epithelium would main-
tain the steep gradient of transfer of antibodies from the se-
NS1 and NS2 proteins. Among the respiratory viruses, RSV
is one of the most effective in blocking synthesis of host type I
IFN in infected individuals (58). RSV has two IFN antagonists,
NS1 and NS2, which are encoded from promoter-proximal
genes to ensure high expression. These inhibit the induction of
IFN-?/? by blocking the activation of IFN regulatory factor 3
and by inhibiting type I IFN-induced signaling through the
JAK/STAT pathway, which otherwise amplifies the IFN re-
sponse, upregulates IFN-stimulated genes, and establishes an
antiviral state (95, 141). One or both of the NS proteins target
STAT2 for proteasomal degradation, thus blocking signaling
from type I IFN (34, 95). Infection of STAT1 knockout mice
with RSV resulted in a Th2-biased response and increased lung
pathology compared to that of wild-type animals despite a
similar level of replication (32). This suggests that, in the nat-
ural host, antagonism of the IFN response by NS1 and NS2
may increase the Th2/Th1 balance.
The level of activation of NF-?B in epithelial cells in re-
sponse to RSV infection was greatly reduced by deleting the
NS2 gene (141). How the expression of NS2 increases the
activation of this transcription factor is unknown, although it
might be related to the recent finding that the expression of
NS1 and/or NS2 activates the phosphoinositide 3-kinase
(PI3K) pathway (11). This is an intracellular signaling pathway,
orchestrated by increased phosphorylation of the phospholipid
phosphatidylinositol on the inner face of the plasma mem-
brane, which enhances cell survival, among other effects. Sup-
pression of the expression of NS1 and/or NS2 by short inter-
fering RNAs or by viral gene deletion suppressed activation of
the PI3K pathway and resulted in accelerated apoptosis of
RSV-infected cells and a reduction in virus yield (11). By
activating the PI3K pathway, NS1 and NS2 increased the sur-
vival time of the infected cell and increased the yield of prog-
Recombinant BRSV lacking the NS1 and NS2 proteins was
highly attenuated but more immunogenic in bovines than the
wild-type virus (152). Improved immunogenicity might be due
to increased IFN signaling creating an adjuvant effect or more
rapid apoptosis leading to efficient cross-priming and antigen
presentation. Increased IFN signaling and apoptosis also
would attenuate viral replication.
G protein. The G protein has a number of features with roles
in immune evasion and attenuation of the immune response,
and these roles continue to be elucidated. G is an unusual viral
neutralization antigen in that very few individual G-specific
monoclonal antibodies efficiently neutralize infectivity; neu-
tralization requires multiple antibodies (103). The ectodomain
of G is extensively decorated with carbohydrate side chains and
contains many more potential acceptor sites for O-linked sug-
ars. Heterogeneity in the placement and structure of the sugar
side chains could introduce antigenic diversity by creating sub-
populations that differ in the efficiency of binding by particular
antibodies. In addition, the extensive sheath of sugar side
chains probably helps shield the polypeptide backbone from
As another immune evasion mechanism, the secreted form
of G (sG) was recently shown to act as a decoy that helps shield
virus from neutralization by RSV-specific antibodies (A.
Bukreyev, L. Yang, J. Fricke, B. R. Murphy, and P. L. Collins,
unpublished data). In cell culture, sG is secreted rapidly and in
considerable quantity such that, at 24 h postinfection, it ac-
counts for 80% of the released G protein compared to 20%
contained in progeny virus (67). This early, abundant expres-
sion suggests that, in vivo, sG might flood the local environ-
ment of the infected cell and saturate RSV-specific antibodies.
This provides the first description of a mechanism by which
RSV might evade neutralizing antibodies more efficiently than
other respiratory viruses.
The cystine noose in the G ectodomain contains a CX3C
motif embedded in a region that has limited sequence relat-
edness with the CX3C chemokine fractalkine (151). Frac-
talkine/CX3CL1 is produced in secreted and membrane-bound
forms that function as chemoattractant and cell adhesion mol-
ecules to mediate the influx of CX3CR1?leukocytes, which
include subsets of NK cells and CD4?and CD8?T lympho-
cytes. RSV G, which also is produced in secreted and mem-
brane-bound forms, was shown to have fractalkine-like che-
moattractant activity in vitro (151). In vivo, there is evidence
that G has the effect of a fractalkine antagonist. In mice,
infection with a mutant RSV in which the CX3C motif had
been ablated by a single-amino-acid substitution, or one that
lacked G protein altogether, was associated with a substantial
increase in pulmonary NK cells and CD4?and CD8?T cells
compared to wild-type RSV (63). Furthermore, when RSV-
specific responses were evaluated, ablation of the CX3C motif
in G was associated with a substantial increase in RSV-specific
pulmonary CD8?CTL that were functional in an in vitro
cell-killing assay. These data suggest that the RSV G protein
substantially reduces recruitment and functionality of pulmo-
nary CX3CR?leukocytes, including RSV-specific CTLs, pre-
sumably by competitively inhibiting the functions of host frac-
talkine. However, it should be noted that a second study found
the opposite effect, namely, that the G protein enhanced the
pulmonary CD8?T-cell response to RSV in mice, an effect
that depended on the conserved cystine noose (16). In any
event, deletion of the cystine noose and fractalkine domain
from RSV had little effect on the efficiency of viral replication
in mice, suggesting that their quantitative impact on virus rep-
lication might not be great (146).
The G protein also influences the innate response of epithe-
lial cells and antigen-presenting cells to RSV infection. Infec-
tion of human epithelial cells and monocytes with an RSV
mutant that does not make sG resulted in increased activation
of NF-?B and increased expression of inflammatory mediators,
such as IL-6, IL-8, and RANTES, compared to infection with
wild-type virus (5). This result suggests that sG normally down-
regulates this host innate response. A second study confirmed
that G strongly inhibits the NF-?B-mediated inflammatory re-
sponse to RSV in human monocytes and showed that this
effect required the cystine noose domain (119). Interestingly,
G also was found to suppress the inflammatory response to
known agonists of TLR2, TLR4, and TLR9 (119). Thus, the G
protein appears to act as a general TLR antagonist to down-
regulate inflammatory responses by a mechanism that is not
VOL. 82, 2008 MINIREVIEW2049
The G glycoprotein influences the pattern of lymphocyte
cytokine expression in murine models and could potentially
induce similar effects in selected humans. Immunization of
BALB/c mice with a recombinant vaccinia virus expressing
RSV G induces a Th2 CD4?T-lymphocyte response that leads
to pulmonary eosinophilia following RSV challenge (113). This
is the result of a V?14-restricted clonal response to a single
epitope in G (amino acids 185 to 198) (154). Interestingly,
immunization with vaccinia virus expressing sG induced even
greater airway eosinophilia postchallenge than vaccinia ex-
pressing wild-type G or membrane-anchored G (73). Vaccinia
virus expressing sG directly induced IL-5 and IL-13, producing
pulmonary eosinophilia, and enhanced mucus production post-
challenge, providing another indication that sG alters the host
response (73). Whether selected humans have biased re-
sponses to G or to other individual RSV proteins is not known
but is a potential factor in primary infections associated with
Studies with PVM, the murine relative of RSV, provided
evidence of an additional role for the G protein in pathogen-
esis (86). In the respiratory tracts of mice, wild-type PVM
replicates efficiently, causing severe disease and death. Virus
from which the G gene had been deleted replicated poorly and
did not cause disease. Virus from which the first 34 amino acids
of G (comprising the complete cytoplasmic domain) had been
deleted replicated as efficiently as the wild-type virus but, sur-
prisingly, was nonpathogenic at comparable doses. None of the
viruses produced a sG, and PVM G does not contain a CX3C
motif, indicating that these factors were not involved. This is
the first report of a disease-attenuating mutation in a pneumo-
virus that cannot be explained simply on the basis of a reduc-
tion in viral load. It may be that the cytoplasmic tail itself is an
important factor in virulence, perhaps initiating intracellular
signaling pathways that remain to be identified.
F protein. Even though the F protein has two cleavage sites,
activation appears to be readily achieved by ubiquitous cellular
proteases. There is no evidence that cleavage activation is a
limiting factor in RSV tissue tropism or pathogenesis, in con-
trast to well-known models, such as the paramyxovirus New-
castle disease virus and avian influenza A virus. In the case of
BRSV, the p27 fragment that is released by cleavage at the two
sites was found to be related by sequence and function to
tachykinins, a family of neuropeptides that promote airway
hypersensitivity and inflammation (166). However, there ap-
pears to be no similarity between the p27 fragment of human
RSV and tachykinins.
The RSV F protein has been shown to bind to, and initiate
signaling through, TLR4 and its accessory protein CD14 (90).
This effect presumably would oppose that of the TLR4 antag-
onism noted above for the G protein, suggesting that there is
a balance in RSV infection. TLR4/CD14 also binds lipopoly-
saccharide and surfactant protein A, among other ligands, and
is abundant on monocytes and DCs. Human airway epithelial
cell lines normally express very low levels of TLR4, but its
expression is increased in response to RSV infection, which
would provide for amplification of the inflammatory response
to infection (108). Interestingly, contact between a number of
different respiratory viruses or bacteria and the mouse epithe-
lium results in rapid inhibition of Na?transport, resulting in
apical fluid accumulation (89). This might be an epithelial
mechanism to dilute and remove irritants but might also con-
tribute to virus spread and disease. In the case of RSV, but not
the other pathogens, this phenomenon appears to be initiated
by ligation of the F protein with TLR4.
The overall effect of TLR4 on RSV pathogenesis is unclear.
Silencing the TLR4 gene in mice appeared to have no discern-
ible effect on viral replication, disease, or the host response
during infection with RSV or with its murine counterpart PVM
(33, 37). In humans, several studies have investigated a possi-
ble link between pediatric RSV disease and two coding poly-
morphisms in TLR4 that are associated with TLR4 hypore-
sponsiveness and increased susceptibility to bacterial infection
(6, 115, 126, 144). Tal et al. found that these two polymor-
phisms indeed were associated more frequently with RSV
bronchiolitis than with controls (144), consistent with a pro-
tective role for TLR4. However, Paulus et al. did not confirm
this association (115), and Puthothu et al. provided evidence of
an effect in the opposite direction, namely, that the same TLR4
polymorphisms might be associated with reduced rather than
increased RSV disease (126). Interestingly, in a recent study, a
cohort of largely premature, high-risk infants with confirmed
RSV disease was found to have remarkably high frequencies of
the two TLR4 polymorphisms (6). The authors suggest that
TLR4 hyporesponsiveness may be associated with increased
susceptibility to both premature birth (perhaps due to in utero
infection) and RSV disease.
An analysis of the antibody response to RSV infection in
humans provided evidence that the F protein is presented to
the immune system in two versions that induce antibody re-
sponses of comparable magnitudes, namely, a mature form, as
found in virions, and incorrectly folded forms that lack impor-
tant neutralization epitopes (132). The latter might represent
immature protein released from lysed cells or denatured ma-
ture protein, possibly related to the noted physical instability of
RSV. The substantial antibody response against incorrectly
folded forms raises the possibility that they might have the
effect of diverting and reducing immune recognition of con-
formationally correct protein. Whether this is unique to RSV
among respiratory viruses is not known.
SH protein. The SH protein does not seem to play a major
quantitative role in RSV replication and pathogenesis, since
deletion of SH from recombinant RSV had little effect on virus
production in vitro and resulted in only a small decrease in
replication in chimpanzees. Furthermore, the deletion of SH
from an attenuated vaccine candidate did not confer a further
attenuating effect in seronegative children (78). Molecular
modeling suggests that the SH protein may be an ion channel-
forming viroporin (84). Viroporins have been identified for a
number of viruses and play roles in virus assembly and release
as well as in pathogenesis and cytotoxicity. SH has been shown
to form pentamers and, when expressed in bacteria, to alter
membrane permeability (22, 117). Recent evidence indicates
that the expression of SH delays apoptosis, probably by inhib-
iting signaling from tumor necrosis factor ? (45). Combined
with the antiapoptotic effect of the NS proteins, this might
increase virus replication by prolonging cell survival and might
also delay antigen processing through cross-priming.
Effects on macrophages and DCs. Alveolar macrophages
represent one of the first lines of host defense against respira-
tory infection and are active in phagocytosis, secretion of mi-
crobicides and inflammatory cytokines, and antigen presenta-
tion. Myeloid DCs serve as major antigen-presenting cells, and
plasmacytoid DCs are a major source of IFN-?/?. Myeloid and
plasmacytoid DCs, and indeed all circulating immune cells, are
recruited to the respiratory mucosa during infection with RSV
and other respiratory viruses, and there is evidence of RSV
infection of DCs in vivo (Fig. 2) (50, 72). In vitro, RSV can
infect alveolar macrophages and DCs (from cord blood or
peripheral blood) at efficiencies of up to 50% and 30%, re-
spectively, although the efficiency of infection of DCs usually is
much lower (7, 56, 104). Infection or uptake can induce DC
maturation based on changes in the expression of surface
markers and in the secretion of chemokines and cytokines. For
the most part, this depends on viral infectivity.
RSV appears to interfere with the functions of macrophages
and DCs in a number of ways. First, RSV-infected monocyte-
derived myeloid DCs make very little IFN-?/? relative to that
made by HMPV-infected cells (56), and RSV blocks the in-
duction of plasmacytoid DC maturation and IFN production in
response to TLR7 and TLR9 agonists (134). Second, com-
pared to influenza virus and HPIV3, RSV induces a somewhat
different spectrum of cytokines from cord blood macrophages
and DCs; specifically, there was a reduced level of IL-12 and a
greater secretion of IL-10, IL-11, and prostaglandin E2, which
might suppress T-cell activation and favor an increased Th2/
Th1 balance (7, 114). Third, RSV infection of cord blood or
monocyte-derived myeloid DCs decreased their capacity to
activate CD4?T cells (7, 29). Surprisingly, part of this effect
was mediated by IFN-? and -? (19). Fourth, DCs persist and
even increase in number at the respiratory mucosa of infants
for up to 8 weeks following the resolution of disease, a finding
that also has been observed with the mouse model (10, 50).
This raises the possibility of a defect in the trafficking of DCs
from the lung to lymphoid tissue in response to RSV infection.
These observations suggest that RSV alters DC biology to
reduce the influence of IFNs, shift the Th2/Th1 balance, and
limit the maturation and possibly the mobility of these impor-
tant cells. These effects are compounded in the neonate be-
cause of the immaturity of the antigen-presenting cells. These
effects might alter the immune response quantitatively and
RSV is a highly infectious and prevalent virus. More than
other respiratory viruses, RSV infection can occur very early in
life despite maternal antibodies, and reinfection can readily
occur throughout life without significant antigenic change.
These capabilities likely involve a number of factors, some of
which are not unique to RSV. These include high infectivity,
tropism to the superficial respiratory epithelium, low invasive-
ness, the antibody decoy activity of sG, expression of type I
IFN antagonists, expression of fractalkine and TLR antago-
nists, inhibition of apoptosis by multiple viral proteins, the
unusual antigenic properties of G, a modest level of antigenic
variability, interference with normal macrophage and DC func-
tion, and possible additional deficiencies in the protective im-
mune response. Thus, RSV infects an anatomical site where
host defense is reduced in effectiveness and employs factors
that blunt the host response and provide for immune evasion.
Figure 3 depicts a speculative model of the impact of major
host and viral factors in RSV pathogenesis.
The unusual ability of the virus to evade maternal antibodies
and efficiently infect infants early in life is a major factor in
RSV disease. The young infant is less able to tolerate a severe
respiratory infection due to its small size and narrow airways.
This is aggravated by the tropism of RSV to the small bron-
chioles, which are particularly prone to obstruction. The young
infant also is less able to control infection because of immu-
nologic immaturity and the immunosuppressive effect of ma-
ternal antibodies. RSV colludes with the inherent vulnerabili-
ties of the neonatal immune system to induce an immune
response that is inadequate and short-lived, at least early in
life, with a particularly deficient IFN component. It seems
reasonable to suspect that neonatal infection of certain indi-
viduals may, depending on timing and genetic background,
meditate long-term pathogenic changes to the developing lung
and host response. Such an effect may not necessarily be
unique to RSV, but RSV is more able than other respiratory
viruses to infect the vulnerable neonate. It is possible that the
immunological imprint of this first infection is related to the
lifelong susceptibility to RSV reinfection.
There is a broad heterogeneity of RSV disease in primary
infection, encompassing upper respiratory tract symptoms,
lower respiratory tract symptoms of varying levels of severity
and with or without wheezing, death in rare cases, airway
reactivity that can last through childhood, and possible allergic
hypersensitivity. This does not seem to involve substantial dif-
ferences in virulence among circulating strains of RSV, but
host factors certainly are involved. As already noted, these
include conspicuous factors affecting the ability to endure and
control a respiratory infection, such as bronchopulmonary dys-
plasia and immunodeficiency. Other likely factors of a more
subtle nature include a predisposition for airway hypersensi-
tivity, a predilection for insufficient (e.g., reduced function of
TLR4 or surfactants) or exaggerated (e.g., increased expres-
sion of IL-4 or IL-8) immune responses, and unusually narrow
airways (98). The ongoing identification of genetic polymor-
phisms associated with increased (or decreased) risk of severe
RSV disease illustrates that numerous host factors contribute
to the variability in host susceptibility and pathogenesis. Even-
tually, genetic polymorphism analysis may help identify the risk
of serious disease.
The relative contribution of viral versus various host factors
to RSV pathogenesis remains controversial. Major proposed
features include direct viral cytopathology, exaggerated CTL
responses, imbalanced Th2/Th1 responses, exaggerated in-
flammatory responses, and insufficient or altered responses
due to young age or viral factors. Various studies have tended
to emphasize selected individual factors. Pathogenesis may
indeed be simple in some cases: for example, in some cases, the
virus might simply outrace host defense and quickly overwhelm
the young infant, such that viral cytopathology is predominant
(159). In most cases, however, there may not be a single pre-
dominant factor. Instead, there may be different relative con-
tributions from the various interacting viral and host factors
that will depend on the speed and magnitude of viral replica-
tion, the effectiveness of the host response, underlying predis-
positions toward aberrant, exaggerated, or deficient aspects of
the host response, maturational state, and other factors. Thus,
VOL. 82, 2008 MINIREVIEW2051
pathogenesis usually is multifactorial and varied (Fig. 3). A
better understanding of the viral and host determinants of
RSV disease will be important for designing vaccines and ther-
apeutic agents. In particular, defining the consequences of
primary infection at a time of developmental and immunolog-
ical immaturity may be a key to the riddle of RSV pathogen-
We thank Brian Murphy for helpful discussions. Only a subset of
relevant references could be accommodated, and we regret the many
The authors’ research was supported by the Intramural Research
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