Human parainfluenza virus serotypes differ in their kinetics of replication
and cytokine secretion in human tracheobronchial airway epithelium
Anne Schaap-Nutta, Rachael Liesmanb,c, Emmalene J. Bartletta, Margaret A. Scullb,c,
Peter L. Collinsa, Raymond J. Picklesb,c, Alexander C. Schmidta,n
aLaboratory of Infectious Diseases, RNA Viruses Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-2007, USA
bCystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill. Chapel Hill, NC 27599-7248, USA
cDepartment of Microbiology and Immunology, University of North Carolina at Chapel Hill. Chapel Hill, NC 27599-7248, USA
a r t i c l e i n f o
Received 21 May 2012
Returned to author for revisions
15 June 2012
Accepted 20 August 2012
Available online 7 September 2012
Human parainfluenza virus
Human airway epithelium
a b s t r a c t
Human parainfluenza viruses (PIVs) cause acute respiratory illness in children, the elderly, and
immunocompromised patients. PIV3 is a common cause of bronchiolitis and pneumonia, whereas
PIV1 and 2 are frequent causes of upper respiratory tract illness and croup. To assess how PIV1, 2, and
3 differ with regard to replication and induction of type I interferons, interleukin-6, and relevant
chemokines, we infected primary human airway epithelium (HAE) cultures from the same tissue
donors and examined replication kinetics and cytokine secretion. PIV1 replicated to high titer yet did
not induce cytokine secretion until late in infection, while PIV2 replicated less efficiently but induced
an early cytokine peak. PIV3 replicated to high titer but induced a slower rise in cytokine secretion. The
T cell chemoattractants CXCL10 and CXCL11 were the most abundant chemokines induced. Differences
in replication and cytokine secretion might explain some of the differences in PIV serotype-specific
pathogenesis and epidemiology.
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PIV infections are a common cause of acute respiratory illness
(ARI) in all age groups. In healthy young adults, illness is typically
mild, self-limited, and restricted to the upper respiratory tract. In
infants and young children, the elderly, and patients with cardi-
opulmonary disease or immunodeficiency, however, PIVs can
causeLRTI, including croup, bronchiolitis, and pneumonia
(Karron and Collins, 2007). Globally, LRTI is the most common
cause of under-five mortality, and viral LRTI represents a large
share of this burden of disease (Nair et al., 2010). PIVs and
respiratory syncytial virus (RSV) are the most frequently detected
viruses in lung tissue specimens from infants who died of LRTI (do
Carmo Debur et al., 2010).
The PIVs are enveloped, cytoplasmic viruses of Family Para-
myxoviridae with single-stranded negative-sense RNA genomes
of approximately 15 kb. Four different PIV serotypes exist, but
PIV4 is generally thought to be infrequently associated with
severe disease and its epidemiology is less well characterized
(Weinberg et al., 2009). PIV3, like RSV (another member of the
family), causes bronchiolitis and pneumonia in young infants
while PIV1 and PIV2 are best known for epidemics of croup (Marx
et al., 1997). Although PIV1 and PIV2 disease is seen most
commonly in 1- to 5-year-olds, hospitalization rates for all three
PIVs are highest in the first six months of life, with bronchiolitis,
fever/possible sepsis, URTI, pneumonia, croup, and apnea as the
most frequent discharge diagnoses (Weinberg et al., 2009).
PIV mortality is highest in bone marrow transplant (BMT)
patients, and PIVs and RSV are reported to be the most frequent
viral etiologies of respiratory illness in both pediatric and adult
Srinivasan et al., 2011). Immunohistochemistry (IHC) and virology
studies provide evidence that PIV replicates predominantly in
respiratory epithelial cells and that, in general, infection is
restricted to the respiratory tract (Bartlett et al., 2008; Schaap-
Nutt et al., 2010c; Zhang et al., 2005). Only in severely immuno-
compromised patients, such as patients with severe combined
immunodeficiency or following BMT, has systemic spread repro-
ducibly been detected by IHC (Madden et al., 2004).
The histopathology of viral bronchiolitis and pneumonia is not
known to have clear virus-specific differences, and there is wide
overlap between PIV and RSV lung pathology (do Carmo Debur
et al., 2010). Fatal RSV LRTI is dominated by mononuclear
cell infiltrates and strong neutrophil movement toward the
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nCorrespondence to: Laboratory of Infectious Diseases, RNA Viruses Section,
National Institute of Allergy and Infectious Diseases, National Institutes of Health,
50 South Drive, Room 6515, MSC 8007, Bethesda, MD 20892-2007, USA.
Fax: þ1 301 480 1268.
E-mail address: firstname.lastname@example.org (A.C. Schmidt).
Virology 433 (2012) 320–328
bronchiolar epithelium (Johnson et al., 2007; Welliver et al.,
2008). In a child who died from trauma one day after being
diagnosed with RSV LRTI, inflammatory infiltrates were found
around bronchial and pulmonary arterioles and consisted mostly
of monocytes, neutrophils, and double-negative T cells. Neutro-
phils were concentrated between arterioles and airways whereas
mononuclear cells were found in airways and lung parenchyma
(Johnson et al., 2007). Although RSV has often been described as
unique in that it induces a weak T helper type 1 (Th1) response
and a bias towards Th2, more recent studies suggest that this
observation might not be RSV specific but a consequence of
infection very early in life. For example, mucosal interleukin
(IL)-4, CCL4, and eotaxin concentrations were reported not to
differ significantly between RSV and PIV or influenza-infected
infants r3 months of age (Kristjansson et al., 2005).
PIVs encode one or more proteins that function to block cellular
innate responses to viral infection and gene expression, thus helping
the virus remain undetected in epithelial cells for as long as possible
and facilitating efficient virus replication. PIV1 and PIV3 (both of
genus Respirovirus) encode a nested set of C proteins, whereas PIV2
(genus Rubulavirus) encodes a V protein bearing a characteristic
cysteine-rich zinc finger. Although the V and C proteins are
unrelated in sequence or mechanism of action, both proteins block
the induction of type I interferons (IFN) as well as IFN-induced
signaling (Bartlett et al., 2010; Boonyaratanakornkit et al., 2011,
2009; Schaap-Nutt et al., 2010a,b; Schomacker et al., 2012). The
innate immune response to PIV infection helps restrict viral replica-
tion but also is thought to contribute to disease. Therefore, an
understanding of the cytokine response of epithelial cells—the cells
that support PIV replication—is important to understanding PIV
pathogenesis. Local production of inflammatory cytokines has been
described in case series of children with PIV disease, and nasal wash
IFN (Hall et al., 1978) and chemokines such as IL-8/CXCL8,
MIP1aþ1b/CCL3þ4, RANTES/CCL5, and CXCL9 (El Feghaly et al.,
2010; Gern et al., 2002). IL-8 and IP-10 concentrations were found to
have a positive correlation with PIV disease (El Feghaly et al., 2010;
Gern et al., 2002). PIV viral load also has been reported to correlate
with severity of illness (Utokaparch et al., 2011). However, the
kinetics of PIV replication and cytokine secretion have not, to our
knowledge, been compared in a single study in primary respiratory
epithelial cells. While increases in nasal wash (i.e., lumenal or apical)
cytokine concentrations are consistent with the neutrophil and
monocyte infiltrations seen in histopathology, chemotaxis and
transmigration of white cells from the blood stream likely depend
on local basolateral (rather than apical) cytokine concentrations,
which cannot be measured in vivo in humans. In addition, the
timing of the inflammatory response relative to virus replication is
impossible to establish in naturally acquired infection, and experi-
mental primary infection of PIV-naı ¨ve infants and young children
with wild-type PIV would be unethical. An additional layer of
complexity is that nasal wash and tracheal lavage fluids do not
permit the assessment of the relative contribution of hematopoietic
versus epithelial cells to cytokine production.
To examine PIV replication and apical and basolateral cytokine
secretion simultaneously in a well-controlled setting that mimics
infection in humans as closely as possible, we used an ex vivo
model of fully differentiated human ciliated airway epithelium
(HAE). HAE cells from human donors post mortem were grown on
a filter support at an air–liquid interface to yield an epithelium
that closely mimics the morphological and physiological char-
acteristics of HAE in vivo (Pickles et al., 1998). We have previously
shown that PIVs infect fully differentiated ciliated cells but not
basal cells or goblet cells, and they do so without causing
extensive cytopathic effect (Bartlett et al., 2008; Schaap-Nutt
et al., 2010c; Zhang et al., 2005, 2011). Here, we compared
replication and apical and basolateral cytokine secretion in
replicate HAE cultures from two donors infected with PIV1,
PIV2, or PIV3 in order to understand the contribution of bronchial
epithelial cells to the immune response and to PIV pathogenesis,
and to test whether the PIV serotypes differ in the inflammatory
response they induce.
2. Results and discussion
Side-by-side comparison of PIV replication in tracheobronchial
PIV1, 2, and 3 replication kinetics were compared in parallel in
human tracheobronchial airway epithelium cultures. For each
virus, three cultures from each of two individual donors were
infected apically, i.e., from the side that would correspond to the
lumenal surface. Virus stocks used in these infections were
inoculated at low multiplicity of infection (MOI) from low passage
viruses to minimize contamination by defective interfering (DI)
particles. Virus particles were purified from the cell culture
supernatant by centrifugation and banding in discontinuous
sucrose gradients to remove cytokines and other molecules
produced by the infected cultures. The ratio between the infec-
tivity titer and the titer of physical particles measured by
hemagglutination assay (Section 3) was similar for each of the
viruses, indicating that none of the viruses contained a dispropor-
tionate content of inactivated particles or DI particles.
Virus release into the apical and basolateral compartments was
monitored daily over the course of seven days after infection
(Fig. 1A). For all three viruses, apical wash titers peaked on day
2 or 3 post-infection (pi) and then plateaued or decreased slightly
over the next several days (Fig. 1A). PIV1- and PIV3-infected
epithelium yielded much higher apical wash titers (mean peak
values of 108.9and 107.9TCID50/ml, respectively) than PIV2-
infected cells (105.3TCID50/ml). Previous studies in HAE suggested
that PIV1 replicated better than PIV2, but those studies involved
different donors and could not be directly compared (Bartlett et al.,
2008; Schaap-Nutt et al., 2010c). PIV3 replication in HAE was
previously shown to peak at day 2 pi, though at a lower peak titer
than in the present experiment (Palermo et al., 2009). PIV1, 2, and
3 exhibit similar growth kinetics in standard cell lines, reaching
peak titers of 108–109log10TCID50/ml (see Supplemental Fig. S1).
Since high titers of PIV and RSV replication in vivo generally
correlates with the severity of disease and contributes to transmis-
sibility (Karron et al., 1997; Utokaparch et al., 2011), the lower
level of replication observed with PIV2 could explain why this virus
causes the least burden of disease of these three serotypes
(Weinberg et al., 2009). For any serotype, virus replication was
similar in the two donors examined (Fig. 1A).
Fig. 1. (A) Replication of PIV1, PIV2, and PIV3 in HAE. HAE were infected via the apical surface and titers of virus shed into the apical compartment were determined at the
indicated times post-infection (pi). Virus titers shown are the means of three cultures per individual tissue donor 7SE (standard error). The limit of detection was 1.2 log10
TCID50/ml. (B) Apical cytokine secretion patterns of PIV-infected HAE. After apical infection, samples from the apical compartment were analyzed for protein
concentrations of secreted IFN-a(2a), IFN-b, IL-6, MCP-1 (CCL2), RANTES (CCL5), IP-10 (CXCL10), and I-TAC (CXCL11) by sandwich immunoassay using MesoScale
Discovery cytokine detection assays. Mean cytokine concentrations are expressed in pg/ml 7SE (triplicate cultures from each donor). Solid lines (
concentrations for PIV-infected cultures, and dashed lines (
) show cytokine concentrations from mock-infected cultures from the same donor. The lower limit of
detection was 2.4 pg/ml. Cytokine concentrations in infected culture fluids were compared to those in mock-infected cultures using two-way analysis of variance (ANOVA)
with Bonferroni post-test; an asterisk (*) indicates a significant difference, Po0.05.
) show mean cytokine
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En face immunostaining for PIV antigens showed a significant
number of cells staining positive for each of the three PIVs
through day 7 pi (25% HPIV1þ, 9% HPIV2þ, and 14% HPIV3þ),
and the ranking of PIV positive cells for each serotype correlated
with the ranking of virus titers in the apical compartment
(PIV14PIV34PIV2, data not shown). However, the difference
in the number of infected cells (3-fold between PIV1 and PIV2)
did not explain the much larger difference in peak virus titers
(4000-fold between PIV1 and PIV2), suggesting that the virus
yield per infected cell is the major determinant of the peak
Previous studies of PIV infection of HAE indicated that PIVs are
primarily released from the apical surface of ciliated HAE, and this
directional budding was proposed to help limit PIV replication to
the respiratory tract (Bartlett et al., 2008; Palermo et al., 2009;
Schaap-Nutt et al., 2010c; Zhang et al., 2005, 2011). Our study
confirmed that apically infected HAE cultures release virus from
the apical/lumenal surface only. None of the three PIVs could be
detected in the basolateral compartment (data not shown).
Although our data suggest that polarized budding and possibly
the innate epithelial immune response contribute to the absence
of viremia and systemic spread in infected individuals, it remains
to be elucidated whether the systemic spread seen in severely
immunodeficient patients is due to a leaky epithelium (possibly
as a result of the adaptive immune response) or the ability of the
virus to leave the respiratory tract within leukocytes, e.g. within
mucosal dendritic cells (Le Nouen et al., 2009).
2.1. Cytokine secretion from tracheobronchial epithelium in
response to PIV infection
One of the principal triggers of the cellular innate response to
viral infection is the detection of viral nucleic acid by cytosolic
pattern recognition receptors such as RIG-I and MDA5, which
initiate signaling cascades that lead to the activation of cellular
transcription factors and expression of type I IFNs, resulting in the
induction of an antiviral state in the infected cell as well as
neighboring cells, and expression of pro-inflammatory cytokines
(Boonyaratanakornkit et al., 2011; Schaap-Nutt et al., 2011). Side-
by-side comparison of cytokine secretion showed that PIV2
induced significant apical and basolateral IFN-a secretion by
day 2 pi, while PIV3 took three days to induce basolateral IFN-
a, and PIV1 did not induce much apical IFN-a at all (Fig. 1B and
Fig. 2). This was particularly surprising because PIV2 titers on day
2 pi were approximately 1000-fold lower than those of PIV1 and
PIV3 (Fig. 1A). Whether the relative restriction of PIV2 is a viral
property (i.e., a lack of viral fitness), or an effect of the early IFN-a
response on PIV2 replication, or both, cannot be deduced from
this study. However, using PIV1 mutants that cannot inhibit the
IFN response as well as wild-type PIV1, we previously observed
that the type I IFN response can restrict PIV1 replication in HAE
approximately 100-fold (Bartlett et al., 2008). Similar to our
results, PIV3 was previously shown to induce type I IFN release
at the basolateral surface, while UV-inactivated PIV3 did not
induce IFN (Zhang et al., 2011).
IFN-b induction was modest in HAE, regardless of the PIV
serotype (Fig. 1B and Fig. 2). PIV1 did not induce a significant
amount of IFN-b in either donor. Compared to mock-infected
cells, PIV2 and PIV3 induced 3.5-fold and 2.3-fold rises in apical
IFN-b, respectively, but only in donor 2 was this rise statistically
significant. We and others had previously reported that the PIV1 C
proteins inhibit the induction of type I IFNs in HAE (Bartlett et al.,
2008) and showed that in the respiratory A549 cell line this
phenotype is due to the inhibition of viral dsRNA synthesis, which
results indelayed activation
(Boonyaratanakornkit et al., 2011). Although viral dsRNA accu-
mulation was not examined here, the lack of type I IFN induction
by PIV1 suggests that the underlying mechanism in HAE might be
the same (Bartlett et al., 2008). For PIV2, the binding of the viral V
protein to MDA5 is known to play a role in inhibiting IFN-b
expression (Schaap-Nutt et al., 2011).
IL-6 plays an important role in acute and chronic inflammation
since it controls the shift between the recruitment of neutrophils
(early) and monocytes (late) to the site of inflammation (Silver
and Hunter, 2010). In children with PIV-positive ARI, IL-6 con-
centrations were found to be significantly elevated in nasal wash
fluid (El Feghaly et al., 2010), and elevated serum IL-6 has been
associated with prolonged hospital stays for RSV bronchiolitis
(Vieira et al., 2010). In our study, IL-6 was significantly elevated
only in HAE infected with PIV2 or PIV3 (except for a single time
point for PIV1), and the kinetics of basolateral IL-6 secretion were
similar to those observed for IFN-a: in PIV2 infected cells,
basolateral IL-6 rose early and then waned whereas concentra-
tions in PIV3 infected cells increased gradually through day 5 pi
(Fig. 2). For PIV1, we previously observed that the viral C proteins
strongly inhibit the activation of transcription factors such as IRF3
and NF-kB that are needed for both IL-6 and IFN-a expression
(Boonyaratanakornkit et al., 2011; Van Cleve et al., 2006).
Since the peak apical IL-6 secretion in donor 2 HAE was
significantly higher than in donor 1 HAE, and because a poly-
morphism in the IL-6 promoter at position ?174 was reported to
be associated with IL-6 expression and with a number of inflam-
matory diseases (Fishman et al., 1998), we genotyped the two
donors for this polymorphism. Donor 1 had a G/C genotype, while
donor 2 (with higher levels of IL-6 in the apical compartment) had
a G/G genotype (data not shown). The C allele at ?174 has been
associated with lower IL-6 expression, more severe symptoms
following rhinovirus and RSV infection, and an increased fre-
quency of otitis media (Alper et al., 2009; Doyle et al., 2010). In
general, HAE from donor 2 secreted higher levels of most of the
cytokines examined than HAE from donor 1 did, suggesting a
stronger pro-inflammatory response by donor 2. Although the
genotype of donor 1 could explain the lower apical secretion of IL-
6 by HAE cells of donor 1, it is important to point out that our
results do not determine an association between PIV-induced IL-6
secretion and the examined polymorphism; a study of that type
would require a very large sample size and would likely not be
We also examined the kinetics of expression of two CC
chemokines that are thought to play a role in viral ARI
(McNamara et al., 2005): MCP-1/CCL2, which acts as a macro-
phage chemoattractant, and RANTES/CCL5, which attracts mono-
cytes, T-helper cells and eosinophils (Fig. 1B and Fig. 2). Apically,
MCP-1 was not significantly induced by any of the PIVs (Fig. 1B),
similar to what was reported for MCP-1 in PIV-infected children
(El Feghaly et al., 2010). Basolateral MCP-1 concentrations were
elevated on days 2 and 3 in PIV2-infected epithelium and through
day 5 in PIV3-infected HAE (Fig. 2). RANTES was secreted by the
of the MDA5/IFN pathway
Fig. 2. Basolateral cytokine secretion patterns of PIV-infected HAE cells. After apical infection, cytokine concentrations in the basolateral compartment of HAE cultures
were determined for the indicated proteins, as described in Fig. 1B. Mean cytokine concentrations are expressed in pg/ml 7SE (triplicate cultures from each donor). Solid
) show mean cytokine concentrations for PIV-infected cultures, and dashed lines (
donor. The lower limit of detection was 2.4 pg/ml. The amount of I-TAC/CXCL11 secreted basolaterally from HPIV3-infected cultures from donor 1 was above the limit of
detection for this assay (4105pg/ml) at day 5 pi in all three replicates and is therefore reported as ‘‘ADL’’ (above detection limit). Cytokine concentrations in infected
culture fluids were compared to those in mock-infected cultures using two-way analysis of variance (ANOVA) with Bonferroni post-test; an asterisk (*) indicates a
significant difference, Po0.05.
) show cytokine concentrations from mock-infected cultures from the same
A. Schaap-Nutt et al. / Virology 433 (2012) 320–328
HAE in response to all three of the viruses, both apically and
basolaterally. Basolateral RANTES secretion kinetics followed the
kinetics of the majority of cytokines, i.e., it peaked early in PIV2
infected HAE, increased steadily in PIV3 infected cells, and was
not significantly elevated in PIV1-infected cells until day 5 pi
(Fig. 2). Peak RANTES concentrations ranged from 580 to 2130 pg/
ml apically (10- to 30-fold over mock) and from 660 to 3220 pg/
ml (17- to 85-fold over mock) basolaterally. Previously, a six-fold
rise in RANTES levels was detected basolaterally in RSV-infected
HAE at 2 day pi, while no significant rise was detected in the
apical supernatants (Mellow et al., 2004). A separate study in
RSV-infected bronchial epithelium found much lower peak con-
centrations than in the present study (Oshansky et al., 2010), but
this difference is likely due to the different time periods examined
(1 day versus 5 days). In our hands, most cytokines did not peak
until 2 day pi, and for PIV1 and PIV3 the concentrations increased
until the last day of sampling (day 5). We feel that the longer
follow-up is meaningful since severe PIV and RSV disease is
generally not seen until 2 to 3 day past the onset of URTI
symptoms, which is likely 5–7 days pi. Our results do agree with
findings in the nasal wash fluid of children naturally infected with
RSV (Becker et al., 1997; Noah et al., 1995).
Finally, we examined the kinetics of expression of three CXC
chemokines (IL-8/CXCL8 [not shown], IP-10/CXCL10, and I-TAC/
CXCL11), based on previous clinical reports on their induction
during viral ARI (McNamara et al., 2005) (Fig. 1B and Fig. 2). IP-10
and I-TAC are structurally and functionally related chemokines
that attract CXCR3-positive activated Th1 cells to the mucosa
(Groom and Luster, 2011). In addition to attracting Th1 cells,
these CXCR3 ligands also antagonize CCR3-mediated Th2 cell
migration, further enhancing Th1 polarization of effector T cell
recruitment (Loetscher et al., 2001; Sallusto et al., 1998; Xanthou
et al., 2003). With regards to respiratory illness, IP-10 has been
suggested as a biomarker for viral (as opposed to non-viral) ARI,
and our data indicate that all three PIV serotypes are potent
inducers of IP-10 (Bafadhel et al., 2011; Sumino et al., 2010). CXC
chemokine concentrations were much higher than those of the CC
chemokines, with basolateral IP-10 and I-TAC concentrations
above 100 and 50 ng/mL, respectively. Again, the kinetics of
basolateral secretion differed by PIV serotype, with PIV2 inducing
an early peak, PIV3 causing a slow rise, and PIV1 remaining
undetected until day 5 pi. IP-10 concentrations as high as we
observed were reported in BAL fluid from RSV-infected infants
with severe bronchiolitis, and it was speculated that neutrophils
might be the source of this cytokine since they were the
predominant cell type in BAL fluid (McNamara et al., 2005). Our
data indicate that the airway epithelium itself is an important
source of IP-10 following PIV infection, and that neither neutro-
phils nor IFN-g (the best known inducer of IP-10) from immune
cells are needed for induction. IP-10 and I-TAC were predomi-
nantly secreted into the basolateral compartment (Fig. 1B and
Fig. 2), in line with the idea that CXCR3 ligands facilitate
transmigration of Th1 cells across the endothelium and into
inflamed tissue (Xie et al., 2003). High levels of IP-10 and I-TAC
induction were reported in response to infection with either RSV
or influenza A viruses in human airway epithelial cells, also with
marked basolateral polarization (Ioannidis et al., 2012). Potent I-
TAC induction was also recently reported for influenza A virus
infection of primary, differentiated type II alveolar cells in culture
(Wang et al., 2011), supporting the notion that CXCR3 ligands are
the most abundant chemokines in viral ARI and an important
determinant of a Th1-dominated adaptive immune response.
IL-8, the third CXC chemokine examined here, is a neutrophil
chemoattractant and is elevated during RSV and PIV ARI in
children (El Feghaly et al., 2010; Larranaga et al., 2009;
McNamara et al., 2005). Unfortunately, in our study, IL-8
concentrations were very high in all cultures, infected or not,
from day 1 pi onwards (not shown), which might have been
induced by manipulation of the cultures as has been previously
reported (Mellow et al., 2004).
In summary, our study represents the first side-by-side com-
parison of PIV serotype-specific induction of cytokines in primary
well-differentiated human airway epithelium. We detected
serotype-specific differences in virus replication as well as in
the kinetics of cytokine induction. The kinetics of the innate
immune responses suggested that (i) PIV1 is better able than PIV2
and PIV3 to stay undetected for several days post-infection; (ii)
PIV2 is less able than PIV1 and PIV3 to inhibit an early immune
response, and (iii) PIV3 induces a slow but steady increase in
basolaterally-secreted cytokines. We report that IFN-g from
immune cells is not necessary for the induction of IP-10 in PIV
infection, and our findings confirm previous clinical reports that
CXCR3 ligands such as IP-10 and I-TAC are dominant chemokines
in PIV infection, and likely mediate recruitment of Th1 cells into
the infected epithelium. With regard to the potential clinical
implications of the differences in cytokine secretion kinetics, one
could speculate that the ability of PIV1 to stay undetected for so
long contributes to its ability to re-infect PIV1-experienced
children and adults more efficiently than PIV2 and PIV3
(Clements et al., 1991; Smith et al., 1966; Tremonti et al., 1968).
PIV2 stands out in that an early innate immune response is
followed by restricted replication, a finding that could explain
why PIV2 circulates less widely and is responsible for less disease
than PIV1 and PIV3. PIV3 was notable for its ability to induce a
steadily increasing inflammatory response over several days,
which fits well with the observation that PIV3 is a more frequent
etiology of bronchiolitis and pneumonia than PIV1 and 2.
3. Materials and methods
3.1. Cells and viruses
Human tracheobronchial epithelial cells were isolated from
airway specimens of patients without underlying lung disease by
the UNC Cystic Fibrosis Center Tissue Culture Core, which
obtained airway specimens with informed consent under Uni-
versity of North Carolina at Chapel Hill Institutional Review
Board-approved protocols from the National Disease Research
Interchange (NDRI, Philadelphia, PA) or as excess tissue following
lung transplantation. Primary cells were expanded on plastic for
one passage and plated at a density of 3?105cells per well on
permeable Transwell-Col (12 mm-diameter) supports. HAE cul-
tures were grown at an air–liquid interface for 4–6 weeks to form
differentiated, polarized cultures, as previously described (Pickles
et al., 1998).
Recombinant (r) PIVs were derived from clinical isolates: rPIV1
was from strain Washington/20993/1964 (Bartlett et al., 2005);
rPIV2 was from strain V9412-6 (V94) (Skiadopoulos et al., 2003);
and rPIV3 was from the JS strain (Durbin et al., 1997). rPIV1 was
constructed from the biologically-derived PIV1 wild-type (wt)
strain Washington/20993/1964, which was isolated in primary
African green monkey (AGM) kidney cells from a clinical sample
from a child with upper respiratory tract illness and subsequently
passaged two additional times in primary AGMs kidney cells
(Murphy et al., 1975) and once in LLC-MK2 cells (Bartlett et al.,
2005). rPIV1, previously referred to as HPIV1LLC1 (GenBank ID:
AF457102), has a wt phenotype in AGMs (Bartlett et al., 2005).
rPIV2 was constructed from the biologically-derived HPIV2
strain V9412-6 (V94), kindly provided by Peter Wright of Van-
derbilt University. The V94 strain (Genbank ID: AF533010) was
isolated in Vero cells from the nasal wash specimen of an infected
A. Schaap-Nutt et al. / Virology 433 (2012) 320–328
infant and was biologically cloned and amplified on Vero cells for
a total of nine passages (Tao et al., 2000), followed by one passage
on LLC-MK2 cells (Skiadopoulos et al., 2003). rPIV2, previously
referred to as rV94(15T), was derived from an antigenomic cDNA
copy of the HPIV2 V94 genome and has a wt phenotype in AGMs
(Nolan et al., 2007). rPIV2 differs from HPIV2 V94 by the addition
of a phenotypically silent NotI restriction site in the upstream
non-translated region of the N gene (HPIV2 nucleotide positions
149-156) and a silent C to T substitution at position 6265 in the
coding region of the F gene (Nolan et al., 2007).
rPIV3 was constructed from HPIV3 JS strain (GenBank ID:
Z11575) and differs at seven nucleotide positions: six silent
changes as well as one coding change at amino acid 263 of the
HN gene (Durbin et al., 1997). The HPIV3 JS strain was isolated in
primary bovine embryonic kidney cells from a clinical isolate
(Wash/47885/57) from an infant with lower respiratory tract
illness, and the virus was subsequently biologically cloned and
passaged in primary AGM or rhesus monkey kidney cells (Belshe
and Hissom, 1982; Stokes et al., 1992; Stokes et al., 1993).
All three sucrose-purified virus stocks used for HAE infections
were inoculated from the second low MOI passage (‘‘working
pool’’) virus following plaque purification (HPIV3) or terminal
dilution (HPIV1 and HPIV2) of virus recovered from cDNA. Virus
titers are expressed as 50% tissue culture infectious dose (TCID50)
per ml and were determined by titration on LLC-MK2 cells. LLC-
MK2 cells were infected with serial 1/10 dilutions of virus at 32 1C
and hemadsorption with guinea pig red blood cells (gp RBCs) was
performed at day 7 post-infection. Titers of sucrose-purified virus
stocks were 7.7 log10 TCID50/ml (HPIV1), 8.1 log10 TCID50/ml
(HPIV2), and 9.8 log10 TCID50/ml (HPIV3). The ratio between
infectious viral particles and total viral particles was also calcu-
lated to confirm that the virus preparations did not have high
concentrations of DI particles, as previously described (Johnston,
1981; Yount et al., 2006). To determine total particle titers in
virus preparations, we used a hemagglutination (HA) assay based
on the binding of gp RBCs to the external HN protein of HPIV
particles. Viruses were diluted 1/2 in 0.5% gp RBCs and incubated
1 h at 4 1C. HA of RBCs indicates the presence of virus particles.
The infectivity to HA ratios were similar for all three viruses
(1.6?105for HPIV1, 2.0?105for HPIV2, and 3.1?105for HPIV3)
and indicate a low level of DI particle contamination.
3.2. Viral infection of HAE
Prior to infection, the apical surfaces of HAE cultures were
washed with phosphate-buffered saline (PBS) and fresh medium
was supplied to the basolateral compartments. PIVs diluted in
culture medium were applied to the apical surface at a MOI of
5.0 TCID50/cell (3?105cells per well) in a 200 ml inoculum. After
incubation for 2 h at 37 1C, the inoculum was removed, and cells
were washed three times for 5 min each with PBS and then
incubated at 37 1C. Virus and cytokines released into the apical
compartment were harvested by performing apical washes with
425 ml of media for 30 min at 37 1C. Basolateral samples were
collected directly from the basolateral compartment, and the
removed volume (500 ml out of 1 ml total volume) was replaced
with fresh media. Samples were collected at 2 h and on days 1–7
post-infection and stored at ?80 1C until analysis.
3.3. Cytokine measurements
Cytokine concentrations were determined using an electro-
chemiluminescence multiplex system, Sector Imager 2400 from
MesoScale Discovery, according to the manufacturer’s instruc-
tions. Custom human multiplex cytokine tissue culture kits (for
IFN-a(2a), IL-6, IL-8, IP-10, I-TAC, MCP-1, and RANTES) and
singleplex tissue culture kits for human IFN-b and human IP-10
were purchased from MesoScale. Cytokine concentrations were
determined based on a standard curve generated on each plate
using the manufacturer-supplied cytokine standards and ana-
lyzed using the Discovery Workbench 3.0 software (MesoScale).
3.4. Analysis of IL-6 G–174C polymorphism
Genomic DNA was isolated from HAE cells, and Custom Taq-
Man Genotyping Assays were designed using primer and probe
sequences 5’–TAGCCTCAATGACGACCTAAGCT (forward primer),
5’–GGGCTGATTGGAAACCTTATTAAG (reverse primer), TGTCTTG-
CGATGCTA (VIC probe, G allele), and TGTCTTGCCATGCTA (FAM
probe, C allele). Genotyping was performed using the TaqMan
Universal PCR Master mix according to the manufacturer’s pro-
tocol. Samples were run on a 7900HT fast real-time PCR system
and quantitative analysis was performed using the SDS 2.3 soft-
ware (all from Applied Biosystems).
We thank Susan Burkett, Sonja Surman, and Emerito Amaro-
Carambot for technical assistance. We also thank the directors
and teams of the UNC Cystic Fibrosis Center Tissue Culture Core,
the Morphology and Morphometry Core, and the Michael Hooker
Microscopy Facilityfor supplying
This work was supported by the National Institutes of Health
(Grant no. R01 HL77844 to R.J.P. and 5-T32-AI007419) and the
Intramural Research Program of the National Institute of Allergy
and Infectious Diseases at the National Institutes of Health. The
views expressed in this report are the personal opinions of the
authors and are not the official opinion of the National Institutes
of Health or the Department of Health and Human Services. The
funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
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