Kinetics of viral replication and induction of host responses in ferrets differs
between ocular and intranasal routes of inoculation
Jessica A. Belser, Taronna R. Maines, Kortney M. Gustin, Jacqueline M. Katz, Terrence M. Tumpeyn
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
a r t i c l e i n f o
Received 16 October 2012
Returned to author for revisions
20 December 2012
Accepted 18 January 2013
Available online 13 February 2013
a b s t r a c t
While influenza viruses are typically considered respiratory pathogens, the ocular system represents a
secondary entry point for virus to establish a productive respiratory infection and the location for rare
instances of virus-induced conjunctivitis. We used the ferret model to conduct a side-by-side
comparison of virus infectivity, kinetics of viral replication, and induction of host responses following
inoculation by either the intranasal or ocular routes with two viruses, A/Netherlands/230/03 (H7N7)
and A/Panama/2007/99 (H3N2). We show that ocular inoculation resulted in delayed virus replication
and reduced levels of proinflammatory cytokine and chemokine transcript in respiratory tract but not
ocular tissues compared with intranasally inoculated animals. We identified numerous proinflamma-
tory mediators with known roles in ocular disease elicited in ferret eye tissue following influenza virus
infection. These findings provide a greater understanding of the modulation of host responses following
different inoculation routes and underscore the risk associated with ocular exposure to influenza
Published by Elsevier Inc.
The eye represents a potential site of influenza virus replica-
tion as well as a gateway for establishment of a respiratory
infection, even among viruses which do not display an ocular
tropism (Belser et al., 2013). While local host responses represent
the first line of defense against influenza virus infection, in vitro
and in vivo studies have been largely restricted to inoculation by
the respiratory route and analysis of cytokines solely within cell
types of the respiratory tract, limiting our understanding of
disease progression following non-respiratory infection routes
(de Jong et al., 2006; Maines et al., 2012; Skoner et al., 1999;
Svitek et al., 2008). The appearance of concurrent mild influenza-
like illness and conjunctivitis in humans infected by H7 viruses in
particular necessitates a greater understanding of the host
responses occurring in ocular tissue and the modulation of these
responsesfollowing ocular exposure
Koopmans et al., 2004).
We recently observed that despite efficient virus replication in
upper respiratory tract tissues, ferrets inoculated by the ocular
route displayed reduced clinical signs of infection and less
efficient transmission of virus by respiratory droplets compared
with ferrets inoculated by the intranasal route (Belser et al.,
2012). However, it was unknown if ocular inoculation results in
delayed kinetics of virus replication in ferret respiratory and
ocular tissues compared with intranasal inoculation and the effect
this might have on the induction of host responses. We inoculated
ferrets (Triple F Farms, 8–9 months old and serologically negative
to currently circulating influenza viruses) either intranasally (i.n.,
1 ml total volume) or ocularly (o.c., 100 ml of virus placed on the
surface of the right eye and massaged across the surface of the eye
within the conjunctival sac as described previously (Belser et al.,
2012)) with 106EID50 of either the highly pathogenic avian
influenza H7N7 virus A/Netherlands/230/03 (NL/230), or the
H3N2 human influenza virus A/Panama/2007/99 (Panama).
Tissues from the respiratory tract and ocular system were
collected on days 0.5, 2, and 6 post-inoculation (p.i.) to measure
both viral mRNA and infectious virus throughout the acute phase
of infection as previously described (Maines et al., 2012). Peak
influenza M1 gene mRNA levels were delayed in the nasal
turbinates for NL/230 and Panama viruses following ocular
inoculation compared with i.n. delivered virus, despite compar-
able viral titers detected in nasal washes, which are representa-
tive of infectious virus present throughout the upper respiratory
tract of an infected animal (Fig. 1A, Table 1). While viral
transcripts detected in the nasal turbinates were significantly
higher at early (12 h) timepoints in i.n. inoculated ferrets com-
pared with ocular inoculation, o.c. inoculated ferrets achieved
significantly higher viral transcript levels at later timepoints (day
6 p.i. NL/230, day 2 p.i. Panama, po0.005). In contrast, infectious
virus and viral mRNA levels were rarely detected in trachea or
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/yviro
0042-6822/$-see front matter Published by Elsevier Inc.
nCorresponding author at: Influenza Division, MS G-16, 1600 Clifton Rd. NE,
Atlanta, GA 30333, USA. Fax: þ1 404 639 2350.
E-mail address: email@example.com (T.M. Tumpey).
Virology 438 (2013) 56–60
lung tissues from o.c. inoculated ferrets (Fig. 1A, Table 1). It
should be noted that replication-independent drainage of virus
inoculum in i.n. and i.o. inoculated ferrets, an inherent limitation
of all in vivo models using a liquid suspension inoculum, could
contribute to the kinetics of virus dissemination observed here
(Belser et al., 2012). Future studies utilizing aerosol-based inocu-
lum delivery systems may allow for a more precise examination
of this property (Gustin et al., 2011).
With few exceptions, infectious influenza virus was detected
bilaterally by day 2 p.i. in all ocular tissues tested of one or more
ferrets regardless of virus subtype or inoculation route (Table 1).
In contrast to respiratory tract tissues, levels of viral mRNA
measured in the eye, conjunctiva, and conjunctival washes
were significantly greater in Panama virus-inoculated ferrets
compared with NL/230 virus-inoculated ferrets (Fig. 1B); inocu-
lation route did not affect virus detection or recovery in these
tissues. The detection of infectious virus present in both the
eye and surrounding conjunctiva in ferrets regardless of virus
subtype or inoculation route likely reflects a combination of
tissue-specific virus replication and circulation of virus from the
proximally located nasal cavity via the nasolacrimal ducts
(Belser et al., 2012; Chentoufi et al., 2010). Lower viral titers in
the eye and conjunctiva compared with viral mRNA in these
tissues is likely due to unreleased or defective viral particles in
infected cells, highlighting the importance of utilizing assays
which detect both RNA and infectious virus to assess virus
replication (Brooke et al., in press). In summary, we found that
infection by the ocular route resulted in a virus infection which
was more restricted to nasal turbinates and not trachea com-
pared with intranasal delivery. Ferrets inoculated o.c. exhibited
delayed peak virus replication in respiratory tract but not ocular
Fig. 1. Detection of influenza virus in respiratory tract and ocular tissues of ferrets inoculated by the intranasal or ocular routes. Ferrets were inoculated with 106EID50of
NL/230 or Panama virus delivered i.n. or i.o. Viral mRNA levels were measured in (A) respiratory tract samples (nasal turbinates, trachea, lungs) and (B) ocular samples
(right eye, right conjunctiva, and conjunctival wash) of ferrets (n¼2 per time point) by real-time RT-PCR and are presented as mean fold change7standard deviation
compared with values in mock-infected animals. Viral mRNA levels were measured in triplicate as previously described with the exception of ocular samples, for which
total RNA was extracted from clarified homogenates using a QIAamp Viral RNA kit (Qiagen) (Maines et al., 2012). Eye and conjunctiva represent the right tissue. * po0.05,
** po0.005 between intranasal and ocular samples taken from ferrets inoculated with homologous virus; y po0.05, yy po0.005 between NL/230 and Panama infected
ferrets inoculated by the same route determined by Student’s t test.
J.A. Belser et al. / Virology 438 (2013) 56–60
The delay in establishment of peak virus replication in
respiratory tissues in o.c. inoculated ferrets prompted us to
examine if coincident delays in induction of host responses were
also present in these ferrets. Intranasal inoculation with NL/230
or Panama virus resulted in earlier detection of IL-6, CXCL8, and
CXCL10 transcript in the nasal turbinates compared with ocular
inoculation, which resulted in delayed peak detection of cytokine
transcripts in this tissue, from day 2 to 6 for NL/230 virus and 0.5
to 2 for Panama virus (Fig. 2A). The absence of significant
upregulation of cytokine transcripts in trachea or lung tissue
following ocular inoculation was in agreement with the decreased
viral load observed in these tissues (Table 1, Fig. 1A, and data not
shown). Notably, reduced and delayed detection of IL-6 in the
nasal turbinates of o.c. inoculated ferrets supports previous
studies in the ferret model which have identified a role for this
cytokine in transmission and disease severity (Belser et al., 2012;
Kang et al., 2011; Maines et al., 2012; Svitek et al., 2008). Delays
in peak induction of proinflammatory cytokines in the upper
respiratory tract of o.c. inoculated ferrets supports an association
between the reduced clinical signs (including sneezing and
rhinorrhea) observed following ocular inoculation and could offer
an explanation for the poor virus transmissibility by respiratory
droplets observed following inoculation by the ocular route or i.n.
with a reduced (100 ml) initial virus inoculum (Belser et al., 2012).
While we and others have examined in vitro and ex vivo
induction of cytokines and chemokines following influenza virus
infection of numerous ocular cell types (Belser et al., 2011b; Chan
et al., 2010; Michaelis et al., 2009), the induction of these
transcripts following in vivo ocular infection have not been
previously examined. Furthermore, it was unknown if intranasal
infection was capable of eliciting host innate immune responses
in ocular tissue. Extracted RNA from clarified eye and conjunctival
tissue homogenates was measured for the expression of a panel of
proinflammatory cytokine and chemokines, with numerous cyto-
kine transcripts upregulated in the ferret eye following influenza
virus infection regardless of the inoculation route (Fig. 2B). Of
note, ocular inoculation resulted in significantly elevated levels of
IL-1a, a proinflammatory cytokine rapidly synthesized and
released following corneal trauma in the mouse and following
viral infection of human corneal epithelial cells, compared with
intranasal inoculation with either NL/230 or Panama viruses
(Chang et al., 2002; Lausch et al., 1996). Ocular inoculation with
Panama virus further induced significantly higher levels of several
chemokines (notably CXCL9-11) compared with intranasal inocu-
lation with this virus (po0.05, Fig. 2B); previous studies have
demonstrated a link between IL-1a induction and subsequent
synthesis of CXC chemokines in corneal cells (Yan et al., 1998).
IFNb and TNFa transcripts were also upregulated in influenza
virus-infected eyes; IL-10 transcript was detected in the eye of
naı ¨ve ferrets but substantial changes in transcript levels were not
observed following virus infection (data not shown). In contrast to
studies demonstrating induction of IL-6 in human and mouse
ocular tissue following stimulation, we found IL-6 transcript to be
constitutively expressed in the ferret eye but 42-fold induction
was not detected following virus inoculation by either route (data
not shown) (Belser et al., 2011b; Gamache et al., 1997; Kumar
et al., 2006; Lausch et al., 1996). IFNg, IL-1b, IL-4, and IL-12p40
transcript were not consistently detected in the eyes of ferrets
(data not shown).
The surrounding conjunctiva represents an additional ocular
source of proinflammatory mediators (Gamache et al., 1997).
Numerous cytokine transcripts were detected in this tissue,
including IFNa, IFNb, TNFa, IL-1a, IL-6, CXCL8, CXCL10, how-
ever significant increases in these transcript levels following
virus infection were not observed (data not shown). As sup-
ported by previous in vitro studies in human ocular cell types,
these results suggest that the eye, and not surrounding con-
junctiva, represents the primary source of inducible proinflam-
matory cytokines and chemokines in the ocular system
following influenza virus infection (Belser et al., 2011b). While
the increased levels of transcript observed were relatively
modest (most within 5-fold of mock), they are comparable to
the changes in magnitude of cytokines in these tissues follow-
ing trauma, stimulation, or viral infection shown in previous
Detection of influenza virus in respiratory tract and ocular tissues of ferrets inoculated by intranasal or ocular routes.
TissueDay p.i.NL/230 i.n.a
NL/230 o.c.Panama i.n.Panama o.c.
aRoute of inoculation: i.n. (106EID50/ml); o.c. (106EID50/100 ml). n¼6 ferrets for each virus and inoculation route (n¼2 per time point), with 2–4 samples tested for
bViral titers expressed per gram of tissue (trachea and lung) or ml (all other samples)7standard deviation. The mean viral titer of all ferrets with positive virus
isolation (denoted in parentheses) is shown. Eye and conjunctiva represent both right and left tissues. Trachea is inclusive of independent sections removed proximal to
the larynx and bronchi. Limit of virus detection is 1.5 (respiratory tissues) or 0.8 (ocular tissues) log10EID50/ml following serial titration in eggs (Maines et al., 2005).
J.A. Belser et al. / Virology 438 (2013) 56–60
studies (Gamache et al., 1997; Kumar et al., 2006; Lausch et al.,
While several cytokines have been implicated in the pathogenesis
of inflammatory eye disease, comparatively little is known about
the pathobiology of conjunctivitis and other ocular complications
caused by influenza virus infection (Wakefield and Lloyd, 1992).
Furthermore, the induction of innate responses following influenza
virus infection in immune privileged tissues such as the eye, which
limit self-damaging immune-mediated inflammation reactions, are
not well studied (Belser et al., 2011b; Holan, 2006; Niederkorn, 2006;
Stevenson et al., 1997). We identified elevated levels of numerous
cytokines and chemokines in the eyes of ferrets inoculated i.n. or o.c.
(Fig. 2B). Interestingly, we observed a greater induction of chemo-
kines in the ferret eye following o.c. inoculation with an H3N2 and
not H7N7 virus, despite the association of H7 and not H3 viruses with
ocular disease. This could be attributed to higher levels of viral mRNA
detected in H3N2 ocular tissue or the comparatively reduced ability
of HPAI H7N7 viruses to elicit host innate immune responses
compared with other virus subtypes (Belser et al., 2011a,2011b;
Friesenhagen et al., 2012). Despite the immune privileged state of the
anterior segment of the eye, local protective immunological reactions
are known to occur following wounding or stimulation (Forrester and
Xu, 2012; Holan, 2006); future work is needed to ascertain the
potential functional roles of these inflammatory mediators in influ-
enza virus-induced ocular infection.
Here, we show that inoculation of ferrets with human or avian
influenza virus by the ocular route results in an infection that is
generally restricted to upper respiratory tract tissues, with delays
in virus replication compared with intranasal inoculation. We
identified a delayed and weakened induction of proinflammatory
cytokines and chemokines in the nasal turbinates following
ocular inoculation, and confirmed that the cytokine response in
respiratory tract tissues is driven by viral replication. Further-
more, we demonstrate for the first time the elicitation of numer-
ous cytokines and chemokines associated with ocular disease in
influenza virus infected ferret eyes. Delayed kinetics of virus
replication, dissemination of virus, and induction of innate
immune responses following ocular compared with traditional
intranasal inoculation shown in this study are in agreement with
previous work which demonstrates that modulation of inocula-
tion route and volume is sufficient to cause altered disease
presentation and virus transmissibility in the ferret model
(Belser et al., 2012; Bodewes et al., 2011; Gustin et al., 2011).
Presence of both virus and proinflammatory cytokines in ocular
tissue following intranasal inoculation in ferrets and elevated
detection of viral and proinflammatory cytokine and chemokine
transcript in the eye of ferrets inoculated with either H3N2 or
H7N7 subtype viruses speaks to the capacity of multiple virus
subtypes to use the eye to infect, even those not typically
associated with an ocular tropism (Belser et al., 2013).
Fig. 2. Detection of proinflammatory cytokine and chemokine mRNA in ferret respiratory and ocular tissue following influenza virus infection. Ferrets were inoculated
with 106EID50of NL/230 or Panama virus delivered i.n. or o.c. Cytokine and chemokine mRNA expression in nasal turbinates (A) or the right eye (B) of ferrets (n¼2 per
time point) was measured in triplicate as previously described by real-time RT-PCR and presented as the mean fold changeþstandard deviation compared with mock-
infected animals (Maines et al., 2012). *, po0.05, ** po0.005 between intranasal and ocular samples taken from ferrets inoculated with homologous virus determined by
Student’s t test.
J.A. Belser et al. / Virology 438 (2013) 56–60
The authors thank Shannon Emery for expertise in real-time
RT-PCR analysis. K.M.G. received financial support from the Oak
Ridge Institute for Science and Education. The findings and
conclusions in this report are those of the authors and do not
necessarily reflect the views of the funding agency.
Belser, J.A., Bridges, C.B., Katz, J.M., Tumpey, T.M., 2009. Past, present, and possible
future human infection with influenza virus A subtype H7. Emerg. Infect. Dis.
Belser, J.A., Gustin, K.M., Maines, T.R., Pantin-Jackwood, M.J., Katz, J.M., Tumpey,
T.M., 2012. Influenza virus respiratory infection and transmission following
ocular inoculation in ferrets. PLoS Pathogens 8, e1002569.
Belser, J.A., Rota, P.A., Tumpey, T.M., 2013. Ocular tropism of respiratory viruses.
Microbiol. Mol. Biol. Rev. 77 (1).
Belser, J.A., Zeng, H., Katz, J.M., Tumpey, T.M., 2011a. Infection with highly
pathogenic H7 influenza viruses results in an attenuated proinflammatory
cytokine and chemokine response early after infection. J. Infect. Dis. 203,
Belser, J.A., Zeng, H., Katz, J.M., Tumpey, T.M., 2011b. Ocular tropism of influenza A
viruses: identification of H7 subtype-specific host responses in human
respiratory and ocular cells. J. Virol. 85, 10117–10125.
Bodewes, R., Kreijtz, J.H., van Amerongen, G., Fouchier, R.A., Osterhaus, A.D.,
Rimmelzwaan, G.F., Kuiken, T., 2011. Pathogenesis of Influenza A/H5N1 virus
infection in ferrets differs between intranasal and intratracheal routes of
inoculation. Am. J. Pathol. 179, 30–36.
Brooke, C.B., Ince, W.L., Wrammert, J., Ahmed, R., Wilson, P.C., Bennink, J.R.,
Yewdell, J.W., 2013. Most influenza A virions fail to express at least one
essential viral protein. J.Virol., http://dx.doi.org/10.1128/JVI.02284-12, in
Chan, M.C., Chan, R.W., Yu, W.C., Ho, C.C., Yuen, K.M., Fong, J.H., Tang, L.L., Lai,
W.W., Lo, A.C., Chui, W.H., Sihoe, A.D., Kwong, D.L., Wong, D.S., Tsao, G.S., Poon,
L.L., Guan, Y., Nicholls, J.M., Peiris, J.S., 2010. Tropism and innate host
responses of the 2009 pandemic H1N1 influenza virus in ex vivo and in vitro
cultures of human conjunctiva and respiratory tract. Am. J. Pathol. 176,
Chang, C.H., Huang, Y., Issekutz, A.C., Griffith, M., Lin, K.H., Anderson, R., 2002.
Interleukin-1alpha released from epithelial cells after adenovirus type 37
infection activates intercellular adhesion molecule 1 expression on human
vascular endothelial cells. J. Virol. 76, 427–431.
Chentoufi, A.A., Dasgupta, G., Nesburn, A.B., Bettahi, I., Binder, N.R., Choudhury,
Z.S., Chamberlain, W.D., Wechsler, S.L., BenMohamed, L., 2010. Nasolacrimal
duct closure modulates ocular mucosal and systemic CD4(þ) T-cell responses
induced following topical ocular or intranasal immunization. Clin. Vacc.
Immunol.: CVI 17, 342–353.
de Jong, M.D., Simmons, C.P., Thanh, T.T., Hien, V.M., Smith, G.J., Chau, T.N., Hoang,
D.M., Chau, N.V., Khanh, T.H., Dong, V.C., Qui, P.T., Cam, B.V., do, Ha, Guan, Q.,
Peiris, Y., Chinh, J.S., Hien, N.T., Farrar, J., T.T., 2006. Fatal outcome of human
influenza A (H5N1) is associated with high viral load and hypercytokinemia.
Nat. Med. 12, 1203–1207.
Forrester, J.V., Xu, H., 2012. Good news-bad news: the Yin and Yang of immune
privilege in the eye. Front. Immunol. 3, 338.
Friesenhagen, J., Boergeling, Y., Hrincius, E., Ludwig, S., Roth, J., Viemann, D., 2012.
Highly pathogenic avian influenza viruses inhibit effective immune responses
of human blood-derived macrophages. J. Leukocyte Biol. 92, 11–20.
Gamache, D.A., Dimitrijevich, S.D., Weimer, L.K., Lang, L.S., Spellman, J.M., Graff, G.,
Yanni, J.M., 1997. Secretion of proinflammatory cytokines by human conjunc-
tival epithelial cells. Ocular Immunol. Inflammation 5, 117–128.
Gustin, K.M., Belser, J.A., Wadford, D.A., Pearce, M.B., Katz, J.M., Tumpey, T.M.,
Maines, T.R., 2011. Influenza virus aerosol exposure and analytical system for
ferrets. Proc. Natl. Acad. Sci. USA 108, 8432–8437.
Holan, V., 2006. Corneal stromal cells selectively inhibit the production of certain
anti-inflammatory cytokines. Exp. Rev. Clin. Immunol. 2, 101–108.
Kang, Y.M., Song, B.M., Lee, J.S., Kim, H.S., Seo, S.H., 2011. Pandemic H1N1 influenza
virus causes a stronger inflammatory response than seasonal H1N1 influenza
virus in ferrets. Arch. Virol. 156, 759–767.
Koopmans, M., Wilbrink, B., Conyn, M., Natrop, G., van der Nat, H., Vennema, H.,
Meijer, A., van Steenbergen, J., Fouchier, R., Osterhaus, A., Bosman, A., 2004.
Transmission of H7N7 avian influenza A virus to human beings during a large
outbreak in commercial poultry farms in the Netherlands. Lancet 363,
Kumar, A., Zhang, J., Yu, F.S., 2006. Toll-like receptor 3 agonist poly(I:C)-induced
antiviral response in human corneal epithelial cells. Immunology 117, 11–21.
Lausch, R.N., Chen, S.H., Tumpey, T.M., Su, Y.H., Oakes, J.E., 1996. Early cytokine
synthesis in the excised mouse cornea. J. Interferon Cytokine Res. 16, 35–40.
Maines, T.R., Belser, J.A., Gustin, K.M., van Hoeven, N., Zeng, H., Svitek, N., von
Messling, V., Katz, J.M., Tumpey, T.M., 2012. Local innate immune responses
and influenza virus transmission and virulence in ferrets. J. Infect. Dis. 205,
Maines, T.R., Lu, X.H., Erb, S.M., Edwards, L., Guarner, J., Greer, P.W., Nguyen, D.C.,
Szretter, K.J., Chen, L.M., Thawatsupha, P., Chittaganpitch, M., Waicharoen, S.,
Nguyen, D.T., Nguyen, T., Nguyen, H.H., Kim, J.H., Hoang, L.T., Kang, C., Phuong,
L.S., Lim, W., Zaki, S., Donis, R.O., Cox, N.J., Katz, J.M., Tumpey, T.M., 2005. Avian
influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit
increased virulence in mammals. J. Virol. 79, 11788–11800.
Michaelis, M., Geiler, J., Klassert, D., Doerr, H.W., Cinatl Jr., J., 2009. Infection of
human retinal pigment epithelial cells with influenza A viruses. Invest.
Ophthalmol. Vis. Sci. 50, 5419–5425.
Niederkorn, J.Y., 2006. See no evil, hear no evil, do no evil: the lessons of immune
privilege. Nat. Immunol. 7, 354–359.
Skoner, D.P., Gentile, D.A., Patel, A., Doyle, W.J., 1999. Evidence for cytokine
mediation of disease expression in adults experimentally infected with
influenza A virus. J. Infect. Dis. 180, 10–14.
Stevenson, P.G., Hawke, S., Sloan, D.J., Bangham, C.R., 1997. The immunogenicity of
intracerebral virus infection depends on anatomical site. J. Virol. 71, 145–151.
Svitek, N., Rudd, P.A., Obojes, K., Pillet, S., von Messling, V., 2008. Severe seasonal
influenza in ferrets correlates with reduced interferon and increased IL-6
induction. Virology 376, 53–59.
Wakefield, D., Lloyd, A., 1992. The role of cytokines in the pathogenesis of
inflammatory eye disease. Cytokine 4, 1–5.
Yan, X.T., Tumpey, T.M., Kunkel, S.L., Oakes, J.E., Lausch, R.N., 1998. Role of MIP-2 in
neutrophil migration and tissue injury in the herpes simplex virus-1-infected
cornea. Invest. Ophthalmol. Vis. Sci. 39, 1854–1862.
J.A. Belser et al. / Virology 438 (2013) 56–60